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Molecular characterization of photoreceptor peripherin-2 and rom-1 complexes and role in disc morphogenesis… Loewen, Christopher Jereme Ronald 2001

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MOLECULAR CHARACTERIZATION OF PERIPHERIN-2 A N D  ROM-1  MORPHOGENESIS  A N D  P H O T O R E C E P T O R  COMPLEXES A N D RETINAL  ROLE IN  DEGENERATION  by Christopher Jereme Ronald Loewen B . S c . ( H o n o u r s ) , University of British C o l u m b i a  A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F THE REQUIREMENTS FOR THE DEGREE Doctor of P h i l o s o p h y in THE FACULTY OF GRADUATE STUDIES D e p a r t m e n t of B i o c h e m i s t r y a n d M o l e c u l a r Biology  W e accept this thesis as conforming To the required  standard  THE UNIVERSITY OF B t a f S H COLUMBIA July  2001  © Christopher J.R. Loewen  5  ~zooi  DISC  in presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  Department of  ^ I O J ^ ' S K ?  trfohovbc  The university of British Columbia Vancouver, Canada  hlol&sy  11  ABSTRACT Peripherin-2  and  rom-1  are  homologous  integral  membrane  proteins of the t e t r a s p a n i n s u p e r f a m i l y that f o r m m u l t i s u b u n i t in t h e r i m s o f p h o t o r e c e p t o r o u t e r s e g m e n t d i s c s . for formation  and  forms  P e r i p h e r i n - 2 is c r i t i c a l  m a i n t e n a n c e of rod a n d c o n e d i s c s , w h i l e  i n v o l v e d in r e g u l a t i o n o f t h i s p r o c e s s . various  complexes  of  human  retinal  rom-1  is  M u t a t i o n s in p e r i p h e r i n - 2 c a u s e degeneration  pigmentosa (RP) and macular degeneration (MD).  including  retinitis  Peripherin-2 and rom-  1 c o n t a i n s e v e n h i g h l y c o n s e r v e d c y s t e i n e s in t h e i n t r a d i s c a l l o o p r e g i o n , t w o o f w h i c h in p e r i p h e r i n - 2 a r e l i n k e d t o R P . Each  of  these  cysteines  in  peripherin-2  has  been  individually  r e p l a c e d b y s e r i n e t o d e t e r m i n e its r o l e in f o l d i n g a n d s u b u n i t a s s e m b l y . S i x o f t h e s e v e n c o n s e r v e d c y s t e i n e r e s i d u e s in t h e  intradiscal loop  of  p e r i p h e r i n - 2 are essential for proper core t e t r a m e r f o r m a t i o n with r o m - 1 . T h e y likely constitute t h r e e i n t r a m o l e c u l a r disulfide b o n d s crucial to p r o p e r f o l d i n g of t h e s u b u n i t s .  M u t a t i o n s at C 1 6 5 a n d C 2 1 4 c a u s e R P .  T h e r e m a i n i n g c y s t e i n e , C 1 5 0 , is n o t i n v o l v e d in t e t r a m e r f o r m a t i o n , is s o l e l y r e s p o n s i b l e f o r d i s u l f i d e - m e d i a t e d o l i g o m e r i z a t i o n o f into higher o r d e r c o m p l e x e s . disulfide-mediated segments. higher-order  Core tetramer  oligomerization  RP-causing mutations oligomer  the  formation  and  for  and  tetramers  formation  is r e q u i r e d  targeting  to  in p e r i p h e r i n - 2  rod  prevent tetramer  cause mistargeting  to  but  rod  for  outer and inner  Ill segments.  D i s u l f i d e - m e d i a t e d o l i g o m e r i z a t i o n p l a y s a r o l e in d i s c r i m a n d  incisure formation and involves both noncovalent and covalent  (disulfide  bonds)  disulfide-  interactions.  mediated  Rom-1  oligomerization  by  functions  in  inhibiting  disulfide-linking  H u m a n RP caused by mutations  the  regulation  of of  tetramers.  in p e r i p h e r i n - 2 r e s u l t s f r o m ( 1 )  having  d e c r e a s e d l e v e l s o f p r o t e i n in t h e o u t e r s e g m e n t s , a n d ( 2 ) b y i n t e r f e r e n c e w i t h t h e f u n c t i o n o f W T p r o t e i n in o u t e r s e g m e n t s in a d o m i n a n t - n e g a t i v e manner.  iv  TABLE OF CONTENTS Abstract  ii  List of T a b l e s  ix  L i s t of F i g u r e s  x  L i s t of A b b r e v i a t i o n s  xiii  Acknowledgements  xvii  CHAPTER 1 - INTRODUCTION 1.1  1  T H E EYE  1  1.1.1  Rod and cone photoreceptors  1  1.1.2  Phototransduction  5  1.1.3  Disc morphogenesis  1.2  and shedding  IDENTIFICATION OF PERIPHERIN-2 AND R O M - 1  1.2.1  Initial  characterization  1.2.2  Initial  characterization  1.2.3  Tetraspanin  1.3  Retinal  1.3.2  Mutations  of peripherin-2  14  of rom-1  19  superfamily  21 22  diseases  and disease  in peripherin-2  genes  22  that cause retinal  disease  MOLECULAR CHARACTERIZATION OF PERIPHERIN-2 AND R O M - 1  1.4.1  Oligomeric  1.4.2  Molecular  1.4.3  Role for peripherin-2  in membrane  1.4.4  Role for peripherin-2  in flattening  1.5  14  HUMAN DISEASE  1.3.1  1.4  8  structure  and size  basis for digenic  ANIMAL MODELS  1.5.1  The rds mouse  1.5.2  Rom-1  knockout  25 28  28  RP  30 fusion membrane  31 vesicles  33 35  35 mouse  36  1.5.3  P216L  1.5.4  Peripherin-2:rom-1  1.5.5  Gene  1.6  peripherin-2  therapy  transgenic chimeric  mouse  37  mouse  37  on the rds mouse  39  THESIS INVESTIGATIONS  40  C H A P T E R 2 - R O L E O F C Y S T E I N E S IN  PERIPHERIN-2  STRUCTURE  42  2.1  INTRODUCTION  42  2.2  MATERIALS AND METHODS  44  2.2.1  Site-directed  mutagenesis  44  2.2.2  Heterologous  expression  45  2.2.3  Immunoprecipitation  2.2.4  Calculation  2.2.5  SDS-PAGE  2.3  and velocity  of sedimentation and Western  coefficients  47 48  blotting  49  RESULTS  49  2.3.1  Location  2.3.2  Expression  and disulfide  2.3.3  Interaction  with rom-1  2.3.4  Velocity  2.4  sedimentation  of cysteine  residues  sedimentation  49  dimerization  50 53  under  reducing  conditions  DISCUSSION  55 59  CHAPTER 3 - DISULFIDE-MEDIATED OLIGOMERIZATION OF PERIPHERIN-2 A N D ROM-1  66  3 . 1 INTRODUCTION  66  3.2  67  MATERIALS AND METHODS  3.2.1  ROS preparations  3.2.2  Dimer  3.2.3  DTT and NEM treatment  reduction  and monoclonal kinetics  Heterologous  67 67  of ROS and  purification 3.2.4  antibodies  immunoaffinity 68  COS-1 cell expression  69  vi 3.2.5  Velocity  3.2.6  Glutaraldehyde  3.2.7  Quantification  3.2.8  Subunit  crosslinked 3.2.9 3.3  sedimentation  70  crosslinking  70  of peripherin-2  composition  and rom-1  of disulfide-linked  in ROS and  71 glutaraldehyde  dimers  72  SDS-PAGE  and  Western  blotting  73  RESULTS  73  3.3.1  Reduction  3.3.2  Velocity  non-reducing 3.3.3  sedimentation  in ROS membranes of ROS peripherin-2  73 and rom-1  under  conditions  Velocity  peripherin-2  of peripherin-2  74  sedimentation  expressed  of peripherin-2,  in COS-1  cells  rom-1,  under  and  C150S-  non-reducing  conditions 3.3.4  79 Crosslinking  of ROS peripherin-2  under  nonreducing  conditions 3.3.5 rom-1  82 Denaturation,  subunits  isolation,  from  3.3.6  Disulfide  3.3.7  Crosslinking  and quantitation  of peripherin-2  ROS  dimer  formation  84 between  of peripherin-2  peripherin-2  and rom-1  under  and rom-1  87  DISCUSSION  89  C H A P T E R 4 - M O L E C U L A R A N A L Y S I S O F D I G E N I C RP 4.1  INTRODUCTION  4.2  MATERIALS AND METHODS  101  Plasmids  4.2.2  Hydrodynamic  characterization  4.2.3  Solubilization,  immunoprecipitation,  sedimentation  and heterologous  analysis  SDS-PAGE  99 99  4.2.1  4.2.4  86  reducing  conditions 3.4  and  and  of COS-1 Western  COS-1  cell expression  of L185P and  cell expressed blotting  peripherin-2  101 102  velocity proteins  104 105  Vll  4.3  RESULTS  4.3.1  Molecular  reducing 4.3.2  106  mass of L185P peripherin-2  Velocity  Velocity  nonreducing 4.3.4  4.4  106  sedimentation  of L185A-peripherin-2  of L185P-peripherin-2 sedimentation  and  L188P-rom-  with L188P-rom-l  of LI 85P peripherin-2  Ill  under  conditions  115  Oligomerization  complexes 4.3.5  disul'fide-  conditions  1, and interaction 4.3.3  under  by velocity  Interaction  of WT- and  L185P-peripherin-2:rom-1  sedimentation  117  of L185P-peripherin-2  with peripherin-2-1D4  DISCUSSION  ..119 121  CHAPTER 5 - EXPRESSION OF PERIPHERIN-2 V A R I A N T S T R A N S G E N I C XENOPUS  IN  LAEVIS  130  5.1  INTRODUCTION  130  5.2  MATERIALS AND METHODS  133  5.2.1  Molecular  biology  5.2.2  Monoclonal  5.2.3  Transgenesis,  5.2.4  Immuno-EM  5.2.5  Immunocytochemistry,  antibody  133 production  GFP screening,  135 and tadpole  rearing  136 con focal and  deconvolution  microscopy  137  5.2.6  Transfections  5.2.7  Biochemistry  5.3  and plasmids  138 138  RESULTS  140  5.3.1  Cloning  5.3.2  Molecular  5.3.3  Immunolocalization  5.3.4  Expression  Xenopus  135  rods  X. laevis peripherin-2 characterization  of Xenopus  of endogenous  of Xenopus  140 peripherin-2-GFP  peripherin-2  peripherin-2-GFP  in  ...140 141  transgenic 144  Vlll 5.3.5  Expression  of bovine  peripherin-2-GFP  in  transgenic  Xenopus 5.3.6 C214S 5.3.7  146 Transgenic  expression  of the Retinitis  Pigmentosa-causing  mutant Transgenic  148 expression  of the digenic  RP-causing  L185P  mutant  155  5.3.8  Transgenic  expression  of the RP-causing  5.5.9  Transgenic  expression  of Cl 50S peripherin-2  5.3.10  Expression  levels of transgenes  5.4  P216L  mutant  DISCUSSION  CHAPTER 6 - CONCLUSION  ....155 160 162 164 179  6.1  SUMMARY  179  6.2  ORGANIZATION OF PERIPHERIN-2 COMPLEXES IN THE D i s c RIM  182  6.3  CONSIDERATIONS FOR THE MOLECULAR BASIS FOR D i s c MORPHOGENESIS . 1 8 5  6.4  FUTURE DIRECTIONS  REFERENCES  193 196  LIST OF T A B L E S  T a b l e 1. P r o t e i n s i n v o l v e d w i t h r e t i n a l d i s e a s e s  24  Table 2. Peripherin-2 mutations associated with h u m a n retinopathies ...26 Table 3. S y n t h e t i c oligonucleotides used for P C R m u t a g e n e s i s  46  Table 4. S e d i m e n t a t i o n coefficients for peripherin-2 a n d peripherin2:rom-l complexes T a b l e 5. V e l o c i t y s e d i m e n t a t i o n of complexes  58 peripherin-2 and rom-1 core 113  LIST OF  FIGURES  F i g 1.  S t r u c t u r e of t h e v e r t e b r a t e e y e a n d retina  2  Fig 2 .  Vertebrate photoreceptor cells  4  Fig 3 .  M e c h a n i s m of p h o t o t r a n s d u c t i o n  6  Fig 4 .  Disc m o r p h o g e n e s i s  11  Fig 5.  Disc shedding and phagocytosis  13  Fig 6.  I m m u n o l o c a l i z a t i o n of p e r i p h e r i n - 2 to t h e d i s c r i m  16  Fig 7 .  Topological m o d e l for p e r i p h e r i n - 2 a n d r o m - 1  18  Fig 8 .  S u b u n i t a s s e m b l y model for digenic RP  32  Fig 9.  L o c a t i o n o f c y s t e i n e r e s i d u e s in p e r i p h e r i n - 2  43  Fig 1 0 .  E x p r e s s i o n a n d d i m e r i z a t i o n of W T a n d m u t a n t  Fig 1 1 .  C o a s s e m b l y of p e r i p h e r i n - 2 c y s t e i n e m u t a n t s w i t h r o m - 1  Fig 1 2 .  V e l o c i t y s e d i m e n t a t i o n a n a l y s i s of p e r i p h e r i n - 2 c y s t e i n e  peripherin-2....51  mutants Fig 1 3 .  57  R a t e o f r e d u c t i o n o f d i s u l f i d e - l i n k e d p e r i p h e r i n - 2 d i m e r s in R O S membranes  Fig 1 4 .  75  V e l o c i t y s e d i m e n t a t i o n a n d W e s t e r n blot a n a l y s i s of p e r i p h e r i n - 2 and rom-1 from reduced and nonreduced ROS membranes  Fig 1 5 .  54  77  V e l o c i t y s e d i m e n t a t i o n a n d W e s t e r n blot a n a l y s i s of heterologously expressed peripherin-2 and rom-1 from reduced a n d n o n r e d u c e d C O S - 1 cell m e m b r a n e s  Fig 1 6 .  81  C r o s s - l i n k i n g of p e r i p h e r i n - 2 a n d r o m - 1 f r o m r e d u c e d a n d nonreduced ROS membranes  83  Fig 1 7 .  A n a l y s i s o f s u b u n i t s i n v o l v e d in d i s u l f i d e - l i n k e d  dimers  Fig 1 8 .  G l u t a r a l d e h y d e crosslinking of s u b u n i t s within the core complex  Fig 1 9 .  85  88  Disulfide-mediated oligomerization model for peripherin-2 a n d rom-1  92  xi Fig 2 0 .  T o p o l o g i c a l m o d e l of p e r i p h e r i n - 2 a n d r o m - 1 s u b u n i t s  Fig 2 1 .  H y d r o d y n a m i c a n a l y s i s of C O S - 1 cell e x p r e s s e d L 1 8 5 P peripherin-2 mutant under reducing conditions  Fig 2 2 .  100  107  I m m u n o p r e c i p i t a t i o n , v e l o c i t y s e d i m e n t a t i o n a n d c r o s s l i n k i n g of the L185P peripherin-2 complex under reducing conditions.... 110  Fig 2 3 .  V e l o c i t y s e d i m e n t a t i o n of L 1 8 5 A p e r i p h e r i n - 2 a n d L 1 8 8 P r o m - 1 a n d c o p r e c i p i t a t i o n of L 1 8 5 P p e r i p h e r i n - 2 a n d L 1 8 8 P r o m - 1 under reducing conditions  Fig 2 4 .  114  V e l o c i t y s e d i m e n t a t i o n of L 1 8 5 P a n d W T p e r i p h e r i n - 2 u n d e r nonreducing conditions  Fig 2 5 .  V e l o c i t y s e d i m e n t a t i o n of W T p e r i p h e r i n - 2 : r o m - l  116 and  L 1 8 5 P : r o m - l complexes under nonreducing conditions Fig 2 6 .  Immunoprecipitation  118  a n d v e l o c i t y s e d i m e n t a t i o n of L 1 8 5 P  peripherin-2:WT peripherin-2-lD4 complexes under reducing and nonreducing conditions Fig 2 7 .  120  M o d e l for t h e o l i g o m e r i c s t r u c t u r e of p e r i p h e r i n - 2 a n d t h e L 1 8 5 P mutant  124  Fig 2 8 .  Topological model for X e n o p u s peripherin-2  132  Fig 2 9 .  M o l e c u l a r characterization of X e n o p u s p e r i p h e r i n - 2 - G F P fusion protein  Fig 3 0 .  E n d o g e n o u s X e n o p u s peripherin-2 localizes to rod a n d cone outer segments  Fig 3 1 .  145  I m m u n o - E M r e v e a l s t a r g e t i n g of X e n o p u s p e r i p h e r i n - 2 - G F P fusion protein to disc r i m s a n d i n c i s u r e s  Fig 3 3 .  143  X e n o p u s p e r i p h e r i n - 2 - G F P fusion protein targets to rod outer segment membranes  Fig 3 2 .  142  147  B o v i n e p e r i p h e r i n - 2 - G F P fusion protein targets to rod outer segment m e m b r a n e s and colocalizes with endogenous Xenopus peripherin-2  149  Xll  Fig 3 4 .  C 2 1 4 S p e r i p h e r i n - 2 m i s t a r g e t s to the rod inner s e g m e n t a n d cell b o d y , a n d d o e s not c a u s e m i s t a r g e t t i n g of e n d o g e n o u s peripherin-2  Fig 3 5 .  151  I m m u n o - E M r e v e a l s a c c u m u l a t i o n of X e n o p u s C 2 1 4 S p e r i p h e r i n 2 - G F P f u s i o n p r o t e i n a t t h e c o n n e c t i n g c i l i u m in t h e i n n e r segment  Fig 3 6 .  N o n - r e d u c i n g velocity s e d i m e n t a t i o n a n a l y s i s of C 2 1 4 S  152 and  P216L peripherin-2 Fig 3 7 .  L 1 8 5 P p e r i p h e r i n - 2 - G F P targets to the rod o u t e r s e g m e n t , the rod i n n e r s e g m e n t , a n d t h e cell b o d y  Fig 3 8 .  159  C 1 5 0 S p e r i p h e r i n - 2 - G F P targets to R O S , but d o e s not localize to disc incisures  Fig 4 1 .  158  I m m u n o - E M of rods e x p r e s s i n g X e n o p u s P 2 1 6 L peripherin-2-GFP  Fig 4 0 .  156  P 2 1 6 L p e r i p h e r i n - 2 - G F P t a r g e t s to the rod o u t e r s e g m e n t a n d causes rod degeneration  Fig 3 9 .  154  161  O v e r e x p r e s s i o n o f t h e X e n o p u s f u s i o n p r o t e i n s is n o t t h e c a u s e o f d e r e a l i z a t i o n t o t h e r o d i n n e r s e g m e n t o r r o d d e g e n e r a t i o n .. 1 6 3  Fig 4 2 .  M o l e c u l a r rationale for t a r g e t i n g of p e r i p h e r i n - 2 to R O S . .  177  Fig 4 3 .  O r g a n i z a t i o n o f p e r i p h e r i n in t h e d i s c r i m  183  Fig 4 4 .  Z i p p e r i n g m o d e l f o r d i s c m o r p h o g e n e s i s in r o d s  187  xm LIST OF  ABBREVIATIONS  ADRP  a u t o s o m a l d o m i n a n t retinitis  AMD  age-related macular dystrophy  ARRP  a u t o s o m a l recessive retinitis  BEM  Bull's-eye maculopathy  BME  pigmentosa  pigmentosa  (3-mercaptoethanol  bp  base pairs  BPD  butterfly-shaped pigment  BSA  bovine serum albumin  dystrophy  c  .conserved  C2-3  c y t o p l a s m i c loop joining t r a n s m e m b r a n e s e g m e n t s 2 a n d 3  CaM  calmodulin  cc  connecting cilium  CD  cone dystrophy  cDNA  D N A reverse transcribed from an m R N A template (coding DNA)  cGMP  guanosine 3',5' -cyclic monophosphate  CRD  cone-rod dystrophy  CSNB  congenital stationary night blindness  del  deletion  DNA  deoxyribonucleic acid  dpf  days post  DTT  fertilization  dithiothreitol  ECL  enhanced chemiluminescence  EM  electron microscopy  EMBL  European Molecular Biology Laboratory  ER  endoplasmic reticulum  ERG  electroretinogram  Fs  frameshift  xiv FTIR  Fourier transform infrared spectroscopy  GAP  GTPase activating  GARP  g l u t a m i c acid rich protein  GC  guanylate cyclase  GCAP  guanylate cyclase activating  GDP  guanosine 3'-diphosphate  GFP  green fluorescent protein  GST  glutathiones-transferase  GTP  guanosine 3'-triphosphate  protein  protein  Ig  immunoglobulin  IS  inner segment  kDa  kilodalton  I  lens  Ll-2  intradiscal loop joining t r a n s m e m b r a n e s e g m e n t s 1 a n d 2  L3-4  intradiscal loop between t r a n s m e m b r a n e s e g m e n t s 3 and 4  LCA  Leber's congenital amaurosis  LUV  large unilamellar vesicle  M II  metarhodopsin  Ml  transmembrane segment 1  M2  .transmembrane segment 2  M3  transmembrane segment 3  M4  transmembrane segment 4  MAb  monoclonal antibody  MBP  maltose binding  MD  macular dystrophy  mi  mitochondrion  M  relative molecular weight  r  mRNA  messenger RNA  n  nucleus  II  protein  XV  nc  nonconserved  NEM  N-ethyl  OS  outer segment  PAGE  p o l y a c r y l a m i d e gel electrophoresis  PBS  phosphate buffered saline  PCR  polymerase chain reaction  PD  pattern dystrophy  PDE  phosphodiesterase  PDI  protein disulfide isomerase  phag  phagosomes  PMSF  phenyl methyl sulfonyl fluoride  pp  pseudopodia  r.  retina  RCD  rod-cone dystrophy  rds  retinal degeneration slow  REMI  restriction endonuclease mediated  RK  rhodopsin kinase  RNA  ribonucleic acid  ROS  rod outer s e g m e n t  RP  retinitis p i g m e n t o s a  RPA  retinitis punctata albescens  RPE  retinal pigment  epithelium  S  S v e d b e r g (x I O '  1 3  s o,w  standardized velocity sedimentation coefficient  SDS  sodium dodecyl sulfate  stop  stop cod on  T  transducin  TM4SF  t r a n s m e m b r a n e four superfamily  TR  T e x a s red  2  maleimide  integration  s)  Tris  Tris  [hydroxymethyl]aminomethane  w/w  weight per weight  WGA  wheat germ agglutinin  WT  wild type  XVII  ACKNOWLEDGEMENTS I w o u l d like to give m y s i n c e r e s t gratitude to B o b M o l d a y for his excellent supervision throughout t h e course of m y PhD a n d for allowing his lab to be such a d y n a m i c a n d enjoyable place to work. I r e m e m b e r o n e of the first things y o u said to m e , B o b , a s a y o u n g m a n of t w e n t y thinking a b o u t a c a r e e r i n s c i e n c e , d u r i n g t h a t f i r s t i n t e r v i e w . It w a s t h a t s c i e n c e is n o t a n i n e - t o - f i v e j o b a n d t h a t h a r d w o r k is n o t n e c e s s a r i l y a s u b s t i t u t e f o r smart work. Taking that to heart I set m y work schedule, ten to three, a l w a y s k e e p i n g in m i n d h o w c l e v e r m y e x p e r i m e n t s w e r e . A l a s , I l e a r n e d s o o n e n o u g h t h a t w h a t y o u r e a l l y m e a n t w a s t h a t s c i e n c e is at least a n i n e - t o - f i v e j o b . I ' m g l a d I f i g u r e d it o u t ( a n d j u s t i n t i m e ) b e c a u s e h e r e I a m t o d a y , a P h D . N e v e r t h e l e s s , it h a s b e e n a t r u l y g r e a t e x p e r i e n c e . A l i t t l e a d v i c e f r o m a f e l l o w P h D , if y o u ' r e s t i l l g i v i n g t h a t s p e e c h t o s t u d e n t s l i k e m e - s t r e s s t h e at least p a r t ! It w i l l c e r t a i n l y d o t h e m s o m e good! Thanks Bob, y o u have been a great mentor. ...and of c o u r s e m y t h a n k s g o t o t h e rest o f t h e M o l d a y l a b , p a s t a n d present, for keeping things interesting, to s a y the least. I apologize ( a g a i n ) f o r t h e d i s h e s , t h e t i m e r , a n d t h e R a m o n e s ( L a u r i e ) . It h a s b e e n a p l e a s u r e w o r k i n g w i t h all of y o u . I w a n t t o e s p e c i a l l y t h a n k A n d y G o l d b e r g a n d O r s o n Moritz for their g u i d a n c e a n d p a t i e n c e a n d for all t h e effort t h e y p u t into this project. W e ' v e h a d a lot of f u n . A big t h a n k y o u t o Laurie Molday for doing so m u c h (everything) to keep the lab running smoothly a n d for teaching m e m a n y of the techniques I know today. Also thanks to Jinhi A h n , m y papers a n d thesis have far fewer typographical and grammatical errors - thank you. I w o u l d like to t h a n k m y f r i e n d s a n d f a m i l y , w h o h a v e b e e n a n d will c o n t i n u e to b e a l a r g e f o r c e in m y life, f o r all t h e i r s u p p o r t . Y o u k n o w w h o y o u a r e . S o m a n y w o n d e r f u l p e o p l e , I'm g l a d w e c o u l d a l l m e e t ( l i k e l y a t 1846 W12th Ave.). I want to especially thank m y parents, A g n e s , Gary, and Paul, a n d m y siblings S i m o n e , S h a n e , a n d Nicole, for giving love a n d e n c o u r a g e m e n t along the w a y , a n d for always r e m e m b e r i n g to ask, " S o a r e y o u still w o r k i n g o n t h o s e e y e b a l l s ? " . Y e s . Lastly, to m y beautiful wife S a r a h , I dedicate this thesis. Y o u have given m e unconditional love a n d support, a n d for that y o u have m y heart. Grow o l d a l o n g w i t h m e , t h e b e s t is y e t t o b e . T h a n k y o u f o r e v e r y t h i n g m y love, y o u are an inspiration.  Chapter 1 Introduction  1  CHAPTER 1 - INTRODUCTION 1.1  THE EYE The vertebrate  e y e is a s o p h i s t i c a t e d o p t i c a l d e v i c e d e s i g n e d b y  evolution over millions of years.  It f u n c t i o n s t o p r o j e c t  a n d focus the  i m a g e s f r o m o u r universe onto a delicate disc of neural tissue called t h e r e t i n a (Fig 1).  Light focused by t h e lens onto t h e retina at t h e back of  t h e e y e is c a p t u r e d c h e m i c a l l y b y t h e r o d a n d c o n e p h o t o r e c e p t o r s . vertebrate  retina  s y n a p s e s (Fig 1).  consists of three  layers  The  of cells a n d t w o layers  T h e photoreceptors transmit their information  of  (colour,  intensity, contrast) to the bipolar, a m a c r i n e , a n d horizontal cells of the inner retina.  T h e s e cells integrate the signals f r o m t h e entire retina a n d  transmit t h e m to the ganglion cells which c o m p r i s e t h e optic nerve.  As  m a n y a s f i f t y p h o t o r e c e p t o r s c a n s y n a p s e w i t h a s i n g l e b i p o l a r c e l l in t h e c o m p l e x p r o c e s s of i m a g e c o d i n g . A single g a n g l i o n cell c a n receive input from up to 75,000 rod photoreceptors.  It is t h e n t h e r e s p o n s i b i l i t y o f t h e  visual cortex of the brain to decode these integrated signals a n d recreate the i m a g e originally focused o n t h e retina (reviewed by Kolb, 1994).  1.1.1  Rod and cone The  two major  photoreceptors classes  of photoreceptors,  rods  and cones, are  r e s p o n s i b l e f o r v i s i o n in d i m l i g h t a n d c o l o u r v i s i o n , r e s p e c t i v e l y .  There  2  F i g 1. S t r u c t u r e of t h e v e r t e b r a t e e y e a n d r e t i n a . ( A ) L i g h t e n t e r s t h e e y e b y p a s s i n g t h r o u g h t h e t r a n s p a r e n t t i s s u e called t h e c o r n e a a n d is f o c u s e d by t h e lens onto t h e retina at t h e b a c k of t h e e y e . T h e r e , t h e p h o t o r e c e p t o r cells detect t h e light and t r a n s m i t a signal v i a t h e optic nerve to t h e brain. T h ec h o r o i d , t h e v a s c u l a r layer w h i c h p r o v i d e s nutrients to the retina, lies a d j a c e n t to t h e s c l e r a , a hard p r o t e c t i v e o u t e r layer, a n d t h e retinal p i g m e n t e p i t h e l i u m ( R P E ) which is in i n t i m a t e c o n t a c t with t h e p h o t o r e c e p t o r s . ( B ) T h e R P E is r e s p o n s i b l e f o r m a i n t e n a n c e o f t h e r o d ( d a r k blue) a n d cone ( r e d , g r e e n , a n d blue) photoreceptors which c o m p r i s e t h e o u t e r n u c l e a r layer. P h o t o r e c e p t o r s s y n a p s e w i t h t h e n e u r o n s of t h e i n n e r n u c l e a r l a y e r ( h o r i z o n t a l , bipolar, a n d a m a c r i n e cells) w h i c h in t u r n s y n a p s e with ganglion cells w h o s e axons comprise t h e optic nerve. Modified from Webvision (www.webvision.med.utah.edu).  Chapter 1 Introduction  3  a r e t h r e e s u b - c l a s s e s of c o n e s in h u m a n s t h a t r e s p o n d t o r e d , g r e e n a n d blue w a v e l e n g t h s of light.  Rods dominate the peripheral retina a n d are  a b s e n t in t h e f o v e a , a s m a l l r e g i o n o f t h e c e n t r a l r e t i n a w h i c h is t h e f o c a l point of t h e lens.  Cones are thinly dispersed throughout the peripheral  retina a n d a r e concentrated visual acuity.  in t h e f o v e a , w h i c h  is r e s p o n s i b l e f o r h i g h  T h e ratio o f r o d s to c o n e s in t h e h u m a n  retina  is 2 0 : 1  (Osterberg, 1935). Although  rods a n d cones vary  called rods a n d cones), they  in s h a p e a n d s i z e ( a n d a r e h e n c e  have a similar cellular organization.  c o n t a i n a s y n a p t i c t e r m i n a l , cell b o d y , i n n e r s e g m e n t , a n d o u t e r r e g i o n ( F i g 2).  T h e outer segment contains 5 0 0 - 2000  discs  separate  that  are  from  the  plasma  rim  and lamellar  proteins. which  regions,  e a c h of which  with the long  rods  l o c a t e d in t h e l a m e l l a r r e g i o n s . connecting  cilium, which  h a s its o w n c o m p l e m e n t  incisures  The number  of  T h e o u t e r s e g m e n t is s u p p o r t e d b y t h e  i s o l a t e s it f r o m  nucleus, and the synaptic terminus (reviewed  of  The opsin photopigments are  the mitochondria,  e n d o p l a s m i c reticulum of t h e inner s e g m e n t .  transmission  but  Discs a r e c o m p r i s e d of  axis of t h e photoreceptor.  incisures varies from species to species.  nerve  in  In r o d s , large i n v a g i n a t i o n s of t h e disc r i m p r o d u c e  align  segment  membraneous  membrane  c o n t i n u o u s w i t h t h e p l a s m a m e m b r a n e in c o n e s .  They  T h e cell b o d y c o n t a i n s t h e  is t h e s i t e o f g l u t a m a t e by  Golgi, and  Massey  &  release and  Maguire,  1995).  A  B asma embrane  disc rim  Rod  Cone  c  Fig 2 . Vertebrate photoreceptor cells. ( A ) R o d a n d c o n e n e u r o n s a r e c o m p r i s e d of a n outer s e g m e n t which contains a stack of m e m b r a n o u s discs. T h e s e d i s c s a r e s e p a r a t e f r o m t h e p l a s m a m e m b r a n e in r o d s a n d c o n t i n u o u s w i t h it i n c o n e s . T h e o u t e r s e g m e n t is s e p a r a t e d f r o m t h e i n n e r s e g m e n t b y a non-motile connecting cilium. T h e inner s e g m e n t contains t h e organelles, the cell b o d y c o n t a i n s t h e n u c l e u s , a n d t h e s y n a p t i c t e r m i n u s is t h e site of neurotransmitter release and signalling. (B) Diagram of a frog rod outer s e g m e n t illustrating t h e s h a p e a n d organization of discs a n d t h e p r e s e n c e of incisures. ( C ) E l e c t r o n m i c r o g r a p h of a longitudinal s e c t i o n of a r h e s u s monkey rod outer segment; X 75,600. Modified from Dose (1996) (A); Steinberg (1980) (B&C).  Chapter 1 Introduction  1.1.2  5  Phototransduction Phototransduction  c e l l s ( F i g 3).  o c c u r s in t h e o u t e r  s e g m e n t s of rod a n d cone  I n r o d s it b e g i n s w h e n a p h o t o n o f l i g h t i s o m e r i z e s t h e 1 1 -  c / ' s - r e t i n a l c h r o m o p h o r e o f r h o d o p s i n t o i t s all-trans a  conformational  metarhodopsin  II  change ( M II)  in  rhodopsin  (reviewed  isomer.  and  by Koch,  the  This leads to  production  of  1 9 9 5 ; Palczewski, 1994;  Polans, Baehr & Palczewski, 1 9 9 6 ; Pugh & Lamb, 1993).  T h i s s i g n a l is  amplified through the activation of the G-protein transducin (T p ), which a  e x c h a n g e s bound G D P for G T P a n d dissociates into T single  metarhodopsin  II  molecule  can activate  a  and T  between  P y  y  subunits.  100 and 500  molecules of transducin ( G r a y - K e l l e r , B i e r n b a u m & B o w n d s , 1 9 9 0 ) . activated T stimulates complex  A  The  subunit binds phosphodiesterase (PDE) stoichiometrically a n d  a  the conversion  can catalyze  of  cGMP  the hydrolysis  to  GMP.  O n e activated  of up to  1000 cGMP  PDE-T  a  molecules  r e s u l t i n g in a n a m p l i f i c a t i o n of 1 - 5 x 1 0 m o l e c u l e s o f c G M P p e r s e c o n d 5  per  rhodopsin  cGMP  result  conductance  molecule in  closing  (Yee & Liebman, of  of t h e p l a s m a  hyperpolarization  the  1978).  cGMP-gated  membrane  to  Decreased levels of  channels  sodium  and  reduced  and calcium.  of t h e cell r e s u l t s in t h e i n h i b i t i o n o f  The  neurotransmitter  (glutamate) release at the synaptic terminal. M e c h a n i s m s exist to reset the photoreceptors physiological r e s p o n s e to light.  to allow for a fast  A c t i v e m e t a r h o d o p s i n II i s i n a c t i v a t e d b y  Disk Membrane  Plasma Membrane  Fig 3 . Phototransduction. Light initiates t h e i s o m e r i z a t i o n of 11-c/s retinal to all-trans retinal which converts r h o d o p s i n into t h e activated m e t a r h o d o p s i n II s t a t e ( M I I ) . T r a n s d u c i n e x c h a n g e s b o u n d G D P f o r G T P and T a activates P D E which converts c G M P to G M P . D e c r e a s e d levels of c G M P r e s u l t in c l o s i n g o f t h e c h a n n e l a n d h y p e r p o l a r i z a t i o n o f t h e r o d cell. P h o t o r e c o v e r y is i n i t i a t e d b y s h u t t i n g o f f t h e v i s u a l c a s c a d e b y ( 1 ) phosphorylation of rhodopsin a n d binding of arrestin, (2) G A P assisted hydrolysis of G T P by T a a n d dissociation f r o m P D E , a n d (3) r e a s s o c i a t i o n of T a w i t h T(3y t o f o r m t h e i n a c t i v a t e d t r a n s d u c i n h e t e r o t r i m e r . Low intracellular calcium resulting from channel closure leads to (1) G C A P a s s i s t e d a c t i v a t i o n o f g u a n y l a t e c y c l a s e a n d a n i n c r e a s e in c G M P c o n c e n t r a t i o n , (2) dissociation of calmodulin ( C a M ) f r o m t h e c h a n n e l a n d increased channel sensitivity to cGMP. A s c G M P levels increase, the c h a n n e l s r e o p e n a n d t h e r o d c e l l is r e t u r n e d t o t h e d e p o l a r i z e d s t a t e . T h e i n c r e a s e i n c a l c i u m r e s u l t s in i n a c t i v a t i o n o f g u a n y l a t e c y c l a s e a n d d e p h o s p h o r y l a t i o n of r h o d o p s i n . Solid arrows s h o w t h e p h o t o e x c i t a t i o n p r o c e s s a n d dashed arrows s h o w t h e p h o t o r e c o v e r y p r o c e s s . M o d i f i e d f r o m Molday (1998).  Chapter 1 Introduction rhodopsin binding  kinase  of  7 (RK) catalyzed  arrestin.  This  phosphorylation  reaction  in t u r n  is  and the  subsequent  regulated  by  calcium  concentrations t h r o u g h t h e interaction of R K with t h e c a l c i u m - a c t i v a t e d protein  recoverin.  T h e all-trans-retinal  chromophore  is r e d u c e d t o a l l -  trans-retinol b y retinal d e h y d r o g e n a s e a n d is r e l e a s e d f r o m t h e inactive phosphorylated rhodopsin. or arrestin tp opsin.  This prevents the binding of transducin, R K ,  R h o d o p s i n is r e g e n e r a t e d u p o n  dephosphorylation  b y p r o t e i n p h o s p h a t a s e 2 A a n d r e b i n d i n g o f 1 1 - c / s - r e t i n a I. The stimulation of PDE by T activity of T PDE  a  a  is r e g u l a t e d b y t h e intrinsic  a n d by GTPase-activating proteins (GAPs).  or GAPs  significantly  i n c r e a s e s its intrinsic  GTPase  Binding of T  GTPase  activity  a  to  and  r e s u l t s in t h e h y d r o l y s i s o f G T P , d i s s o c i a t i o n f r o m P D E , a n d r e b i n d i n g of T . P y  Transducin a n d P D E are returned  to their  resting  states and are  ready for subsequent activation. P h o t o a c t i v a t i o n r e s u l t s in a d e c r e a s e in i n t r a c e l l u l a r c a l c i u m . T h e closing of the c G M P channels d u e to low levels of c G M P influx of c a l c i u m into t h e outer s e g m e n t . continues to extrude  calcium from  prevents the  A Na/Ca-K exchanger however,  t h e cell resulting  in a d e c r e a s e in  c a l c i u m f r o m 5 0 0 t o 5 0 n M . T h e e n z y m e g u a n y l a t e c y c l a s e ( G C ) , w h i c h is inactive  at  high  calcium  concentrations  (dark  state),  is a c t i v a t e d  g u a n y l a t e c y c l a s e a c t i v a t i n g p r o t e i n s ( G C A P s ) in l o w c a l c i u m . calcium  binding  proteins  that  activate  G C in t h e i r  by  GCAPs are  unliganded  state.  Chapter 1 Introduction  8  G u a n y l a t e c y c l a s e c o n v e r t s G T P into c G M P t h e r e b y increasing t h e level of c G M P in t h e c e l l .  T h i s in t u r n r e o p e n s t h e c h a n n e l s a n d r e s t o r e s t h e  resting m e m b r a n e potential.  T h e c G M P - g a t e d c h a n n e l is a l s o  regulated  by t h e calcium-binding protein calmodulin ( H s u & Molday, 1 9 9 3 ) . calcium  concentrations  (and  low  cGMP  concentrations),  In low  calmodulin  dissociates f r o m t h e c h a n n e l . This increases t h e a p p a r e n t affinity of the channel  for cGMP  and allows  it t o o p e n  at low c G M P  concentrations.  T h e s e c o n c e r t e d e f f e c t s r e s u l t in t h e f a s t r e c o v e r y o f t h e c e l l t o t h e d a r k state a n d cessation of the neural signal.  1.1.3  Disc morphogenesis  and shedding  Rod a n d cone photoreceptors continually renew the photosensitive membranes  of their  outer  segments  (Young,  1976).  Completely new  outer s e g m e n t s a r e g e n e r a t e d o n a v e r a g e e v e r y t e n d a y s at a rate of o n e hundred discs per d a y or maximally one new disc every 9 min (Besharse &  Defoe,  1998;  Besharse,  Hollyfield  &  Rayborn,  1977).  m o r p h o g e n e s i s occurs at t h e base or proximal e n d of t h e outer and  discs  migrate  towards  t h e tip w h e r e  p h a g o c y t o s i s by t h e retinal  pigment  epithelium  relationship  exists  between  disc  dependent  upon  the  photoreceptors  distally  intimate  (Kaplan, Iwata & Sterrett,  of  1990).  and shedding the  segment  shedding  (RPE)occurs.  morphogenesis interaction  disc  Disc  and  A close that  R P E with  is the  A s i n g l e rat R P E cell  Chapter 1 Introduction  9  can phagocytize as many as 30,000 discs per d a y , a prodigious  effort.  G e n e r a l l y , d i s c s h e d d i n g is a d i u r n a l p r o c e s s t h a t o c c u r s a t t h e o n s e t o f light after a period of d a r k n e s s a n d involves t h e a c t i o n s of m e l a t o n i n , d o p a m i n e , a n d a photoreceptor circadian clock (Besharse & Defoe, 1998). W h y then do photoreceptors a n d RPE cells e x p e n d so m u c h energy on m e m b r a n e synthesis a n d renewal?  It h a s b e e n k n o w n s i n c e a n t i q u i t y ,  in P l a t o ' s P h a e d o , t h a t S o c r a t e s a d v i s e d a g a i n s t s t a r i n g d i r e c t l y i n t o t h e s u n b e c a u s e of a c u t e light d a m a g e to t h e e y e ( D u k e - E l d e r , 1 9 5 4 ) .  In  fact, d a m a g e to the photoreceptors a n d R P E cells of t h e retina by acute a n d c h r o n i c light e x p o s u r e h a s b e e n s h o w n to o c c u r in a n i m a l m o d e l s a n d varies with wavelength ( L a n u m , 1 9 7 8 ;Oranisciak & Winkler, 1 9 9 4 ; R e m e et al.,  1998).  T h e r e is a l s o a p o s i t i v e c o r r e l a t i o n b e t w e e n light e x p o s u r e  a n d t h e o c c u r r e n c e of a g e - r e l a t e d m a c u l a r d e g e n e r a t i o n in h u m a n s , t h e most common form  of b l i n d n e s s in t h e e l d e r l y  K l e i n , 1 9 9 3 ; T a y l o r et al., has  been  shown  photo-oxidation  1 9 9 0 ; T a y l o r e t al.,  for t h e first t i m e and aggregation  that  of a  ( C r u i c k s h a n k s , Klein & 1992).  V e r y r e c e n t l y , it  causes  retinal-catalyzed  light number  of  photoreceptor  disc  proteins including A B C R , peripherin-2 a n d r o m - 1 ( S u n & Nathans, 2001), thus providing a m o l e c u l a r rationale for light d a m a g e .  It t h e r e f o r e s e e m s  quite clear that outer s e g m e n t m e m b r a n e s a n d their constituent are highly susceptible to light-damage a n d must be rapidly to m a i n t a i n p r o p e r v i s i o n .  proteins  regenerated  Chapter 1 Introduction An  10  elegant  morphogenesis observations  model  has  of  for  been  rods  the  mechanism  proposed  and cones  based  on  in t h e r h e s u s  (Steinberg, Fisher & Anderson, 1980).  of  vertebrate  electron  disc  microscopic  monkey  and  squirrel  At the base of the outer  segment  a d j a c e n t t o t h e c o n n e c t i n g c i l i u m , m e m b r a n e o u s e v a g i n a t i o n s o c c u r in a sequential  manner growing  t h e o u t e r s e g m e n t (Fig 4). at  nodes between  radially a s they a r e displaced distally  along  A s e c o n d p r o c e s s , r i m f o r m a t i o n , is initiated  evaginations  on the connecting  cilium  and proceeds  o u t w a r d a l o n g t h e p l a n e o f t h e d i s c c i r c u m s c r i b i n g it. A t t h e o p p o s i t e e n d of t h e d i s c t o t h e c o n n e c t i n g c i l i u m , a m e m b r a n e f u s i o n e v e n t o c c u r s in r o d s t h a t s e a l s t h e d i s c a n d s e p a r a t e s it f r o m t h e p l a s m a m e m b r a n e . precise  molecular  mechanisms  determined, however. disc morphogenesis  of disc  morphogenesis  have  The  yet to be  Studies e x a m i n i n g t h e effects of cytochalasin D on  have revealed that membrane  evagination  a n d disc  f o r m a t i o n a r e d e p e n d e n t o n t h e a c t i v i t y of f i l a m e n t o u s a c t i n in t h e c i l i u m , but disc d i s p l a c e m e n t 1998).  Breaking  is n o t ( H a l e , F i s h e r & M a t s u m o t o ,  the  photoreceptor  R P E cell  1996; Kaplan,  contact  by  retinal  detachment also prevents m e m b r a n e evagination a n d disc formation, but not  displacement  Sterrett, RPE  1990).  is a n a c t i v e  (Hale,  Fisher & Matsumoto,  1 9 9 1 ; Kaplan, Iwata  This suggests that displacement process  independent  of discs towards  of disc f o r m a t i o n .  m i c r o t u b u l e s in d i s c d i s p l a c e m e n t h a s b e e n s u g g e s t e d .  Also,  A role  &  the for  membrane-  11  Fig 4 . D i s c m o r p h o g e n e s i s . ( A ) T h e s e q u e n c e o f m e m b r a n e e v a g i n a t i o n a s o b s e r v e d b y e l e c t r o n m i c r o s c o p y a n d in d i a g r a m . T h e c o n t i n u o u s p r o c e s s o f b u d d i n g a n d d i s p l a c e m e n t r e s u l t s in a s t e a d y s u p p l y o f n a s c e n t d i s c s t o t h e r i m f o r m i n g r e g i o n . ( B ) A d i a g r a m o f r i m f o r m a t i o n i l l u s t r a t i n g d i s c s in both longitudinal a n d cross section. T h e rim forming region (1) between the vitreal a n d scleral f a c e s of n a s c e n t d i s c s g r o w s o u t w a r d ( 2 - 3 ) a r o u n d t h e c i r c u m f e r e n c e of t h e disc. Meeting of t h e t w o s i d e s of t h e g r o w i n g rim (4) r e s u l t s in m e m b r a n e f u s i o n a n d s e p a r a t i o n o f t h e d i s c f r o m t h e p l a s m a m e m b r a n e . Modified from Steinberg (1980).  Chapter 1 Introduction impermeable suggest  a  12  sulfhydryl  role  modifying  for disulfide  reagents  bonding  inhibit  in d i s c  disc formation  morphogenesis  and  (Wetzel,  Parsons & Besharse, 1994). The current model for disc shedding a n d phagocytosis involves two inter-related processes (Besharse & Defoe, 1998). distal tip of t h e outer membrane  segment  in c l o s e c o n t a c t  fusion events presumably  Discs located near the with the R P E undergo  with the plasma m e m b r a n e .  c u l m i n a t e s i n t h e s h e d d i n g o f p a c k e t s o f d i s c s ( F i g 5).  This  At the same time,  actin-filled p r o c e s s e s f r o m R P E cells called p s e u d o p o d i a penetrate into t h e o u t e r s e g m e n t in r e g i o n s o f f u s i o n a n d e n g u l f t h e d i s c p a c k e t s .  What  f o l l o w s is a s e q u e n c e of i n g e s t i o n a n d d i g e s t i o n of t h e R O S m e m b r a n e s within  the  R P E cell  components,  and the  including  photoreceptor.  It  retinal  recycling and  of  some  of  docosahexanoic  is u n c l e a r a t t h e p r e s e n t t i m e w h i c h  the  membrane  acid,  to  the  of these  cells  initiates this process since both disc s h e d d i n g a n d p h a g o c y t o s i s s e e m to occur concomitantly. not constitutively  B e c a u s e R P E cells in c o n t a c t w i t h p h o t o r e c e p t o r s d o  phagocytize discs, and photoreceptors  in c o n t a c t  with  RPE cells defective for pseudopodia o u t g r o w t h d o not undergo s h e d d i n g , there m u s t be a delicate interplay governing initiation of these processes. During disc shedding, adhesion between significantly  increased.  this, but there  RPE and photoreceptor  c e l l s is  It is u n c l e a r w h a t r e c e p t o r s a r e r e s p o n s i b l e f o r  is e v i d e n c e f o r r o l e s o f a n F  c  -immunoglobulin  receptor  13  A  1  2  3  4  5  Fig 5 . D i s c s h e d d i n g a n d p h a g o c y t o s i s . ( A ) E l e c t r o n m i c r o g r a p h s o f r o d photoreceptors s h o w i n g (1) RPE p h a g o s o m e s (phag) containing discs a n d (2) penetration of t h e o u t e r s e g m e n t by p s e u d o p o d i a ( p p ) of t h e R P E ( I S , i n n e r s e g m e n t ; O S , o u t e r s e g m e n t ) . ( B ) D i a g r a m o f t h e e v e n t s o c c u r i n g in phagocytosis of discs. Initiation of p s e u d o p o d i a growth occurs concomitantly with photoreceptor disc-plasma m e m b r a n e fusion (1-2). P e n e t r a t i o n of t h e d i s c s t a c k ( 3 - 4 ) r e s u l t s in r e l e a s e a n d i n t e r n a l i z a t i o n of a packet of discs (5) by the R P E . Modified from S t e i n b e r g (1977) ( A ) ; Matsumoto (1987) (B).  Chapter 1 Introduction  14  S y s t e m ( E l n e r et al., system  (Boyle  et  1 9 8 1 ; Laird & Molday, 1 9 8 8 ) , a m a n n o s e  al.,  1991),  and a  CD36-integrin  receptor  receptor  system  (Ryeom, Sparrow & Silverstein, 1996).  1.2  IDENTIFICATION OF PERIPHERIN-2 AND ROM-1 Peripherin-2  homologous  (formerly  tetraspanning  known  as peripherin/rds)  integral  membrane  and rom-1 are  proteins  that  form  multisubunit c o m p l e x e s at t h e rims of rod a n d cone o u t e r s e g m e n t s discs (Molday,  1998).  membrane  R o l e s f o r t h e s e c o m p l e x e s in d i s c m o r p h o g e n e s i s a n d  fusion  B a t t a g l i a et al.,  have  been  proposed  1 9 9 8 ; W r i g l e y e t al.,  2000).  are responsible for autosomal dominant  et al.,  (Arikawa  Mutations  1 9 9 2 ; Boeszein  peripherin-2  r e t i n a l d y s t r o p h i e s ( F a r r a r e t al.,  1991a).  1.2.1  Initial characterization  of  peripherin-2  Peripherin-2 w a s identified using biochemical a n d i m m u n o c h e m i c a l approaches  (Connell & Molday,  1 9 9 0 ; Molday,  Hicks & Molday,  1987).  T h e protein w a s first identified o n W e s t e r n blots of bovine R O S using t w o m o n o c l o n a l a n t i b o d i e s ( P e r 2 B 6 a n d P e r 3 B 6 ) p r o d u c e d in m i c e with  total  ROS membranes  (Molday,  Hicks  &  Molday,  immunized  1987).  Since  peripherin-2 co-migrates with rhodopsin (approx. 35 k D a ) on S D S - P A G E , it w a s n o t d e t e c t e d  o n C o o m a s s i e o r s i l v e r s t a i n e d g e l s e v e n t h o u g h it  Chapter 1 Introduction  15  constitutes approximately & Molday, 1996b).  3 % of total R O S m e m b r a n e protein  Further biochemical analysis of peripherin-2  (Goldberg revealed  t h a t it f o r m s d i s u l f i d e l i n k e d d i m e r s a n d is g l y c o s y l a t e d ( C o n n e l l e t al., 1991;  Molday, Hicks & Molday, 1987).  C o o m a s s i e stained S D S gels of  p u r i f i e d p e r i p h e r i n - 2 s h o w e d t h a t it a l s o m i g r a t e s a s a d o u b l e t , the  possibility of a n interaction  with another  protein.  indicating  Immunoelectron  m i c r o s c o p y o f b o v i n e r e t i n a s u s i n g t h e s e m o n o c l o n a l a n t i b o d i e s ( F i g 6) showed specific disc rim a n d incisure labeling similar to w h a t had been observed for a 2 9 0 k D a protein from frog photoreceptors (Papermaster et al., 1 9 7 8 ) .  P e r i p h e r i n w a s n a m e d f o r its u n i q u e location a t t h e p e r i p h e r y  of  This  discs.  localization  is v e r y  different  than  the localization  of  rhodopsin along the flattened lamellar region of t h e disc (Molday, Hicks & Molday, 1987). Peripherin-2 w a s cloned by t w o different laboratories using different approaches. anti-peripherin  In o u r l a b , a A , g t l l monoclonal  expression library  antibodies  (Connell  w a s screened with  & Molday,  c D N A encodes a protein of 3 4 6 a m i n o acids a n d hydropathy four t r a n s m e m b r a n e segments.  1990). plots  The predict  A large 141 a m i n o acid intradiscal  loop  connects t r a n s m e m b r a n e s e g m e n t s three a n d four a n d a 6 0 a m i n o acid C - t e r m i n a l tail e x t e n d s f r o m t h e fourth t r a n s m e m b r a n e s e g m e n t ( F i g 7). The m e m b r a n e topology w a s confirmed in-part using the pre-embedding labeling of isolated R O S for analysis by electron  microscopy.  Labeling  16  •*vv  • .-V;  F i g 6 . I m m u n o l o c a l i z a t i o n of p e r i p h e r i n - 2 t o t h e d i s c r i m . ( A & B ) Electron m i c r o g r a p h s of isolated R O S discs s h o w i n g i m m u n o g o l d labeling of peripherin-2 specifically at the disc r i m . ( C ) I m m u n o g o l d labeling of r h o d o p s i n s h o w s l a m e l l a r b u t n o t r i m l o c a l i z a t i o n . B a r s : A & C = 0 . 1 p.m, B = 0.05 p m . Modified from Molday (1987).  Chapter 1 Introduction  17  with the Per2B6 monoclonal antibody showed that the epitope  contained  in t h e C - t e r m i n u s w a s l o c a t e d o n t h e c y t o p l a s m i c s i d e o f t h e m e m b r a n e (Connell & Molday, 1990).  Peripherin-2 contains 13 cysteines, six which  are not c o n s e r v e d , a n d seven which are highly conserved a m o n g species and with r o m - 1 (Molday, 1994).  A l l c o n s e r v e d c y s t e i n e s a r e l o c a t e d in  the intradiscal (extracellular equivalent) loop.  A consensus sequence for  N - l i n k e d g l y c o s y l a t i o n is a l s o p r e s e n t w i t h i n t h e i n t r a d i s c a l l o o p a t N 2 2 9 . No  homology  found  with a n y existing  at the time,  tetraspanin  s e q u e n c e s in t h e E M B L d a t a b a s e w a s  but peripherin-2  has since  been  included  f a m i l y o f p r o t e i n s t o w h i c h it s h a r e s o n l y l i m i t e d  homology.  Using a subtractive genetic approach, Travis et a l . (1989) the g e n e defect responsible for t h e phenotype of t h e retinal s l o w (rds)  m o u s e at approximately the s a m e time.  the heterozygous  rds  mouse  exhibits  membranes (Sanyal & Jansen, 1981).  whorls  identified  degeneration  T h e h o m o z y g o u s rds  m o u s e is c h a r a c t e r i z e d by t h e a b s e n c e of r o d a n d c o n e o u t e r and  in t h e  segments  of disorganized  disc  A slow degeneration of t h e retina  occurs such that after a year only a few photoreceptors  remain.  T h e RDS g e n e w a s f o u n d t o b e 9 2 . 5 % i d e n t i c a l t o b o v i n e a n d t h e r e f o r e r e p r e s e n t s t h e m o u s e o r t h o l o g ( C o n n e l l e t al.,  peripherin  1991).  The  s a m e g e n e a p p e a r s to e n c o d e f o r p e r i p h e r i n - 2 in r o d s a n d c o n e s s i n c e (1) in  t h e rds  mouse  neither  rod nor cone  outer  segments  develop,  monoclonal antibodies to the C-terminus of bovine peripherin  (2)  label both  18  L3-4  Fig 7 .  T o p o l o g i c a l m o d e l for p e r i p h e r i n - 2 a n d r o m - 1 .  Both proteins  contain four t r a n s m e m b r a n e segments ( M 1 - M 4 ) , cytoplasmic N and C t e r m i n i , a n d a large intradiscal loop (L3-4) of a p p r o x . 1 4 0 a m i n o acids. P e r i p h e r i n - 2 c o n t a i n s a n N - l i n k e d o l i g o s a c c h a r i d e c h a i n ( h e x a g o n s ) t h a t is n o t p r e s e n t in r o m - 1 . C o n s e r v e d c y s t e i n e s a n d s t r e t c h e s of c o n s e r v e d amino acids between peripherin-2 and rom-1 are indicated. Modified from Molday (1994).  Chapter 1 Introduction rods  and cones  peripherin  19  (Arikawa  et  al.,  l a b e l s c o n e s b y in situ  1992),  a n d (3)  hybridization  the cDNA  ( U e h a r a e t al.,  for rod 1993).  S i n c e t h e s e e f f o r t s , p e r i p h e r i n - 2 o r t h o l o g s h a v e b e e n i d e n t i f i e d in h u m a n , r a t , c a t , d o g , c h i c k e n , a n d f r o g , b u t n o t in t h e f r u i t f l y , t h e w o r m , o r yeast  (BLAST  chromosome  search). 6pl2  The  b y in situ  human  gene  hybridization  has  (Travis  been e t al.,  localized  to  1991) and  contains three exons (Kajiwara e t a / . , 1991).  1.2.2  Initial characterization The  using  a  human  of rom-1  r o m - 1 c D N A w a s initially  differential  screening  approach  cloned by genetic (Bascom  et  al.,  methods  1992b).  It  encodes a protein of 3 5 1 a m i n o acids. T h e r o m - 1 s e q u e n c e w a s found to be 3 5 % identical to bovine peripherin-2 a n d h y d r o p a t h y  plots predict a  topology similar to peripherin-2 with four t r a n s m e m b r a n e segments, an i n t r a d i s c a l l o o p , a n d a l o n g C - t e r m i n a l t a i l (Fig 7).  T h e intradiscal loop  regions of peripherin-2 a n d r o m - 1 are more highly conserved exhibiting 4 7 % identity.  T h e s e v e n cysteines within this loop a r e also c o n s e r v e d .  S e v e r a l s e q u e n c e s a r e i d e n t i c a l in p e r i p h e r i n - 2 a n d r o m - 1 i n c l u d i n g R Y L D and DGVPFSCCNPXSPRPC (Bascom, Schappert & Mclnnes, 1993). R o m - 1 differs  from  consensus  peripherin-2  in  that  sequence and has more  peripherin-2).  it  lacks  an  N-linked  glycosylation  acidic residues ( p l 5.2 v s 8.2 for  Chapter 1 Introduction Rom-1  was  20 localized  to  the  rim  region  of  R O S discs  by  immunoelectron microscopy with polyclonal antibodies, but no labeling of cones  was observed  (Bascom  e t al., 1 9 9 2 b ) .  R o m - 1 w a s found  to  coprecipitate with peripherin-2 w h e n purified using t h e anti-peripherin-2 monoconal antibody Per2B6. linked  homodimers  (Bascom  eta/.,  when  1992b).  L i k e p e r i p h e r i n - 2 , it m i g r a t e d a s d i s u l f i d e analyzed  by  two-dimensional  Disulfide-linked heterodimers  of  SDS-PAGE peripherin-2  a n d r o m - 1 w e r e n o t o b s e r v e d a n d a s a r e s u l t it w a s c o n c l u d e d t h a t t h e two  proteins associated noncovalently to form a multisubunit  The h u m a n r o m - 1 gene localizes to c h r o m o s o m e l l q l 3  complex.  ( B a s c o m e t al.,  1992a). The  presence  of r o m - 1 in c o n e s  and the predicted  membrane  t o p o l o g y f o r r o m - 1 w e r e c o n f i r m e d in s t u d i e s u s i n g a n t i b o d i e s g e n e r a t e d to specific s e q u e n c e s of bovine r o m - 1 (Moritz & M o l d a y , 1 9 9 6 ) .  Bovine  rom-1 w a s found to be 8 4 % identical to h u m a n r o m - 1 a n d w a s localized to rod a n d c o n e outer s e g m e n t disc rims by i m m u n o e l e c t r o n m i c r o s c o p y . Pre-embedding  immunoelectron  labeling  of  isolated  R O S discs  with  polyclonal a n t i b o d i e s to t h e C - t e r m i n a l tail of r o m - 1 c o n f i r m e d t h a t this r e g i o n is l o c a t e d o n t h e c y t o p l a s m i c s i d e o f t h e d i s c .  Proteolytic studies  in c o n j u n c t i o n w i t h a n t i b o d i e s g e n e r a t e d t o t h e c y t o p l a s m i c  C-terminus  and  topological  to  the intradiscal  loop  region  of  r o m - 1 verified  organization of r o m - 1 (Moritz & Molday, 1996).  the  Co-immunoprecipitation  Chapter 1 Introduction  2 1  experiments demonstrated the presence of a p e r i p h e r i n - 2 : r o m - l  complex  in b o v i n e R O S .  1.2.3 Tetraspanin superfamily Peripherin-2 a n d r o m - 1 a r e m e m b e r s of a family of proteins as the tetraspanin or t r a n s m e m b r a n e four superfamily proteins  are characterized  by  the  presence  domains  and an extended  extracellular  of  four  (or intradiscal  known  (TM4SF). These transmembrane in t h e c a s e  of  peripherin-2 a n d r o m - 1 ) loop connecting t r a n s m e m b r a n e s e g m e n t s three and  four  homology  (reviewed between  by Wright peripherin-2  & Tomlinson,  1994).  There  is  and rom-1 and the tetraspanin  little group  b e y o n d t h e o v e r a l l t o p o l o g y a n d t h e e x i s t e n c e of t h r e e s h o r t m o t i f s in t h e loop region involving c y s t e i n e s . In c o n t r a s t , p e r i p h e r i n - 2 a n d r o m - 1 h a v e e x t e n d e d c y t o p l a s m i c C - t e r m i n a l r e g i o n s t h a t a r e a b s e n t in m o s t tetraspanin  proteins.  No structural  information  other  is a v a i l a b l e o n a n y o f  these proteins at this time ( B L A S T c o n s e r v e d - d o m a i n d a t a b a s e search). H o w e v e r , it is k n o w n t h a t o t h e r m e m b e r s o f t h e t e t r a s p a n i n f a m i l y  form  oligomeric structures similarly to peripherin-2 a n d r o m - 1 . The tetraspanin family comprises a group of at least 19 cell-surface p r o t e i n s . It i n c l u d e s t h e l e u k o c y t e c e l l s u r f a c e m a r k e r s C D 9 , C D 3 7 , C D 5 3 , C D 6 3 , C D 8 1 , C D 8 2 , a n d C D 1 5 1 w h i c h a r e t h o u g h t t o b e i n v o l v e d in B-cell maturation,  signal t r a n s d u c t i o n , cell a d h e s i o n , f u s i o n , a n d cell  motility.  Chapter 1 Introduction  22  C D 6 3 a n d C D 8 1 have been identified o n t h e surface of s o m e and  leukemia  cells, respectively,  ( A t k i n s o n et al.,  and m a y be involved  1 9 8 4 ; O r e n e t al.,  1990).  in  melanoma proliferation  S o m e of these proteins  been s h o w n to modulate integrin signaling.  have  This suggests that they  play  a role in t h e r e g u l a t i o n o f cell m o t i l i t y ( B e r d i t c h e v s k i & O d i n t s o v a , 1 9 9 9 ; Yauch  & Hemler,  2000).  Other tetraspanins  interact w i t h Ig s u p e r f a m i l y  have  been  proteins, the neural protein  suggested  to  LI, and MHC I  a n d M H C II m o l e c u l e s , a m o n g o t h e r s ( Y a u c h & H e m l e r , 2 0 0 0 ) . The  function  Drosophila,  of  has been  Latebloomer  one  tetraspanin  defined  is e x p r e s s e d  at  n e u r o n a l a x o n s a n d is i n v o l v e d and their efferent muscles. nervous  system  protein,  latebloomer  (Kopczynski, Davis & G o o d m a n , the  growth  in f o r m i n g  cone  of  developing  from 1996). motor  contacts between the axons  Fruit flies t h a t lack this protein d i s p l a y s l o w e d  development,  although  these  contacts  do  eventually  form.  1.3 1.3.1  HUMAN  DISEASE  Retinal diseases  and disease  genes  Retinal diseases e n c o m p a s s a heterogenous  group  of dystrophies  and a r e diagnosed by clinicians according to their hereditary pattern a n d clinical s y m p t o m s .  Autosomal dominant,  autosomal  recessive, and X -  linked f o r m s have been identified a n d involve a n u m b e r of different g e n e s  Chapter 1 Introduction (Table 1).  23  Clinical s y m p t o m s are described by fundoscopic  in w h i c h a n o p h t h a l m o s c o p e is u s e d t o o b s e r v e a n a t o m i c a l  examination abnormalities  in t h e r e t i n a , t h e p i g m e n t e p i t h e l i u m a n d t h e r e t i n a l b l o o d v e s s e l s , a n d by e l e c t r o r e t i n o g r a p h y electroretinograms  which utilizes a contact  (ERGs)  c o n e s to f l a s h e s of light. retinal  degenerations  degeneration (MD). Retinitis  which  monitor  lens electrode to record  the responses  of  rods a n d  B y clinical diagnosis, t h e m o s t c o m m o n forms of are  retinitis  pigmentosa  (RP)  and  macular  Few treatments are currently available.  pigmentosa  affects  approximately  1  in  3700  people  ( B o u g h m a n , C o n n e a l l y & N a n c e , 1 9 8 0 ) . It is c h a r a c t e r i z e d b y o n s e t i n t h e s e c o n d o r t h i r d d e c a d e of life, n i g h t b l i n d n e s s , a p r o g r e s s i v e loss in t h e p e r i p h e r a l v i s u a l f i e l d , s u b s e q u e n t g r a d u a l l o s s in c e n t r a l v i s u a l f u n c t i o n , abnormal  electroretinograms,  patches (reviewed  and development  by (Henkenlively,  are usually  pigmentary 1993)).  In  in t h e i r fifties a n d s i x t i e s ) E R G  responses are absent, severe attenuation a n d patients  retinal  1 9 8 8 ; K a j i w a r a et al.,  the later stages of t h e disease (patients  occurred,  of  of retinal  completely  classified genetically a s autosomal dominant,  blood  blind.  vessels has  R P is  further  recessive, or X-linked, and  based o n t h e rate of onset (rapid o r slow). Macular  degeneration  is c h a r a c t e r i z e d  by loss  of central  vision  r e s u l t i n g f r o m d e g e n e r a t i o n o f c o n e s in t h e m a c u l a r r e g i o n of t h e retina (Bressler, Bressler & Fine, 1988).  Age-related M D , which affects up to  24  Chapter 1 Introduction Table 1 P r o t e i n s I n v o l v e d in R e t i n a l D i s e a s e s Protein cGMP-gated channel ( a ) rhodopsin p h o s p h o d i e s t e r a s e (a,|3) arrestin rhodopsin kinase transducin ( a ) RPGR peripherin-2  C e l l u l a r localization rods rods rods rods rods rods rods rods a n d c o n e s  rom-1 ABCR  rods a n d c o n e s rods a n d c o n e s  guanylate cyclase GCAP1 CRX XLRS1  rods a n d c o n e s rods a n d c o n e s rods a n d c o n e s rods a n d c o n e s , extracellular rods a n d c o n e s , R P E RPE a n d Muller RPE RPE Bruch's membrane  myosin VIIA CRALBP bestrophin RPE65 TIMP3 a  ARRP  - autosomal  dominant  retinitis  recessive retinitis pigmentosa,  CSNB  Disease ARRP ADRP, ARRP, CSNB ARRP, CSNB CSNB CSNB CSNB X - l i n k e d RP A D R P , digenic A D R P , M D , PD Digenic A D R P Stargardt's MD, A M D , ARRP, CRD LCA, CRD Cone dystrophy LCA, CRD X-linked retinoschisis 3  Usher syndrome ARRP Best's MD LCA, ARRP Sorby's MD  pigmentosa, ADRP -  congenital  -  autosomal  stationary  night  blindness, M D - macular dystrophy, P D - pattern dystrophy, A M D - a g e related  macular  dystrophy,  C R D - cone-rod  dystrophy,  congenital amaurosis; table modified from Molday (1998).  LCA -  Leber's  Chapter 1 Introduction  25  1 0 % o f p e o p l e o v e r f i f t y ( 1 1 m i l l i o n N o r t h A m e r i c a n s ) is d i v i d e d i n t o t w o g r o u p s , a dry f o r m w i t h a d e f i n e d h e r e d i t a r y c o m p o n e n t , a n d a wet The dry form  a c c o u n t s f o r 8 0 % o f c a s e s a n d is c h a r a c t e r i z e d  presence  drusen  of  irregularities  in t h e  characterized  by  (proteo-lipid  deposits  R P E ,and geographic  serious  detachment  of  in  Bruch's  atrophy. the  form.  by the  membrane),  The wet form  R P E resulting  is  from  neovascularization of t h e underlying choroid ( S p a l t o n , Hitchings & Hunter, 1994). are  In c h i l d r e n , S t a r g a r d t ' s d i s e a s e a n d f u n d u s f l a v i m a c u l a t u s ,  rare  autosomal  recessive  forms  of  macular  which  degeneration,  are  characterized by central visual dysfunction, t h e p r e s e n c e of perimacular yellow  deposits, and atrophy  1994).  of t h e R P E ( S p a l t o n , Hitchings &  Hunter,  M u t a t i o n s in A B C R h a v e b e e n l i n k e d to b o t h o f t h e s e d i s e a s e s a s  well a s t h e d r y form  of A M D (Allikmets  et al.,  1 9 9 7 a ; A l l i k m e t s et  al.,  1997b).  1.3.2 Mutations in peripherin-2 that cause retinal disease After resulted turned  determining  in retinal their efforts  that  a mutation  degeneration to identifying  for h u m a n retinal disease.  in t h e g e n e  in t h e r d s m o u s e , mutations  for  peripherin-2  many  researchers  in p e r i p h e r i n - 2  responsible  T o date over thirty mutations have been found  a n d a r e s u m m a r i z e d in T a b l e 2 .  These mutations are responsible for a  h e t e r o g e n e o u s group of a u t o s o m a l d o m i n a n t diseases w h i c h include R P ,  Chapter 1 Introduction  26  Table 2 Peripherin-2 Mutations A s s o c i a t e d with H u m a n Retinopathies Location Disease Reference Mutation RPA (Kajiwara etal., 1993) Ml Trp25rs (Fishman e r a / . , 1997) Ml CD Ser27Phe Ll-2 ADRP Arg46stop (Meins etal., 1993) (Jacobson e r a / . , 1994) M2 PD AMet67 ADRP ACysll8/119 M3 (Farrar er al., 1991b; Wells e r a / . , 1993) L3-4 ADRP (Kajiwara e t a / . , 1992) Leul26Arg MD (Keen e r a / . , 1994; Kim e r a / . , 1995) L3-4 Tyrl40rs Tyrl41Cys L3-4 ADRP (Sohocki etal., 2001) MD L3-4 (Trujillo e r a / . , 2001) Tyrl41His MD Argl42Trp L3-4 (Hoyng etal., 1996) Lysl53Arg L3-4 ADRP, RCD, PD (Jacobson etal., 1994) ALysl53/154 L3-4 ADRP, MD (Weleber e r a / . , 1993) L3-4 ADRP Cysl65Tyr (Souied etal., 1998) (Nichols e r a / . , 1993b) L3-4 BPD Glyl67Asp L3-4 MD Argl72Glu (Wells etal., 1993) L3-4 MD Argl72Trp (Jacobson etal., 1994; Wells etal., 1993) ADRP L3-4 Aspl73Val (Gaining etal., 1994) L3-4 ADRP Glnl78Arg (Sohocki etal., 2001) L3-4 ADRP Trpl79Arg (Bareil etal., 2000) L3-4 CRD Tyrl84Ser (Wada etal., 1995) L3-4 ADRP Leul85Pro (Kajiwara, Berson & Dryja, 1994; Kajiwara et al., 1991) (Jacobson e t a / . , 1994) L3-4 RCD ALysl93 L3-4 CRD Val200Gly (Nakazawa etal., 1996) ADRP L3-4 Gly208Asp (Trujillo etal., 1998) L3-4 ADRP Pro210Arg (Gorin etal., 1995) ADRP Pro210Ser L3-4 (Kemp etal., 1994) L3-4 ADRP Phe211Leu (Ekstrom etal., 1998) ADRP L3-4 Ser212Gly (Farrar etal., 1992) ADRP Cys214Ser L3-4 (Saga etal., 1993) L3-4 MD Cys214Tyr (Trujillo etal., 2001) L3-4 ADRP Pro216Leu (Kajiwara etal., 1991) L3-4 ADRP Pro216Ser (Fishman etal., 1994) ADRP L3-4 APro219 (Kajiwara etal., 1991) ADRP, BEM L3-4 Asn244Lys (Kikawa etal., 1994) ADRP, BEM L3-4 Asn244His (Nakazawa etal., 1994) MD L3-4 Tyr258stop (Wells etal., 1993) M4 ADRP Gly266Asp (Kajiwara etal., 1992) BPD C-term Ser299r~s (Nichols etal., 1993a) C-term ADRP (Gruninq etal., 1994) Leu307rs a fs - f r a m e s h i f t , stop - s t o p c o d o n ; s e e F i g 7; R P A - R e t i n i t i s p u n c t a t a a l b e s c e n s , C D - cone d y s t r o p h y , A D R P - a u t o s o m a l d o m i n a n t retinitis pigmentosa, PD- pattern dystrophy, M D - macular dystrophy, RCD - rodcone dystrophy, B P D - Butterfly-shaped pigment dystrophy, C R D- conerod d y s t r o p h y , B E M - Bull's-eye m a c u l o p a t h y 3  15  0  b  c  Chapter 1 Introduction MD,  a n d pattern  27 dystrophies  like  butterfly-shaped  pigment  dystrophy,  fundus flavimaculatus, cone-rod dystrophy, a n d Bull's Eye maculopathy (reviewed  by  Molday,  1998).  Mutations  in p e r i p h e r i n - 2  account  for  approximately 5 % of individuals with autosomal d o m i n a n t RP. The majority  of disease-causing mutations  are located within the  large highly c o n s e r v e d intradiscal loop of peripherin-2. of  the  missense type  substitutions.  a n d result  However, mutations  in  proteins  Most of these are  containing  amino  acid  h a v e a l s o b e e n f o u n d t h a t result in  amino acid deletions a n d protein truncations.  S i n c e d e c r e a s e d levels of  p e r i p h e r i n - 2 r e s u l t i n p h o t o r e c e p t o r d e g e n e r a t i o n i n t h e rds m o u s e , it is likely that s o m e of t h e s e m u t a t i o n s , especially t h e t r u n c a t i o n s , also result in  decreased  levels  of functional  protein  in t h e o u t e r  segment.  A  d o m i n a n t - n e g a t i v e e f f e c t r e s u l t i n g f r o m t h e p r e s e n c e o f m u t a n t p r o t e i n in t h e o u t e r s e g m e n t s , h o w e v e r , m a y a l s o c o n t r i b u t e t o t h e h e t e r o g e n e i t y in disease type a n d a g e of onset. It is i n t e r e s t i n g t h a t s o m e p e r i p h e r i n - 2 m u t a t i o n s cones, while  others  affect  rods.  A mutation  primarily  affect  at Arg 172, for example,  r e s u l t s in M D w h e r e c o n e s b u t n o t r o d s a r e a f f e c t e d , b u t a m u t a t i o n A s p l 7 3 p r i m a r i l y a f f e c t s r o d s a n d r e s u l t s in R P .  at  S i n c e t h e s a m e g e n e is  likely p r e s e n t in b o t h r o d s a n d c o n e s , this m e a n s t h a t r o d s c a n t o l e r a t e the  172 mutation,  but not the 173 mutation, a n d vice versa for cones.  T h e s e m u t a t i o n s either m u s t affect t h e protein's structure differently  in  Chapter 1 Introduction rods a n d cones,  28  or disrupt  protein  interactions  specific for these  cell  types. The only  mutations  found  in r o m - 1 c o n c l u s i v e l y l i n k e d to  retinal  d i s e a s e h a v e b e e n i n v o l v e d in a novel f o r m of d i g e n i c R P t h a t requires a L 1 8 5 P m u t a t i o n in p e r i p h e r i n - 2 in a d d i t i o n t o a null o r G 1 1 3 E m u t a t i o n in rom-1  ( B a s c o m e t al.,  Dryja,  1994).  heterozygotes) individuals  1 9 9 3 ; Dryja  Individuals display  w h o inherit  et al.,  w h o inherit  the autosomal only  1 9 9 7 ; Kajiwara, Berson &  both  mutant  dominant  the peripherin-2  (single heterozygotes) are essentially normal.  alleles  (double  RP phenotype,  while  or rom-1 mutant  allele  Even though rom-1 forms  a n o l i g o m e r i c c o m p l e x w i t h p e r i p h e r i n - 2 a n d is s i m i l a r i n i t s s u b c e l l u l a r l o c a l i z a t i o n , t o p o l o g y , a n d s e q u e n c e c o n s e r v a t i o n , its lack of i n v o l v e m e n t in r e t i n a l d i s e a s e i m p l i e s a d i f f e r e n t ,  p o s s i b l y n o n - d o m i n a n t role f o r this  s u b u n i t in o u t e r s e g m e n t m o r p h o g e n e s i s .  1.4  MOLECULAR  CHARACTERIZATION  OF PERIPHERIN-2  AND ROM-1 1.4.1 Oligomeric structure and size To further characterize the molecular properties of t h e peripherin 2:rom-l velocity  complex,  Goldberg et al. (1996)  sedimentation  complexes.  to define  the size  employed  the technique  and stoichiometry  of  of  these  Experiments were performed under mildly disulfide-reducing  Chapter 1 Introduction conditions  29  (1 m M D T T ) to p r e v e n t  Immunoaffinity-purified,  secondary oxidation  detergent-solubilized  of t h e protein.  peripherin-2: rom-1  complex from bovine R O S sedimented as a single peak indicating these  proteins  Molday,  form  1995).  a c o m p l e x of a discrete size  that  (Goldberg, Moritz  H y d r o d y n a m i c characterization of this  complex,  &  which  c o m b i n e s v e l o c i t y s e d i m e n t a t i o n m e a s u r e m e n t s i n H2O a n d D2O a n d g e l exclusion  chromatography  to  determine  molecular  mass,  gave  an  e s t i m a t e d m o l e c u l a r m a s s o f 1 3 5 , 0 0 0 k D a . T h i s v a l u e is i n r e a s o n a b l e agreement with a tetrameric  protein  (152,000  k D a calculated from the  primary sequences). A heterologous expression s y s t e m w a s d e v e l o p e d to e x a m i n e the a s s e m b l y of peripherin-2 a n d r o m - 1 s u b u n i t s into o l i g o m e r i c c o m p l e x e s . When  individually  expressed  in C O S - 1 c e l l s ,  peripherin-2  and rom-1  f o r m e d d i s u l f i d e - l i n k e d d i m e r s , but d i d n o t a s s o c i a t e w h e n s o l u b i l i z e d cell extracts  were  rhodopsin  mixed  which  prior  targets  to  immunoprecipitation.  to the p l a s m a m e m b r a n e  In  when  contrast  to  e x p r e s s e d in  C O S - 1 cells, peripherin-2 a n d r o m - 1 localized to internal m e m b r a n e s a n d were  absent  from  the plasma  membrane  C O S - 1 cells,  specifically  to  form  information  r e q u i r e d f o r s u b u n i t a s s e m b l y is c o n t a i n e d in t h e a m i n o a c i d  complex  rom-1  thus  did  When co-  in  heteromeric  and  cells.  expressed  a  peripherin-2  of t h e s e  showing  associate that  the  Chapter 1 Introduction sequence  30  of these  proteins  and that  c o - a s s e m b l y in t h e e n d o p l a s m i c  r e t i c u l u m o f c e l l s is r e q u i r e d f o r a s s o c i a t i o n . W h e n i n d i v i d u a l l y e x p r e s s e d in C O S - 1 c e l l s , o r w h e n c o - e x p r e s s e d and  purified,  tetrameric  peripherin-2  protein  and  rom-1 sedimented  similarly  from R O S under reducing conditions.  to  These  the  results  indicate that peripherin-2 a n d r o m - 1 can form homotetrameric c o m p l e x e s in t h e a b s e n c e o f t h e o t h e r s u b u n i t a n d t h a t o l i g o m e r i c a s s e m b l y o f t h e s e proteins  can occur  in C O S - 1 cells.  Based  previous studies by B a s c o m et a l . ( 1 9 9 2 ) ,  on these  results  a model w a s developed for  n a t i v e p e r i p h e r i n - 2 : r o m - l c o m p l e x in w h i c h a d i s u l f i d e - l i n k e d of  peripherin-2  associates  and on  noncovalently  with  h o m o d i m e r of r o m - 1 to form a heterotetrameric  a  homodimer  disulfide-linked  p r o t e i n (Fig 8).  In t h e  a b s e n c e of r o m - 1 , peripherin-2 disulfide-linked h o m o d i m e r s associate to form a homotetrameric  protein.  1.4.2 Molecular basis for digenic RP Using  the heterologous  expression system  effects of t h e L185P mutation assembly  a n d interaction  Molday, 1996a).  with  in p e r i p h e r i n - 2 rom-1 were  described above, the  o n its s t r u c t u r e , determined  (Goldberg  T h e authors found that the L185P mutation  homotetramer formation  subunit &  prevented  a n d r e s u l t e d in a p r o t e i n t h a t s e d i m e n t e d a t a  slower rate than W T . T h e protein did form disulfide-linked d i m e r s .  When  31  Chapter 1 Introduction co-expressed  with  rom-1,  L185P  sedimented as a tetrameric presence  of  rom-1,  heterotetrameric subunit  assembly  protein.  L185P  protein  co-assembled and co-  T h e s e r e s u l t s i n d i c a t e t h a t in t h e  peripherin-2  similarly  model  peripherin-2  to  WT.  for digenic  can  assemble  Based  on these  RP was developed  to  m o l e c u l a r i n t e r a c t i o n s w i t h g e n o t y p e a n d p h e n o t y p e (Fig 8). stresses  the  peripherin-2  functional  importance  of  having  containing  tetramers  in  ROS  homotetramers  of  demonstrated  for  peripherin-2 the  first  time  function how  a  results  a  correlate  This model  sufficient and  into  levels  predicts  normally.  This  disease-causing  of that  study  mutations  in  p e r i p h e r i n - 2 affect its s t r u c t u r e .  1.4.3  Role for peripherin-2 A potential  has  been  Battaglia  role for p e r i p h e r i n - 2  examined et  in membrane  al.,  in  vitro  1998  fusion  in d i s c - p l a s m a m e m b r a n e  ( B o e s z e - B a t t a g l i a e t al.,  2000).  When  1 9 9 7 ; Boesze-  peripherin-2-enriched  unilamellar vesicles (LUVs) were incubated with R O S plasma vesicles,  fusion  occurred  in  a  calcium  dependent  increased with phosphorylated peripherin-2. trypsinization of peripherin-2. acids  311-325  inhibited  fusion  large  membrane  manner,  and was  Fusion w a s decreased upon  A peptide (PP-5) corresponding to amino  of t h e c y t o p l a s m i c between  fusion  C-terminal  disc a n d plasma  region  membranes  of  peripherin-2  and  promoted  32  Genotype  Complex  Phenotype  WT Per WT Rom  L185P Per WT Rom  WT Per Null Rom  L185P Per Null Rom  Fig 8 . S u b u n i t a s s e m b l y m o d e l f o r d i g e n i c R P . N o r m a l i n d i v i d u a l s h a v e a full c o m p l e m e n t o f W T p e r i p h e r i n - 2 ( d a r k f i l l ) - r o m - l ( l i g h t fill) t e t r a m e r s . T h o s e w i t h o n l y a n L 1 8 5 P m u t a t i o n in p e r i p h e r i n - 2 a r e b o r d e r l i n e " n o r m a l " b e c a u s e L 1 8 5 P p e r i p h e r i n - 2 ( s p o t t e d fill) is p r e d i c t e d t o a s s e m b l e i n t o f u n c t i o n a l t e t r a m e r s w i t h r o m - 1 . T h o s e w i t h o n l y a null m u t a t i o n in r o m - 1 are " n o r m a l " because peripherin-2 h o m o t e t r a m e r s a r e predicted to c o m p e n s a t e for reduced levels of r o m - 1 . Only individuals with a n L 1 8 5 P p e r i p h e r i n - 2 m u t a t i o n a n d a r o m - 1 null e x h i b i t t h e d i s e a s e p h e n o t y p e b e c a u s e L 1 8 5 P p e r i p h e r i n - 2 is i n c a p a b l e o f s u p p o r t i n g R O S m o r p h o g e n e s i s . Therefore d e c r e a s e d levels of tetramers are predicted to contribute to the RP disease phenotype. Modified from Molday (1998).  Chapter 1 Introduction  33  vesicle aggregation and m e m b r a n e depolarization. the N - a n d C - t e r m i n i had no effect. amphipathic peptides.  alpha  and  This peptide can be m o d e l e d as an  shares  similarities  to  other  fusogenic  F T I R s t u d i e s c o n f i r m its a l p h a h e l i c a l n a t u r e a n d g e l e x c l u s i o n  chromatography Mutation  helix  Other peptides from  of  e x p e r i m e n t s s u g g e s t t h a t it m a y  PP-5,  which  disrupts  its  exist as a  amphipathic  nature,  tetramer. inhibited  m e m b r a n e depolarization, but not vesicle a g g r e g a t i o n , a n d disruption  of  its a l p h a h e l i c a l n a t u r e h a d n o e f f e c t s . It  is t h e r e f o r e  possible that  in  photoreceptors,  a  tetrameric  C-  t e r m i n a l region of peripherin-2 p r o m o t e s fusion b e t w e e n disc a n d p l a s m a m e m b r a n e s a t t h e d i s t a l t i p a n d is r e g u l a t e d t h r o u g h This  process must  be  highly  regulated  phosphorylation.  since disc shedding  occurs  in  s p e c i f i c c e l l s a t s p e c i f i c t i m e s a n d o n l y in t h e d i s t a l t i p r e g i o n o f r o d s a n d cones.  P e r i p h e r i n - 2 m a y a l s o b e i n v o l v e d in f u s i o n e v e n t s t h a t r e s u l t in  disc c l o s u r e d u r i n g disc m o r p h o g e n e s i s . T h e s e h y p o t h e s e s h a v e yet to be t e s t e d in vivo.  M u t a t i o n s that affect the C - t e r m i n u s of peripherin-2  are  k n o w n to c a u s e R P .  1.4.4  Role for peripherin-2  in flattening membrane  vesicles  I n a r e c e n t s t u d y b y W r i g l e y e t al. ( 2 0 0 0 ) , t h e e f f e c t o f  peripherin-2  o n t h e s h a p e of p a n c r e a t i c m i c r o s o m a l v e s i c l e s w a s e x a m i n e d by e l e c t r o n microscopy.  When  peripherin-2  is e x p r e s s e d in  vitro  under  disulfide  Chapter 1 Introduction  -  34  r e d u c i n g c o n d i t i o n s in t h e p r e s e n c e o f m i c r o s o m a l v e s i c l e s , t y p i c a l vesicles are observed. significant regions  fraction  of  high  round  When expressed under nonreducing conditions a  of the vesicles exhibit  curvature  similar  to  a flattened  disc  rims  of  appearance ROS.  with  Rhodopsin  e x p r e s s e d u n d e r t h e s a m e c o n d i t i o n s d i d n o t r e s u l t in v e s i c l e f l a t t e n i n g . Expression flattening prevents  of  the  RP-causing  C165Y  and P216L  of vesicles as did expression disulfide-dimerization  of  mutants  of t h e C 1 5 0 S  peripherin-2  prevented  mutant  (Goldberg,  which  Loewen  &  Molday, 1998). It a p p e a r s , t h e r e f o r e , t h a t a f u n c t i o n o f p e r i p h e r i n - 2 m a y a l s o b e t o bind  m e m b r a n e s in a d i s u l f i d e - d e p e n d e n t  manner.  This function m a y  serve to bring adjacent m e m b r a n e e v a g i n a t i o n s t o g e t h e r at t h e base of the outer s e g m e n t during disc rim formation a n d m a y be responsible for the high degree of curvature associated with the rim region of discs. T h e inability  of t h e d i s e a s e - c a u s i n g m u t a t i o n s ,  which  are located  in t h e  intradiscal r e g i o n , to flatten m e m b r a n e s s u p p o r t s a role f o r p e r i p h e r i n - 2 in  membrane  however  that  adhesion these  in  photoreceptors.  experiments  have  been  It  is  important  conducted  in a  to  note  cell-free  s y s t e m a n d t h a t t h e p r e s e n c e o f p e r i p h e r i n - 2 in t h e f l a t t e n e d v e s i c l e s w a s not verified.  Chapter 1 Introduction 1.5  35  ANIMAL MODELS  1.5.1  The rds mouse A  role for p e r i p h e r i n - 2  in p h o t o r e c e p t o r  disc morphogenesis and  h u m a n r e t i n a l d i s e a s e o r i g i n a t e d f r o m s t u d i e s i n t h e rds m o u s e , a n a t u r a l peripherin-2 gene knock-out.  M i c e h o m o z y g o u s f o r t h e rds m u t a t i o n  fail  to d e v e l o p p h o t o r e c e p t o r o u t e r s e g m e n t s a n d t h e p h o t o r e c e p t o r s u n d e r g o slow degeneration over the period of about 1984; Sanyal & Jansen, 1981).  a year (Jansen &  Sanyal,  H e t e r o z y g o u s rds m i c e e x h i b i t s h o r t e n e d ,  highly disorganized outer s e g m e n t s consisting of whorls of m e m b r a n e s . The  gene  r e s p o n s i b l e f o r t h e rds  phenotype  w a s identified  i n t h e rds  m o u s e a n d it w a s f o u n d t o c o n t a i n a l a r g e i n s e r t i o n o f f o r e i g n D N A t h a t r e s u l t s in a p r e m a t u r e s t o p c o d o n ( T r a v i s e t a / . , 1 9 8 9 ) . was shown to encode for the Therefore,  reduced  levels  degeneration and death.  The normal gene  peripherin-2 protein (Connell e t a / . , 1991). of  peripherin-2  result  in  photoreceptor  Adding back a W T transgene restores  normal  p h o t o r e c e p t o r m o r p h o g e n e s i s a n d r e s c u e s t h e rds p h e n o t y p e ( T r a v i s al., disc  1992). rims  et  T h e s e results a n d the unique location of peripherin-2 at the indicate  that  peripherin-2  plays  an  essential  role  in  disc  m o r p h o g e n e s i s , a p r o c e s s t h a t is n e c e s s a r y f o r p h o t o r e c e p t o r v i a b i l i t y .  Chapter 1 Introduction 1.5.2  36  Rom-1 knockout  mouse  To e x a m i n e the function (2000)  o f r o m - 1 i n p h o t o r e c e p t o r s , C l a r k e e t al.  inactivated t h e g e n e by replacing t h e first e x o n with a n e o m y c i n  resistance  gene,  and examined  heterozygous mice.  t h e effects  in b o t h  homozygous and  A m a r k e d contrast b e t w e e n t h e p h e n o t y p e of r o m - 1  n u l l m i c e a n d h o m o z y g o u s rds  m i c e is o b s e r v e d .  Photoreceptor  outer  s e g m e n t s d e v e l o p in r o m - 1 null m i c e a n d a p p e a r fairly n o r m a l , e x c e p t f o r occasional disc disorganization months.  a n d the presence of longer discs at 18  T h e s e abnormalities disappear at later a g e s .  A l s o , t h e length of  t h e o u t e r s e g m e n t is d e c r e a s e d a n d t h e t h i c k n e s s o f t h e n u c l e a r l a y e r is r e d u c e d .  Heterozygous mice appear normal.  rom-1  appears to play a minor  unlike  peripherin-2  formation.  photoreceptor  which  is  regulatory  absolutely  role in d i s c required  for  Therefore,  morphogenesis, outer  segment  T h i s r e s u l t is c o n s i s t e n t w i t h t h e l a c k o f i n v o l v e m e n t o f r o m - 1  in h u m a n d i s e a s e . B i o c h e m i c a l a n a l y s i s of r o m - 1 null m i c e r e v e a l e d t h a t t h e a b s e n c e of r o m - 1 h a d no effect o n t h e e x p r e s s i o n level o r s u b c e l l u l a r localization of r h o d o p s i n , A B C R ,  or peripherin-2.  Interestingly,  in t h e a b s e n c e of  r o m - 1 , p e r i p h e r i n - 2 f o r m e d h o m o t e t r a m e r s s i m i l a r l y t o w h a t is o b s e r v e d in t r a n s f e c t e d C O S - 1 c e l l s . homotetramer  formation  This supports t h e i m p o r t a n c e of peripherin-2 in d i s c  morphogenesis.  These  results  p r e d i c t e d i n t h e m o l e c u l a r m o d e l f o r d i g e n i c R P b y G o l d b e r g e t al.  were (1996)  Chapter 1 Introduction  37  w h e r e i n d i v i d u a l s w i t h a null m u t a t i o n  in r o m - 1 a r e n o r m a l b e c a u s e of  p e r i p h e r i n - 2 h o m o t e t r a m e r i z a t i o n (Fig 8).  1.5.3 P216L peripherin-2 transgenic mouse T o d e v e l o p a m o u s e m o d e l f o r h u m a n R P , K e d z i e r s k i et al.  (1997)  e x a m i n e d t h e e f f e c t o f e x p r e s s i o n o f t h e P 2 1 6 L m u t a n t p r o t e i n o n W T , rds heterozygous,  and  rds  homozygous  mouse  backgrounds.  P216L  p e r i p h e r i n - 2 w a s u n a b l e t o r e s c u e t h e rds p h e n o t y p e i n t h e h o m o z y g o u s or heterozygous backgrounds. heterozygous  state  without.  This  negative  effect.  degeneration  more  implies  that  The  effect  a n d therefore  peripherin-2.  severe the  P216L  Thus  the  of this could effects  with  the phenotype  the  P216L  P216L  mutation  transgene  also  on a W T background  dominant-negative transgene  is  Interestingly,  providing  mutant. not of  caused  a  than  dominant  photoreceptor  evidence for the  T h e authors did not tag the  distinguish this  transgene  exerts  further  in t h e rds  it  mutation  from on  its  endogenous subcellular  localization a n d biochemical properties were not e x a m i n e d .  1.5.4 Peripherin-2:rom-l chimeric mouse To  isolate  the functions  of the C-terminal  r e g i o n s o f p e r i p h e r i n - 2 , K e d z i e r s k i e t al. ( 1 9 9 9 )  a n d intradiscal  loop  constructed a chimeric  rom-1 molecule that contained the intradiscal loop region of peripherin-2.  Chapter 1 Introduction The  ability  of  38  this  molecule  to  restore  photoreceptor  function  was  a d d r e s s e d in vivo b y t r a n s g e n i c e x p r e s s i o n i n t h e rds m o u s e b a c k g r o u n d . In  the  heterozygous  photoreceptor between  rds  background,  defect completely;  h o m o z y g o u s rds  e x p r e s s i n g it.  the  however,  chimera  no difference  rescued  the  was observed  mice expressing the chimera a n d those not  Biochemical analysis of transgenic m i c e revealed that t h e  ratio of p e r i p h e r i n - 2  t o r o m - 1 i n R O S is a p p r o x i m a t e l y  2 . 5 , that the  t r a n s g e n e is g l y c o s y l a t e d a n d i n t e r a c t s w i t h b o t h p e r i p h e r i n - 2 a n d r o m - 1 , but d o e s not f o r m disulfide-linked h e t e r o d i m e r s . peripherin-2  homo-oligomerization  oligomerization  with  rom-1,  is  and that  T h e authors claim that  stronger peripherin-2  than and  heterorom-1 are  quantitatively associated. This study highlights the functional importance a n d independence of both  the  intradiscal  loop  region  photoreceptor disc morphogenesis. the  homozygous  rds  phenotype  and  the  C-terminal  region  to  T h e inability of t h e c h i m e r a to rescue identifies  the  importance  of the C -  t e r m i n a l r e g i o n a n d s u p p o r t s in vitro d a t a s u g g e s t i n g a r o l e f o r t h i s r e g i o n in m e m b r a n e f u s i o n d u r i n g d i s c f o r m a t i o n ( B o e s z e - B a t t a g l i a et al.,  1998).  The  of t h e  authors  membranes  did not extensively in t h e s e  peripherin-2 function  that  functions  animals. in  m a y not  protein be  not  characterize It  the morphology  is p o s s i b l e t h a t targeting present  to in  the C-terminus  the outer  rom-1.  of  segment,  Rescue  of  a  the  Chapter 1 Introduction  39  h e t e r o z y g o u s rds m o u s e b y t h e c h i m e r a h i g h l i g h t s t h e d o m i n a n t of  the  intradiscal  importance  loop  of  peripherin-2  of loop-mediated  and  peripherin-2  further  subunit  function  implicates  interactions  the  in d i s c  morphogenesis.  1.5.5  Gene therapy To  gene  on the rds mouse  investigate t h e possibility of t r e a t m e n t of retinal  therapy,  A N et  al.  (2000)  examined  the effects  of  diseases by intraocularly  injected recombinant adeno-associated virus carrying the gene encoding peripherin-2  in t h e null  o f t h e rds  background  observed a 3 0 % rod transduction  efficiency.  peripherin-2  segment  re-established  outer  stacks of discs similarly to W T .  mouse.  T h e authors  Photoreceptors expressing structures  which  contained  E x p r e s s i o n of r o m - 1 a n d rhodopsin w a s  also restored a n d localization of these proteins to outer s e g m e n t s w a s established. ERG  T h e p r e s e n c e of p h a g o s o m e s in R P E c e l l s a n d r e s t o r a t i o n o f  responses  photoreceptor demonstrates differentiated  in  treated  metabolism  retinal  and  indicate  mature  a  reconstitution  phototransduction.  for t h e first t i m e that function photoreceptor  photoreceptor specific gene. linked  animals  cells  of  This  study  can be restored  in fully  by  re-introduction  of  a  Hence, gene therapy to treat peripherin-2-  disease m a y be possible, but other  factors  need to be  Chapter 1 Introduction  40  d e f i n e d i n c l u d i n g t h e stability of long t e r m e x p r e s s i o n a n d t h e effects o n cone photoreceptors.  1.6  THESIS At the  role  of  b e g i n n i n g of this project,  cysteine  peripherin-2 formed  INVESTIGATIONS  and  residues rom-1  disulfide-linked  and  very  disulfide  little w a s k n o w n bonds  complexes.  It  was  homodimers  and  in  the  known  that  contained  c y s t e i n e r e s i d u e s in t h e i n t r a d i s c a l l o o p r e g i o n .  about  the  structure  of  each  seven  protein  conserved  M u t a t i o n s in t w o o f t h e  i n t r a d i s c a l c y s t e i n e s h a d b e e n linked to h u m a n R P . H e n c e , t h e initial goal of  this  thesis work  residues  in  presented unique  peripherin-2  determine  on  its  the  importance  oligomeric  of the  structure.  The  cysteine results  in t h i s t h e s i s p r o v i d e c o m p e l l i n g e v i d e n c e f o r a c e n t r a l  role  complexes  w a s to  of  disulfide-mediated  in  photoreceptor  oligomerization  disc  of  morphogenesis,  peripherin-2 and  identify  and core a  d i f f e r e n t i a l r e g u l a t o r y r o l e f o r r o m - 1 in t h i s p r o c e s s . T h e h y p o t h e s i s t h a t h u m a n d i s e a s e r e s u l t s f r o m d e c r e a s e d l e v e l s o f p e r i p h e r i n - 2 c o m p l e x e s in rod outer s e g m e n t s has been c o n f i r m e d  in vivo.  C h a p t e r 2 o f t h i s t h e s i s d e s c r i b e s a m u t a t i o n a l s t u d y in w h i c h e a c h of t h e t h i r t e e n c y s t e i n e r e s i d u e s of p e r i p h e r i n - 2 w a s r e p l a c e d by s e r i n e . T h e ability of t h e s e proteins to f o r m disulfide d i m e r s , a s s e m b l e into core  Chapter 1 Introduction  41  tetramers, and interact with rom-1 was e x a m i n e d .  This work has been  published elsewhere (Goldberg, Loewen & Molday, 1998). C h a p t e r 3 d e s c r i b e s the c h a r a c t e r i z a t i o n of p e r i p h e r i n - 2 a n d complexes under nonreducing conditions using a novel velocity  sedimentation  formation  is  technique.  determined.  This  The work  importance has  been  of  rom-1  two-dimensional disulfide  published  dimer  elsewhere  (Loewen & Molday, 2000). Chapter 4 e x a m i n e s the structural  determinants  of c o r e  tetramer  formation and disulfide-mediated oligomerization by analyzing the mutation oligomers  in p e r i p h e r i n - 2 in  preventing  responsible for disease  is  digenic RP.  proposed.  This  L185P  A role for work  these  has  been  C h a p t e r 5 p r e s e n t s the results of a collaborative w o r k with  David  published elsewhere (Loewen, Moritz & Molday, 2001).  P a p e r m a s t e r ' s g r o u p at t h e U n i v e r s i t y of C o n n e c t i c u t w h i c h e x a m i n e s the effects  of  e x p r e s s i o n of  RP-causing  p h o t o r e c e p t o r s in t r a n s g e n i c Xenopus of the t r a n s g e n e s a n d the  mutations laevis.  effects on rod  T h i s m a n u s c r i p t is in p r e p a r a t i o n .  in  peripherin-2  on  rod  The subcellular localization morphology  are  determined.  Chapter 2 Role of Cysteines CHAPTER  2  -  42  ROLE  OF  CYSTEINES  IN  PERIPHERIN-2  STRUCTURE 2.1  INTRODUCTION Peripherin-2  conserved  contains  among  species  thirteen  cysteine  or  rom-1 and  with  residues.  c y t o p l a s m i c a n d t r a n s m e m b r a n e r e g i o n s (Fig 9). conserved  among  species  and with  intradiscal loop region of t h e molecule.  are  Six are  located  in  not the  Seven are completely  rom-1 and are  located  in t h e  M u t a t i o n s in t w o o f t h e c o n s e r v e d  cysteines (C165Y and C214S) are responsible for autosomal dominant RP ( s e e T a b l e 2 in I n t r o d u c t i o n ) . In  this  study,  the importance  of the cysteine  residues  on the  structure a n d subunit a s s e m b l y of peripherin-2 h a s been e x a m i n e d . of  the  13 cysteine  replaced  with  residues  a serine  in  residue  bovine  peripherin-2  by site-directed  was  Each  individually  mutagenesis, and the  r e s u l t i n g m u t a n t s w e r e e x p r e s s e d i n d i v i d u a l l y o r t o g e t h e r w i t h r o m - 1 in COS-1  cells.  SDS-polyacrylamide  immunoprecipitation, evaluate  the  ability  a n d velocity of  these  gel  sedimentation mutants  to  were form  electrophoresis, carried  out to  disulfide-linked  h o m o d i m e r s , associate with r o m - 1 , a n d a s s e m b l e into t e t r a m e r s . The  results  of this  study  suggest that  (1) the conserved C 1 5 0  r e s i d u e is r e q u i r e d f o r i n t e r m o l e c u l a r d i s u l f i d e b o n d i n g b u t n o t s u b u n i t assembly;  (2) the six other conserved cysteine residues are crucial for  43  L3-4  Fig 9 . L o c a t i o n of c y s t e i n e s in bovine p e r i p h e r i n - 2 .  Nonconserved  c y s t e i n e s (open circles) are at a m i n o acid positions 7 2 , 8 1 , 1 0 5 , 1 1 8 , 119, and 3 0 1 ; conserved cysteines (shaded circles) are present within t h e intradiscal L 3 - 4 loop at positions 1 5 0 , 1 6 5 , 1 6 6 , 2 1 3 , 2 1 4 , 2 2 2 , a n d 250.  Chapter 2 Role of Cysteines proper  folding  intramolecular  44  and subunit disulfide  assembly,  bonds;  possibly  through  a n d (3) the misfolding  formation  of  a n d defective  s u b u n i t a s s e m b l y of t h e C 1 6 5 S a n d C 2 1 4 S m u t a n t s is r e s p o n s i b l e f o r a f o r m of m o n o g e n i c a u t o s o m a l d o m i n a n t retinitis p i g m e n t o s a . Most of these results are published elsewhere (Goldberg, Loewen & Molday, 1998).  A l l e x p e r i m e n t s d e s c r i b e d in t h i s c h a p t e r w e r e  by  except:  the author  (1) site-directed  mutagenesis;  performed  (2) analysis of  cysteine mutants by reducing a n d nonreducing S D S - P A G E (Fig 10).  2.2  MATERIALS AND METHODS  2.2.1  Site-directed Plasmids  Diego,  mutagenesis  composed  of t h e p c D N A I / A M P v e c t o r  C A ) a n d the full-length  (pcPER)  coding  or rom-1 (pcROM) were  used  (Invitrogen, S a n  regions for bovine for heterologous  peripherin-2 (COS-1)  expression as previously described (Goldberg, Moritz & Molday, 1995). PCR-based 1989)  method  w a s used  of  primer-directed  to generate  mutagenesis  missense mutations  (Nelson  in w h i c h  &  cell A  Long,  individual  cysteine residues were replaced with serine residues; subcloned portions of t h e W T p c P E R w e r e u s e d a s t e m p l a t e s . Plasmid pHindbg w a s constructed by ligating a 2 6 0 base-pair (bp) HindlU/Bglll  f r a g m e n t of t h e peripherin-2 coding region ( 5 ' region) into a  r V / n c f l l l / E c o R I - d i g e s t e d B l u e s c r i p t II  K S + plasmid (Stratagene, La Jolla,  Chapter 2 Role of Cysteines CA)  45  u s i n g a s y n t h e t i c BgM/EcoRI  AATTCGGTGACCA-3'). b p Bglll/Sacll directly was  adapter (5'-GATCTGGTCACCG-3\  Plasmid p B g S k s w a s constructed by ligating a 4 1 6  fragment of the peripherin-2 coding region (central  i n t o t h e B l u e s c r i p t II K S + p l a s m i d p o l y l i n k e r .  constructed  by  5'-  ligating  a  521 bp  Sacll/Xhol  part)  Plasmid pScXho fragment  of t h e  p e r i p h e r i n - 2 c o d i n g r e g i o n ( 3 ' r e g i o n ) d i r e c t l y i n t o t h e B l u e s c r i p t II S K + plasmid  polylinker.  Vancouver,  BC)  Sense-strand  and  the  synthetic  templates  used  oligonucleotides for  the  (NAPS,  corresponding  m u t a g e n e s i s r e a c t i o n s a r e s h o w n i n T a b l e 3. PCR products obtained essentially as described 1989) were digested with the appropriate  restriction  (Nelson & enzymes,  Long,  purified  f r o m a g a r o s e gels, a n d cloned into their respective t e m p l a t e vectors ( s e e a b o v e a n d T a b l e 3).  T h e desired mutations were selected by complete  ( s i n g l e - s t r a n d ) d i d e o x y - s e q u e n c i n g with a S e q u e n a s e T 7 kit ( U S B ) . mutagenized peripherin-2  regions gene  were  a n d final  subsequently  subcloned  expression constructs  back were  into  The  the WT  confirmed  by  restriction m a p p i n g .  2.2.2  Heterologous  expression  C O S - 1 cells ( c a . 2 x 1 0 / 6 0 m m dish) were singly transfected with 5  expression plasmid (12 ug) or cotransfected with a mixture of t w o  46  Chapter 2 Role of Cysteines Table 3 Synthetic Oligonucleotides Used for PCR Mutagenesis Subclone  Mutation  Synthetic  oligonucleotide  pHindBg  C72S  5' - C T G T C C T C T G T C T T C A A T T C T C T G G - 3'  pBgSks  C81S C105S C118S C119S C150S C165S C166S C213S C214S  5'-GGCAAGATCTCTTACGACGCCC-3' 5' - G C C G T G T C T G T C C T C T T C A A C G - 3' 5'-GGCCCTCAGCTGCTTCCTCCTGC-3' 5'-GCCCTCTGCAGCTTCCTCCTGCG-3' 5'-CCAGGCCGGTCTTTCATGAAG-3' 5' - G A T C G A A T T C A A G A G C T G C G G - 3' 5'-CGAATTCAAGTGCAGCGGCAAC-3' 5'-CCCTTCAGCTCCTGCAACCC-3' 5'-TTCAGCTGCAGCAACCCCAAC-3'  pScXho  C222S C250S C301S  5'-CCGCGGCCCTCGATCCAGTACC-3' 5'-CTGCGTGGCAGCAGGGCC-3' 5'-GACCCTGAGTCCGAGAGTGAGG-3'  Chapter 2 Role of Cysteines  47  expression  p l a s m i d s ( 6 pg  essentially  a s described (Chen & O k a y a m a , 1 9 8 7 ; G o l d b e r g , Moritz  Molday, 1995).  each)  using the calcium phosphate  method &  A t 7 2 h post-transfection, o n e dish of cells w a s w a s h e d  t w i c e w i t h P B S a n d s o l u b i l i z e d w i t h 3 0 0 - 5 0 0 pl o f 1 % T r i t o n X - 1 0 0 i n PBS buffer containing P M S F (pH 7 . 4 ) . For velocity s e d i m e n t a t i o n studies, ImM  D T T w a s i n c l u d e d in t h e s o l u b i l i z a t i o n buffer.  centrifuged at 9 0 , 0 0 0 x g for 30 m i n at 4° C  Cell extracts  were  in a B e c k m a n T L A - 4 5 r o t o r  and t h e s u p e r n a t a n t s (detergent solubilized fraction) w e r e retained on ice until u s e d .  T y p i c a l l y 1 5 - 3 0 pl o f e x t r a c t w a s s u b j e c t e d t o S D S - P A G E  under reducing a n d nonreducing conditions (presence or absence of 4 % 2-mercaptoethanol),  and  peripherin-2  expression  was  detected  on  Western blots.  2.2.3  Immunoprecipitation Homomeric  complexes  peripherin-2  were  isolated  immunoprecipitation  and velocity and from  sedimentation  heteromeric COS-1  cell  peripherin-2:rom-l extracts  by  procedure (Goldberg & Molday, 1996a).  an  Triton X -  1 0 0 - s o l u b i l i z e d c e l l e x t r a c t s ( 1 0 0 pl ) w e r e i n c u b a t e d w i t h 2 5 - 5 0 pl o f anti-peripherin-2  M A b P e r 2 B 6 coupled to S e p h a r o s e beads.  T h e matrix  was w a s h e d three t i m e s with e x c e s s buffer to r e m o v e u n b o u n d  proteins,  and t h e bound peripherin-2 c o m p l e x w a s eluted with a n e x c e s s of 2 B 6  Chapter 2 Role of Cysteines peptide  48  corresponding  to  the C-terminal  nine  amino  acids  of  bovine  peripherin-2. For complex  velocity  sedimentation  studies,  the  purified  peripherin-2  w a s applied to 5 - 2 0 % ( w / w ) sucrose gradients  in P B S buffer  c o n t a i n i n g 0 . 1 % T r i t o n X - 1 0 0 , P M S F a n d 1 m M D T T a n d c e n t r i f u g e d in a Beckman TLS-55 fractions  were  rotor  for 16 h at 50,000  collected from  the tube  rpm a n d 4° C.  bottom  and equivalent  were subjected to S D S - P A G E under reducing conditions. profiles  of fractionated  sucrose gradients  Four-drop  were  volumes  Sedimentation  determined  by  western  blotting with P e r 2 B 6 M A b a n d laser densitometry, according to Goldberg etal.  (1995).  2.2.4 Calculation of sedimentation coefficients Estimates  of  values  S2o,w  were  made  as  described  (Goldberg, Moritz & Molday, 1995), but with the following all  partial  (determined Molday,  specific  volume  for the  1996b)),  bovine  initial  values  were  assumed  peripherin-2:rom-1  sample  radius  {r ) mm  to  modifications: be  complex  was taken  originally  0.83  (Goldberg  and  gradient height w a s measured as 29 m m .  &  as 47.5 m m  ( B e c k m a n T L S - 5 5 r o t o r r in c o r r e c t e d f o r s a m p l e a n d g r a d i e n t m  ml/g  volume),  F o r a review of velocity  sedimentation analysis see Clarke & Smigel (1989).  Chapter 2 Role of Cysteines 2.2.5  SDS-PAGE  49  and Western  blotting  2 5 pl s a m p l e s c o n t a i n i n g a p p r o x i m a t e l y 1.5 p g o f t o t a l p r o t e i n w e r e denatured with a n equal v o l u m e of S D S cocktail ( 4 % S D S , 0 . 0 2 M TrisHCl,  pH 6.8, 4 0 % sucrose, 0 . 0 1 % Bromophenol  (nonreducing) were  or presence  applied  to  8%  (reducing)  or  10%  electrophoresis, the proteins  were  blue  in t h e a b s e n c e  of 5 % p-mercaptoethanol,  S D S polyacrylamide transferred  and  gels.  After  to I m m o b i l o n - P  using a  BioRad s e m i d r y transfer apparatus a n d t h e blots w e r e labeled with t h e anti-peripherin-2  MAb Per2B6 or the anti-rom-1 M A b R o m l C 6  Hicks & Molday, 1 9 8 7 ; Moritz & Molday,  1996) and sheep  (Molday,  anti-mouse  immunoglobulin-peroxidase for detection by ECL.  2.3  RESULTS  2.3.1  Location  of cysteine  residues  Previous studies have demonstrated expression  system  peripherin-2  (Goldberg  1995).  This  for  system  t h e utility of a  mutagenesis-based  & Molday,  1996a;  has now been  heterologous  structural  studies  Goldberg, Moritz  used  to  examine  of  &  Molday,  the  relative  i m p o r t a n c e of t h e v a r i o u s c y s t e i n e r e s i d u e s f o u n d in b o v i n e p e r i p h e r i n - 2 . Fig (Connell  9 &  depicts Molday,  the  current  1 9 9 0 ; Molday,  topological  model  for  1 9 9 4 ) in t h e o u t e r  peripherin-2 segment  disc  m e m b r a n e a n d highlights the position a n d conservation of the 13 cysteine  Chapter 2 Role of Cysteines  50  r e s i d u e s p r e s e n t in t h e b o v i n e s e q u e n c e .  R e s i d u e s s h a d e d in b l a c k a r e  a b s o l u t e l y c o n s e r v e d in b o t h p e r i p h e r i n - 2  a n d its h o m o l o g o u s  rom-1,  a c r o s s all s p e c i e s e x a m i n e d  to  date  (Bascom  et  subunit,  al.,  1992b;  Bascom, Schappert & Mclnnes, 1 9 9 3 ; Begy & Bridges, 1 9 9 0 ; Connell & Molday,  1 9 9 0 ; G o r i n et al.,  1 9 9 3 ; K e d z i e r s k i e t al.,  Kedzierski & Travis, 1 9 9 5 ; Ray, Acland & Aguirre, 1 9 8 9 ; T r a v i s e t al.,  1996; Moghrabi,  1 9 9 6 ; Travis et  1 9 9 1 ) (see sections 1.2.2 a n d 1.2.3).  al.,  These seven  c o n s e r v e d cysteines a r e confined to the large intradiscal L 3 - 4 loop joining the third a n d fourth t r a n s m e m b r a n e s e g m e n t s .  Nonconserved residues  a r e p r e s e n t in b o t h t h e p u t a t i v e t r a n s m e m b r a n e s e g m e n t s a n d s e g m e n t s e x p o s e d o n t h e c y t o p l a s m i c side of t h e m e m b r a n e .  2.3.2 Expression and disulfide dimerization In  order  properties  to assess  of peripherin-2,  peripherin-2  t h e effect  of the various  each of t h e 13 cysteine  w a s individually  substituted  with serine.  mutations  on the  r e s i d u e s in  bovine  As shown  in Fig  10A, all o f t h e v a r i a n t s c o u l d b e e x p r e s s e d in C O S - 1 cells a n d m i g r a t e d as  monomers  (apparent  reducing conditions. among  experiments  systems),  none  M  r  3 5 K ) on S D S - p o l y a c r y l a m i d e gels  under  A l t h o u g h relative levels of protein e x p r e s s i o n varied (a  common  of the mutants  property  showed  of  transient  consistently  transfection  altered  levels of  expression relative to W T . T h e s e observations indicate that none of t h e  51  Non-conserved  Conserved  monomer  B top  dimer monomera o, o, c\ a c» a % % % % % % > ^ ^ ^> ^> * * % \ % % % % ^% %%% %  Fig 1 0 . E x p r e s s i o n a n d d i m e r i z a t i o n of W T a n d m u t a n t p e r i p h e r i n - 2 . C O S - 1 cells transfected with plasmids containing t h e indicated mutants were detergent-solubilized 72 h posttransfection, a n d approximately 1.5 p g o f t o t a l p r o t e i n / l a n e w a s s u b j e c t e d t o S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s in t h e p r e s e n c e ( A ) o r a b s e n c e ( B ) o f r e d u c i n g agent (2-mercaptoethanol). Western blots w e r e labeled with antiperipherin-2 M a b Per2B6 for chemiluminescent detection (see Methods sections 2.2.2 and 2.2.5)  Chapter 2 Role of Cysteines cysteine  residues  52  are  absolutely  required  for  the  biosynthesis  of  the  p e r i p h e r i n - 2 p o l y p e p t i d e in C O S - 1 c e l l s . The under  mobilities  nonreducing  variants  to  panel),  form  a  mutants  of  the  peripherin-2  conditions  to  disulfide-linked  substantial migrated  portion  mutants  assess  the  dimers.  of  the  at a p p r o x i m a t e l y  were  ability  of  As shown  WT  twice  and the  in  also these  rod  photoreceptor  Molday,  1987).  The  panel),  on  other  the  outer  segment  conserved hand,  Peripherin-2 with mutations  showed  at positions  10B  nonconserved  mutants  of  the  peripherin-2  (Molday,  (Figure  distinctly  (left  cysteine  molecular weight  membranes  cysteine  cysteine  Fig  m o n o m e r i c f o r m of p e r i p h e r i n - 2 , a s p r e v i o u s l y r e p o r t e d for from  compared  different  Hicks  10B,  right  behaviors.  165, 166, 213, 214, 222,  and  2 5 0 f o r m e d m a i n l y d i m e r s a n d a g g r e g a t e s f o u n d at the top of the gel. contrast,  the  conserved C150S  mutant  migrated  solely as a  &  In  monomer  under n o n r e d u c i n g , as well as reducing, conditions. T h e s e results indicate t h a t n o n c o n s e r v e d c y s t e i n e m u t a n t s , like W T p e r i p h e r i n - 2 , are c a p a b l e of f o r m i n g C165S,  C166S,  dimers  under  C213S,  C214S,  C222S,  dimers  in  presence or  a b s e n c e of  addition  C 1 5 0 S to f o r m  reducing  agents  have  also  form  folding  of t h e  in  mutants  t e n d e n c y to f o r m larger a g g r e g a t e s , m o s t likely d u e to i m p r o p e r T h e inability  but  C250S  strong  mutants.  conditions  and  Conserved  a  of t h e s e  nonreducing  disulfide-linked dimers.  disulfide-linked  suggests that  this  Chapter 2 Role of Cysteines  53  c y s t e i n e r e s i d u e is e s s e n t i a l f o r i n t e r m o l e c u l a r d i s u l f i d e b o n d Three nonconserved cysteine mutants  formation.  (C72S, C118S, and C119S), two  conserved cysteine mutants (C165S and C214S), a n d the intermolecular disulfide dimerization-defective mutant ( C 1 5 0 S ) were selected for further study.  2.3.3  Interaction  with rom-1  Co-immunoprecipitation peripherin-2  assembles with  studies  have  previously  r o m - 1 in m e m b r a n e s  established  of both  that  vertebrate  p h o t o r e c e p t o r s a n d t r a n s i e n t l y c o t r a n s f e c t e d C O S - 1 c e l l s ( B a s c o m e t al., 1992b; G o l d b e r g , Moritz & Molday, 1 9 9 5 ; Moritz & Molday, 1996).  This  procedure h a s n o w been used to assess t h e ability of t h e n o n c o n s e r v e d and conserved peripherin-2 cysteine mutants to assemble with W T rom-1. C O S - 1 cells were transiently rom-1  plasmids  and  immunoprecipitated the  cell e x t r a c t s  the  cotransfected with  detergent-solubilized  cell  with a P e r 2 B 6 - S e p h a r o s e matrix.  and unbound  labeled with anti-peripherin-2  and bound fractions  peripherin-2 and extracts  were  W e s t e r n blots of were  subsequently  a n d anti-rom-1 antibodies to assess the  interaction of W T a n d m u t a n t peripherin-2 with r o m - 1 . 11A, p e r i p h e r i n - 2 i m m u n o r e a c t i v i t y  A s s h o w n in Fig  w a s o b s e r v e d in t h e b o u n d  fraction  ( l a n e c ) b u t n o t in t h e u n b o u n d f r a c t i o n ( l a n e b) in e v e r y c a s e , i n d i c a t i n g that the Per2B6-Sepharose matrix quantitatively  precipitated W T and the  54  WT  C118S  C150S  C214S  WT  C118S  C150S  C214S  peripherin  B  rom-1  a  Fig 1 1 .  b  c  a b c  a  b  c  a  b  c  C o a s s e m b l y of p e r i p h e r i n - 2 c y s t e i n e m u t a n t s w i t h r o m - 1 .  C O S - 1 cells were cotransfected with wild-type (WT) or m u t a n t peripherin-2 p l a s m i d a n d W T r o m - 1 p l a s m i d . D e t e r g e n t - s o l u b i l i z e d e x t r a c t s (a) w e r e a d s o r b e d to P e r 2 B 6 - S e p h a r o s e , a n d after r e m o v a l of t h e u n b o u n d f r a c t i o n s ( b ) , t h e b o u n d f r a c t i o n s (c) w e r e e l u t e d w i t h t h e 2 B 6 c o m p e t i n g p e p t i d e . S a m p l e s w e r e a n a l y z e d on W e s t e r n blots labeled w i t h e i t h e r t h e p e r i p h e r i n - 2 Mab p e r 2 B 6 (A) or the r o m - 1 Mab R o m l C 6 (B) (see M e t h o d s sections 2 . 2 . 2 , 2.2.3and2.2.5).  Chapter 2 Role of Cysteines various  cysteine  55  peripherin-2  mutants  from  C O S - 1 cell  A n a l o g o u s w e s t e r n blots labeled with a n a n t i - r o m - 1  antibody  extracts. R o m1C6  (Fig 11B) f u r t h e r s h o w e d t h a t o v e r 9 0 % o f t h e r o m - 1 c o p r e c i p i t a t e d w i t h WT  peripherin-2  (second panel). and  (first  panel)  and the  nonconserved  C 1 1 8 S mutant  Similar results were obtained for the nonconserved C 7 2 S  C 1 1 9 S mutants  (data  not  shown).  Interestingly,  r o m - 1 also  c o p r e c i p i t a t e d w i t h t h e C 1 5 0 S d i s u l f i d e - d i m e r - d e f e c t i v e m u t a n t (Fig 1 1 B , third panel, lane c).  In contrast,  r o m - 1 did not coprecipitate with the  C 2 1 4 S (Fig 1 1 B , f o u r t h p a n e l ) o r C 1 6 5 S m u t a n t s ( d a t a n o t s h o w n ) b u t i n s t e a d w a s p r e s e n t o n l y i n t h e u n b o u n d f r a c t i o n (Fig 1 1 B , f o u r t h p a n e l , lane b). These studies indicate that the interaction of peripherin-2 with r o m 1  requires  cysteines at conserved  positions  nonconserved postions 7 2 , 118, or 1 1 9 . serine  at  the  conserved  position  165 a n d 2 1 4 but not at  R e p l a c e m e n t of cysteine with  150  prevents  homodimerization but does not prevent peripherin-2 from  disulfide-linked noncovalently  associating with r o m - 1 .  2.3.4  Velocity sedimentation Previous  complex from  studies  have  under reducing  shown  that  the  conditions WT  peripherin-2:rom-l  rod o u t e r s e g m e n t a n d C O S - 1 cell m e m b r a n e s s e d i m e n t s  as a heterotetrameric  complex  (Goldberg & Molday, 1996b; Goldberg,  Chapter 2 Role of Cysteines  56  Moritz & Molday, 1995). assembles  In t h e a b s e n c e of r o m - 1 , W T p e r i p h e r i n - 2 self-  into a h o m o t e t r a m e r i c  protein.  T h i s f o r m o f t h e p r o t e i n is  thought to c o m p e n s a t e for reduced levels of the heterotetrameric in  i n d i v i d u a l s w h o inherit a null  allele of r o m - 1 (Goldberg  protein  & Molday,  1996a). W e have e x a m i n e d the sedimentation behavior of several conserved and nonconserved peripherin-2 cysteine mutants or with r o m - 1 .  expressed  individually  T h e sedimentation profiles a n d s e d i m e n t a t i o n coefficients  a r e g i v e n i n Fig 12 a n d T a b l e 4.  The nonconserved cysteine  mutants  exhibited sedimentation profiles a n d sedimentation coefficients similar to WT  peripherin-2  in  both  the  presence  and  absence  of  rom-1.  C150S  mutant  also  had a  Interestingly,  the disulfide-dimer-defective  sedimentation  profile a n d coefficient s i m i l a r t o W T in t h e p r e s e n c e a n d  absence  of  rom-1.  In  contrast,  the  conserved  C214S  and  C165S  m u t a n t s , which failed to a s s e m b l e with r o m - 1 , s e d i m e n t e d m o r e  slowly  t h a n W T p e r i p h e r i n - 2 w h e n s i n g l y e x p r e s s e d i n C O S - 1 c e l l s (Fig 12 a n d Table  4).  mutants  In  addition,  sedimented  a significant,  near  or at  but variable, fraction  the bottom  of  these  of t h e centrifuge  tube,  presumably due to aggregation. These cysteine assembly.  results  residues  indicate with  that  serine  replacement  does  not  affect  of  the  nonconserved  peripherin-2  subunit  In c o n t r a s t , r e p l a c e m e n t of t h e c o n s e r v e d c y s t e i n e s (with t h e  57  50  H  WT  25  50  C118S  25 X  I  0  o  C150S  ifro  50  ^  25 H  o  *J  i\  50 H  C165S  25  *4 10  15  20  5  10  15  20  % s u c r o s e (w/w)  Fig 1 2 .  V e l o c i t y s e d i m e n t a t i o n a n a l y s i s of p e r i p h e r i n - 2  cysteine  mutants. C O S - 1 cells w e r e transfected with p e r i p h e r i n - 2 wild-type (WT) or mutant plasmids alone ( C 1 1 8 S , C 1 5 0 S , or C 1 6 5 S ) or together with rom-1 (WT + r o m - 1 , C 1 1 8 S + r o m - 1 , or C 1 5 0 S + rom-1). Peripherin-2-containing c o m p l e x e s w e r e purified on a P e r 2 B 6 - S e p h a r o s e matrix a n d s e d i m e n t e d t h r o u g h a 5 - 2 0 % s u c r o s e g r a d i e n t in t h e p r e s e n c e o f 1 m M DTT. F r a c t i o n s were a s s a y e d on w e s t e r n blots for peripherin-2 with P e r 2 B 6 antibody for singly transfected cells and with R o m l C 6 antibody for co-transfected cells and quantified by laser densitometry (see Methods section 2.2.3). Velocity s e d i m e n t a t i o n s t u d i e s of the C 1 6 5 S a n d C 2 1 4 S m u t a n t s c o - e x p r e s s e d with r o m - 1 w e r e not carried out since these m u t a n t s do not a s s o c i a t e with r o m - 1 . T h e d a s h e d line i n d i c a t e s t h e s e d i m e n t a t i o n position of W T c o m p l e x .  Chapter 2 Role of Cysteines  58  Table 4 Sedimentation Coefficients for Peripherin-2 a n d P e r i p h e r i n - 2 : r o m - l Complexes  1  S20.W (S) Conservation/location  Variant  - Rom-1  + Rom-1  WT  5.4 + 0.1 (n = 3)  5.0 ± 0 . 4 (n = 4 )  C72S  5.1 ± 0 . 4 (n = 3)  4 . 8 ± 0.1 (n = 3)  nc/M2  C118S  5.4 ± 0.1 (n = 3)  5.1 ± 0.1 (n = 3)  nc/M3  C119S  5.5 ± 0 . 2 (n = 3)  5.2 ± 0 . 1 (n = 3)  nc/M3  C150S  5.2 ± 0 . 3 (n = 3)  5.0 ± 0 . 2 (n = 3)  C/L3-4  C165S  3.9 + 0.1 (n = 3)  not a s s e m b l e d  C/L3-4  C214S  3.9 ± 0 . 2 (n = 3)  not a s s e m b l e d  C/L3-4  3  •  2  -  S e d i m e n t a t i o n coefficients e s t i m a t e d a s d e s c r i b e d in t h e M a t e r i a l s a n d M e t h o d s are g i v e n f o r W T a n d m u t a n t p e r i p h e r i n - 2 a n d p e r i p h e r i n - 2 - r o m - l c o m p l e x e s ± S . D . (n e q u a l s t h e n u m b e r of i n d e p e n d e n t e x p e r i m e n t s ) . V a l u e s f o r W T , C 7 2 S , C 1 1 8 S , C 1 1 9 S , and C150S mutants are similar; the WT peripherin-2-rom-l c o m p l e x f r o m rod o u t e r s e g m e n t s h a s been s h o w n to be a t e t r a m e r ( G o l d b e r g 81 M o l d a y , 1 9 9 6 b ) . V a l u e s f o r C 1 6 5 S a n d C 2 1 4 S e x p r e s s e d in t h e a b s e n c e of r o m 1 a r e c o n s i d e r a b l y less t h a n t h a t f o r t h e t e t r a m e r i c f o r m of p e r i p h e r i n - 2 . c, conserved; nc, nonconserved; M2, transmembrane segment 2; M3, t r a n s m e m b r a n e s e g m e n t 3 ; L 3 - 4 , i n t r a d i s k a l loop j o i n i n g transmembrane s e g m e n t s 3 a n d 4 ( s e e Fig 9 ) . 2  T h e s e d i m e n t a t i o n b e h a v i o u r of W T p e r i p h e r i n - 2 a n d p e r i p h e r i n - 2 - r o m - l h a s been c h a r a c t e r i z e d p r e v i o u s l y ( G o l d b e r g , Moritz & M o l d a y , 1 9 9 5 ) . S e d i m e n t a t i o n coefficients f o r t h e s e s p e c i e s h a v e been r e c a l c u l a t e d f r o m t h e original d a t a using new v a l u e s f o r (1) t h e partial specific v o l u m e of t h e p r o t e i n - d e t e r g e n t - c o m p l e x a n d (2) s u c r o s e g r a d i e n t g e o m e t r y , a s d e s c r i b e d in t h e M e t h o d s . W e c o n s i d e r t h a t t h e s e a d j u s t m e n t s m o r e a c c u r a t e l y reflect a b s o l u t e s o,w v a l u e s a s measured. 3  2  Chapter 2 Role of Cysteines exception  of cysteine  assemble  into  59 at position  tetramers,  150) result  presumably  in p r o t e i n s  d u e to  improper  that  fail  folding.  to  The  C 1 5 0 S m u t a n t is u n i q u e in t h a t r e p l a c e m e n t o f t h i s c o n s e r v e d c y s t e i n e affects d i s u l f i d e - l i n k e d d i m e r i z a t i o n b u t d o e s n o t affect its a s s e m b l y into homo- and heterotetramers.  2.4  DISCUSSION In t h i s s t u d y t h e c o n t r i b u t i o n of t h e v a r i o u s c y s t e i n e r e s i d u e s t o t h e  folding  and subunit  individually  a s s e m b l y of peripherin-2  replacing  each  cysteine  with  a  has been determined serine  residue.  by  T h e 13  c y s t e i n e r e s i d u e s in b o v i n e p e r i p h e r i n - 2 fall into t w o g r o u p s a c c o r d i n g t o their c o n s e r v a t i o n a n d effect o n protein structure. six  nonconserved  cysteine  residues  G r o u p I consists of the  present  in  the  putative  t r a n s m e m b r a n e a n d cytoplasmic s e g m e n t s of peripherin-2 (positions 7 2 , 81,  1 0 5 , 1 1 8 , 1 1 9 , and 3 0 1 ) , while  conserved cysteines found  group  in t h e i n t r a d i s c a l  II  contains  loop  seven  (positions  highly  150,165,  166, 2 1 3 , 2 1 4 ,222, and 250). Substitution formation  of  polyacrylamide  of group  disulfide-linked gel  Immunoprecipitation of  these  I cysteines has no apparent  mutants  homodimers  electrophoresis  under  as  effect  analyzed  nonreducing  on the  by S D S conditions.  studies a n d velocity s e d i m e n t a t i o n a n a l y s e s of three (C72S,  C118S,  and C119S)  indicate  that,  like W T  Chapter 2 Role of Cysteines peripherin-2, they  60  a s s e m b l e into heterotetramers  or homotetramers  in  the p r e s e n c e o r a b s e n c e of r o m - 1 , respectively.  It is l i k e l y t h a t t h e o t h e r  three nonconserved cysteines behave similarly.  O n this basis, o n e c a n  conclude  that  the  nonconserved  cysteine  residues  do  not  play  an  i m p o r t a n t role in t h e f o l d i n g o r s u b u n i t a s s e m b l y o f p e r i p h e r i n - 2 a n d a r e not likely t o b e i n v o l v e d in i n t r a - o r i n t e r m o l e c u l a r d i s u l f i d e b o n d i n g . T h e s e v e n c o n s e r v e d c y s t e i n e r e s i d u e s o f g r o u p II a p p e a r t o p l a y a n i m p o r t a n t role in t h e s t r u c t u r e o f p e r i p h e r i n - 2 . II c y s t e i n e r e s i d u e s ( p o s i t i o n s required Individual  for  proper  folding  S i x of the seven group  165, 166, 2 1 3 , 2 1 4 , 2 2 2 , and 250) are and  subunit  assembly  of  peripherin-2.  r e p l a c e m e n t o f t h e s e c y s t e i n e r e s i d u e s w i t h s e r i n e r e s u l t s in  m u t a n t proteins t h a t d o not a s s e m b l e into a native-like t e t r a m e r . S D S - P A G E and velocity sedimentation studies reveal that these  Both  mutants  exist a s a mixture of large protein aggregates a n d a distinct species, most likely a d i m e r .  T h e intradiscal c o m p a r t m e n t  in w h i c h t h e s e c o n s e r v e d  c y s t e i n e s r e s i d e i s t o p o l o g i c a l ^ s i m i l a r t o t h e e x t r a c e l l u l a r s p a c e a n d is expected  to  maintain  a  nonreducing  environment.  Therefore,  it  is  possible t h a t s o m e o r all of t h e s e s i x c y s t e i n e r e s i d u e s participate  in  intramolecular disulfide bonds. The  presence  of  intramolecular  disulfide  bonds  in  peripherin-2  p r e v i o u s l y h a s b e e n s u g g e s t e d f r o m t h e s t u d i e s o f B a s c o m et al. ( 1 9 9 2 b ) . Intramolecular  disulfide  bonds  in i n t e g r a l  membrane  polypeptides are  Chapter 2 Role of Cysteines  61  known to be important for both proper folding a n d subunit a s s e m b l y . For e x a m p l e , vertebrate rod a n d cone opsins have a n intramolecular disulfide bond b e t w e e n t h e third t r a n s m e m b r a n e helix a n d s e c o n d intradiscal loop t h a t is e s s e n t i a l f o r p r o p e r p r o t e i n f o l d i n g , t r a n s p o r t , s t a b i l i t y , a n d l i g h t dependent  activation  of transducin  S a k m a r & Ostrer, 1997).  (Karnik  &  Khorana,  1990; Kazmi,  A s s e m b l y of the G L U T - 1 glucose  transporter  into t e t r a m e r s also d e p e n d s strongly o n t h e p r e s e n c e of i n t r a m o l e c u l a r d i s u l f i d e b o n d s l o c a t e d in t h e e x t r a c e l l u l a r d o m a i n s ( Z o t t o l a e t a / . , 1 9 9 5 ) . In  the  case  of  peripherin-2,  the  misfolding  and abnormal  subunit  a s s e m b l y o b s e r v e d f o r t h e s i x g r o u p II c y s t e i n e m u t a n t s m a y r e s u l t f r o m the  disruption  cysteine  residues with  contribute  to  peripherin-2 The  of intramolecular  the  more  disulfide  bonds.  R e p l a c e m e n t of t h e  polar serine residues, however,  abnormal  structural  properties  observed  m a y also in  these  mutants.  cysteine  at  position  1 5 0 is  unique  c o n s e r v e d c y s t e i n e r e s i d u e s in t h a t t h e C 1 5 0 S  among mutant  the  group  II  is i n c a p a b l e o f  f o r m i n g d i s u l f i d e - l i n k e d h o m o d i m e r s b u t d o e s a s s e m b l e i n t o a t e t r a m e r in the  presence  or  absence  sedimentation analysis.  of  rom-1  as  subunit  intermolecular  association disulfide  by  velocity  T h i s s i n g l e c y s t e i n e , t h e r e f o r e , is n e c e s s a r y a n d  sufficient for disulfide-linked h o m o d i m e r i z a t i o n . and  determined  of  peripherin-2  bond; instead subunit  Interestingly, the folding does  not  require  this  a s s e m b l y appears to be  Chapter 2 Role of Cysteines mediated  primarily  62  through  noncovalent  s u r p r i s i n g r e s u l t is n o t u n p r e c e d e n t e d . cleavage  of  the  intersubunit  monooxygenase stoichiometry concludes  does  not  or activity  that  a  lack  (Ishida of  destabilizes the tetrameric  that  e t al.,  bridges  1996).  proteins  somewhat  bovine  dopamine-  tetrameric  a result that  also  bonds  significantly  might  be predicted  Structural stability of other  has also been shown to be dependent  presence of intersubunit  subunit  T h e s a m e study  disulfide  from purely thermodynamic considerations. multisubunit  in  enzyme's  intersubunit structure,  This  Recent studies have shown that  disulfide  affect  interactions.  c o v a l e n t b o n d s ( R e z n i k e t al.,  upon the  1996).  These  considerations suggest that the intermolecular disulfide bond at position 150  of peripherin-2  m a y contribute  to the stability  of t h e  tetrameric  protein complex. A l t e r n a t i v e l y , t h e s e d i s u l f i d e b o n d s m a y n o t b e d i r e c t l y i n v o l v e d in tetramer formation  b u t instead m a y link t h e t e t r a m e r s into h i g h e r o r d e r  species that are not observed under the conditions used for the velocity sedimentation  experiments  reported  here.  Studies  addressing  this  p o s s i b i l i t y a r e r e p o r t e d in t h e f o l l o w i n g c h a p t e r . The  C214S  mutation  monogenic  form  progressive  degeneration  missense mutation  of  in  autosomal of  peripherin-2 dominant  photoreceptors  has RP  been  linked  characterized  (Saga  e t al.,  to  a  by  a  1993).  in a n o t h e r c o n s e r v e d c y s t e i n e r e s i d u e , C 1 6 5 Y ,  A has  Chapter 2 Role of Cysteines  63  also been reported to cause RP (Souied e t a / . , 1998).  Studies presented  here indicating that these m u t a n t s a r e misfolded a n d incapable of forming heterotetramers  with  rom-1 are consistent  with  their  p h o t o r e c e p t o r d e g e n e r a t i o n a n d s u p p o r t t h e subunit autosomal  dominant  involvement  assembly  RP (Goldberg & Molday, 1996a).  in  model for  In t h e s i m p l e s t  case, t h e disease state c a n result from d e c r e a s e d levels of peripherin-2c o n t a i n i n g t e t r a m e r s in i n d i v i d u a l s h e t e r o z y g o u s f o r t h e C 2 1 4 S o r C 1 6 5 Y mutations.  R e d u c e d l e v e l s o f p e r i p h e r i n - 2 h a v e b e e n s h o w n t o r e s u l t in  shortened, highly disorganized outer s e g m e n t s a n d lead to slow degeneration 1985).  in  heterozygous  rds  mice  (Hawkins,  Jansen  &  retinal Sanyal,  Haploinsufficient levels of peripherin-2-containing tetramers  have  b e e n s u g g e s t e d t o a c c o u n t , a t l e a s t in p a r t , f o r t h e d i g e n i c f o r m o f R P resulting f r o m c o i n h e r i t a n c e of a L 1 8 5 P p e r i p h e r i n - 2 g e n e a n d a null allele of r o m - 1 ( G o l d b e r g , Moritz & M o l d a y , 1 9 9 5 ) . peripherin-2-containing  tetramers  A l t h o u g h r e d u c e d levels of  c a n lead to a disease p h e n o t y p e , the  presence of the misfolded C 2 1 4 S or C 1 6 5 Y mutants to t h e d i s e a s e p h e n o t y p e in a d o m i n a n t n e g a t i v e Missense mutations  manner.  conserved cysteine residues are  to  mutations  i n t h e n o n c o n s e r v e d c y s t e i n e s a r e e x p e c t e d t o h a v e l i t t l e , if  missense  autosomal  contribute  predicted  a n y , effect.  cause  in t h e o t h e r  m a y also  dominant  F i s h m a n e t al. ( 1 9 9 7 )  mutation  RP.  In  contrast,  missense  have reported that individuals with a  in t h e n o n c o n s e r v e d  cysteine  of codon  72 (C72S  Chapter 2 Role of Cysteines mutation) are normal.  64 These authors have ascribed this mutation  as a  polymorphism. A u t o s o m a l dominant RP has also been linked to a deletion at t h e n o n c o n s e r v e d 1 1 8 o r 1 1 9 position of p e r i p h e r i n - 2 1991b). either  mutation  ( F a r r a r et  al.,  Studies presented here showing that a serine substitution of  these  nonconserved  positions  has  no  obvious  effect  at on  peripherin-2 structure or a s s e m b l y suggests that the deletion of a n amino a c i d , a n d n o t t h e a b s e n c e o f a c y s t e i n e r e s i d u e , is p r i m a r i l y for t h e d i s e a s e .  responsible  I n d e e d , preliminary heterologous e x p r e s s i o n studies of  the C118del (C119del) peripherin-2 variant  i n d i c a t e t h a t t h i s m u t a n t is  h i g h l y m i s f o l d e d ( C . L o e w e n a n d R. M o l d a y , u n p u b l i s h e d r e s u l t s ) .  The  shortening of t h e polypeptide c h a i n , a n d not loss of cysteine functionality, appears to prevent t h e proper folding of this mutant. In c o n c l u s i o n , t h i s m u t a g e n e s i s s t u d y i n d i c a t e s t h a t t h e c o n s e r v e d c y s t e i n e r e s i d u e s within t h e large intradiscal loop play a n i m p o r t a n t role in  the structure  essential through  for their  of peripherin-2.  proper  protein  involvement  S i x of these  folding  cysteine  and subunit  in i n t r a m o l e c u l a r  residues are  assembly,  disulfide  bond  formation.  A b n o r m a l folding a n d subunit a s s e m b l y of t h e C 2 1 4 S a n d C 1 6 5 S a p p e a r to be responsible for the m o n o g e n i c form of a u t o s o m a l RP linked to t h e s e mutations. 150  is r e q u i r e d  possibly  mutants dominant  T h e conserved cysteine residue at position  for disulfide-dependent  homodimerization  b u t is n o t  Chapter 2 Role of Cysteines  required for hetero- or homotetramer formation.  65  S i n c e r o m - 1 is h i g h l y  h o m o l o g o u s to peripherin-2, w e expect that c o n s e r v e d cysteine residues in t h e l a r g e i n t r a d i s c a l l o o p o f r o m - 1 l i k e w i s e p l a y a n e s s e n t i a l r o l e in t h e folding a n d subunit a s s e m b l y of this subunit.  Chapter 3 Disulfide-Mediated Oligomerization  CHAPTER  3  -  DISULFIDE-MEDIATED  PERIPHERIN-2 A N D 3.1  OLIGOMERIZATION  OF  ROM-1  INTRODUCTION  Identification disulfide  dimer  disulfide  bonding  into  66  the  of  Cysl50  formation  as the  and  in t e t r a m e r  function  of  the  cysteine  exclusion formation  disulfide  of  residue  a  role  prompted  dimer.  responsible  for  intermolecular  further  Since  for  investigation  previous  velocity  sedimentation studies were conducted under mildly reducing conditions, the  presence  demonstrated. been  analyzed  membranes identifies  by  the  of  the  disulfide  dimer  in  the  tetramer  was  never  T h e r e f o r e , the o l i g o m e r i c structure of t h e s e proteins has from a  reduced  novel  contribution  and  nonreduced  two-dimensional of  ROS and  separation  intermolecular  C O S - 1 cell  technique  disulfide  bonds  which to  the  structure. The  results  of  this  study  suggest  that  peripherin-2  a s s o c i a t e n o n c o v a l e n t l y to f o r m a m i x t u r e of h o m o - a n d core  proteins.  A  significant  fraction  of  and  rom-1  heterotetrameric  peripherin-2-containing  core  c o m p l e x e s further links together t h r o u g h intermolecular disulfide bonds at Cysl50  to f o r m  higher order oligomers.  T h e s e results lead to a  new  m o d e l f o r t h e o l i g o m e r i c s t r u c t u r e o f p e r i p h e r i n - 2 c o n t a i n i n g p r o t e i n s in R O S a n d p r o v i d e i n s i g h t i n t o t h e p o s s i b l e r o l e o f t h e s e c o m p l e x e s in o u t e r s e g m e n t m o r p h o g e n e s i s and retinal d e g e n e r a t i o n .  Chapter 3 Disulfide-Mediated Oligomerization  67  Most of t h e s e results have b e e n published e l s e w h e r e ( L o e w e n Molday, 2000).  A l l e x p e r i m e n t s d e s c r i b e d in t h i s c h a p t e r w e r e  &  performed  by t h e author.  3.2  MATERIALS AND METHODS  3.2.1  ROS preparations  and monoclonal  antibodies  R O S were isolated from frozen bovine retina centrifugation Monoclonal  by sucrose  gradient  as previously described (Molday, Hicks & Molday,  antibodies  have been reported  to  peripherin-2  (Per2B6)  1987).  and rom-1 (RomlC6)  (Molday, Hicks & Molday, 1 9 8 7 ; Moritz & Molday,  1996).  3.2.2  Dimer reduction  kinetics  ROS were washed three times by low speed centrifugation dim  red  light  with  10  volumes  of  10  m M Tris-HCl,  resuspended at a protein concentration of 2 m g / m l  pH  under  7.4, and  in t h e s a m e buffer.  R e d u c t i o n in t h e p r e s e n c e o r a b s e n c e of 1 % T r i t o n X - 1 0 0 w a s initiated a t 25°C  by the addition  of D T T , cysteine o r glutathione  to yield  a  final  concentration of 10 m M . Aliquots w e r e r e m o v e d at 5 m i n intervals a n d a d d e d to a n equal v o l u m e of stop buffer ( 2 % Triton X - 1 0 0 , 1 2 0 m M N E M , 0.2 m g / m l PMSF, l O m M Tris-HCl, pH 7.4). T h e s a m p l e s were  centrifuged  at 9 0 , 0 0 0 x g f o r 3 0 m i n a n d t h e s u p e r n a t a n t s w e r e s u b j e c t e d to S D S -  Chapter 3 Disulfide-Mediated Oligomerization  68  PAGE under nonreducing conditions for analysis by W e s t e r n blotting as described rom-1  below.  F o r k i n e t i c a n a l y s i s , t h e d e c r e a s e in p e r i p h e r i n - 2  d i m e r as a function of time w a s quantified  exposed film with a laser densitometer.  or  by scanning the E C L  Kinetics w e r e fitted to a single  e x p o n e n t i a l d e c a y u s i n g S i g m a Plot (Jandel Scientific).  3.2.3  DTT and NEM treatment  of ROS and  immunoaffinity  purification R O S w e r e w a s h e d t h r e e t i m e s in P B S ( 0 . 0 1 M s o d i u m  phosphate,  0 . 1 5 M NaCI, p H 7 . 4 ) a n d t h e final pellet w a s r e s u s p e n d e d at a protein concentration of 2 m g / m l DTT.  After 9 0 m i n . at 25°C, R O S s a m p l e s were solubilized by the drop-  wise addition Triton  in P B S in t h e p r e s e n c e o r a b s e n c e of 1 0 m M  of a n equal v o l u m e  X - 1 0 0 and 0.2 mg/ml  of ice cold solubilization  P M S F in P B S ) c o n t a i n i n g  obtain a protein concentration of 1 m g / m l .  buffer  (2%  1 0 0 m M N E M to  T h e solution w a s centrifuged  at 9 0 , 0 0 0 x g f o r 3 0 m i n to r e m o v e a n y residual i n s o l u b l e m a t e r i a l a n d t h e supernatant  (solubilized  R O S ) w a s used  either  directly  for  velocity  sedimentation m e a s u r e m e n t s a n d crosslinking studies or for purification of p e r i p h e r i n - 2 a n d r o m - 1 . Peripherin-2:rom-1  complex w a s typically  purified  from Triton X -  100 solubilized R O S on a Per2B6-Sepharose immunoaffinity previously described (Goldberg, Moritz & Molday, 1995).  matrix  as  B r i e f l y , 2 0 0 ul  Chapter 3 Disulfide-Mediated Oligomerization  69  o f s o l u b i l i z e d R O S w e r e i n c u b a t e d w i t h 5 0 pi o f m a t r i x f o r 1 h a t 4 ° C in a Millipore Ultrafree M C 0 . 4 5 p m filter unit. three  times  with  0.4  ml  centrifugation (10,000 x g, 4 and the bound  of 0  The matrix w a s then  solubilization  buffer  by  low  C , microfuge) to remove unbound  peripherin-2:rom-l  complex w a s eluted  washed speed protein,  w i t h 2 0 0 pi o f  solubilization buffer containing 0.1 m g / m l of t h e 2 B 6 c o m p e t i n g  peptide  (DAGQAPAAG).  3.2.4  Heterologous pcPER  COS-1 cell  (wild-type  expression  peripherin-2),  pcPER-C150S  (C150S  mutant  p e r i p h e r i n - 2 ) , a n d p c R O M ( w i l d - t y p e r o m l ) p l a s m i d s u s e d for C O S - 1 cell transfections Molday,  have  been  previously  1 9 9 8 ; G o l d b e r g , Moritz  e x p r e s s i o n , C O S - 1 cells (~6 x 1 0  & 5  described Molday,  (Goldberg,  1995).  Loewen  &  For heterologous  cells/100 m m dish) were  transfected  with 3 0 pg of plasmid using the calcium phosphate m e t h o d a n d harvested 72 h post-transfection as described (Goldberg, Moritz & Molday,  1995).  T h e c e l l s w e r e w a s h e d t w i c e w i t h P B S a n d i n c u b a t e d w i t h 2 7 0 pi P B S containing 0.1 m g / m l P M S F a n d either 10 m M D T T o r 4 0 m M N E M .  After  9 0 m i n a t 2 5 ° C , t h e c e l l s w e r e s o l u b i l i z e d b y t h e a d d i t i o n o f 3 0 pi o f 1 0 % Triton  X - 1 0 0 and incubated  centrifuged  for  20  min on  ice.  T h e solution  was  at 9 0 , 0 0 0 x g for 10 m i n a n d the supernatant w a s collected  a n d m a i n t a i n e d o n ice until u s e .  70  Chapter 3 Disulfide-Mediated Oligomerization  3.2.5  Velocity  sedimentation  T r i t o n X - 1 0 0 s o l u b i l i z e d protein ( 1 0 0 pl) f r o m  DTT or N E M treated  R O S o r C O S - 1 cell m e m b r a n e s (1 m g / m l total p r o t e i n ) o r  immunoaffinity-  purified p e r i p h e r i n - 2 : r o m - l (0.01 m g / m l ) w a s applied to 5 - 2 0 % ( w / w ) 2 m l s u c r o s e g r a d i e n t s p r e p a r e d in P B S a n d c o n t a i n i n g 0 . 1 % T r i t o n X - 1 0 0 . Routinely, samples. 55  1 m M DTT w a s included  in t h e g r a d i e n t s  for  DTT-treated  A f t e r c e n t r i f u g a t i o n f o r 1 2 h a t 5 0 , 0 0 0 r p m in a B e c k m a n T L S -  rotor at 4°C, the bottom  four-drop  fractions  were  of t h e centrifuge t u b e w a s punctured a n d  collected  by  gravity  flow.  Fractions  incubated with 4 0 m M N E M for 3 0 m i n at 25°C to block free  were  sulfhydryl  groups and neutralize any remaining DTT. T h e samples were then added to  an equal  volume  of S D S cocktail  in t h e a b s e n c e ( o r p r e s e n c e ) of  reducing agent a n d analyzed by S D S - P A G E a n d Western blotting.  3.2.6  Glutaraldehyde  crosslink!ng  Reduced or nonreduced, Triton X - 1 0 0 solubilized R O S(0.1 m g / m l ) or purified mM  NEM  peripherin-2:rom-l and  subsequently  c o m p l e x (4 p g / m l ) incubated  glutaraldehyde for 1 5 - 3 0 m i n at 37°C. volume  of S D S cocktail  SDS-PAGE.  containing  with  w a s treated 0.001%  or  with 50 0.01%  S a m p l e s were a d d e d to a n equal  p-mercaptoethanol  for analysis by  Chapter 3 Disulfide-Mediated Oligomerization  3.2.7  71  Quantification of peripherin-2 and rom-1 in ROS Peripherin-2 and r o m - 1 subunits, used as standards for quantitative  analysis, w e r e isolated a s follows: 1 ml of D T T - t r e a t e d R O S m e m b r a n e s ( 5 - 1 0 m g / m l t o t a l p r o t e i n ) w a s s o l u b i l i z e d w i t h in a n e q u a l v o l u m e of 1 % S D S in P B S c o n t a i n i n g 1 0 0 m M N E M a n d P M S F . 30  min at 100,000 x g, the supernatant  containing bound  2 % Triton  X - 1 0 0 and PMSF.  After centrifugation for  w a s diluted  10-fold  Peripherin-2 w a s selectively  t o 2 0 0 pi P e r 2 B 6 - S e p h a r o s e a n d r o m - 1 w a s b o u n d  RomlC6-Sepharose.  in P B S  t o 2 0 0 pi  A f t e r e x t e n s i v e w a s h i n g in t h e s a m e buffer, b o u n d  p r o t e i n w a s e l u t e d w i t h 1 m l of 2 % S D S in P B S c o n t a i n i n g P M S F f o r 15 min at 37°C.  T h e purity of t h e proteins w a s c o n f i r m e d by S D S - P A G E a n d  the protein concentration w a s d e t e r m i n e d by t h e m e t h o d of K a p l a n a n d Pedersen  (Kaplan  &  Pedersen,  1985).  Under  these  conditions,  the  purified preparation of the peripherin-2 subunit lacked r o m - 1 a n d purified rom-1  subunit  w a s free  of  peripherin-2  as determined  by  Western  blotting. The  a m o u n t o f p e r i p h e r i n - 2 a n d r o m - 1 in b o v i n e R O S c o u l d n o t b e  d i r e c t l y d e t e r m i n e d f r o m W e s t e r n b l o t s o f R O S s i n c e r h o d o p s i n is k n o w n to block t h e e l e c t r o - t r a n s f e r of p e r i p h e r i n - 2 a n d to a lesser e x t e n t r o m - 1 . As  a result,  the peripherin-2  complex from  nonreduced, Triton X - 1 0 0  solubilized R O S w a s quantitatively bound to a P e r 2 B 6 - S e p h a r o s e c o l u m n  Chapter 3 Disulfide-Mediated Oligomerization  72  a s d e s c r i b e d in s e c t i o n 3 . 2 . 3 s u c h t h a t n o p e r i p h e r i n - 2 w a s d e t e c t e d in the  unbound  fraction.  After  the matrix  was thoroughly  bound peripherin-2 complex w a s quantitatively with  2% SDS.  peripherin-2  The bound  and unbound  and rom-1 standards,  were  W e s t e r n blotting u n d e r reducing conditions. and from  rom-1 was determined Western  blots.  from  eluted from  fractions,  analyzed  the column  along  with  the  by S D S - P A G E  and  T h e a m o u n t of peripherin-2  laser densitometry  Values interpolated  washed, the  from  of t h e E C L signal  standard  curves  were  reported a s a n a v e r a g e of 3 o r m o r e e x p e r i m e n t s a n d correlated with t h e p r o t e i n c o n t e n t in R O S ( K a p l a n & P e d e r s e n , 1 9 8 5 ) .  3.2.8  Subunit  crosslinked  composition  of disulfide-linked  and  glutaraldehyde  dimers  To d e t e r m i n e t h e c o m p o s i t i o n of disulfide-linked s p e c i e s , 0.1 ml of nonreduced  R O S (1 m g / m l  v o l u m e of denaturing  buffer  total  protien)  were  s o l u b i l i z e d in a n e q u a l  consisting of 1 % S D S , 1 0 0 m M N E M a n d  P M S F in P B S . T h e s o l u t i o n w a s t h e n d i l u t e d 1 0 - f o l d w i t h P B S c o n t a i n i n g 2%  Triton X - 1 0 0 a n d PMSF and the peripherin-2 a n d r o m - 1 containing  complexes  were  selectively  RomlC6-Sepharose  adsorbed  to  either  a s d e s c r i b e d in s e c t i o n 3 . 2 . 3 .  Per2B6-Sepharose or Bound protein w a s  e l u t e d w i t h 2 % S D S in P B S a n d a n a l y z e d u n d e r n o n r e d u c i n g by S D S - P A G E a n d W e s t e r n blotting.  conditions  Chapter 3 Disulfide-Mediated Oligomerization  73  To e x a m i n e subunit interactions within the core tetramer, Triton X 100  solubilized  DTT-reduced  and  ROS  Per2B6-purified were  crosslinked  d e s c r i b e d in s e c t i o n 3 . 2 . 6 . then  treated  with  peripherin-2:rom-l  SDS  with  0.01%  glutaraldehyde  The immunoaffinity-purified denaturing  buffer  and  complex  from as  complexes were  isolated  on  Per2B6-  S e p h a r o s e a s d e s c r i b e d in s e c t i o n 3 . 2 . 3 .  3.2.9  SDS-PAGE and Western blotting S a m p l e s w e r e d e n a t u r e d with a n e q u a l v o l u m e of S D S cocktail  (4%  S D S , 0 . 0 2 M T r i s - H C l , p H 6 . 8 , 4 0 % s u c r o s e , 0 . 0 1 % B r o m o p h e n o l b l u e in the  absence  (nonreducing)  mercaptoethanol,  and  polyacrylamide gel.  20  pi  or  presence  were  applied  (reducing) to  an  8%  of or  After electrophoresis, the proteins were  5% 10%  pSDS  transferred  to I m m o b i l o n - P using a BioRad s e m i d r y transfer a p p a r a t u s a n d the  blots  were labeled with the Per2B6 or R o m l C 6 monoclonal antibodies (Molday, Hicks &  Molday,  1987;  Moritz & Molday,  1996)  and sheep  anti-mouse  i m m u n o g l o b u l i n - p e r o x i d a s e for detection by E C L (see section 2.2.5).  3.3 3.3.1  RESULTS Reduction of peripherin-2 Previous  studies  have  in ROS  shown  that  membranes a  substantial  portion  of  peripherin-2 and rom-1 from R O S migrates as disulfide-linked dimers on  Chapter 3 Disulfide-Mediated Oligomerization  74  n o n r e d u c i n g S D S p o l y a c r y l a m i d e g e l s ( B a s c o m et al., 1 9 9 2 b ; C o n n e l l & Molday,  1 9 9 0 ; Molday, Hicks & Molday, 1 9 8 7 ; Travis, Sutcliffe & B o k ,  1991).  T o d e t e r m i n e if t h e i n t e r m o l e c u l a r d i s u l f i d e b o n d r e s p o n s i b l e f o r  these dimers can be reduced within the m e m b r a n e  environment, ROS  w e r e treated with 10 m M D T T for various t i m e s a n d t h e d i s a p p e a r a n c e of peripherin-2 d i m e r w a s monitored o n W e s t e r n blots of nonreducing S D S gels.  As shown  i n Fig 1 3 A  &  B, p e r i p h e r i n - 2 - c o n t a i n i n g  dimer was  exponentially reduced to m o n o m e r by DTT with a half-time of 9.8 ± 0.7 min.  A similar rate of reduction w a s observed for Triton X - 1 0 0 solubilized  peripherin-2 a n d for m e m b r a n e shown).  In c o n t r a s t ,  bound a n d solubilized r o m - 1 (data not  glutathione  e v e n in t h e p r e s e n c e o f T r i t o n  w a s ineffective  X-100,  as a reducing  agent  presumably d u e to either  lower  reactivity or inaccessibility of this reagent to the intermolecular bond  (Fig  detergent  13B).  Cysteine, on the other  solubilized  peripherin-2,  but  at  hand, a  w a s able  slower  rate  disulfide  to  reduce  than  that  observed for DTT (data not shown).  3.3.2  Velocity  sedimentation  under non-reducing  of ROS peripherin-2  and rom-1  conditions  A two dimensional separation technique w a s devised to assess the contribution  of intermolecular disulfide b o n d s to t h e o l i g o m e r i c  of t h e p e r i p h e r i n - r o m - 1  complex from  ROS membranes.  structure  In t h e first  75  dimer  monomer  B  120 100  5  80  o O  60  0  o  0—e  o  o  40 20 0 0  10  20  30  40  50  60  70  80  90  T i m e (min)  Fig 1 3 .  Rate of r e d u c t i o n of d i s u l f i d e - l i n k e d p e r i p h e r i n - 2 d i m e r s in  ROS m e m b r a n e s . B o v i n e R O S m e m b r a n e s w e r e i n c u b a t e d w i t h 10 m M D T T o r 1 0 m M g l u t a t h i o n e in t h e p r e s e n c e o r a b s e n c e o f T r i t o n X - 1 0 0 . A f t e r various t i m e s , the reaction w a s stopped by the addition of N E M and peripherin-2 dimer reduction was analyzed on a 1 0 % S D S - p o l y a c r y l a m i d e gel u n d e r n o n r e d u c i n g conditions. (A) W e s t e r n blot labeled with the P e r 2 B 6 antibody s h o w i n g the t i m e - d e p e n d e n t reduction of p e r i p h e r i n - 2 d i m e r by DTT. ( B ) T i m e c o u r s e f o r t h e r e d u c t i o n o f p e r i p h e r i n - 2 d i m e r b y 1 0 m M D T T (•) a n d 1 0 m M g l u t a t h i o n e in t h e p r e s e n c e o f T r i t o n X - 1 0 0 ( o ) . R a t e o f reduction w a s quantified by laser d e n s i t o m e t r y of W e s t e r n blots. DTT i n d u c e d d e c r e a s e in d i m e r w a s fitted a s a s i n g l e e x p o n e n t i a l d e c a y w i t h a h a l f - t i m e o f 9 . 8 ± 0 . 7 m i n (n = 3 ) ( s e e M e t h o d s s e c t i o n 3 . 2 . 2 ) .  Chapter 3 Disulfide-Mediated Oligomerization  76  dimension, velocity sedimentation w a s used to resolve oligomeric  forms  of Triton X - 1 0 0 solubilized p e r i p h e r i n - 2 - r o m l c o m p l e x e s f r o m n o n r e d u c e d (-DTT)  and reduced  (+DTT)  membranes.  In  the second  dimension,  fractions from the velocity sedimentation w e r e treated with N E M to block free  sulfhydryl  groups  and subjected to S D S - P A G E  under  nonreducing  conditions for detection of disulfide-linked d i m e r s by W e s t e r n blotting. Fig 1 4 A & B s h o w t h e t w o d i m e n s i o n a l a n a l y s i s o f purified  peripherin-2-roml  membranes.  Both  complex  peripherin-2  from  immunoaffinity  reduced  (+DTT)  ROS  a n d r o m - 1 co-sedimented as a single  s p e c i e s ( f r a c t i o n s 9 - 1 2 ) w i t h a s e d i m e n t a t i o n c o e f f i c i e n t s o,w o f ~ 5 . 1 S , 2  a value that h a s been previously reported to c o r r e s p o n d to a peripherin2:rom-l  tetramer  (Goldberg,  Molday,  1996b).  Further  Loewen  analysis  by  &  Molday,  SDS-PAGE  1998; Goldberg under  &  nonreducing  conditions indicated that this species lacked disulfide-linked dimers. Peripherin-2 containing proteins from nonreduced R O S , treated with NEM to prevent s e c o n d a r y sulfhydryl oxidation, s h o w e d a m o r e b e h a v i o r (Fig 14C & D). two  rom-1  sedimentation.  containing  complex  Three peripherin-2 containing components and components  C o m p o n e n t a (fractions  were  resolved  by  velocity  9 - 1 2 ) sedimented at the s a m e  rate a s t h e D T T r e d u c e d c o m p l e x a n d like this c o m p l e x c o n s i s t e d solely of peripherin-2  and  rom-1  monomers.  when  analyzed  on  77  Reduced  Non-reduced  dimer  LU O 1  2  3  4  5  B  6  7 8 9 10 11 12 13  14  15  16  1  2  3  4  5  6  7  8  9  10  11  12  13  7 8  9  10  11 12  13  14  15  16  W  Q w  Reduced  dimer  1  2  3  4  5  6  7 8 9 10 11 12 13  14  15  16  1  2  (bottom) ^  3  4  5  6  14 15  16  (top) Sedimentation Fractions  Fig 1 4 . Velocity peripherin-2 a n d  s e d i m e n t a t i o n a n d W e s t e r n blot a n a l y s i s of rom-1 f r o m r e d u c e d a n d n o n r e d u c e d ROS  membranes. DTT-reduced (A&B) or nonreduced (C&D) R O S m e m b r a n e s w e r e s o l u b i l i z e d in T r i t o n X - 1 0 0 c o n t a i n i n g N E M . P e r i p h e r i n - 2 c o n t a i n i n g c o m p l e x e s w e r e isolated on a P e r 2 B 6 - S e p h a r o s e affinity matrix and subjected to velocity s e d i m e n t a t i o n on a s u c r o s e gradient. Fractions were t r e a t e d with N E M a n d s u b j e c t e d to S D S - g e l e l e c t r o p h o r e s i s u n d e r n o n r e d u c i n g conditions. W e s t e r n blots w e r e labeled for P e r i p h e r i n - 2 with the Per2B6 antibody (A&C) and Rom-1 with the R o m l C 6 antibody (B&D). T h r e e p e r i p h e r i n - 2 c o n t a i n i n g c o m p o n e n t s ( a , b, a n d c ) a n d t w o R o m - 1 c o n t a i n i n g c o m p o n e n t s ( a a n d b) w e r e r e s o l v e d f r o m n o n r e d u c e d R O S m e m b r a n e s (see Methods sections 3.2.3 and 3.2.5).  Chapter 3 Disulfide-Mediated Oligomerization nonreducing faster  rate  7.2 S) characteristic  2  component  C o m p o n e n t b (fractions  S D S gels. (s o,w =  78  contained  both  monomers  5-8) s e d i m e n t e d at a  of a larger  oligomer.  and disulfide-linked  This  dimers  of  peripherin-2 a n d r o m - 1 proteins when analyzed on nonreducing S D S gels. Peripherin-2 containing dimers appeared as a single band, whereas r o m - 1 containing d i m e r s w e r e resolved into a doublet. rom-1  doublet  co-migrated  with  the  T h e upper band of the  peripherin-2  band  and  corresponds to disulfide-linked p e r i p h e r i n - 2 : r o m - l heterodimer. intense  lower  band  of  the  rom-1 doublet  lacked  likely  T h e less  peripherin-2,  and  therefore, m o s t likely c o r r e s p o n d s to disulfide-linked r o m - 1 h o m o d i m e r s . Component  c  (fractions  1-3)  sedimented  characteristic of a higher-order oligomer. lacked  with  an  s o,w  Interestingly,  of  2  this  r o m - 1 a n d consisted exclusively of disulfide-linked  homodimers.  >11  S  component peripherin-2  Similar results were obtained w h e n detergent  solubilized  R O S w e r e s u b j e c t e d to velocity s e d i m e n t a t i o n w i t h o u t prior isolation of the peripherin-2 complexes except that c o m p o n e n t c w a s spread evenly  throughout  the  lower  fractions  of  the  gradient  more  indicative  of  oligomers of various sizes (data not shown). The  relative  components  amounts  resolved  of  peripherin-2  by velocity  sedimentation  Western blotting a n d laser densitometry. fractions  subjected  to  SDS-PAGE  a n d r o m - 1 in  under  were  the  three  determined  by  Analysis w a s performed on the reducing  conditions  in  which  Chapter 3 Disulfide-Mediated Oligomerization  79  peripherin-2 and rom-1 migrated as monomers.  A p p r o x i m a t e l y , 3 5 % of  p e r i p h e r i n - 2 w a s p r e s e n t in t h e c o r e c o m p l e x ( c o m p o n e n t a ) , 2 5 % in t h e intermediate oligomer  oligomer  (component  (component c).  b)  a n d 4 0 % in t h e  In t h e c a s e  of r o m - 1 ,  higher  order  5 6 % of r o m - 1 w a s  p r e s e n t i n c o m p o n e n t a a n d 4 4 % in c o m p o n e n t b. These results indicate that peripherin-2 a n d r o m - 1 interact  through  noncovalent bonds to form core h o m o m e r i c a n d heteromeric c o m p l e x e s , presumably tetramers. through  A significant portion of t h e s e c o m p l e x e s interacts  intermolecular disulfide bonds to f o r m larger oligomers, a large  f r a c t i o n o f w h i c h is d e v o i d o f r o m - 1 .  3.3.3  Velocity  peripherin-2  sedimentation expressed  in  of peripherin-2, COS-1  cells  rom-1, under  and C150Snon-reducing  conditions P r e v i o u s l y , p e r i p h e r i n - 2 a n d r o m - 1 s e p a r a t e l y e x p r e s s e d in C O S - 1 cells  were  shown  to  s e l f - a s s e m b l e into  sedimented as a tetramer  under  mildly  a  multisubunit  complex  reducing conditions  that  (Goldberg,  Loewen & Molday, 1 9 9 8 ; Goldberg & Molday, 1 9 9 6 b ; G o l d b e r g , Moritz & Molday,  1995).  We have  now used  the two-dimensional  separation  technique to analyze for the disulfide-linked oligomerization of individually expressed  peripherin-2  and rom-1.  As shown  i n Fig 1 5 A  & B,  both  peripherin-2 and rom-1 from DTT-treated membranes sedimented as a  Chapter 3 Disulfide-Mediated Oligomerization  80  single species as previously shown (Goldberg, Moritz & Molday, Further analysis by S D S - P A G E  under  nonreducing  conditions  1995).  indicated  that these complexes lacked disulfide-linked dimers. The  velocity s e d i m e n t a t i o n profile of p e r i p h e r i n - 2 f r o m n o n r e d u c e d  ( - D T T ) C O S - 1 cell m e m b r a n e s s h o w e d l a r g e r p e r i p h e r i n - 2 o l i g o m e r s in addition  to  the core  complex  (Fig  15D).  The oligomers  consisted  exclusively of disulfide-linked peripherin-2 dimers w h e n analyzed by S D S PAGE under nonreducing conditions. COS-1  cell m e m b r a n e s s h o w e d  In c o n t r a s t , r o m - 1 f r o m n o n r e d u c e d  little t e n d e n c y  to form  disulfide-linked  oligomers, but instead sedimented primarily as the core complex lacking i n t e r m o l e c u l a r d i s u l f i d e b o n d s (Fig 15E). C y s t e i n e a t p o s i t i o n 1 5 0 in t h e l a r g e i n t r a d i s c a l l o o p o f p e r i p h e r i n - 2 has  been reported to be responsible for disulfide-linked dimerization of  peripherin-2 (Goldberg, Loewen & Molday, 1998) (see section 2.3.2). further  examine  sedimentation peripherin-2  the  role  of  this  behavior a n d disulfide mutant  was examined.  cysteine  in  oligomerization,  linked dimerization As shown  To the  of the C 1 5 0 S  i n Fig 15C  &  F, t h e  C 1 5 0 S m u t a n t f r o m r e d u c e d ( + D T T ) a n d n o n r e d u c e d ( - D T T ) C O S - 1 cell m e m b r a n e s sedimented as a single core c o m p l e x devoid of intermolecular disulfide  bonds.  These studies indicate that C 1 5 0 mediates  l i n k e d o l i g o m e r i z a t i o n Of p e r i p h e r i n - 2 c o r e c o m p l e x e s .  disulfide-  81  Reduced  Non-reduced  dimer  1  2  3  4  5  6  7 8 9 10 11 12 13 14 15 16  1  2  3  4  5  Reduced  6  7  8  9 10 11 12 13 14 15 16  LU  Non-reduced  O  £  dimer  CO Q CO O) c  o  I 1  2  3  4  5  6  7 8  9 10 11 12 13 14 15 16  1  2  3  4  5  6  7 8  9 10 11 12 13 14 15 16  13 -D  0) i—  i  C  o Reduced  1  2  3  4  5  6  Non-reduced  7 8 9 10 11 12 13 14 15 16  (bottoms-  1  2  3  Sedimentation Fractions  4  5  6  7  8  9 10 11 12 13 14 15 16  (top)  Fig 1 5 . V e l o c i t y s e d i m e n t a t i o n a n d W e s t e r n blot a n a l y s i s of heterologously expressed peripherin-2 and rom-1 from reduced and n o n r e d u c e d C O S - 1 cell m e m b r a n e s . P e r i p h e r i n - 2 ( A & D ) , R o m - 1 ( B & E ) , a n d C 1 5 0 S ( C & F ) p e r i p h e r i n - 2 w e r e i n d i v i d u a l l y e x p r e s s e d in C O S - 1 c e l l s . Cells pretreated with DTT (A,B&C) or without DTT (D,E&F) were solubilized with Triton X - 1 0 0 containing N E M a n d subjected to velocity s e d i m e n t a t i o n . Fractions w e r e a n a l y z e d on nonreducing S D S gels for detection of peripherin-2 with Per2B6 antibody (A,C,D&F) and R o m - 1 with R o m l C 6 antibody (B&E) (see Methods sections 3.2.4 and 3.2.5).  Chapter 3 Disulfide-Mediated Oligomerization 3.3.4  Crosslinking  of  ROS  82  peripherin-2  under  nonreducing  conditions Covalent  crosslinking  was  used  to  further  analyze  subunit  associations a n d disulfide-linked oligomerization of peripherin-2 a n d - r o m 1.  In t h e s e s t u d i e s , D T T - r e d u c e d a n d n o n r e d u c e d R O S m e m b r a n e s w e r e  s o l u b i l i z e d in T r i t o n X - 1 0 0 , purified o n a P e r 2 B 6 - S e p h a r o s e m a t r i x a n d crosslinked with glutaraldehyde for analysis by S D S - P A G E under reducing conditions. peripherin-2 dimers.  W e s t e r n b l o t s in Fig 16A s h o w t h a t a s u b s t a n t i a l p o r t i o n o f and rom-1 from  reduced  membranes  w a s crosslinked to  Only a faint band corresponding to a t e t r a m e r  w a s detected  w h e n a relatively high glutaraldehyde concentration ( 0 . 0 1 % ) In  contrast,  NEM-treated  crosslinking of peripherin-2 membranes  crosslinked multimers  produced  and rom-1 from  a series of high  was used.  nonreduced,  molecular  weight  i n a d d i t i o n t o m o n o m e r s a n d d i m e r s (Fig  16B).  These results indicate that peripherin-2 a n d r o m - 1 from D T T reduced R O S m e m b r a n e s preferentially crosslink into d i m e r s , w h e r e a s t h e protein  from  n o n r e d u c e d m e m b r a n e s crosslink into larger m u l t i m e r i c s p e c i e s , a result that  is c o n s i s t e n t  with  the presence of large  velocity sedimentation experiments.  oligomers  observed  in  83  A  a  b  B  c  a  b  c  Fig 1 6 . C r o s s - l i n k i n g of peripherin-2 a n d r o m - 1 f r o m r e d u c e d a n d nonreduced ROS membranes. R O S m e m b r a n e s w e r e i n c u b a t e d in t h e p r e s e n c e ( A ) o r a b s e n c e ( B ) of 1 0 m M D T T a n d s o l u b i l i z e d in T r i t o n X - 1 0 0 containing N E M . Peripherin-2 containing c o m p l e x e s w e r e purified on P e r 2 B 6 - S e p h a r o s e a n d cross-linked with 0 % (lane a ) , 0 . 0 0 1 % (lane b), and 0 . 0 1 % glutaraldehyde (lane c). Western blots w e r e labeled for peripherin-2 with the Per2B6 antibody.  Chapter 3 Disulfide-Mediated Oligomerization 3.3.5  Denaturation,  isolation,  and rom-1 subunits  from ROS  To  determine  immunoaffinity and  t h e ratio  and quantitation  of peripherin-2  of  peripherin-2  a n d r o m - 1 in  R O S , an  based method w a s developed to separate both covalently  noncovalently-linked  disulfide  84  dimers,  subunits.  ROS samples  After  were  reduction  denatured  by D T T to in  disrupt  S D S to  disrupt  noncovalent associations, diluted with Triton X - 1 0 0 to decrease theS D S concentration,  and subjected  to affinity  chromatography  with  Per2B6-  Sepharose or R o m l C 6 - S e p h a r o s e matrix to isolate peripherin-2 a n d r o m 1 respectively. (Fig 17). was  P e r i p h e r i n - 2 w a s p r e s e n t o n l y in t h e b o u n d f r a c t i o n a n d r o m - 1  found  17A).  Purity w a s assessed by S D S - P A G E a n d W e s t e r n blotting  in t h e u n b o u n d  Similarly,  unbound  immunoaffinity  of P e r 2 B 6 - S e p h a r o s e  rom-1 was present  R o m l C 6 - S e p h a r o s e matrix the  fraction  fraction.  only  matrix  in t h e b o u n d  (Fig  fraction  of  (Fig 17B) a n d p e r i p h e r i n - 2 w a s d e t e c t e d in  Therefore,  the  purified  fractions  of  these  matrices contained only peripherin-2 or r o m - 1 and were  used as standards for quantitation. T h e a m o u n t o f p e r i p h e r i n - 2 a n d r o m - 1 in R O S w a s d e t e r m i n e d b y quantifying unbound  the  fraction  amount  each  protein  of a P e r 2 B 6 - S e p h a r o s e  under non-denaturing 3.2.7).  of  Peripherin-2  conditions (data w a s only  present matrix  in  the  bound  purification  and  performed  not s h o w n ; s e e Methods section  present  in  the  bound  fraction  and  85  Rom-1 purification  Peripherin-2 purification  Per2B6  a  b  c  Per2B6  Rom1C6  a  b  B Rom1C6  a  c  Rom1C6  b  ^ Rom1C6  • a  b  c  a  b  c  c  Per2B6  a  b  c  Per2B6  ,  —  a  b  c  a  b  c  Fig 1 7 . A n a l y s i s of s u b u n i t s involved in d i s u l f i d e - l i n k e d d i m e r s . D T T r e d u c e d (A&.B) o r n o n r e d u c e d ( C & D ) R O S m e m b r a n e s w e r e d e n a t u r e d in 0 . 5 % S D S to d i s r u p t n o n c o v a l e n t i n t e r a c t i o n s . A f t e r d i l u t i o n w i t h Trition X 100, peripherin-2 containing complexes were isolated on a Per2B6S e p h a r o s e matrix (A&C) and rom-1 containing c o m p l e x e s w e r e isolated on a R o m l C 6 - S e p h a r o s e m a t r i x ( B & D ) . E q u i v a l e n t v o l u m e s of t h e initial e x t r a c t ( l a n e a ) , t h e u n b o u n d fraction ( l a n e b), a n d t h e S D S e l u t e d f r a c t i o n ( l a n e c) w e r e a n a l y z e d on W e s t e r n blots labeled with P e r 2 B 6 or R o m l C 6 antibody (see Methods section 3.2.8).  Chapter 3 Disulfide-Mediated Oligomerization constituted 2 . 1 % ± 0 . 3  86  ( n = 6 ) of t h e R O S protein by weight.  Rom-1 was  i p r e s e n t in b o t h t h e b o u n d a n d u n b o u n d f r a c t i o n s a n d t o g e t h e r c o m p r i s e d 1 . 1 % ± 0 . 2 ( n = 6 ) of t h e total R O S protein. rom-1  w a s detected  lacking peripherin-2  in t h e u n b o u n d migrated  A p p r o x i m a t e l y 1 4 % of total  fraction.  This r o m - 1 component  solely as m o n o m e r s by SDS-PAGE  nonreducing conditions (data not shown).  under  T h e inability of r o m - 1 to form  d i s u l f i d e - l i n k e d h o m o d i m e r s i n t h e a b s e n c e o f p e r i p h e r i n - 2 is c o n s i s t e n t w i t h t h e b e h a v i o r o f r o m - 1 e x p r e s s e d in C O S - 1 cells.  3.3.6  Disulfide The  dimer formation  subunit  composition  between  of  peripherin-2  disulfide-linked  reduced R O Smembranes w a s determined  and rom-1  dimers  from n o n -  using the SDS-denaturation-  p u r i f i c a t i o n m e t h o d d e s c r i b e d in s e c t i o n 3 . 3 . 5 , b u t r e d u c t i o n b y D T T w a s omitted.  Fig 17C s h o w s  Western  blots  of the unbound  f r a c t i o n s f r o m P e r 2 B 6 - S e p h a r o s e a n d Fig 17D f r o m The  unbound  fraction  and  a smaller amount  RomlC6-Sepharose.  of P e r 2 B 6 - S e p h a r o s e contained  devoid of peripherin-2.  of disulfide-linked  and bound  rom-1 monomer  rom-1 homodimer,  T h e bound fraction contained both  m o n o m e r a n d d i s u l f i d e - l i n k e d d i m e r (* in Fig 1 7 C ) .  but w a s  peripherin-2  This dimer  migrated  m o r e s l o w l y t h a n t h e r o m - 1 d i s u l f i d e - l i n k e d h o m o d i m e r in t h e u n b o u n d fraction.  It  most  likely  represents  a  mixture  of  disulfide-linked  peripherin-2 homodimer and peripherin-2:rom-l heterodimer that are not  Chapter 3 Disulfide-Mediated Oligomerization r e s o l v e d in t h i s g e l s y s t e m .  87  A n additional  rom-1 containing  band was  r o u t i n e l y o b s e r v e d a b o v e t h e d i m e r in t h e b o u n d f r a c t i o n (? in Fig 1 7 C ) . T h e n a t u r e o f t h i s s p e c i e s is n o t k n o w n a t t h e p r e s e n t t i m e . fraction  from  monomer fraction  the  RomlC6-Sepharose  and disulfide-linked exhibited  bands  disulfide-linked dimer. this  dimer  These  band  results  bridges between  homodimer,  corresponding  contained  peripherin-2  but no r o m - 1 .  The bound  to  rom-1  monomer  and  a  S i n c e p e r i p h e r i n - 2 w a s a l s o p r e s e n t in t h e latter,  appears  indicate  matrix  The unbound  to  that  contain  oligomers  peripherin-2:rom-1 are generated  peripherin-2-peripherin-2  subunits,  heterodimer.  through  disulfide  peripherin-2:rom-l  subunits a n d r o m l - r o m - 1 subunits of the core c o m p l e x e s .  3.3.7  Crosslinking  of peripherin-2  and rom-1 under  reducing  conditions The  subunit  peripherin-2 using  the  composition  and rom-1 from  of g l u t a r a l d e h y d e - c r o s s l i n k e d d i m e r s DTT treated  SDS-denaturation-purification  peripherin-2:rom-1 glutaraldehyde  complex  prior  to  from  R O S w a s also technique.  reduced  denaturation  investigated  Per2B6-purified  R O S w a s crosslinked  with  SDS  of  and  with  subsequent  purification with P e r 2 B 6 - S e p h a r o s e .  A s s h o w n i n Fig 1 8 B , t h e u n b o u n d  fraction  matrix  of  the  Per2B6-Sepharose  contained  primarily  rom-1  88  a b c  Fig 1 8 .  a b c  G l u t a r a l d e h y d e c r o s s l i n k i n g of s u b u n i t s  w i t h i n t h e core  complex. Immunoaffinity purified peripherin-2 containing c o m p l e x from DTT-reduced R O S membranes was cross-linked with 0 . 0 1 % glutaraldehyde. T h e c o m p l e x w a s treated with S D S to disrupt n o n c o v a l e n t interactions a n d purified on a P e r 2 B 6 - S e p h a r o s e matrix. T h e extract (lane a ) , u n b o u n d (lane b) a n d S D S - e l u t e d f r a c t i o n ( l a n e c ) w e r e a n a l y z e d o n W e s t e r n b l o t s l a b e l e d with Per2B6 (A) or R o m l C 6 (B) antibody under reducing conditions (see Methods sections 3.2.6and 3.2.8).  Chapter 3 Disulfide-Mediated Oligomerization monomer.  89  This fraction of r o m - 1 w a s not associated with  (compare  to  Fig 1 8 A , lane  b).  The bound  fraction  peripherin-2  contained  both  p e r i p h e r i n - 2 m o n o m e r a n d c r o s s l i n k e d d i m e r (Fig 1 8 A , l a n e c ) . R o m - 1 w a s a l s o d e t e c t e d in t h e d i m e r b a n d i n d i c a t i n g t h a t a t l e a s t a f r a c t i o n of t h i s d i m e r is c o m p o s e d o f p e r i p h e r i n - 2 : r o m - l  heterodimer.  Thus, within  t h e core c o m p l e x e s , p e r i p h e r i n - 2 c a n be c o v a l e n t l y c r o s s l i n k e d to itself o r to r o m - 1 to g e n e r a t e d i m e r s .  G l u t a r a l d e h y d e , h o w e v e r , is i n e f f e c t i v e i n  crosslinking two rom-1 subunits.  3.4  DISCUSSION The  peripherin-2:rom-l  previously peripherin-2  thought  to  and  rom-1  heterotetrameric  complex  consist  protein  that  of  of  R O S disc  disulfide-linked  interact  1992b;  This model w a s based on the findings that  fraction  of  and  homodimers  on  nonreducing  peripherin-2  by  immunoaffinity  solubilized properties  with  migrates  S D S gels;  peripherin-2: rom-1 consistent  rom-1  a  2)  complex tetrameric  1)  as  rom-1  chromatography;  to  Goldberg  1996b).  complex.  form &  of a  Molday,  a substantial disulfide-linked  co-purifies  and  possesses  was  homodimers  noncovalently  ( B a s c o m e t al.,  peripherin-2  membranes  3)  with  detergent  hydrodynamic However,  the  h y d r o d y n a m i c e x p e r i m e n t s w e r e c a r r i e d o u t in t h e p r e s e n c e o f D T T a n d  Chapter 3 Disulfide-Mediated Oligomerization  90  the e x i s t e n c e of disulfide-linked d i m e r s u n d e r t h e s e conditions w a s not determined. Velocity sedimentation  measurements  this s i m p l e m o d e l is n o t c o r r e c t . disulfide-mediated  reported  here indicate  that  Instead, o u r results suggest a novel  oligomerization  model  as  depicted  in  Fig  19.  P e r i p h e r i n - 2 a n d r o m - 1 in R O S m e m b r a n e s i n t e r a c t n o n c o v a l e n t l y to f o r m multisubunit  core  complexes.  A  major  portion  of t h e  peripherin-2-  containing c o m p l e x e s links together via intermolecular disulfide bonds to form intermediate and higher order oligomers. The  core  multisubunit  complex  is a m i x t u r e  proteins.  peripherin-2:rom-l  of h o m o m e r i c  Peripherin-2-peripherin-2  heteromeric  core  proteins  species a n d readily form disulfide-linked core protein constitutes only about  has  been  previously  heteromeric  homomeric  are the most  oligomers.  and  abundant  Rom-1 homomeric  1 0 % of t h e s e c o m p l e x e s a n d s h o w s  little c a p a c i t y t o f o r m d i s u l f i d e - l i n k e d complex  and  oligomers.  estimated  to  T h e size of the core be  a  tetramer  by  hydrodynamic measurements (Goldberg & Molday, 1996b).  S u b u n i t s of  the  dimers  complex,  glutaraldehyde that  the  large  interactions 1996a).  however,  preferentially  crosslink  and other crosslinking agents. intradiscal  (Goldberg,  loop  Loewen  is  involved  & Molday,  into  by  Previous studies  indicate  in  subunit  noncovalent  1998; Goldberg  &  It i s p o s s i b l e t h a t r e a c t i v e g r o u p s i n t h i s s e g m e n t a r e n o t  Molday,  Chapter 3 Disulfide-Mediated Oligomerization  9 1  Fig 1 9 . D i s u l f i d e - m e d i a t e d o l i g o m e r i z a t i o n m o d e l f o r p e r i p h e r i n - 2 and rom-1. P e r i p h e r i n - 2 ( d a r k fill) a n d r o m - 1 ( w h i t e fill) i n t e r a c t noncovalently via their large intradiscal loops to produce homotetrameric and heterotetrameric core c o m p l e x e s . T h e C y s - 1 5 0 of peripherin-2 a n d c o r r e s p o n d i n g C y s - 1 5 3 of r o m - 1 located within t h e large intradiscal loop of t h e s e p r o t e i n s a r e in t h e i r r e d u c e d f o r m ( S H ) . A l a r g e p o r t i o n of t h e p e r i p h e r i n - 2 h o m o t e t r a m e r s link t o g e t h e r t h r o u g h C y s - 1 5 0 m e d i a t e d intermolecular disulfide bonds to form intermediate size h o m o - o c t a m e r s . These oligomers c a n further associate to form higher order disulfidel i n k e d h o m o - o l i g o m e r s . A l l s u b u n i t s in t h e p e r i p h e r i n - 2 h o m o - o l i g o m e r s contain intermolecular disulfide bonds since only disulfide-linked dimers are observed on nonreducing S D S gels. A significant portion of the peripherin-2-rom-l heterotetramers are linked together via intermolecular disulfide bonds to form h e t e r o - o c t a m e r s . Only s o m e of the subunits within these hetero-octamers are disulfide bonded since both m o n o m e r and disulfide-linked dimers are observed on nonreducing S D S gels. Intermolecular disulfide bonds c a n form between t w o peripherin-2 subunits, two r o m - 1 subunits or a peripherin-2 and r o m - 1 subunit. The hetero-octamers do not form higher order oligomers. Rom-1 h o m o t e t r a m e r s p r e s e n t in r e l a t i v e l y l o w a m o u n t s d o n o t r e a d i l y f o r m disulfide-linked oligomers.  92  Core Complexes  Intermediate Disulfide-linked Oligomers  Homo-octamers  Higher Order Disulfide-linked Homo-Oligomers  Homo-oligomers  Hetero-octamers  Chapter 3 Disulfide-Mediated Oligomerization accessible  for efficient  crosslinking  93 of t h e s u b u n i t s  into  tetramers  by  glutaraldehyde. T w o classes of peripherin-2-containing velocity  sedimentation  under  oligomers are observed by  nonreducing  conditions.  One  class  d e s i g n a t e d a s c o m p o n e n t b (Fig 14) i s i n t e r m e d i a t e i n s i z e a n d c o n t a i n s both peripherin-2 and r o m - 1 subunits.  O n l y s o m e o f t h e s u b u n i t s in t h e  h e t e r o - o l i g o m e r s p a r t i c i p a t e in i n t e r m o l e c u l a r d i s u l f i d e b o n d s s i n c e b o t h monomers conditions. the  core  and dimers  are observed by S D S - P A G E  under  nonreducing  T h e s i z e o f this o l i g o m e r is e s t i m a t e d t o b e t w i c e t h e s i z e o f complex  coefficients  to  based  molecular  on  the  weights  relationship (Martin  &  of  the  Ames,  sedimentation  1961) and the  a s s u m p t i o n that both species bind similar a m o u n t s of detergent per core complex.  T h e intermolecular disulfide b o n d s that link t h e  peripherin-2  containing core complexes together are formed between two peripherin-2 subunits, a peripherin-2 The  second,  large  and a rom-1 subunit,  class  of  oligomers  is  or two rom-1 subunits.  composed  exclusively  p e r i p h e r i n - 2 s u b u n i t s , all o f w h i c h p a r t i c i p a t e in i n t e r m o l e c u l a r  of  disulfide  bonds. Treatment breakdown  of R O S m e m b r a n e s with  of the oligomers  intermolecular  disulfide  bonds  into  core  DTT results  complexes  are readily  agent a n d essential for oligomerization.  in t h e c o m p l e t e  indicating  accessible to this  Quantitative  that  the  reducing  analysis indicates  Chapter 3 Disulfide-Mediated Oligomerization  94  t h a t p e r i p h e r i n - 2 is p r e s e n t a t a l m o s t t w i c e t h e c o n c e n t r a t i o n o f r o m - 1 in R O S , a f i n d i n g t h a t is c o n s i s t e n t w i t h t h e p r e s e n c e o f a s i g n i f i c a n t a m o u n t of  peripherin-2  analysis. rom-1  homo-oligomers  by  velocity  sedimentation  Earlier studies failed to detect t h e presence of peripherin-2 a n d  homotetrameric  Moritz & Molday, smaller  detected  amounts  c o r e c o m p l e x e s i n R O S ( B a s c o m e t al.,  1996).  This  of peripherin-2  m a y be d u e to t h e inability  1992b;  to  detect  a n d r o m - 1 in t h e p r e s e n c e o f  large  a m o u n t s of rhodopsin by W e s t e r n blotting a n d / o r t h e u s e of less sensitive a n t i b o d i e s in t h e s e s t u d i e s . P e r i p h e r i n - 2 a n d r o m - 1 e x p r e s s e d in C O S - 1 c e l l s s h o w a pattern of disulfide-mediated peripherin-2  core  complex  oligomerization. interacts  through  A major  fraction  intermolecular  related of t h e disulfide  bonds to form intermediate a n d higher order oligomers of various sizes. A s in t h e c a s e o f R O S , a l l t h e s u b u n i t s i n t h e s e o l i g o m e r s p a r t i c i p a t e in intermolecular disulfide bond formation.  R o m - 1 e x p r e s s e d in C O S - 1 cells  also s e l f - a s s e m b l e s into a core c o m p l e x , tendency  to  form  disulfide-linked  but this  oligomers,  as  protein  shows  observed  Mutagenesis studies have confirmed that disulfide-linked  little  in R O S .  oligomerization  o f p e r i p h e r i n - 2 is m e d i a t e d b y C 1 5 0 , p r e s e n t w i t h i n t h e l a r g e i n t r a d i s c a l loop of t h e protein. likely to participate  T h e c o r r e s p o n d i n g C 1 5 3 r e s i d u e i n r o m - 1 is a l s o in i n t e r m o l e c u l a r d i s u l f i d e b o n d f o r m a t i o n  heterotetrameric core complexes.  between  H o w e v e r , in t h e a b s e n c e o f p e r i p h e r i n -  Chapter 3 Disulfide-Mediated Oligomerization  95  2, t w o C 1 5 3 r e s i d u e s o f r o m - 1 s h o w little t e n d e n c y to f o r m i n t e r m o l e c u l a r disulfide  bonds,  possibly  d u e to  limited  accessibility  or  unfavorable  alignment of these groups. These studies taken together  indicate that peripherin-2 exhibits a  strong t e n d e n c y to form disulfide-linked oligomers of various sizes.  The  a s s o c i a t i o n of p e r i p h e r i n - 2 w i t h r o m - 1 in t h e c o r e c o m p l e x limits t h e size of  the  oligomers  and  the  number  of  intermolecular disulfide bond formation.  subunits  that  participate  in  Thus, r o m - 1 can be considered  as a negative m o d u l a t o r of peripherin-2 o l i g o m e r i z a t i o n . Intermolecular  disulfide bonds are known  t o b e i m p o r t a n t in t h e  assembly of multisubunit proteins a n d higher order oligomeric complexes. T h e light a n d h e a v y c h a i n s of i m m u n o g l o b u l i n s a n d t h e a a n d p subunits of t h e insulin disulfide  receptor  bonding  are joined  is i m p o r t a n t  by disulfide  bonds.  Intermolecular  in c a p s i d a s s e m b l y a n d d i s a s s e m b l y of  p a p i l l o m a v i r u s ( B e a r d e t al., 1 9 9 8 ) , s t a b i l i z a t i o n o f v a c c i n i a v i r u s ( L o c k e r &  Griffiths,  matrix  1999) and oligomerization  protein  involved  in  of t e n a s c i n - C , a n  embryogenesis  extracellular  and tumorgensis  (Luczak,  Redick & Schwarzbauer, 1998). We  speculate  peripherin-2  plays  that an  the  disulfide-mediated  important  morphogenesis a n d stabilization.  role  in  oligomerization  outer  segment  T h i s is b a s e d o n t h e f o l l o w i n g .  of disc The  c y s t e i n e r e s i d u e s r e s p o n s i b l e f o r i n t e r m o l e c u l a r d i s u l f i d e b o n d s ( C 1 5 0 in  Chapter 3 Disulfide-Mediated Oligomerization peripherin-2  and  C 1 5 3 in  rom-1)  96 are  conserved  in  all  vertebrate  peripherin-2 and r o m - 1 proteins analyzed to date (Goldberg, Loewen & Molday,  1998).  Disulfide  dimerization  of  property  o f t h e s e p r o t e i n s ( B a s c o m e t al.,  peripherin-2  is a  general  1 9 9 2 b ; C o n n e l l e t al., 1 9 9 1 ;  Kedzierski e t a / . , 1 9 9 6 ; Molday, Hicks & Molday, 1 9 8 7 ; Travis, Sutcliffe & Bok, 1991). formation  (Wetzel,  isomerase, disulfide  Sulfhydryl agents have been reported to disrupt new disc  a  Parsons &  protein  bonds,  unpublished).  that  is p r e s e n t In  one  Besharse, 1994),  functions  in  the  a n d protein  making  and breaking  in R O S d i s c s ( C . L o e w e n  model,  peripherin-2  disulfide  and  of  a n d R. M o l d a y ,  rom-1  containing  h o m o m e r i c a n d heteromeric core c o m p l e x e s a r e envisioned to a s s e m b l e in  the  endoplasmic  reticulum  membrane  of  photoreceptors  t r a n s l o c a t e in v e s i c l e s t o t h e b a s e of o u t e r s e g m e n t s . proteins  may  prevent  disulfide-linked  and  Specific chaperone  oligomerization  from  occurring  during this trafficking process. A t the base of the outer s e g m e n t ,  protein  disulfide i s o m e r a s e w o u l d catalyze d i s u l f i d e - m e d i a t e d oligomerization of peripherin-2  containing  across juxtaposed together idea that new.  homomeric  newly forming  and  heteromeric  core  complexes  disc m e m b r a n e s to effectively  zipper  t h e r i m region a s part of outer s e g m e n t m o r p h o g e n e s i s . T h e membrane  proteins  T h e major structural  tetrameric complex.  can mediate  membrane  a d h e s i o n is n o t  p r o t e i n , P o , o f p e r i p h e r a l n e r v e m y e l i n is a  T h e extracellular d o m a i n s of t h e s e c o m p l e x e s from  Chapter 3 Disulfide-Mediated Oligomerization opposing  membranes  membrane  adhesion  interact (Shapiro  with e t al.,  97 each  other  1996).  to  In  mediate  this  case,  myelin however,  protein-protein associations occur through noncovalent interactions. An  alternative  mechanism  would  involve  disulfide-linked  oligomerization of peripherin-2 containing core c o m p l e x e s laterally within a membrane.  T h e s e oligomers could be envisioned to initiate t h e disc rim  curvature or promote interactions with other outer s e g m e n t proteins that participate  in o u t e r  segment formation.  Efforts  are now underway  to  e x a m i n e m o r e directly t h e role of d i s u l f i d e - m e d i a t e d o l i g o m e r i z a t i o n of p e r i p h e r i n - 2 in d i s c m o r p h o g e n e s i s . P e r i p h e r i n - 2 a n d r o m - 1 differ significantly w i t h r e s p e c t to their role in  outer  segment  morphogenesis.  Peripherin-2  is e s s e n t i a l f o r  s e g m e n t morphogenesis since h o m o z y g o u s rds mice lacking this fail t o f o r m o u t e r s e g m e n t s ( S a n y a l & J a n s e n ,  1981).  with  slightly  enlarged  oligomerization  discs  supports  the dominant  segment disc morphogenesis.  mice produce outer  ( C l a r k e e t al., role  2000). of  protein  R o m - 1 , on the  other h a n d , a p p e a r s to regulate t h e fine structure of t h e outer discs since h o m o z y g o u s r o m - 1 knockout  outer  segment segments  Disulfide-mediated  peripherin-2  in  In addition to being m o r e a b u n d a n t  outer than  r o m - 1 in R O S , p e r i p h e r i n - 2 is r e q u i r e d f o r t h e f o r m a t i o n o f i n t e r m e d i a t e and higher order disulfide-linked oligomers, a process that m a y be crucial for disc m o r p h o g e n e s i s a s discussed a b o v e .  R o m - 1 , on the other hand,  Chapter 3 Disulfide-Mediated Oligomerization does  not form  higher  order  98  oligomers,  and therefore  considered to be essential for disc m o r p h o g e n e s i s . serve  to  limit  the  formation  of  higher  order  would  not be  R o m - 1 m a y simply  peripherin-2  oligomer  formation through interactions with peripherin-2 a n d thereby regulate the size of t h e discs during outer s e g m e n t m o r p h o g e n e s i s . A  relatively  large  number  of  missense  mutations  in t h e  large  intradiscal loop of peripherin-2 have been linked to a variety of h u m a n retinal d e g e n e r a t i v e diseases.  Previous studies have indicated that some  of t h e s e m u t a t i o n s affect protein folding a n d s u b u n i t a s s e m b l y ( G o l d b e r g , Loewen & Molday, 1 9 9 8 ; Goldberg & Molday, 1996a).  A consequence of  the misfolding of this large loop m a y be t h e inability of t h e peripherin-2 mutants  to  form  intermolecular  required for oligomerization. impact  disulfide  bonds  or other  interactions  S u c h m u t a t i o n s in r o m - 1 w o u l d h a v e less  since disulfide-linked  oligomerization  o f r o m - 1 is n o t c r u c i a l t o  outer segment morphogenesis and structure. In  summary,  associate complexes.  we have shown  noncovalently Peripherin-2  to  form  here that homomeric  containing  peripherin-2 and  complexes  and rom-1  heteromeric  interact  via  core  Cysl50  mediated intermolecular disulfide bonds to f o r m oligomers that m a y play a n i m p o r t a n t r o l e in r o d a n d c o n e o u t e r s e g m e n t m o r p h o g e n e s i s .  Chapter 4 Digenic RP  99  C H A P T E R 4 - M O L E C U L A R A N A L Y S I S OF DIGENIC RP 4.1  INTRODUCTION I n d i v i d u a l s w h o c o - i n h e r i t a L 1 8 5 P p e r i p h e r i n - 2 m u t a t i o n a n d a null  or G 1 1 3 E r o m - 1 mutation are afflicted with retinitis p i g m e n t o s a , w h e r e a s individuals w h o inherit only one defective g e n e are normal (Dryja et 1997; Kajiwara, Berson & Dryja, 1994).  al.,  Biochemical studies have shown  t h a t the L 1 8 5 P p e r i p h e r i n - 2 m u t a n t a s s e m b l e s w i t h r o m - 1 to f o r m core heterotetrameric  c o m p l e x e s , b u t u n l i k e W T p e r i p h e r i n - 2 , it is u n a b l e  to  s e l f - a s s e m b l e into core h o m o t e t r a m e r s ( G o l d b e r g & M o l d a y , 1 9 9 6 a ) (see section  1.4.2).  These findings  have been incorporated  m o d e l in w h i c h d e c r e a s e d l e v e l s o f t e t r a m e r i c Fig  20  s h o w s t h e location of t h e s e m u t a t i o n s  into a disease  c o m p l e x e s result and conserved  in R P .  structural  e l e m e n t s in p e r i p h e r i n - 2 a n d r o m - 1 . This  chapter  focuses  digenic RP and the  on  the  molecular  involved  in  i m p o r t a n c e of d i s u l f i d e - m e d i a t e d o l i g o m e r i z a t i o n  in  maintaining normal photoreceptor function. 2 and several related mutants structure  of  the  core complex  interactions  A n a l y s i s of L 1 8 5 P p e r i p h e r i n -  h a s lead to g r e a t e r u n d e r s t a n d i n g of t h e and  of  the  requirements  for  disulfide-  u s e d in t h e s e s t u d i e s h a v e b e e n  published  mediated oligomerization. M o s t of t h e  methods  elsewhere (Loewen, Moritz & Molday, 2001). this  chapter  were  performed  by  the  A l l e x p e r i m e n t s d e s c r i b e d in  author  except  (1)  hydrodynamic  100  Per2B6  L185P (Peripherin-2)  Fig 2 0 .  T o p o l o g i c a l m o d e l of p e r i p h e r i n - 2 a n d r o m - 1  subunits.  Conserved amino acid segments between peripherin-2 and rom-1 and c o n s e r v e d cysteine residues within the large intradiskal L 3 - 4 loop are s h o w n . T h e locations of t h e L 1 8 5 P p e r i p h e r i n - 2 a n d G 1 1 3 E r o m - 1 m i s s e n s e m u t a t i o n s linked to digenic retinitis p i g m e n t o s a are indicated with a black circle. T h e binding site for the peripherin-2 m o n o c l o n a l a n t i b o d y P e r 2 B 6 a n d the Q 3 4 1 P substitution that abolishes binding are also indicated. M1-M4 i n d i c a t e t r a n s m e m b r a n e s e g m e n t s 1-4.  Chapter 4 Digenic RP  101  analysis of L 1 8 5 P (Fig 2 1 ) ; (2) velocity sedimentation  of L 1 8 8 P r o m - 1  (Fig 2 3 B ) .  4.2 MATERIALS AND METHODS 4.2.1  Plasmids and heterologous COS-1 cell expression pcPer  (WT peripherin-2),  pcROM (WT rom-1)  pcPer-L185P (L185P peripherin-2),  have been described (Goldberg, Loewen & Molday,  1998; G o l d b e r g , Moritz & Molday, 1 9 9 5 ; Moritz & Molday, 1996). L185A  (L185A  constructed pcPerlD4,  peripherin-2),  by  and  PCR-based  containing  a  and pcROM-L188P mutagenesis  RholD4  (L188P  (Nelson  monoclonal  pcPer-  rom-1)  &  Long,  antibody  were 1989).  epitope  tag  T E T S Q V A P A at the C-terminus w a s created by ligating a n oligonucleotide corresponding to the 1 D 4 epitope onto cDNA.  A  Q341P  change  that  the 3' e n d of the  abolishes  the  binding  of  peripherin-2 monoclonal  antibody P e r 2 B 6 w a s also introduced into this construct by site-directed mutagenesis. created  p c L 1 8 5 P - l D 4 ( p c P e r l D 4 with the L185P mutation) w a s  by cloning  the BamHI-SacII  restriction  fragment  from  pcPer-  L 1 8 5 P into B a m H I - S a c I I digested p c P e r l D 4 . C O S - 1 cells (ca. 6 x 1 0 c e l l s / 1 0 0 - m m dish) were transfected with a 5  total of 3 0 pg of p l a s m i d D N A a n d h a r v e s t e d 7 2 h post-transfection as d e s c r i b e d in s e c t i o n 3 . 2 . 4 .  Chapter 4 Digenic RP 4.2.2  102  Hydrodynamic Hydrodynamic  conditions  was  peripherin-2  characterization analysis  carried  (Goldberg  of  out &  of L185P  the  as  L185P  described  Molday,  peripherin-2  mutant  under  previously  1996b).  reducing  for  Briefly,  wild-type  pcPer-L185P  t r a n s f e c t e d c e l l s f r o m o n e 1 0 0 m m d i s h w e r e s o l u b i l i z e d in 3 0 0 - 5 0 0 pl P B S containing 1 % Triton X - 1 0 0 , 2 m M DTT, a n d P M S F , a n d centrifuged at 9 0 , 0 0 0 x g for 30 min at 4°C.  T h e s u p e r n a t a n t w a s u s e d to  determine  the h y d r o d y n a m i c properties of the d e t e r g e n t - s o l u b i l i z e d c o m p l e x . Stokes  radius was  determined  by  calibrated Sephacryl S - 3 0 0 column.  The  gel exclusion c h r o m a t o g r a p h y  on  a  Velocity sedimentation analysis was  p e r f o r m e d b y c e n t r i f u g a t i o n a t 4 ° C in a B e c k m a n S W 5 0 . 1 r o t o r a t 4 9 , 0 0 0 r p m f o r 1 3 h o n s u c r o s e g r a d i e n t s in H 0 a n d 2 8 h f o r g r a d i e n t s in D 0 . 2  Fractions  were  collected  and  2  analyzed on  Western  blots.  Calibration  standards (Stokes radius, sedimentation coefficient ( S v e d b e r g s ) ,  partial  s p e c i f i c v o l u m e ) w e r e c y t o c h r o m e c ( 1 . 7 n m , 1.9 S , 0 . 7 2 8 m l / g ) ,  malate  dehydrogenase (3.5 n m , 4.32 S , 0.734 ml/g), aldolase (4.6 n m , 7.70 S, 0.742 ml/g) 1995;  and apoferritin  (6.7 n m , 17.7 S , 0.73 ml/g)  Clarke & Smigel, 1989;  molecular  mass  of  m o l e c u l a r m a s s of the protein method  al.,  Fasman & Chemical Rubber Company,  1975; Siegel & Monty, 1966; Worthington, The  (Cascio et  the  1993).  detergent-protein  and  the  portion were calculated according to  the  of C l a r k e ( C l a r k e & S m i g e l , 1 9 8 9 ) .  complex  Briefly, the  sedimentation  Chapter 4 Digenic RP coefficients  103  ( s ) , a n d viscosities  sedimentation  positions  (7) a n d densities  (determined  from  (/?) a t t h e a v e r a g e  calibration  standards a n d  i n d i c a t e d b y t h e s u b s c r i p t avg) w e r e d e t e r m i n e d i n H 0 a n d D 0 ( d e n o t e d 2  by  the corresponding  2  subscripts H a n d D ) a n d used to calculate t h e  p a r t i a l s p e c i f i c v o l u m e ( F ) o f t h e c o m p l e x u s i n g E q u a t i o n 1.  v=  The  ( D7D 5  sedimentation  -1) / (p  / S„TJ„  H  coefficient  D  (s ,w)  andp  run temperature  (subscript  Tm  Tm  H  d  ( s u b s c r i p t m). T h e d e n s i t i e s  )-  p  under  determined  T) a n d in s u c r o s e g r a d i e n t s  (p2 ,w)  a n d viscosities  0  ( E q . 1)  )  D  standard  using Equation 2. T h e values  are experimentally  Tm  H  for the complex  20  conditions (20 °C, water) w a s determined u s e d f o r s ,rj ,  I s t]  (S TJ avg ^ ^avg  values at the in H 0 o r D 0 2  (720,^)  of water  s t a n d a r d c o n d i t i o n s o f 2 0 °C a r e 0 . 9 9 8 g c m " a n d 0 . 0 1 0 0 2 g c m " 3  ^2o,w = *r, (7r, ' %o. J d " A o ) / (1 ~ m,m)) l  m  m  F  The molecular mass of the detergent-protein  2  1  under  s" . 1  (Eq • 2)  ; W  complex ( M ) was calculated c  u s i n g t h e s e d i m e n t a t i o n c o e f f i c i e n t (s o,w), t h e p a r t i a l s p e c i f i c v o l u m e ( 7 ) , 2  and t h eStokes radius (a) using Equation 3 (Siegel & Monty, 1966) N is A v o g a d r o ' s n u m b e r .  where  104  Chapter 4 Digenic RP  ( E q . 3)  Finally,  the  molecular  mass  p e r i p h e r i n - 2 c o m p l e x (M )  of  the  protein  value for the partial specific v o l u m e for protein Triton  of  the  was calculated using Equation 4.  p  detergent  portion  X-100  (0.94  ml/g)  were  (0.74 used  An average  ml/g) in  L185P  a n d for  the  the  calculation  (Goldberg & Molday, 1996b).  M  p  4.2.3  Solubilization,  sedimentation  washed  immunoprecipitation,  C O S - 1 cells from  with  (Eq. 4)  c  and  analysis of COS-1 cell expressed  Transfected plates,  = M /(l + (v - 0.74)7(0.94 - v))  PBS, and  100 m m  incubated  a b s e n c e of 2 0 m M DTT for 9 0 min at 25°C.  velocity proteins  dishes were  with  scraped  P B S in t h e  from  presence  or  T h e cells were solubilized with  a n e q u a l v o l u m e ( 1 5 0 pl) o f P B S c o n t a i n i n g 2 % T r i t o n X - 1 0 0 , 8 0 m M N E M , and P M S F (pH 7.4)  for 10 m i n on ice.  In s o m e e x p e r i m e n t s ,  r e p l a c e d w i t h 1 m M D T T in t h e s o l u b i l i z a t i o n b u f f e r . centrifuged fraction)  T h e cell extract  at 9 0 , 0 0 0 x g for 30 m i n at 4°C a n d s u p e r n a t a n t  w a s r e t a i n e d o n ice for a n a l y s i s .  was  (solubilized  Peripherin-2 w a s purified  the solubilized fraction on a P e r 2 B 6 - S e p h a r o s e immunoaffinity p r e v i o u s l y d e s c r i b e d in s e c t i o n 3 . 2 . 3 .  NEM was  from  matrix  as  Chapter 4 Digenic RP  105  Purified peripherin-2 (approximately 5-20% Triton  (w/w) sucrose gradients X - 1 0 0 (Goldberg  included  in  the  reducing conditions. Beckman TLS-55  prepared  & Molday,  gradients  for  1-2 pg p r o t e i n ) w a s a p p l i e d to in P B S a n d c o n t a i n i n g 0 . 1 %  1996a).  DTT-treated  After centrifugation  Routinely, samples  I m M DTT was analyzed  under  for 16 h at 5 0 , 0 0 0  r p m in a  rotor at 4°C, the bottom of t h e centrifuge  tube w a s  punctured and four-drop fractions were collected for analysis by Western blotting.  This procedure w a s used to determine apparent  sedimentation  coefficients for comparative purposes.  4.2.4  SDS-PAGE  and Western  blotting  S a m p l e s ( 2 5 pi c o n t a i n i n g 0 . 1 - 0 . 5 p g p e r i p h e r i n - 2 ) w e r e  denatured  with a n e q u a l v o l u m e of S D S cocktail ( 4 % S D S , 0 . 0 2 M T r i s - H C l , p H 6 . 8 , 2 0 % g l y c e r o l , 0 . 0 1 % b r o m o p h e n o l b l u e ) in t h e a b s e n c e ( n o n r e d u c i n g ) o r presence (reducing)  of 5 % p-mercaptoethanol, a n d applied to 8 or 1 0 %  S D S - p o l y a c r y l a m i d e gels as described (Loewen & Molday, 2000). electrophoresis, the proteins  were  Bio-Rad semidry transfer apparatus.  transferred  to I m m o b i l o n - P  After  using a  Blots were probed with monoclonal  antibodies to peripherin-2 (Per2B6) (Molday, Hicks & Molday, 1987), r o m 1 ( R o m l C 6 ) , o r r h o d o p s i n ( R h o l D 4 ) ( M a c K e n z i e etal., anti-mouse RomlC6  immunoglobulin-peroxidase  monoclonal  antibody  for  was generated  detection  1984) and sheep by  ECL. The  to a G S T - f u s i o n  protein  Chapter 4 Digenic RP  106  c o n t a i n i n g t h e i n t r a d i s c a l loop r e g i o n of r o m - 1 ( M o r i t z & M o l d a y , 1 9 9 6 ) . Crosslinking  was  performed  on  Triton  X - 1 0 0 solubilized samples  with  0 . 0 0 1 % g l u t a r a l d e h y d e for 1 5 m i n at 37°C.  4.3  RESULTS  4.3.1  Molecular mass of L185P peripherin-2 under disulfidereducing conditions Previous  studies  have  shown  that  under  reducing  conditions  ( + D T T ) , t h e s e d i m e n t a t i o n c o e f f i c i e n t o f t h e L 1 8 5 P p e r i p h e r i n - 2 m u t a n t is significantly complex  smaller than  (Goldberg  &  that of the Molday,  tetrameric  1996a).  To  W T peripherin-2 determine  core  how  the  s e d i m e n t a t i o n b e h a v i o u r o f t h i s m u t a n t r e l a t e s t o its m o l e c u l a r m a s s , w e carried out protein  a hydrodynamic  under  reducing  study  conditions.  of t h e  C O S - 1 cell e x p r e s s e d  As shown  in  Fig  21A,  the  L185P L185P  m u t a n t eluted f r o m a calibrated gel exclusion c o l u m n as a single species w i t h a S t o k e s r a d i u s o f 4 . 8 3 ± . 2 9 n m ( n = 5 ) , a v a l u e t h a t is s i g n i f i c a n t l y lower than the value of 6.20 ± .14 n m previously m e a s u r e d for the peripherin-2 coefficient  complex (Goldberg & Molday, 1996b). and  partial  specific  volume  of  the  The  L185P  sedimentation mutant  d e t e r m i n e d b y v e l o c i t y s e d i m e n t a t i o n in s u c r o s e g r a d i e n t s c o n t a i n i n g a n d D 0 (Fig 2  21B).  A single detergent-protein  WT  were H2O  s p e c i e s w a s o b s e r v e d in  e a c h c a s e r e s u l t i n g in a S2o,w v a l u e o f 2 . 7 8 + 0 . 0 6 S ( n = 8 )  a n d a partial  107  Fraction Number  Fig 2 1 . H y d r o d y n a m i c a n a l y s i s of C O S - 1 cell e x p r e s s e d L 1 8 5 P peripherin-2 mutant under reducing conditions. C O S - 1 cells e x p r e s s i n g t h e L 1 8 5 P p e r i p h e r i n - 2 m u t a n t w e r e s o l u b i l i z e d in T r i t o n X - 1 0 0 in t h e p r e s e n c e o f DTT. ( A ) G e l e x c l u s i o n c h r o m a t o g r a p h y o f L 1 8 5 P p e r i p h e r i n 2. Inset s h o w s calibration curve generated from s t a n d a r d s : c y t o c h r o m e c (cyt), malate dehydrogenase (mal), aldolase (aid), and apoferritin (apo) and t h e v o i d (V o ) a n d i n c l u d e d (V t ) v o l u m e s . (B) V e l o c i t y s e d i m e n t a t i o n of L185P peripherin-2 through 5 - 2 0 % sucrose gradients m a d e up with H 0 or D 0 . Peripherin-2 w a s detected on Western blots labeled with the Per2B6 a n t i b o d y a n d quantified by d e n s i t o m e t r y (see M e t h o d s s e c t i o n 4 . 2 . 2 a n d 4.2.4). 2  2  Chapter 4 Digenic RP  108  specific v o l u m e of 0 . 8 2 m l / g . There unpurified  was and  no  difference  between  immunoaffinity  purified  the  sedimentation  complex  (data  profiles  not  of  shown).  F u r t h e r m o r e , since the partial specific v o l u m e of L 1 8 5 P p e r i p h e r i n - 2 w a s similar to the  native complex from  rod outer s e g m e n t s ( 0 . 8 3  ml/g  native complex (Goldberg & Molday, 1996b)), the L185P mutant  for  bound  s i m i l a r a m o u n t s o f d e t e r g e n t a s t h e n a t i v e c o m p l e x i n d i c a t i n g t h a t it w a s not grossly misfolded.  Using the values determined for S t o k e s radius,  s e d i m e n t a t i o n coefficient, and partial specific v o l u m e , a m o l e c u l a r m a s s of  91  kDa  was  calculated  for  the  L185P  detergent-protein  a c c o r d i n g to the m e t h o d of C l a r k e ( C l a r k e & S m i g e l ,  1989).  complex Using an  a v e r a g e value for the partial specific v o l u m e for protein (0.74 m l / g ) the  value  additivity  for of  the  detergent  partial  Triton  X-100  specific v o l u m e s  for  (0.94  ml/g)  protein  and  and  assuming  detergent,  m o l e c u l a r m a s s w a s e s t i m a t e d to be 54 k D a for t h e d e t e r g e n t - f r e e protein complex. (39  kDa)  and  experimental  and  the  L185P  T h i s v a l u e is i n t e r m e d i a t e b e t w e e n a L 1 8 5 P m o n o m e r dimer  value  of  (78 135  kDa), kDa  but  considerably  previously  measured  less for  than the  the native  peripherin-2 t e t r a m e r (calculated Mr = 1 5 2 , 0 0 0 ) from rod outer s e g m e n t s (Goldberg & Molday, 1996b). T o d e t e r m i n e if t h e L 1 8 5 P m u t a n t e x i s t s a s a m o n o m e r o r a d i m e r , a series of c o - i m m u n o p r e c i p i t a t i o n studies with the L 1 8 5 P m u t a n t a n d a  Chapter 4 Digenic RP related  109  L185P-1D4 mutant  recognized  by  monoclonal  were  carried  antibody  out.  Per2B6;  The L185P mutant the  L185P-1D4  is  mutant  contains a 9 amino acid C-terminal epitope tag recognized by monoclonal antibody  R h o l D 4 and a Q341P mutation  ( P a t p o s i t i o n 3 4 1 is f o u n d in  mouse peripherin-2) that abolishes Per2B6 immunoreactivity. The  ability  translationally  of  L185P a n d L 1 8 5 P - 1 D 4 proteins  was determined.  detergent-solubilized immunoprecipitated conditions,  As  shown  in  interact  post-  Figure  22A,  when  mixed  together  L185P and L185P-1D4 were with  L185P-1D4  Per2B6-Sepharose under  mutant  w a s eluted  in  the  to  and  disulfide  reducing  unbound  fraction,  w h e r e a s t h e L 1 8 5 P m u t a n t w a s o n l y p r e s e n t in t h e b o u n d f a c t i o n .  This  result indicates that individually e x p r e s s e d L 1 8 5 P a n d L 1 8 5 P - 1 D 4 proteins do not associate to form a c o m p l e x . that the Per2B6-Sepharose matrix  This experiment also demonstrates selectively binds the L185P  mutant,  but not t h e L 1 8 5 P - 1 D 4 mutant. To determine into a multi-subunit  if L 1 8 5 P a n d L 1 8 5 P - 1 D 4 m u t a n t s  c o m p l e x , C O S - 1 cells were co-transfected with t h e  L185P and L185P-1D4 plasmids. reduce Triton  can co-assemble  intermolecular  disulfide  T h e cells were  bonds  and subsequently  X - 1 0 0 in t h e p r e s e n c e of N E M t o b l o c k  (Loewen & Molday, 2000).  treated  free  with  DTT to  solubilized  sulfhydryl  in  groups  T h e detergent-solubilized extract w a s applied  to a P e r 2 B 6 - S e p h a r o s e c o l u m n a n d t h e u n b o u n d  and bound  (peptide-  110  kDa  Per2B6  Rho1D4  B  Per2B6  Rho1D4  205 116 97 66 45 29  m  monomer  - *  m ^  m m  m  mmm wmmm  kDa 205 116 97 66  dimer  45 29 12.5  10  monomer  2.5  % Sucrose  Fig 2 2 . Immunoprecipitation, velocity sedimentation and c r o s s l i n k i n g of t h e L 1 8 5 P p e r i p h e r i n - 2 c o m p l e x u n d e r reducing conditions. (A) C O S - 1 cells individually e x p r e s s i n g t h e L 1 8 5 P a n d L 1 8 5 P 1 D 4 m u t a n t w e r e r e d u c e d with DTT a n d s o l u b i l i z e d in Triton X - 1 0 0 . T h e extracts were mixed together and immunoprecipitated with Per2B6S e p h a r o s e . T h e C O S - 1 cell e x t r a c t (lane a ) , u n b o u n d f r a c t i o n ( l a n e b) a n d b o u n d , p e p t i d e - e l u t e d fraction (lane c) w e r e a n a l y z e d o n W e s t e r n blots labeled with Per2B6 a n d R h o l D 4 antibodies. (B) C O S - 1 cells coexpressing L 1 8 5 P a n d L 1 8 5 P - 1 D 4 p e r i p h e r i n - 2 w e r e s o l u b i l i z e d in Triton X - 1 0 0 u n d e r reducing conditions, and immunoprecipitated with Per2B6-Sepharose. The C O S - 1 cell extract (lane a ) , u n b o u n d fraction (lane b ) , a n d b o u n d , p e p t i d e eluted fraction (lane c) w e r e a n a l y z e d on W e s t e r n blots l a b e l e d with P e r 2 B 6 or R h o l D 4 antibody. (C) T h e coprecipitated (bound, peptide-eluted) fraction from (B) w a s subjected to velocity s e d i m e n t a t i o n a n d fractions were analyzed on Western blots labeled with R h o l D 4 antibody. The s e d i m e n t a t i o n p o s i t i o n of L 1 8 5 P p e r i p h e r i n - 2 ( a r r o w h e a d ) is s h o w n f o r c o m p a r i s o n . ( D ) W e s t e r n blot of r e d u c e d , i m m u n o a f f i n i t y purified L 1 8 5 P peripherin-2 crosslinked with glutaraldehyde a n d labeled with the Per2B6 a n t i b o d y ( m o l e c u l a r m a s s s t a n d a r d s a r e g i v e n in k D a ) ( s e e M e t h o d s sections 4.2.3 and 4.2.4).  Chapter 4 Digenic RP eluted)  111  fractions were analyzed for L185P a n d L 1 8 5 P - 1 D 4 by Western  blotting.  Fig 2 2 B s h o w s t h a t a l l t h e L 1 8 5 P p r o t e i n b o u n d t o t h e P e r 2 B 6 -  Sepharose  matrix.  Approximately  5 0 % of  the  L185P-1D4  protein  coprecipitated with the L185P peripherin-2. The  isolated  L 1 8 5 P : L 1 8 5 P - 1 D 4 complex w a s further analyzed by  v e l o c i t y s e d i m e n t a t i o n (Fig 22C).  The major complex had an apparent  sedimentation coefficient that w a s similar to the individually e x p r e s s e d L 1 8 5 P (Table 5). of t h e t u b e .  A n additional complex w a s apparent near the bottom  This most  that form during  likely r e p r e s e n t s L 1 8 5 P : L 1 8 5 P - 1 D 4 a g g r e g a t e s  the immunoaffinity  purification  procedure.  F i n a l l y , it  w a s d e t e r m i n e d if t h e i n d i v i d u a l l y e x p r e s s e d a n d p u r i f i e d L 1 8 5 P m u t a n t could  be crosslinked into dimer.  As shown  i n Fig 2 2 D ,  a  significant  fraction of detergent-solubilized L 1 8 5 P peripherin-2 m i g r a t e d a s a d i m e r on a S D S gel after glutaraldehyde crosslinking. Taken together, the hydrodynamic analysis, coimmunoprecipitation, and  crosslinking  studies  provide  strong  evidence  that  the  L185P  peripherin-2 m u t a n t exists as a dimer under disulfide reducing conditions.  4.3.2  Velocity sedimentation  rom-l  and interaction  r  To subunit  further assembly  of L185P-peripherin-2  evaluate of  of L185A-peripherin-2  t h e role  peripherin-2,  of leucine  with  and L188PL188P-rom-l  at position  the sedimentation  185 on the  behaviour  of a  Chapter 4 Digenic RP L185A  mutant  L185A  mutant,  apparent  112  under  disulfide-reducing  like  L185P,  sedimentation  T a b l e 5).  sedimented  coefficient  conditions as a  was analyzed.  single  The  species with  characteristic of a d i m e r  (Fig  an  23A;  This m u t a n t also c o - a s s e m b l e d with r o m - 1 into a tetrameric  core complex (data not shown). Rom-1 (Leul88). an  contains  a  leucine  residue  at  an  equivalent  position  T h e L188P r o m - 1 mutant sedimented a s a single species with  apparent  sedimentation  coefficient  of  3.09  S,  a  value  that  is  considerably less than that of W T r o m - 1 t e t r a m e r a n d consistent with a d i m e r (Fig 2 3 B ; T a b l e 5). T o d e t e r m i n e if L 1 8 5 P p e r i p h e r i n - 2 c a n a s s e m b l e w i t h L 1 8 8 P r o m - 1 , C O S - 1 cells c o - e x p r e s s i n g the L 1 8 5 P peripherin-2 a n d L 1 8 8 P r o m - 1 were treated  with  DTT  immunoprecipitated  and  with  the  detergent-solubilized  Per2B6-Sepharose.  Fig  23C  extract shows  was that  a  significant fraction of L188P r o m - 1 coprecipitated with L 1 8 5 P peripherin2.  This isolated complex sedimented with a n apparent  c o e f f i c i e n t o f 3 . 2 4 S (Table 5), a v a l u e i n t e r m e d i a t e homodimer  (S2o,w  sedimentation  between a L185P  = 3 . 6 4 S ) a n d a L 1 8 8 P h o m o d i m e r (s o,w = 3 . 0 9 S ) , a n d  consistent with heterodimer formation.  2  Chapter 4 Digenic RP  113  Table 5 Velocity S e d i m e n t a t i o n of  Peripherin-2 and Rom-1 Core C o m p l e x e s  Species WT peripherin-2  s o,w ( S )  rom-1 peripherin-2-lD4  4.6 ± 0.3 5.2  L185P peripherin-2 L185A peripherin-2 L185P:L185P-1D4 peripherin-2 L185P peripherin-2:L188P rom-1 L188P rom-1  3.6 ± 0.2 3.7 ± 0.3 3.7 ± 0.2  WT WT  Core complex  2  5.4 ± 0.1  2  4  (n = 3 ) (n = 6) ( n = 1) (n= 3)  1  3  homotetramer homotetramer homotetramer homodimer  (n= 3) (n= 3)  homodimer heterodimer  3.2  (n = 2)  heterodimer  3.1 ± 0 . 2  (n= 5)  homodimer  Proteins e x p r e s s e d in C O S - 1 cells w e r e s o l u b i l i z e d in T r i t o n X - 1 0 0 , a p p l i e d to 5 - 2 0 % s u c r o s e g r a d i e n t s , a n d s e d i m e n t e d for 16 h at 5 0 , 0 0 0 r p m a n d 4°C in a B e c k m a n T L S - 5 5 rotor u n d e r r e d u c i n g c o n d i t i o n s . F r a c t i o n s w e r e a n a l y z e d on W e s t e r n blots. S2o,w v a l u e s are g i v e n in S v e d b e r g s (S) ± S . D . value previously reported (Goldberg, Loewen & Molday, 1998) n is t h e n u m b e r of i n d e p e n d e n t e x p e r i m e n t s v a l u e p r e v i o u s l y r e p o r t e d ( G o l d b e r g & M o l d a y , 1 9 9 6 a ) w a s r e c a l c u l a t e d using c o r r e c t e d p a r a m e t e r s as d e s c r i b e d in G o l d b e r g , L o e w e n & M o l d a y ( 1 9 9 8 ) 1  2  3  4  114  Fig 2 3 . V e l o c i t y s e d i m e n t a t i o n of L 1 8 5 A p e r i p h e r i n - 2 a n d L 1 8 8 P r o m - 1 a n d c o p r e c i p i t a t i o n of L 1 8 5 P p e r i p h e r i n - 2 a n d L 1 8 8 P r o m - 1 under reducing conditions. Velocity s e d i m e n t a t i o n profiles of (A) L 1 8 5 A p e r i p h e r i n - 2 a n d ( B ) L 1 8 8 P r o m - 1 m u t a n t i n d i v i d u a l l y e x p r e s s e d in C O S - 1 cells. Positions of W T peripherin-2 a n d W T r o m - 1 a r e s h o w n for c o m p a r i s o n in t h e r e s p e c t i v e p r o f i l e s . ( C ) D e t e r g e n t - s o l u b i l i z e d e x t r a c t s o f C O S - 1 c e l l s coexpressing L185P peripherin-2 and L188P rom-1 were immunoprecipitated with Per2B6 Sepharose. T h e extract (a), unbound (b), a n d b o u n d , p e p t i d e - e l u t e d fraction (c) w e r e a n a l y z e d o n W e s t e r n blots labeled with Per2B6 and R o m l C 6 antibodies (see Methods section 4.2.3).  Chapter 4 Digenic RP 4.3.3  115  Velocity sedimentation  nonreducing  of L185P peripherin-2  under  conditions  The W T peripherin-2 core tetramer  forms  higher order,  disulfide-  linked oligomers under nonreducing conditions (Loewen & Molday, 2000). T o d e t e r m i n e if t h e L 1 8 5 P p e r i p h e r i n - 2 d i m e r a l s o f o r m s  disulfide-linked  oligomers, C O S - 1 cells expressing t h e L 1 8 5 P m u t a n t w e r e treated N E M a n d s o l u b i l i z e d in T r i t o n X - 1 0 0 in t h e a b s e n c e o f D T T . extract  w a s subjected  analyzed  to  on nonreducing  velocity  sedimentation  T h e soluble  a n d fractions  SDS-polyacrylamide gels  (Fig  velocity s e d i m e n t a t i o n profile s h o w e d t w o distinct s p e c i e s .  with  24A).  were The  O n e species,  a, h a d a n a p p a r e n t s e d i m e n t a t i o n c o e f f i c i e n t o f 3 . 6 2 S , a v a l u e s i m i l a r t o t h e v a l u e f o r r e d u c e d L 1 8 5 P (Table 5); t h i s s p e c i e s w a s c o m p o s e d s o l e l y of m o n o m e r s w h e n a n a l y z e d o n n o n r e d u c i n g S D S g e l s . A s e c o n d s p e c i e s , b, h a d a n a p p a r e n t s e d i m e n t a t i o n c o e f f i c i e n t o f 5 . 2 2 S , a v a l u e t h a t is similar  to that  observed  for W T tetrameric  peripherin-2  (Table 5); t h i s s p e c i e s c o n t a i n e d d i s u l f i d e - l i n k e d d i m e r s . indicate  that  through  intermolecular  However,  this  two L185P peripherin-2  complex  disulfide does  core  dimers  bonds to form not form  the  o b s e r v e d f o r W T p e r i p h e r i n - 2 (Fig 2 4 C & D).  core  T h e s e results  c a n link  a tetrameric higher  species  order  together complex. oligomers  116  B  6  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  13  14  15  7  8  9  10  11  12  13  14  15  Fraction Number  Sedimentation Fractions  5  1  2  3  4  5  6  7  8  9  10  11  12  6  7  8  9  10  11  12  13  14  15  Fraction Number  Sedimentation Fractions  Fig 2 4 . V e l o c i t y s e d i m e n t a t i o n of L 1 8 5 P a n d W T p e r i p h e r i n - 2 u n d e r nonreducing conditions. C O S - 1 cells e x p r e s s i n g either L 1 8 5 P o r W T p e r i p h e r i n - 2 w e r e s o l u b i l i z e d in Triton X - 1 0 0 c o n t a i n i n g N E M a n d s u b j e c t e d to velocity s e d i m e n t a t i o n . Western blots of fractions separated on nonreducing S D Sgels were labeled with the Per2B6 monoclonal antibody a n d quantified by densitometry. (A) Western blot of L 1 8 5 P peripherin-2 with s e d i m e n t a t i o n positions of d i m e r (a) a n d t e t r a m e r (b) s h o w n , a n d (B) c o r r e s p o n d i n g densitometric profiles of t h e m o n o m e r (closed s q u a r e s ) a n d dimer (open circles) bands. (C) W e s t e r n blot of W T p e r i p h e r i n - 2 with s e d i m e n t a t i o n p o s i t i o n s of t e t r a m e r (b) a n d o l i g o m e r (c) s h o w n , a n d ( D ) corresponding profiles of the m o n o m e r a n d d i m e r b a n d s (see Methods section 4.2.3).  Chapter 4 Digenic RP 4.3.4  117  Oligomerization  complexes  of WT- and  by velocity  L185P-peripherin-2:rom-l  sedimentation  Previous studies have shown that both W T and L185P assemble  with  WT  rom-1  to  form  heterotetrameric  reducing conditions (Goldberg & Molday, 1996a).  peripherin-2  complexes  under  T o d e t e r m i n e if t h e s e  complexes can form higher order disulfide-linked oligomers, we examined the  sedimentation  2:rom-l  b e h a v i o u r of i m m u n o a f f i n i t y  and the L185P peripherin-2:rom-l  conditions.  A s s h o w n in Fig  sedimented  as two  complex  distinct  sedimented  as  25A,  WT  complex under  nonreducing  Approximately  heterotetramer  (b  in  (c  in  Fig  25A),  presumably  an  Fig  octamer,  complex  2 5 % of the 25A)  intermolecular disulfide bonds and 7 5 % s e d i m e n t e d as an oligomer  peripherin-  the W T peripherin-2:rom-1  species.  a  purified  total  devoid  of  intermediate  containing  a  high  proportion of i n t e r m o l e c u l a r disulfide b o n d s . T h e s e d i m e n t a t i o n profile of L 1 8 5 P p e r i p h e r i n - 2 : r o m - l s h o w n in Fig 25B.  c o m p l e x is  Three distinct species were o b s e r v e d : a heterodimer  (a) a c c o u n t i n g for a p p r o x i m a t e l y  1 7 % of t h e c o m p l e x a n d c o n s i s t i n g of  m o n o m e r s ; a h e t e r o t e t r a m e r (b) a c c o u n t i n g for 2 3 % of t h e total c o m p l e x and containing both m o n o m e r and disulfide-dimers; and an oligomer  (octamer)  (c)  accounting  containing disulfide-linked dimers. L185P  peripherin-2  mutant  for  60%  of  total  intermediate complex  and  T h e s e r e s u l t s s h o w t h a t m o s t of t h e  assembles  with  rom-1  into  octameric  118  dimer —  monomer 7  8  9 10  11 12  13  14  15  16  17  18 19 20  Sedimentation Fractions  B  dimer  monomer 3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  Sedimentation Fractions  Fig 2 5 . V e l o c i t y s e d i m e n t a t i o n of W T p e r i p h e r i n - 2 : r o m - l a n d L 1 8 5 P : r o m - l complexes under nonreducing conditions. C O S - 1 cells coexpressing either W T peripherin-2 and W T r o m - 1 (A) or L185P and W T r o m - 1 ( B ) w e r e s o l u b i l i z e d in Triton X - 1 0 0 c o n t a i n i n g N E M , purified w i t h Per2B6 S e p h a r o s e , a n d subjected to velocity s e d i m e n t a t i o n . Fractions were analyzed on nonreducing S D S gels a n d Western blots w e r e labeled with the R o m l C 6 monoclonal antibody. Sedimentation positions of dimer (a), t e t r a m e r (b), a n d o c t a m e r (c) a r e s h o w n (see M e t h o d s s e c t i o n 4 . 2 . 3 ) .  Chapter 4 Digenic RP disulfide-linked 2:rom-l  4.3.5  119  complexes similar to that observed for W T peripherin-  complex.  Interaction Individuals  of L185P-peripherin-2 with  autosomal  mutant peripherin-2 alleles. assembles with  with  dominant  RP express  both  T o d e t e r m i n e if L 1 8 5 P p e r i p h e r i n - 2  with W T peripherin-2, the L185P  peripherin-2-lD4  peripherin-2-lD4  containing  mutant  the C-terminal  WT and mutant  was co-expressed  1D4 epitope  Q341P mutation that abolishes Per2B6 immunoreactivity.  and the  Fig 2 6 A s h o w s  that under disulfide reducing conditions over 5 0 % of t h e peripherin-21D4  coprecipitated  sedimentation  with  the  analysis of this  L185P  the  velocity  mutant.  complex showed a mixed  heterodimer (a) a n d heterotetramer conditions,  peripherin-2  ( D ) (Fig 2 6 B ) .  sedimentation  profile  of  Velocity  population  of  Under nonreducing L185P  peripherin-  2 : p e r i p h e r i n - 2 - l D 4 c o m p l e x (Fig 26C) r e v e a l e d t h e p r e s e n c e o f h i g h e r o r d e r disulfide-linked o l i g o m e r s (c) a s well a s h e t e r o t e t r a m e r s  (b) a n d  h e t e r o d i m e r s ( a ) . In control e x p e r i m e n t s , t h e individually e x p r e s s e d W T peripherin-2-lD4  exhibited  the same  sedimentation  behavior  as W T  p e r i p h e r i n - 2 (Table 5), i n d i c a t i n g t h a t a d d i t i o n o f t h e 1 D 4 e p i t o p e h a d n o effect o n s u b u n i t a s s e m b l y .  120  Fig 2 6 . I m m u n o p r e c i p i t a t i o n a n d v e l o c i t y s e d i m e n t a t i o n of L 1 8 5 P peripherin-2:WT p e r i p h e r i n - 2 - l D 4 complexes under reducing and nonreducing conditions. (A) C O S - 1 cells c o e x p r e s s i n g L 1 8 5 P peripherin2 a n d W T p e r i p h e r i n - 2 - l D 4 w e r e r e d u c e d w i t h D T T , s o l u b i l i z e d in T r i t o n X 100, and immunoprecipitated with P e r 2 B 6 - S e p h a r o s e . T h e C O S - 1 cell extract (lane a ) , u n b o u n d fraction (lane b), a n d b o u n d , peptide-eluted fraction (lane c) w e r e a n a l y z e d on W e s t e r n blots l a b e l e d w i t h P e r 2 B 6 or R h o l D 4 antibody. (B) The coprecipitated c o m p l e x from (A) w a s subjected to velocity s e d i m e n t a t i o n a n d fractions w e r e a n a l y z e d o n W e s t e r n blots l a b e l e d w i t h R h o l D 4 a n t i b o d y . ( C ) S a m p l e s w e r e t r e a t e d a s in ( A ) e x c e p t under nonreducing conditions (-DTT), and the purified L185P:WT p e r i p h e r i n - 2 - l D 4 c o m p l e x e s were subjected to velocity s e d i m e n t a t i o n . W e s t e r n blots of fractions f r o m a n o n r e d u c i n g S D S gel w e r e labeled with the R h o l D 4 antibody. S e d i m e n t a t i o n positions of d i m e r ( a ) , t e t r a m e r (b), and o l i g o m e r (c) a r e s h o w n ( s e e M e t h o d s s e c t i o n 4 . 2 . 3 ) .  Chapter 4 Digenic RP 4.4  121  DISCUSSION Previous studies have shown that the  expressed  in  COS-1  considerably  cells  smaller than  has that  a of  L185P peripherin-2  sedimentation the  WT  coefficient  peripherin-2  core  mutant that  is  tetramer  (Goldberg & Molday, 1996a).  W e h a v e d e t e r m i n e d t h e size of this m u t a n t  by  co-immunoprecipitation  hydrodynamic  Although  analysis,  hydrodynamic  analysis  gave  intermediate  between  a monomer  crosslinking  provided  strong  a  molecular  and dimer,  evidence  and  that  crosslinking.  mass  that  was  both coprecipitation the  and  detergent-solubilized  L185P mutant exists as a dimer under disulfide reducing conditions. low  value for  hydrodynamic  the  m o l e c u l a r m a s s of the  L185P dimer  m e a s u r e m e n t s is c o n s i s t e n t w i t h t h e  determined  using  this  technique  (Goldberg  &  by  underestimation  t h e m o l e c u l a r m a s s of t h e n a t i v e t e t r a m e r i c p e r i p h e r i n - 2 : r o m - l reported  The  Molday,  of  complex  1996b).  This  u n d e r e s t i m a t i o n in m o l e c u l a r m a s s c a n a r i s e in p a r t f r o m t h e a s s u m p t i o n that the  protein  binds the s a m e amount  (Clarke & Smigel, 1989).  of d e t e r g e n t  in H2O  and  T h e e s t i m a t e d partial s p e c i f i c v o l u m e of  D2O the  protein c a n also c o n t r i b u t e to a n i n a c c u r a t e v a l u e for t h e m o l e c u l a r m a s s o f t h e p r o t e i n . P a r t i a l s p e c i f i c v o l u m e s in t h e r a n g e o f 0 . 7 1 - 0 . 7 5 are generally used for proteins. not  only  the  carbohydrate  amino in  the  acid case  T h e t r u e v a l u e , h o w e v e r , is d e p e n d e n t  composition of  ml/g  a  of  glycoprotein  the as  protein, well  as  but  also  on  the  possible  Chapter 4 Digenic RP  122  p r e s e n c e of s m a l l a m o u n t s of b o u n d lipid ( C l a r k e & S m i g e l , 1 9 8 9 ) .  Since  L 1 8 5 P p e r i p h e r i n - 2 is g l y c o s y l a t e d in C O S - 1 c e l l s ( d a t a n o t s h o w n ) m a y a l s o c o n t a i n a s m a l l a m o u n t o f t i g h t l y b o u n d l i p i d in t h e solubilized state, the  v a l u e of 0 . 7 4  ml/g  used here  may  and  detergent  represent  an  u n d e r e s t i m a t i o n of the actual v a l u e for the partial specific v o l u m e of the protein. T h e r o l e o f t h e c o n s e r v e d l e u c i n e r e s i d u e a t p o s i t i o n 1 8 5 in s u b u n i t assembly  was  determined  peripherin-2 mutant  by  analyzing several mutants.  b e h a v e d like t h e L 1 8 5 P m u t a n t .  co-expressed  with  rom-1.  This  L185A  It f o r m e d a c o r e  d i m e r w h e n individually e x p r e s s e d , but a s s e m b l e d into a when  The  indicates  heterotetramer that  defective  h o m o t e t r a m e r f o r m a t i o n o f t h e L 1 8 5 P m u t a n t is d u e t o t h e a b s e n c e o f a leucine at position 1 8 5 a n d not the introduction of a proline r e s i d u e . ability  of t h e s e  tetramer  mutants  to  interact  further indicates that the  with  rom-1  mutants  to  are not  form  The  a  native-like  grossly  misfolded.  R o m - 1 has a leucine residue at an equivalent position ( L 1 8 8 ) within a c o n s e r v e d s e g m e n t o f t h e l a r g e i n t r a d i s c a l l o o p (Fig of this l e u c i n e w i t h proline a l s o p r e v e n t e d homotetramers.  rom-1  20).  from  Replacement a s s e m b l i n g into  T o g e t h e r t h e s e s t u d i e s i n d i c a t e t h a t L 1 8 5 in p e r i p h e r i n -  2 a n d L 1 8 8 in r o m - 1 p l a y e s s e n t i a l r o l e s in t h e a s s o c i a t i o n o f d i m e r s i n t o core tetramers.  Chapter 4 Digenic RP  123  T h e fact that these mutants  exist as d i m e r s , a n d not  monomers,  p r o v i d e s additional insight into the q u a t e r n a r y structure of t h e p e r i p h e r i n 2  and  rom-1  core complexes.  The  interactions  responsible for  dimer  formation are distinct from those responsible for t e t r a m e r formation since d i m e r i z a t i o n is n o t d e p e n d e n t o n l e u c i n e a t p o s i t i o n 1 8 5 in ( o r l e u c i n e 1 8 8 in r o m - 1 ) . 2 and  rom-1  leads to  not  known  exhibits  in w h i c h t h e  ' d i m e r - o f - d i m e r s ' (Fig is  O n this basis w e infer that the W T peripherin-  core tetramer  a model  at  the  peripherin-2  27).  pseudo two-fold  core tetramer  symmetry.  c a n be considered as a  The domain responsible for d i m e r  present  time.  This  However,  the  ability  formation of  L185P  p e r i p h e r i n - 2 to a s s o c i a t e with L 1 8 8 P r o m - 1 to f o r m a h e t e r o d i m e r 23C)  s u g g e s t s t h a t t h e d i m e r i z a t i o n d o m a i n of t h e s e s u b u n i t s , like  (Fig the  tetramerization d o m a i n , m u s t be structurally similar. T h e contribution of leucine a n d o t h e r b u l k y h y d r o p h o b i c r e s i d u e s to the  formation  and  d o c u m e n t e d for  maintenance  intracellular  1995));  thrombospondins  protein-protein  proteins  c l a s s l i k e G C N 4 ( O ' S h e a e t al., (Li & R u b i n ,  of  (transcription  factors of the  1997));  proteins  and  (tenascins,  integral  structure  receptor of  the  (Kunishima  dimeric  well bZIP  et  al.,  extracellular  2000)). ligand  laminins,  membrane  (glycophorin A (MacKenzie, Prestegard & Engelman, 1997), glutamate  is  1 9 9 1 ) ; the regulatory s u b u n i t of PKAIIb  extracellular matrix  (Kammerer,  interactions  metabotropic  Recently, the binding  proteins  region  crystal of  the  124  Transmembrane dimerization  C150  Intradiscal  disulfide-linked oligomerization  WT Fig 2 7 .  L185P  Model f o r t h e o l i g o m e r i c s t r u c t u r e of p e r i p h e r i n - 2 a n d t h e  L185P mutant. W T peripherin-2 monomers (transmembrane domain s h o w n a s a cylinder) interact to form a core noncovalent t e t r a m e r with a twofold a x i s of s y m m e t r y . Leucine at position 1 8 5 ( L 1 8 5 ) of t h e intradiskal loop is r e q u i r e d f o r t e t r a m e r f o r m a t i o n ( t e t r a m e r i z a t i o n ) . Dimer formation (dimerization) m a y involve interactions between transmembrane, intradiskal, or cytoplasmic domains (omitted for simplicity). T h e core tetramers link-up through C 1 5 0 - m e d i a t e d intermolecular disulfide bonds to f o r m h i g h e r o r d e r o l i g o m e r s ( o n l y t h e o c t a m e r is s h o w n ) . The L185P peripherin-2 m u t a n t consists of a core d i m e r that c a n link t h r o u g h C 1 5 0 mediated intermolecular disulfide bonds to form a tetramer, but not a higher order oligomer. N u m b e r s o n cylinders illustrate t h e t w o - f o l d s y m m e t r y of t h e c o r e t e t r a m e r a n d t h e c o n c e p t t h a t it c o n s i s t s o f a d i m e r o f d i m e r s (dimerization has been drawn as head-to-head). R o m - 1 also forms homotetramers a n d heterotetramers with peripherin-2, but limits oligomerization to octamers.  Chapter 4 Digenic RP metabotropic  125  glutamate  receptor (mGluR), a seven transmembrane G -  p r o t e i n c o u p l e d r e c e p t o r , h a s b e e n s o l v e d ( K u n i s h i m a e t al.,  2000).  this case, four conserved leucines a n d o n e isoleucine contribute dimer interface.  In  to the  In t h e intradiscal loop r e g i o n of p e r i p h e r i n - 2 a n d r o m - 1 ,  there are 9 conserved leucine a n d several conserved isoleucine residues in a d d i t i o n t o L 1 8 5 . It is p o s s i b l e t h a t a t l e a s t s o m e o f t h e s e r e s i d u e s also contribute Interestingly,  to inter-subunit  interactions of peripherin-2  and rom-1.  m a n y of t h e s e h y d r o p h o b i c r e s i d u e s a r e a l s o c o n s e r v e d in  o t h e r m e m b e r s o f t h e tetraspanin  family of proteins including C D 9 , C D 3 7 ,  CD53, CD63, C D 8 1 / 8 2 , CD151, A 1 5 , and SJ23 (see Wright & Tomlinson (1994) for review; alignment performed using BLAST conserved domain database search).  This leads to the possibility that these proteins also  exist as oligomeric c o m p l e x e s w h o s e subunit interactions are mediated by these hydrophobic residues. P e r i p h e r i n - 2 containing core t e t r a m e r s further link t o g e t h e r C150  mediated  intermolecular  disulfide  oligomers (Loewen & Molday, 2000).  bonds  to  form  through  higher  In this s t u d y , w e h a v e s h o w n t h a t  two  L 1 8 5 P d i m e r s link t o g e t h e r to f o r m a d i s u l f i d e - l i n k e d t e t r a m e r  27),  but this c o m p l e x d o e s not further a s s e m b l e into t h e higher  oligomers tetramer  observed formation  for is  WT-peripherin-2.  not  required  order  for  This  indicates  intermolecular  (Fig order  that  core  disulfide  bond  f o r m a t i o n , b u t it i s r e q u i r e d f o r t h e g e n e r a t i o n o f h i g h e r o r d e r o l i g o m e r s .  Chapter 4 Digenic RP This  finding  peripherin-2  126  together mutants  with  previous  studies  showing  form core noncovalent tetramers,  that  C150S  but not  higher  order oligomers (Loewen & Molday, 2000), indicates that higher oligomerization interactions. oligomerization tetramerization  involves  both  noncovalent  The noncovalent are (Fig  likely 27).  and  interactions  to  be  covalent  involved  similar  to  in  F o r m a t i o n of C 1 5 0 m e d i a t e d  (disulfide)  higher  those  order  order  involved  in  intermolecular  disulfide bonds between adjacent t e t r a m e r s m a y initiate a conformational c h a n g e within the intradiscal loop that facilitates n o n c o v a l e n t interactions responsible for oligomerization. D i s u l f i d e - l i n k e d o l i g o m e r s a p p e a r to b e i m p o r t a n t in o u t e r s e g m e n t disc formation.  In vitro  e x p r e s s i o n of W T p e r i p h e r i n - 2 u n d e r n o n r e d u c i n g  c o n d i t i o n s h a s b e e n r e p o r t e d to r e s u l t in f l a t t e n e d whereas  e x p r e s s i o n of W T p e r i p h e r i n - 2  under  microsomal  reducing  vesicles  conditions  or  e x p r e s s i o n o f C 1 5 0 S p e r i p h e r i n - 2 u n d e r n o n r e d u c i n g c o n d i t i o n s r e s u l t s in r o u n d v e s i c l e s ( W r i g l e y et al., linked L185P tetramers  2000).  It i s n o t k n o w n  if t h e d i s u l f i d e -  c a u s e t h e f l a t t e n i n g o f t h e s e m e m b r a n e s , o r if  higher order oligomers are required for this process. Individuals with a u t o s o m a l d o m i n a n t digenic R P e x p r e s s a W T allele along with the mutant allele.  T o u n d e r s t a n d in m o r e d e t a i l t h e m o l e c u l a r  basis for this d i s e a s e , w e e x a m i n e d t h e interaction of L 1 8 5 P mutant with W T peripherin-2 and W T r o m - 1 .  peripherin-2  C o - e x p r e s s i o n of L 1 8 5 P a n d  Chapter 4 Digenic RP  WT  peripherin-2  127  resulted  in a m i x t u r e  of dimeric  and tetrameric  core  complexes that further interacted through intermolecular disulfide bonds to form form  oligomers.  L185P peripherin-2  predominantly  Molday,  1996a).  core tetramers  also a s s e m b l e d with r o m - 1 to  as previously  shown  Like the W T proteins, the L185P  (Goldberg  &  peripherin-2:rom-1  h e t e r o t e t r a m e r s further a s s o c i a t e d via i n t e r m o l e c u l a r disulfide b o n d s into disulfide  linked  intermediate  oligomers, most  likely o c t a m e r s .  On the  basis of t h e s e results, w e c o n c l u d e that t h e L 1 8 5 P m u t a n t  can interact  with  most  WT  proteins  to  form  higher  order  oligomers  that  likely  contribute to t h e f o r m a t i o n a n d stabilization of o u t e r s e g m e n t discs. A u t o s o m a l d o m i n a n t d i g e n i c R P is a c o m p l e x d i s e a s e r e q u i r i n g t h e c o i n h e r i t a n c e of a L 1 8 5 P p e r i p h e r i n - 2 allele a n d a n effective r o m - 1 null a l l e l e ( D r y j a etal., Dryja, 1994). essentially  1997; Goldberg & Molday, 1996a; Kajiwara, Berson &  Individuals w h o inherit only o n e of these m u t a n t alleles are  normal.  O u r data suggests that the level of  peripherin-2-  c o n t a i n i n g o l i g o m e r s is c r i t i c a l i n t h e m a n i f e s t a t i o n o f t h i s d i s e a s e . the and  L185P peripherin-2 corresponding  mutant  higher  w h o inherit this mutation  is i n c a p a b l e o f f o r m i n g  order  disulfide-linked  Since  homotetramers  oligomers,  individuals  a l o n g w i t h a null allele in r o m - 1 , will h a v e a  level of peripherin-containing o l i g o m e r s below t h e critical threshold level required for the formation disorganized,  unstable  of stable outer s e g m e n t s .  outer  segments  will  lead  T h e p r e s e n c e of to  photoreceptor  Chapter 4 Digenic RP  128  d e g e n e r a t i o n a s o b s e r v e d f o r h e t e r o z y g o u s rds Sanyal,  1985).  peripherin-2 with  WT  In  mice (Hawkins, Jansen &  t h e c a s e of i n d i v i d u a l s w h o  inherit  m u t a t i o n , a significant a m o u n t of the  rom-1,  as  well  as  WT  peripherin-2,  only the  L185P  L 1 8 5 P will a s s e m b l e to  form  'functional'  oligomers.  T h i s w o u l d h a v e t h e effect of raising t h e level of o l i g o m e r s  above  threshold  the  needed  to  form  stable  outer  segments.  These  i n d i v i d u a l s w o u l d e x p e r i e n c e l i t t l e if a n y p h o t o r e c e p t o r d e g e n e r a t i o n a n d have essentially normal vision.  Individuals w h o inherit only a rom-1  null  allele, w o u l d be e x p e c t e d to h a v e n o r m a l levels of p e r i p h e r i n - 2 , a n d half the  level  of  rom-1.  Reduced  levels  of  rom-1,  however,  have  appreciable effect on outer s e g m e n t structure or p h o t o r e c e p t o r  no  viability  a s s h o w n in r e c e n t s t u d i e s o f h e t e r o z y g o u s r o m - 1 k n o c k o u t m i c e ( C l a r k e et al.,  2000).  H e n c e , individuals w h o inherit only a null allele or a G 1 1 3 E  m i s s e n s e m u t a t i o n in r o m - 1 w o u l d h a v e a s u f f i c i e n t l e v e l o f c o n t a i n i n g o l i g o m e r s to s u p p o r t  peripherin-2  photoreceptor outer segment  structure  and photoreceptor viability. The  L185P  peripherin-2  mutant  is  distinct  from  peripherin-2  mutants that cause monogenic autosomal dominant RP. For e x a m p l e , the C 2 1 4 S and C 1 6 5 S peripherin-2 mutants are highly misfolded and do  not  f o r m c o r e t e t r a m e r s o r h i g h e r o r d e r o l i g o m e r s in t h e p r e s e n c e o r a b s e n c e of r o m - 1  (Goldberg, Loewen & Molday, 1998; Loewen & Molday, 2000).  Furthermore,  expression  of  the  C165Y  mutant  fails  to  result  in  the  Chapter 4 Digenic RP  129  f o r m a t i o n o f f l a t t e n e d v e s i c l e s ( W r i g l e y e t al., with  these  functional unstable,  mutations  would  o l i g o m e r s in t h e disorganized  have  a  2000).  significantly  Hence, individuals decreased  p r e s e n c e o r a b s e n c e o f rom-1  outer  segment  discs  and  level  of  resulting  in  photoreceptor  degeneration.  A d o m i n a n t negative effect of t h e s e m i s f o l d e d  mutants,  however,  also contribute  and  may  to  autosomal dominant RP phenotype.  photoreceptor  degeneration  the  Chapter 5 Transgenic Xenopus CHAPTER  5  -  EXPRESSION  XENOPUS  TRANSGENIC 5.1  130 OF  PERIPHERIN-2  VARIANTS  IN  LAEVIS  INTRODUCTION  The them  unique  an excellent  However, their easy  divide  targeting have  left  polarized  system  to  nature  study  of  photoreceptor  protein  and  in  cell  culture  because  cells  membrane  i n t i m a t e a s s o c i a t i o n w i t h R P E c e l l s in t h e  manipulation  maintained. not  highly  these  makes  trafficking.  eye  contacts  prevents are  not  R o d s in c e l l c u l t u r e q u i c k l y l o s e t h e i r o u t e r s e g m e n t s a n d d o (Townes-Anderson,  MacLeish & Raviola,  of m u t a n t r h o d o p s i n s many  questions  1985).  Studies  in M D C K c e l l s h a v e b e e n v a l u a b l e ,  unanswered  (Chuang  &  Sung,  on but  1998).  In  c o n t r a s t , s t u d i e s in t r a n s g e n i c m i c e a n d r a t s h a v e p r o v e n v e r y u s e f u l in understanding  m o r e about the requirements for outer s e g m e n t  of r h o d o p s i n a n d h o w m u t a t i o n s d i s e a s e ( G r e e n e t al., and  lengthy  molecular  time  2 0 0 0 ; S u n g e t al.,  requirements  manipulation  t r a n s g e n i c Xenopus  lead to p h o t o r e c e p t o r  laevis  of  the  of  1994).  these  targeting  degeneration  H o w e v e r , the high cost  studies  transgenes.  preclude  Recent  extensive  studies  h a v e b e e n s u c c e s s f u l in d e f i n i n g a  al.,  2000).  The  low  cost  and  speed  of  this  procedure  using  C-terminal  p e p t i d e s e q u e n c e in r h o d o p s i n t h a t is r e q u i r e d f o r R O S l o c a l i z a t i o n et  and  (Tarn  allows  for  e x t e n s i v e m a n i p u l a t i o n of t r a n s g e n e s a n d g e n e r a t i o n of large n u m b e r s of transgenic lines.  Chapter 5 Transgenic Xenopus  131  U s i n g t h e t r a n s g e n i c Xenopus  technology  in a c o l l a b o r a t i o n  with  David P a p e r m a s t e r ' s lab at t h e University of C o n n e c t i c u t , t h e effects of mutations  in  peripherin-2  on  structure have been examined.  protein  targeting  and  photoreceptor  This technique involves expression of the  protein of interest a s a G F P - f u s i o n protein w h i c h facilitates e a s y a n d rapid screening  for transgenic  animals  and allows  direct  localization  of t h e  protein using microscopy techniques.  T h e t r a n s g e n e is u n d e r c o n t r o l of  the  is  Xenopus  opsin  photoreceptors. integration  promoter  The technique  (REMI)  containing  restriction  of  only  When  mixed restriction  M i c r o i n j e c t i o n o f t h e m i x t u r e i n t o a Xenopus sperm  D N A containing  the  endonuclease  the transgene  are mildly  incorporates into a c o m p l e m e n t a r y  the  in  rod  mediated  into  Xenopus  In R E M I , both t h e s p e r m nuclei a n d t h e  the transgene  endonuclease.  expressed  restriction  is u s e d t o i n c o r p o r a t e  sperm ( A m a y a & Kroll, 1999). plasmid  and  the  transgene  digested together,  with  the  same  the  transgene  s i t e ( s ) in t h e s p e r m D N A . o o c y t e r e s u l t s in ligation of a n d fertilization.  If  the  t r a n s g e n e i n c o r p o r a t e s p r i o r t o t h e f i r s t c e l l d i v i s i o n , t h e n e v e r y c e l l in t h e a n i m a l w i l l h a r b o u r it. G F P h a s been fused to the C - t e r m i n u s of peripherin-2 interference  with protein  translation  and membrane  to  insertion.  prevent Several  d i s e a s e - c a u s i n g m u t a n t s have been constructed to d e t e r m i n e the effects o f t h e s e m u t a t i o n s in vivo  (Fig 28).  The L185P and C214S mutants  have  132  F i g 2 8 . Topological model for Xenopus peripherin-2. L o c a t i o n o f G F P fusion and P e r 5 A l l M A b epitope are shown. L o c a t i o n of C 1 5 0 S a n d R P c a u s i n g m u t a t i o n s a r e g i v e n in o p e n c i r c l e s . A m i n o a c i d s d i f f e r i n g f r o m t h e p u b l i s h e d s e q u e n c e a r e g i v e n in b l a c k c i r c l e s .  Chapter 5 Transgenic Xenopus been  characterized  133  biochemically  in  some  detail  previously  Loewen & Molday, 1 9 9 8 ; Loewen, Moritz & Molday, 2001) 1.4.2,  2.4, and 4.4).  Additionally,  peripherin-2-GFP on  rod  examined  hope  with  the  structure of  (Goldberg,  (see sections  t h e effects of e x p r e s s i o n of and  disc morphogenesis  understanding  more  about  C150S  have the  been  role  of  d i s u l f i d e - m e d i a t e d o l i g o m e r i z a t i o n in t h e s e p r o c e s s e s . All t r a n s g e n i c  animals  used  in t h i s  study  University of C o n n e c t i c u t by the a u t h o r t o g e t h e r Tarn.  Confocal  microscopy  was  performed  were  produced  at  the  with O. Moritz and at  Connecticut Health Sciences C e n t e r Microscopy Unit.  the  University  B. of  All other procedures  w e r e p e r f o r m e d by the a u t h o r at U . B . C . , with the e x c e p t i o n of sectioning for electron m i c r o s c o p y which w a s performed by Laurie Molday.  5.2 MATERIALS AND METHODS Molecular  5.2.1  Xenopus using  biology  laevis  prph-2  w a s cloned from total retinal  sequence-specific primers  Xrds-38 restriction products  (Kedzierski  et  al.,  based on  1996).  the  published  PCR primers  sequenced  entirely  on  both  sequence  contained  sites for cloning into p c D N A I / a m p (Invitrogen). were  R N A by P C R  strands.  for  unique  Cloned PCR The  p e r i p h e r i n - 2 - G F P fusion construct w a s created by P C R r e m o v i n g the  Xenopus prph-  2 s t o p c o d o n a n d c l o n i n g in f r a m e i n t o t h e X h o l a n d E c o R I s i t e s o f p e G F P -  Chapter 5 Transgenic Xenopus N2  (CLONTECH  134  Laboratories, Inc.)  a h e a d of G F P .  This construct  was  u s e d f o r e x p r e s s i o n o f t h e p e r i p h e r i n - 2 - G F P f u s i o n p r o t e i n in C O S - 1 c e l l s . F o r e x p r e s s i o n in t r a n s g e n i c X. laevis, peripherin-2-GFP (including sites of the laevis  from  Xenopus  G F P ) w a s subcloned into the X h o l and  plasmid X O P 1 . 3 G F P - N 1 which contains a portion  opsin promoter  obtained  t h e X h o I - N o t I f r a g m e n t of  s e q u e n c e a n d is b a s e d o n t h e p l a s m i d  CLONTECH  Laboratories,  Inc.  (Tarn  et  NotI  of t h e  peGFP-Nl  al.,  2000).  M u t a g e n e s i s w a s p e r f o r m e d on this construct to introduce individually RP-causing mutations  X.  the  L185P, C 2 1 4 S , and P216L using the Quik Change  PCR-based protocol (Stratagene).  Bovine peripherin-2-GFP and  C214S-  p e r i p h e r i n - 2 - G F P f u s i o n c o n s t r u c t s w e r e c r e a t e d in a s i m i l a r m a n n e r a s Xenopus  peripherin-2-GFP.  constructs peGFP-N3. GFP  was  were  cloned  Stop c o d o n s were r e m o v e d by P C R and  in  frame  the  EcoRI and  BamHI  sites  of  A s u b s e q u e n t EcoRI-NotI f r a g m e n t containing the insert plus subcloned  XOP1.3GFP-N1.  into  the  by  BamHI  and  NotI  sites  of  the  plasmid  All constructs created by P C R w e r e s e q u e n c e d to confirm  that no errors were introduced. linearized  into  the  digestion  G e n e C l e a n Kit ( B i o l O l ) .  with  All t r a n s g e n i c e x p r e s s i o n c o n s t r u c t s w e r e  restriction  enzyme  and  purified  using  the  Chapter 5 Transgenic Xenopus 5.2.2  Monoclonal  135  antibody  production  O l i g o n u c l e o t i d e s w e r e s y n t h e s i z e d t o t h e Xenopus terminal  amino  acid  sequence  peripherin-2 C -  KDTIKSSWELVKSMGKLNKVE  and  were  cloned into t h e P G E X a n d P M A L e x p r e s s i o n v e c t o r s for preparation of G S T and M B P fusion proteins, respectively.  P u r i f i e d G S T f u s i o n p r o t e i n f r o m E.  coli w a s u s e d t o i m m u n i z e S w i s s W e b s t e r m i c e .  The monoclonal  antibody  Per2A5 w a s prepared from a sero-positive mouse as previously described (Mackenzie & Molday, 1982).  S c r e e n i n g w a s performed o n W e s t e r n blots  containing M B P fusion protein a n d by immunofluorescence microscopy of a d u l t Xenopus  5.2.3  retinal cryosections.  Transgenesis,  GFP screening,  and tadpole  rearing  Transgenic frogs were generated using a modified protocol  (Moritz  er a / . , 1 9 9 9 ) b a s e d o n that of Kroll a n d A m a y a (Kroll & A m a y a , 1 9 9 6 ) a n d Amaya  a n d Kroll ( A m a y a & Kroll, 1 9 9 9 ) .  i n c u b a t e d w i t h 0.3x  X. laevis  sperm  nuclei  high speed e g g extract, 0 . 0 5 U restriction  and 1 0 0 - 2 0 0 ng linearized vector D N A . T h e reaction mixture d i l u t e d t o 0.3 embryos  were  n u c l e i / n l a n d 1 0 nl w a s i n j e c t e d kept  a t 1 8 ° C i n O.lx  per egg.  Marc's Modified  were  enzyme, was then  T h e resulting  R i n g e r , 6 % Ficoll  s o l u t i o n f o r 4 8 h a n d t h e n s w i t c h e d t o O.lx G e r h a r t ' s R i n g e r S o l u t i o n ( W u & Gerhart, 1991).  A t 5 - 6 d postfertilization  (dpf) roughly  corresponding  to s t a g e s 4 0 - 4 2 ( N i e u w k o o p & F a b e r , 1 9 9 4 ) , t a d p o l e s w e r e s c r e e n e d for  Chapter 5 Transgenic Xenopus  136  G F P expression using a Leica M Z 8 dissecting m i c r o s c o p e equipped with epifluorescence optics and a G F P filter set. g l a s s P a s t e u r pipettes to i m m o b i l i z e t h e m .  Animals were  placed  into  Tadpoles expressing GFP were  e a s i l y identified by t h e g r e e n f l u o r e s c e n c e e m i t t e d f r o m t h e i r e y e s . A t 14 d p f , t r a n s g e n i c t a d p o l e s w e r e p l a c e d in t a n k s w i t h O . l x G e r h a r t ' s R i n g e r S o l u t i o n a n d r e a r e d a t 1 8 ° C o n a 1 2 / 1 2 h l i g h t / d a r k c y c l e . A d u l t X.  laevis  were obtained from Nasco or Xenopus Express.  5.2.4  Immuno-EM T r a n s g e n i c t a d p o l e s w e r e sacrificed at 14 a n d 28 d.p.f. (stage 4 8 -  6 2 ) a n d f i x e d in 4 % p a r a f o r m a l d e h y d e , 0 . 1 M s o d i u m p h o s p h a t e pH 7.5. White  A f t e r o v e r n i g h t f i x a t i o n , e y e s w e r e e x c i s e d a n d e m b e d d e d in L R according  Company).  to  the  manufacturer's  instructions  (London  Thin sections were labeled overnight with a rabbit  polyclonal antibody  ( C L O N T E C H Laboratories, Inc.)  bovine serum albumin washed  buffer,  three  times  ( B S A ) , 0.1 and  labeled  M Tris, pH 7.4. for  1  h  with  diluted  anti-GFP  1:100  The reactions anti-rabbit  a n t i b o d y c o n j u g a t e d to 10 n m colloidal gold (British BioCell  Resin  in  1% were  secondary  International)  d i l u t e d 1 : 5 in 0 . 1 M T r i s , p H 7 . 4 , 1 % B S A . A m i n i m u m o f t w o t r a n s g e n i c a n i m a l s w a s e x a m i n e d by i m m u n o - E M for each construct.  Chapter 5 Transgenic Xenopus 5.2.5  137  Immunocytochemistry,  confocal  and  deconvolution  microscopy Transgenic tadpoles were sacrificed between  stages 4 8 - 6 2 .  After  i m m o b i l i z i n g t h e t a d p o l e s in 0 . 0 2 % T r i c a i n e , t h e i r e y e s w e r e e x c i s e d a n d fixed  in  4%  overnight.  paraformaldehyde,  sodium  phosphate  buffer,  pH  F i x e d e y e s w e r e e m b e d d e d in O C T t i s s u e e m b e d d i n g  ( T i s s u e - T e k ) a n d f r o z e n in a d r y i c e / i s o p e n t a n e b a t h .  7.5,  medium  Cryostat sections  ( 1 4 p m ) w e r e b l o c k e d ( 1 0 % B S A , 0 . 1 % T r i t o n X - 1 0 0 in P B S ) a n d l a b e l e d overnight  with 0.1 m g / m l T e x a s R e d - c o n j u g a t e d w h e a t g e r m  (TR-WGA)  (Molecular  (Sigma-Aldrich) respectively.  to  Probes) label  2  photoreceptor  mg/ml  Hoescht  membranes  S e c t i o n s w e r e a l s o l a b e l l e d w i t h ant\-Xenopus  monoclonal antibody Per2A5. CaCI  and 0.01  , 1 m M MgCI  2  agglutinin  33342 and  stain nuclei,  peripherin-2  L a b e l i n g w a s d o n e in t h e p r e s e n c e of 1 m M  , 1 % B S A , and 0 . 1 % Triton  X - 1 0 0 in P B S .  Labeled  sections were analyzed using a Z E I S S confocal laser scanning microscope (models  410 and 510).  A minimum  of three  transgenic  examined by confocal microscopy for each construct. for deconvolution  animals was  T h e conditions used  microscopy involved 3 nearest neighbors for low m a g  i m a g e s ( 1 0 X N A 0 . 4 5 ) a n d t h e i m a g e s in t h e Z - s e r i e s w e r e s p a c e d 0 . 5 p m a p a r t in all c a s e s . to d o t h e a n a l y s i s .  Openlab version 2.03 (Improvision)  software w a s used  Chapter 5 Transgenic Xenopus  5.2.6  138  Transfections and plasmids C O S - 1 cells (ca. 6 x 1 0  t o t a l o f 3 0 jig  of p l a s m i d  5  c e l l s / 1 0 0 - m m dish) were transfected with a  D N A by the  harvested 72 h post-transfection u s e d w e r e Xenopus  calcium  phosphate  method  a s d e s c r i b e d in s e c t i o n 3 . 2 . 4 .  peripherin-2-GFP (described  above),  and  Plasmids  pcPer  (bovine  W T peripherin-2 cloned into p c D N A I / a m p ) ( G o l d b e r g , L o e w e n & M o l d a y , 1998), p c P e r - C 2 1 4 S (bovine peripherin-2 with the C 2 1 4 S mutation  cloned  into p c D N A I / a m p ) ( G o l d b e r g , L o e w e n & M o l d a y , 1 9 9 8 ) , a n d p c P e r - P 2 1 6 L (bovine peripherin-2 with the P 2 1 6 L mutation cloned into p c D N A I/amp).  5.2.7  Biochemistry Transfected C O S - 1 cells were scraped f r o m 100 m m dishes, w a s h e d  w i t h P B S , a n d i n c u b a t e d w i t h P B S in t h e p r e s e n c e o r a b s e n c e of 2 0 DTT for 9 0 m i n at 25°C.  T h e cells were solubilized with an equal  ( 1 5 0 ul) o f P B S c o n t a i n i n g 7.4) for 10 m i n o n ice.  with  (pH  T h e cell extract w a s c e n t r i f u g e d at 9 0 , 0 0 0 x g for  r e t a i n e d o n ice for a n a l y s i s . performed  volume  2 % Triton X - 1 0 0 , 8 0 m M N E M , and P M S F  30 m i n at 4°C a n d the s u p e r n a t a n t  was  mM  (detergent  solubilized fraction)  Immunopurification  Per2B6-Sepharose  of b o v i n e  immunoaffinity  was  peripherin-2 matrix  as  p r e v i o u s l y d e s c r i b e d in s e c t i o n 3 . 2 . 3 . For  velocity  sedimentation  experiments  done  under  nonreducing  c o n d i t i o n s , 1 0 0 uJ o f n o n r e d u c e d s o l u b i l i z e d c e l l e x t r a c t w a s a p p l i e d t o 5 -  Chapter 5 Transgenic Xenopus  13 9  2 0 % ( w / w ) s u c r o s e g r a d i e n t s p r e p a r e d in P B S a n d c o n t a i n i n g 0 . 1 % T r i t o n X-100  (Loewen  & Molday, 2000)  (see methods  section  3.2.5).  After  c e n t r i f u g a t i o n f o r 16 h a t 5 0 , 0 0 0 r p m in a B e c k m a n T L S - 5 5 r o t o r at 4 ° C , the bottom of the centrifuge tube w a s punctured a n d four-drop  fractions  were collected for analysis on Western blots. Protein extracts  crosslinking was performed  with  0.005%  glutaraldehyde  on detergent  for  15 m i n at  solubilized 37°C.  cell  For gel  e l e c t r o p h o r e s i s , s a m p l e s w e r e d e n a t u r e d with a n e q u a l v o l u m e of S D S cocktail  (4%  S D S , 0.02M  Tris-HCl,  pH  6.8,  20%  glycerol, 0 . 0 1 %  b r o m o p h e n o l b l u e ) in t h e a b s e n c e ( n o n r e d u c i n g ) o r p r e s e n c e ( r e d u c i n g ) of 5 % p - m e r c a p t o e t h a n o l , a n d applied to 6 % o r 8 % S D S - p o l y a c r y l a m i d e gels.  After electrophoresis, the proteins were transferred to Immobilon-P  using a Bio-Rad semidry transfer apparatus. with  a  monoclonal  antibody  against  Western Blots were  bovine  peripherin-2  (Molday, Hicks & Molday, 1987) and a polyclonal antibody (CLONTECH rabbit  Laboratories Inc.) a n d sheep anti-mouse  immunoglobulin-peroxidase  by E C L .  probed  (Per2B6)  against G F P  or donkey  (Amersham-Pharmacia) for  anti-  detection  Chapter 5 Transgenic Xenopus  140  5.3 RESULTS 5.3.1  Cloning X. laevis Xenopus  Prph-2  peripherin-2  c D N A f r o m a d u l t X. laevis  retina has been cloned  by P C R using p r i m e r s g e n e r a t e d to the p u b l i s h e d s e q u e n c e of X r d s - 3 8 ( K e d z i e r s k i e t al., 1 9 9 6 ) a n d s e q u e n c e d . T h e s e q u e n c e o f Xenopus 2 differed f r o m the published s e q u e n c e . found  at A 7 8 , A 9 2 , D 1 8 7 , F 1 8 8 , and  completely including  conserved mouse,  in  rat,  all  cat,  Five a m i n o acid c h a n g e s were S189.  mammalian dog,  bovine,  These amino  orthologs and  The  nucleotide and  protein  human  sequence have  acids  are  s e q u e n c e d to (Fig  date  28),  t h e r e f o r e m o s t l i k e l y r e p r e s e n t t h e t r u e s e q u e n c e o f Xenopus 2.  Prph-  and  peripherin-  been deposited  in  the  GenBank database.  5.3.2  Molecular characterization Xenopus  and  bovine  of Xenopus  peripherin-2-GFP  peripherin-2-GFP fusion  proteins  were  e x p r e s s e d in C O S - 1 c e l l s in o r d e r t o d e t e r m i n e if G F P f u s e d t o t h e i r C terminus  affects  their  biochemical  properties.  Previous studies  demonstrated that bovine peripherin-2 forms core noncovalent that  associate  intermediate  and  Molday, 2000). same  properties  dimerization,  via  disulfide  bonds  higher-order  between  disulfide-linked  C150  residues  oligomers  have  tetramers to  form  (Loewen  &  The bovine peripherin-2-GFP fusion protein exhibited the as  bovine  crosslinking  peripherin-2  in t h e  when  reduced and  analyzed for nonreduced  disulfide-  states,  and  Chapter 5 Transgenic Xenopus  141  association with peripherin-2 and rom-1  (data not s h o w n ) .  can  little  or  also  behaves  be  concluded  that  G F P has  no  effect  Therefore  on  its  it  structural  properties. Xenopus  peripherin-2-GFP  peripherin-2. present  on  to  bovine  It l o c a l i z e d t o i n t e r n a l v e s i c l e s o f C O S - 1 c e l l s a n d w a s (data  not  p e r i p h e r i n - 2 , it f o r m e d d i s u l f i d e - d i m e r s (Fig  29A,  the  predominantly Crosslinking  similarly  plasma  membrane  shown).  Like  nonreducing  conditions  h i g h e r m o l e c u l a r w e i g h t b a n d s (Fig  29A,  linked  &  oligomer  oligomerization  formation o f Xenopus  bovine (data not s h o w n ) .  (Loewen  revealed  29A,  the  lane c).  presence  of  lane d) i n d i c a t i v e of d i s u l f i d e -  Molday,  2000).  Disulfide-linked  p e r i p h e r i n - 2 - G F P is m e d i a t e d Xenopus  bovine  l a n e b) a n d c r o s s l i n k e d  i n t o d i m e r s u n d e r r e d u c i n g c o n d i t i o n s (Fig under  not  by C 1 5 0 , like  peripherin-2-GFP also interacted  b o v i n e p e r i p h e r i n - 2 in c o - i m m u n o p r e c i p i t a t i o n  e x p e r i m e n t s (Fig  29,  with lane  e).  5.3.3  Immunolocalization of endogenous peripherin-2 T h e cellular a n d s u b c e l l u l a r localization of e n d o g e n o u s  in  Xenopus  tadpoles  was  determined  using  the  P e r 2 A 5 d i r e c t e d t o t h e C - t e r m i n a l t a i l o f Xenopus  monoclonal peripherin-2.  s h o w s specific localization to rod a n d c o n e o u t e r s e g m e n t T h e v e r t i c a l s t r i a t i o n s a r e o b s e r v e d in R O S .  peripherin-2 antibody Fig  30B  membranes.  T h i s p a t t e r n o f l a b e l l i n g is  142  Fig 2 9 .  M o l e c u l a r c h a r a c t e r i z a t i o n of Xenopus p e r i p h e r i n - 2 - G F P  fusion protein. ( A ) X e n o p u s p e r i p h e r i n - 2 - G F P w a s e x p r e s s e d in C O S - 1 cells a n d the cells w e r e treated with (reduced) or without DTT (nonreduced) a n d s o l u b i l i z e d in b u f f e r c o n t a i n i n g Triton X - 1 0 0 a n d N E M . R e d u c e d ( l a n e a ) and n o n r e d u c e d (lane b) extract w a s s u b j e c t e d to S D S - P A G E u n d e r r e d u c i n g and nonreducing conditions respectively. R e d u c e d (lane c) a n d nonreduced (lane d) extract w a s crosslinked with glutaraldehyde a n d s u b j e c t e d to S D S P A G E u n d e r r e d u c i n g conditions. W e s t e r n blot a n a l y s i s w a s p e r f o r m e d with a n t i - G F P a n t i b o d y . ( B ) Xenopus p e r i p h e r i n - 2 - G F P a n d b o v i n e p e r i p h e r i n - 2 w e r e c o e x p r e s s e d in C O S - 1 c e l l s , s o l u b i l i z e d in b u f f e r c o n t a i n i n g Triton X 100 and N E M , and immunoprecipitated with anti-peripherin-2 monoclonal antibody Per2B6 coupled to S e p h a r o s e . T h e peptide-eluted bound fraction was subjected to S D S - P A G E u n d e r reducing conditions a n d t h e W e s t e r n blot was probed with Per2B6 and anti-GFP antibodies (lane e ; bovine peripherin2 (BPer) and Xenopus peripherin-2-GFP (XPerGFP) are labeled) (see Methods sections 5.2.6and 5.2.7).  143  Fig 3 0 . E n d o g e n o u s Xenopus p e r i p h e r i n - 2 l o c a l i z e s to rod a n d c o n e outer segments. C o n f o c a l m i c r o g r a p h s of a n o n - t r a n s g e n i c retina labelled with T R - W G A (A) and a n t i - X e n o p u s peripherin-2 m o n o c l o n a l antibody Per2A5 (B). Nuclei were stained with H o e s c h t 3 3 3 4 2 . In ( A ) , T R - W G A l a b e l l i n g ( r e d ) p r i m a r i l y r e p r e s e n t s o p s i n l o c a l i z a t i o n a n d is g r e a t e s t in r o d a n d c o n e o u t e r s e g m e n t s . (B) P e r i p h e r i n - 2 ( g r e e n ) l o c a l i z e s to i n c i s u r e s (vertical striations m a r k e d with a r r o w h e a d s ) of rod o u t e r s e g m e n t discs a n d to o n e e d g e of c o n e o u t e r s e g m e n t s ( a r r o w ) ( s e e M e t h o d s s e c t i o n 5 . 2 . 5 ) . r o s , r o d o u t e r s e g m e n t ; c o s , c o n e o u t e r s e g m e n t ; is, r o d i n n e r s e g m e n t ; n, n u c l e u s . Bar, 5 f i m .  Chapter 5 Transgenic Xenopus  144  consistent with the localization in  R O S of  Xenopus  ( A r i k a w a e t al.,  of p e r i p h e r i n - 2 to disc r i m s a n d incisures  tadpoles,  similar  to  1 9 9 2 ) a n d a d u l t Xenopus  that  observed  in  ( K e d z i e r s k i e t al.,  mammals 1996).  In  c o n e s , l a b e l l i n g is r e s t r i c t e d t o a l o n g o n e s i d e o f t h e c o n e o u t e r s e g m e n t , a  pattern  consistent  with  localization to  the  rim  region  of c o n e  outer  segment membranes.  5.3.4 Expression of Xenopus peripherin-2-GFP  in transgenic  Xenopusrods The Xenopus  Xenopus  peripherin-2-GFP transgene  is u n d e r  of  the  o p s i n p r o m o t e r , w h i c h d i r e c t s s p e c i f i c e x p r e s s i o n t o r o d c e l l s in  the retina.  Fig 3 1 A  s h o w s rod p h o t o r e c e p t o r - s p e c i f i c e x p r e s s i o n of t h e  t r a n s g e n e ( i n y e l l o w ) i n t h e c o n t e x t o f a w h o l e Xenopus 31B).  control  The  contiguous  row  of  photoreceptor  nuclei  tadpole eye and  long  a p p e a r a n c e of the rod o u t e r s e g m e n t s indicate t h a t retinal is n o t o c c u r i n g .  Fusion protein  cells,  that  indicating  proper  (Fig  uniform  degeneration  is a l s o o b s e r v e d in p h a g o s o m e s o f R P E disc  morphogenesis  and  shedding  has  occurred. Specific targeting o b s e r v e d in Fig  31C.  s e g m e n t s of rods.  of t h e f u s i o n  protein  to  V e r y little f u s i o n p r o t e i n  rod outer  segments  is l o c a t e d in t h e  is  inner  N o n - u n i f o r m o r m o s a i c e x p r e s s i o n o f t h e t r a n s g e n e is  o b s e r v e d a n d v a r i e s f r o m e y e to e y e a n d rod to r o d .  T h i s is a p r o p e r t y  of  145  Fig 3 1 . Xenopus p e r i p h e r i n - 2 - G F P f u s i o n p r o t e i n t a r g e t s to rod outer segment membranes. C r y o s e c t i o n s of w h o l e t a d p o l e e y e s w e r e labelled with T R - W G A (red) and Hoescht 3 3 3 4 2 (blue). (A) Deconvolution e p i f l u o r e s c e n c e m i c r o s c o p y a n d (B) c o r r e s p o n d i n g D I C i m a g e of a retina e x p r e s s i n g Xenopus p e r i p h e r i n - 2 - G F P ( g r e e n a n d y e l l o w ) s h o w s l o c a l i z a t i o n o f t h e f u s i o n p r o t e i n t o r o d p h o t o r e c e p t o r s . F u s i o n p r o t e i n is a l s o p r e s e n t in p h a g o s o m e s of R P E cells (arrow). ( C & D ) C o n f o c a l m i c r o g r a p h s of retinas e x p r e s s i n g Xenopus p e r i p h e r i n - 2 - G F P s h o w o u t e r s e g m e n t t a r g e t i n g o f t h e fusion protein a n d localization to disc incisures ( a r r o w h e a d s ) . A l m o s t no G F P f l u o r e s c e n c e i s d e t e c t e d in t h e i n n e r s e g m e n t s . ( I n s e t ) C r o s s - s e c t i o n of a rod o u t e r s e g m e n t s h o w s disc rim a n d incisure labelling (see M e t h o d s s e c t i o n s 5 . 2 . 5 ) . I, l e n s ; r, r e t i n a ; r p e , r e t i n a l p i g m e n t e p i t h e l i u m ; o s , r o d o u t e r s e g m e n t ; i s , r o d i n n e r s e g m e n t ; n, n u c l e u s . B a r s : ( A & B ) 1 0 0 u m ; ( C ) 1 0 | i m ; ( D ) 5 | i m ; ( I n s e t ) 1 p:m.  Chapter 5 Transgenic Xenopus  146  t h e e x p r e s s i o n s y s t e m a n d is d u e t o d i f f e r e n c e s i n s i t e s o f i n t e g r a t i o n a n d n u m b e r o f t r a n s g e n e s i n c o r p o r a t e d p e r a n i m a l ( M o r i t z e t al., 2001). 3ID  shows targeting  striations)  a n d disc  of t h e fusion rims  (Fig  protein  31D,  observed for endogenous peripherin-2.  to disc incisures  inset),  a  similar  Fig  (vertical  localization as  At higher expression levels, G F P  f l u o r e s c e n c e s a t u r a t e s t h e d i s c r i m a n d t h e s t r i a t e d p a t t e r n is n o l o n g e r observed. Immuno-electron protein  m i c r o s c o p y of t r a n s g e n i c rods c o n f i r m e d  localization to disc  expressing  moderate  rims  levels  a n d i n c i s u r e s (Fig  of p e r i p h e r i n - 2 - G F P  confined to rims a n d incisures.  32).  (Fig  fusion  In  regions  32B,C),  it w a s  I n r e g i o n s e x p r e s s i n g h i g h l e v e l s (Fig  3 2 A ) , f u s i o n p r o t e i n w a s a l s o o b s e r v e d in l a m e l l a r r e g i o n s i n a d d i t i o n t o rims.  High e x p r e s s i o n levels of fusion protein also correlated with s m a l l e r  d i s c s a n d n a r r o w e r o u t e r s e g m e n t d i a m e t e r i n m a n y r o d s (Fig 3 2 A ) .  5.3.5  Expression  of bovine peripherin-2-GFP  in  transgenic  Xenopus To  investigate  localization  of  peripherin-2-GFP  if  the  peripherin-2 was  requirements are  expressed  distribution w a s e x a m i n e d .  for  conserved in  proper across  transgenic  outer species,  tadpoles  segment bovine and  its  Fig 3 3 A s h o w s t h a t t h e b o v i n e f u s i o n p r o t e i n  targeted to rod outer segments and disc incisures.  No fusion protein w a s  147  A  J  Fig 3 2 . I m m u n o - E M r e v e a l s t a r g e t i n g of Xenopus p e r i p h e r i n - 2 - G F P fusion protein to disc rims (arrows) a n d incisures ( a r r o w h e a d s ) . Ultrathin sections of transgenic rods were labeled with a n t i - G F P antibody followed by a gold-conjugated secondary. (A) C o m p o s i t e image of a longitudinal s e c t i o n of a rod e x p r e s s i n g high levels of t r a n s g e n e . (B) Longitudinal s e c t i o n of a rod e x p r e s s i n g m o d e r a t e levels o f t r a n s g e n e . ( C ) C r o s s - s e c t i o n of a rod expressing m o d e r a t e levels of t r a n s g e n e ( s e e M e t h o d s section 5.2.4). Bars: (A) 5 0 0 n m ; ( B ) 2 0 0 n m ; ( C ) 5 0 0 n m .  Chapter 5 Transgenic Xenopus  148  visible in t h e i n n e r s e g m e n t s . antibody  B e c a u s e t h e anti-Xenopus  P e r 2 A 5 did not cross react with bovine p e r i p h e r i n - 2 , t h e effects  of t r a n s g e n e e x p r e s s i o n o n e n d o g e n o u s p e r i p h e r i n - 2 33  (Fig  T h e bovine fusion protein  B-D).  peripherin-2 to incisures of R O S . rod  expressing fusion  illustrates suggests  the that  similar the  protein  fusion  protein.  endogenous  peripherin-2.  Transgenic  C214S  could be examined  co-localized with  endogenous  C o m p a r i n g t h e P e r 2 A 5 labelling of t h e  to the rod not expressing fusion  localization  endogenous  5.3.6  peripherin-2  protein  did  T h e fusion  expression  of  endogenous not  protein  cause is  peripherin-2, mistargeting  likely  of the Retinitis  protein  in  and  of  complex  the with  Pigmentosa-causing  mutant  The C 2 1 4 S mutation  in p e r i p h e r i n - 2  is k n o w n t o c a u s e  autosomal  d o m i n a n t r e t i n i t i s p i g m e n t o s a , w h e r e r o d s a r e p r i m a r i l y a f f e c t e d ( S a g a et al.,  1993).  Previous biochemical studies have s h o w n that this  prevents core tetramer  formation  Loewen & Molday, 1998).  and interaction  with r o m - 1 (Goldberg,  A model describing the molecular  disease has been developed that stresses the importance above-threshold  levels of functional  mutation  of  basis for  maintaining  c o m p l e x e s in t h e o u t e r s e g m e n t s of  rods to prevent disease (Goldberg, Loewen & Molday, 1 9 9 8 ; Goldberg & Molday,  1996a;  Loewen, Moritz  & Molday,  2001).  T h e effects  of the  149  Fig 3 3 . B o v i n e p e r i p h e r i n - 2 - G F P f u s i o n p r o t e i n t a r g e t s to r o d o u t e r segment m e m b r a n e s and colocalizes with e n d o g e n o u s Xenopus peripherin-2. (A) Confocal micrograph of a retina expressing bovine p e r i p h e r i n - 2 - G F P (green) labelled with T R - W G A (red) a n d Hoescht 3 3 3 4 2 (blue). ( B - D ) Confocal m i c r o g r a p h s of a retina e x p r e s s i n g bovine peripherin-2-GFP labelled with Per2A5 monoclonal antibody showing Per2A5 labelling ( B ) , G F P fluorescence ( C ) , a n d t h e c o m b i n e d i m a g e (D) ( s e e Methods section 5.2.5). Fusion protein colocalizes with e n d o g e n o u s X e n o p u s p e r i p h e r i n - 2 to t h e incisures of rod o u t e r s e g m e n t d i s c m e m b r a n e s (yellow), o s , r o d o u t e r s e g m e n t ; is, rod inner s e g m e n t ; n, n u c l e u s . B a r s , 5 l_im.  Chapter 5 Transgenic Xenopus  150  C 2 1 4 S p e r i p h e r i n - 2 m u t a t i o n o n its e x p r e s s i o n a n d s u b c e l l u l a r localization in t r a n s g e n i c Xenopus t h a t Xenopus  rods have been investigated.  C 2 1 4 S - p e r i p h e r i n - 2 - G F P fusion protein  i n n e r s e g m e n t s a n d cell b o d i e s of r o d s .  Fig 3 4 A & B s h o w mistargeted to the  In rods e x p r e s s i n g  moderate  levels of fusion protein, localization w a s confined to inner s e g m e n t s a n d cell b o d i e s , w h e r e a s in r o d s e x p r e s s i n g h i g h e r l e v e l s o f t r a n s g e n e , f u s i o n protein w a s observed additionally near the base.  in o u t e r s e g m e n t s w i t h  Rod degeneration w a s not observed.  Fig 3 4 C s h o w s t h e e x p r e s s i o n o f b o v i n e  C214S-peripherin-2-GFP.  T h e s a m e p h e n o t y p e w a s o b s e r v e d a s w i t h t h e Xenopus mistargeting fusion  to t h e inner s e g m e n t a n d cell b o d y .  protein  did  not  however  p e r i p h e r i n - 2 (Fig 3 4 D & E).  was  localized  to  expressing fusion protein.  cause  fusion  protein;  Mistargeting of the  mistargeting  of  endogenous  Endogenous peripherin-2 did not colocalize  with the bovine fusion protein instead  accumulation  in t h e i n n e r s e g m e n t a n d cell b o d y , b u t  the  outer  segment  similarly  to  rods  not  This data suggests that the fusion protein did  not interact with e n d o g e n o u s peripherin-2. Immuno-electron  microscopy  of  peripherin-2-GFP confirmed fusion protein  rods  expressing  localization to inner  Xenopus segment  m e m b r a n e s a n d s h o w s a c c u m u l a t i o n a t t h e c o n n e c t i n g c i l i u m (Fig Outer  segment  discs  of  rods  a p p e a r e d n o r m a l (Fig 35B).  expressing  the  mutant  fusion  35A). protein  In o u t e r s e g m e n t s , f u s i o n protein localized  151  Fig 3 4 . C 2 1 4 S p e r i p h e r i n - 2 m i s t a r g e t s t o t h e r o d i n n e r s e g m e n t a n d cell b o d y , a n d d o e s not c a u s e m i s t a r g e t t i n g o f e n d o g e n o u s p e r i p h e r i n - 2 . ( A & B ) C o n f o c a l m i c r o g r a p h s o f r e t i n a s e x p r e s s i n g Xenopus C 2 1 4 S - p e r i p h e r i n - 2 - G F P (green) labelled with T R - W G A (red) a n d Hoescht 3 3 3 4 2 (blue). (C) Confocal micrograph of a retina e x p r e s s i n g bovine C 2 1 4 S - p e r i p h e r i n - 2 - G F P (green) labelled with T R - W G A (red) a n d Hoescht 3 3 3 4 2 (blue). ( D & E ) Confocal micrographs of a retina e x p r e s s i n g bovine C 2 1 4 S - p e r i p h e r i n - 2 - G F P (green) labelled with P e r 2 A 5 m o n o c l o n a l antibody (red) a n d H o e s c h t 3 3 3 4 2 (blue) showing P e r 2 A 5 labelling (D) a n d t h e overlap with G F P fluorescence (E) (see Methods section 5.2.5). o s , rod o u t e r s e g m e n t ; is, r o d inner s e g m e n t ; n, n u c l e u s . B a r s : ( A , B , D & E ) 5 u m ; (C)10um.  152  Fig 3 5 . I m m u n o - E M r e v e a l s a c c u m u l a t i o n o f Xenopus C214S p e r i p h e r i n - 2 - G F P f u s i o n p r o t e i n at t h e c o n n e c t i n g c i l i u m in t h e i n n e r segment. Ultrathin sections of transgenic rods w e r e labeled with anti-GFP antibody followed by gold-conjugated secondary. (A) Longitudinal section t h r o u g h a t r a n s g e n i c rod s h o w s fusion protein a c c u m u l a t i o n at t h e c o n n e c t i n g c i l i u m ( c c ) a n d in t h e r o d i n n e r s e g m e n t ( i s ) . ( B ) F u s i o n p r o t e i n accumulates along the connecting cilium edge of t h e outer s e g m e n t but does not disrupt disc structure, o s , rod outer s e g m e n t ; is, rod inner s e g m e n t ; m i , mitochondrion (see Methods section 5.2.4). Bars: (A) 2 0 0 n m ; (B) 1 0 0 n m .  Chapter 5 Transgenic Xenopus in a p u n c t a t e connecting  153  pattern along the edge of the rod continuous  cilium  (Fig  35B).  Labelling  of  disc  incisures  with the was  not  observed. T h e m o l e c u l a r properties of bovine C 2 1 4 S p e r i p h e r i n - 2 h a v e been examined  by  nonreducing  velocity  sedimentation  conditions.  This  e x a m i n e s t h e size of d e t e r g e n t  and  SDS-PAGE  two-dimensional  analysis  separation  technique  solubilized oligomeric complexes  nondenaturing conditions a n d the contribution of intermolecular bonds  to complex  formation.  under  Fig 3 6 A  shows that  core  under  disulfide  noncovalent  tetramers a n d higher order disulfide-linked oligomers do not form for the C214S  mutant.  Instead,  the  C214S  core  complex  sediments  as a  noncovalent d i m e r (a) a n d a s disulfide b o n d e d t e t r a m e r s (b) (see section 4.3.3)  (Loewen, Moritz & Molday, 2001).  also present as faster sedimenting  Considerable aggregation  is  s p e c i e s in t h e s u c r o s e g r a d i e n t .  It  consists of high molecular weight disulfide-linked bands o n the S D S gel ( c o m p a r e t o t h e W T - l i k e p r o f i l e i n Fig 36B). has  been  observed  on  nonreducing  Aggregation of this m u t a n t  SDS  gels  and  by  velocity  sedimentation under reducing conditions previously (Goldberg, Loewen & Molday, 1998).  T h e inability of this m u t a n t to f o r m core t e t r a m e r s a n d  well-defined disulfide-linked oligomers provides a molecular rationale for t h e m i s t a r g e t i n g o f f u s i o n p r o t e i n in t r a n s g e n i c r o d s .  154  aggregate  dimer  monomer 1  2  3  4  5  6  7  8  9  10 11 12 13 14 15 16  Sedimentation F r a c t i o n s  B  dimer  monomer 2  3  4  5  6  7  8  9 10  11 12  13 14  15 16  Sedimentation Fractions  Fig 3 6 .  Velocity sedimentation analysis of C 2 1 4 S  and P216L  peripherin-2 under nonreducing conditions. (A) Bovine C 2 1 4 S p e r i p h e r i n - 2 w a s e x p r e s s e d in C O S - 1 c e l l s , s o l u b i l i z e d i n b u f f e r c o n t a i n i n g Triton X - 1 0 0 a n d N E M , a n d s u b j e c t e d to v e l o c i t y s e d i m e n t a t i o n o n 5 - 2 0 % sucrose gradients under nonreducing conditions. Fractions were collected f r o m t h e t u b e b o t t o m a n d s u b j e c t e d to S D S - P A G E u n d e r n o n r e d u c i n g conditions. W e s t e r n blots w e r e labelled with P e r 2 B 6 antibody. (B) Bovine P 2 1 6 L p e r i p h e r i n - 2 w a s e x p r e s s e d in C O S - 1 c e l l s a n d t r e a t e d a s in ( A ) . S e d i m e n t a t i o n p o s i t i o n s of d i m e r ( a ) , t e t r a m e r ( b ) , a n d o l i g o m e r (c) a r e indicated (see Methods sections 5.2.6 and 5.2.7).  Chapter 5 Transgenic Xenopus  5.3.7  155  Transgenic expression of the digenic RP-causing  L185P  mutant D i g e n i c r e t i n i t i s p i g m e n t o s a is a c o m p l e x d i s e a s e t h a t r e q u i r e s b o t h a n L 1 8 5 P m u t a t i o n in p e r i p h e r i n - 2 a n d a n u l l o r G 1 1 3 E m u t a t i o n in r o m 1.  Only  individuals  phenotype.  that  inherit  both  alleles  display  the  R e c e n t l y , the effects of the L 1 8 5 P m u t a t i o n on  disease  peripherin-2  c o r e a n d o l i g o m e r i c s t r u c t u r e h a v e b e e n c h a r a c t e r i z e d in d e t a i l ( L o e w e n , Moritz & Molday, 2001).  L185P peripherin-2 exists as a core heterodimer.  A significant fraction f u r t h e r a s s o c i a t e s t h r o u g h disulfide b o n d i n g to tetramers.  form  H o w e v e r , t h e s e t e t r a m e r s do not form higher order oligomers  unless they are associated with W T peripherin-2 or r o m - 1 . The L185P mutant subcellular  localization  h a s b e e n e x p r e s s e d in t r a n s g e n i c r o d s a n d has  been  examined  L 1 8 5 P - p e r i p h e r i n - 2 - G F P fusion protein inner s e g m e n t s a n d cell bodies.  (Fig  localized to  37).  The  rod outer  its  Xenopus segments,  T h e d e g r e e of m i s t a r g e t i n g w a s  much  less t h a n o b s e r v e d for t h e C 2 1 4 S m u t a n t , a s s i g n i f i c a n t a m o u n t s of f u s i o n protein  were  observed  in  the  outer  segments.  Localization to  disc  incisures w a s also o b s e r v e d . Rod degeneration w a s not apparent.  5.3.8  Transgenic expression of the RP-causing Fig 3 8 A  s h o w s t h e e x p r e s s i o n o f Xenopus  f u s i o n p r o t e i n in r o d s o f a t r a n s g e n i c r e t i n a .  P216L mutant  P216L-peripherin-2-GFP A dramatic difference was  156  Fig 3 7 .  L 1 8 5 P p e r i p h e r i n - 2 - G F P t a r g e t s to t h e r o d o u t e r s e g m e n t ,  t h e r o d i n n e r s e g m e n t , a n d t h e cell b o d y .  (A&B) Confocal micrographs  of a retina e x p r e s s i n g X e n o p u s L 1 8 5 P - p e r i p h e r i n - 2 - G F P ( g r e e n )  labelled  with T R - W G A (red) a n d Hoescht 3 3 3 4 2 (blue) ( s e e M e t h o d s section 5.2.5). o s , r o d o u t e r s e g m e n t ; i s , r o d i n n e r s e g m e n t ; n , n u c l e u s . B a r s : ( A ) 1 0 jj.m; (B) 2 . 5  urn.  Chapter 5 Transgenic Xenopus  157  o b s e r v e d b e t w e e n t h e cell m o r p h o l o g i e s o f r o d s in t h e p e r i p h e r a l a n d r o d s i n t h e c e n t r a l r e t i n a (Fig 38B).  retina  Peripheral rods were long a n d  u n i f o r m in a p p e a r a n c e a n d t h e f u s i o n p r o t e i n l o c a l i z e d t o o u t e r s e g m e n t s and  incisures similarly  however,  t o W T (Fig  appeared shortened  38C).  a n d highly  Rods  in t h e c e n t r a l  disorganized.  Fusion  retina protein  l o c a l i z e d t o w h o r l s o f m e m b r a n e s i n o u t e r s e g m e n t s b u t w a s n o t f o u n d in i n n e r s e g m e n t s (Fig 3 8 D  & E).  No incisures were visible.  R o d nuclei  w e r e a l s o m o r e d i s o r g a n i z e d in t h e c e n t r a l r e t i n a t h a n in t h e p e r i p h e r a l retina.  The P216L mutation  did not cause mistargeting  of the  fusion  protein to t h e i n n e r s e g m e n t s o r cell b o d i e s of rods a s o b s e r v e d for t h e C214S and L185P mutations.  T h e P216L mutation however, affected R O S  m o r p h o l o g y a n d r e s u l t e d in p h o t o r e c e p t o r d e g e n e r a t i o n . Immuno-electron  m i c r o s c o p y r e v e a l e d t h a t in p e r i p h e r a l r o d s  to d e g e n e r a t i o n , fusion protein 39A).  Disc  degenerating segment  structure rods,  membranes  localized to disc rims a n d incisures  and organization  fusion  protein  (Fig 3 9 B  appeared  colocalized  & C).  normal.  with  A breakdown  whorls  In  (Fig  central  of  of outer  prior  outer  segment  o r g a n i z a t i o n s i m i l a r t o w h a t is o b s e r v e d i n t h e r d s / + h e t e r o z y g o u s m o u s e a n d t h e P 2 1 6 L t r a n s g e n i c m o u s e w a s o b s e r v e d ( K e d z i e r s k i e t al., 1 9 9 7 ; Sanyal & Jansen, 1981). Velocity peripherin-2  sedimentation  and SDS-PAGE  analysis of bovine  u n d e r n o n r e d u c i n g c o n d i t i o n s (Fig 36B)  P216L-  revealed that  this  158  Fig 3 8 . P 2 1 6 L p e r i p h e r i n - 2 - G F P t a r g e t s to t h e r o d o u t e r s e g m e n t and c a u s e s rod degeneration. (A) Deconvolution epifluorescence microscopy a n d (B) corresponding DIC image of a retina expressing Xenopus P 2 1 6 L - p e r i p h e r i n - 2 - G F P ( g r e e n ) l a b e l l e d w i t h T R - W G A ( r e d ) a n d Hoescht 3 3 3 4 2 (blue). R o d s in t h e p e r i p h e r a l r e t i n a a r e m a r k e d w i t h a n a r r o w a n d rods in t h e central retina a r e m a r k e d w i t h a n a r r o w h e a d . C o n f o c a l m i c r o g r a p h s of peripheral (C) a n d central (D&E) regions of t h e s a m e retina s h o w specific t a r g e t i n g of t h e fusion protein to t h e o u t e r s e g m e n t a n d d e g e n e r a t i o n o f t h e c e n t r a l r o d s . N o f u s i o n p r o t e i n is v i s i b l e in t h e i n n e r s e g m e n t o r cell b o d y of either t h e peripheral o r t h e central r o d s (see M e t h o d s section 5 . 2 . 5 ) . o s , r o d outer s e g m e n t ; i s , r o d inner s e g m e n t ; n, n u c l e u s . B a r s : ( A & B ) 2 0 um; ( C ) 5 Lim; ( D ) 1 0 urn; ( E ) 5 um.  159  Fig 3 9 . I m m u n o - E M o f r o d s e x p r e s s i n g Xenopus  P216L peripherin-2-  GFP. ( A ) I m m u n o - E M of a peripheral rod s h o w s t r a n s g e n e disc rims (arrows) a n d incisures (arrowheads). (B&C) Rods retina s h o w highly disorganized shortened outer s e g m e n t s whorls of m e m b r a n e s with transgene labelling t h r o u g h o u t section 5.2.4). Bars, 2 5 0 n m .  localization to of the central consisiting of (see Methods  Chapter 5 Transgenic Xenopus mutation  did not disrupt  oligomerization. Xenopus  segments  protein  core tetramer  formation  or  disulfide-mediated  T h i s r e s u l t e x p l a i n s t h e o b s e r v e d e x p r e s s i o n p a t t e r n in  rods.  oligomerization inner  160  Since remain  intact,  o r cell  in r o d o u t e r  core  formation  the fusion  bodies.  segments  Instead, resulted  and  protein  disulfide-mediated  did not mistarget  the presence of the  in p h o t o r e c e p t o r  to  mutant  degeneration.  This must be due to an additional undefined molecular defect.  5.5.9  Transgenic To  determine  peripherin-2 expressed  in  expression the  protein  Xenopus  role  of Cl50S of  targeting  C150S  peripherin-2  disulfide-mediated and disc  core  tetramers  but  is i n c a p a b l e  (Loewen & Molday, 2000).  morphogenesis,  peripherin-2-GFP  e x a m i n e d the effects by confocal microscopy.  oligomerization  in  transgenic  we  have  rods  and  C150S peripherin-2  of disulfide-mediated  of  forms  oligomerization  Fig 40 s h o w s t h a t C 1 5 0 S p e r i p h e r i n - 2 - G F P  t a r g e t s s p e c i f i c a l l y t o R O S . N o f u s i o n p r o t e i n is v i s i b l e i n i n n e r s e g m e n t s . H o w e v e r , l o c a l i z a t i o n t o d i s c i n c i s u r e s is n o t r e a d i l y o b s e r v e d a s w i t h W T fusion  protein  or endogenous  peripherin-2.  In  some  cases,  a  single  c o l u m n o f l a b e l i n g n e a r t h e c e n t e r o f t h e o u t e r s e g m e n t is o b s e r v e d (Fig 40).  This m a y represent a single, deep incisure. R o d d e g e n e r a t i o n w a s n o t o b s e r v e d in a n y C 1 5 0 S a n i m a l s .  conventional and immuno-electron  Both  microscopy of rods expressing C 1 5 0 S  161  Fig 4 0 . C 1 5 0 S p e r i p h e r i n - 2 - G F P t a r g e t s to R O S , but d o e s not localize to d i s c i n c i s u r e s . ( A ) C o n f o c a l m i c r o g r a p h o f a r e t i n a e x p r e s s i n g Xenopus C 1 5 0 S - p e r i p h e r i n - 2 - G F P (green) labeled with T R - W G A (red) and Hoescht 3 3 3 4 2 ( b l u e ) . F u s i o n p r o t e i n in o u t e r s e g m e n t s is o f t e n o b s e r v e d in a c e n t r a l c a n a l in R O S ( a r r o w s ; * i n d i c a t e s a c r o s s - s e c t i o n o f a R O S ) . T h i s s t r u c t u r e m a y represent a single incisure. (B) Retina e x p r e s s i n g p e r i p h e r i n - 2 - G F P s h o w n for c o m p a r i s o n (see Methods section 5.2.5). o s , rod outer s e g m e n t ; i s , r o d i n n e r s e g m e n t ; n , n u c l e u s . B a r , 5 urn.  Chapter 5 Transgenic Xenopus  162  p e r i p h e r i n - 2 - G F P d i d n o t r e v e a l a n y s i g n i f i c a n t s t r u c t u r a l a b n o r m a l i t i e s in d i s c s o r in o r g a n i z a t i o n o f t h e o u t e r s e g m e n t ( d a t a n o t s h o w n ) .  However,  f e w e r i n c i s u r e s w e r e o b s e r v e d p e r r o d a n d in s o m e r o d s , s m a l l v e s i c l e s were  p r e s e n t a m o n g s t t h e d i s c s in t h e  outer  segment.  These  results  i m p l y t h a t o l i g o m e r i z a t i o n o f p e r i p h e r i n - 2 is i n v o l v e d in r i m a n d i n c i s u r e formation and possibly disc stability.  T h e l a c k o f a d o m i n a n t p h e n o t y p e is  likely t h e result of h a v i n g a n a b u n d a n c e of e n d o g e n o u s W T  peripherin-2  p r e s e n t in t h e s e a n i m a l s .  5.3.10 Expression  levels of  transgenes  To ensure that the effects o b s e r v e d for the t r a n s g e n e s were  not  artefacts of high e x p r e s s i o n levels, w e e x a m i n e d retinas e x p r e s s i n g W T a n d m u t a n t p e r i p h e r i n - 2 - G F P t h a t w e r e p r o c e s s e d in p a r a l l e l b y c o n f o c a l microscopy using the s a m e settings (i.e., laser attenuation, and  contrast).  Therefore  GFP  intensity  and  thus  c o n c e n t r a t i o n is d i r e c t l y c o m p a r a b l e b e t w e e n m i c r o g r a p h s .  brightness,  fusion  Although GFP  i n t e n s i t y is h i g h e s t in r o d s e x p r e s s i n g p e r i p h e r i n - 2 - G F P (Fig little f u s i o n  protein  was  located  in  rod  inner  segments.  41A), In  m i c r o g r a p h s o f r o d s e x p r e s s i n g L 1 8 5 P - p e r i p h e r i n - 2 - G F P (Fig C214S-peripherin-2-GFP  (Fig  41C)  at  lower  levels than  protein  very  contrast, 41B)  in A  and  showed  c o n s i d e r a b l e m i s l o c a l i z a t i o n of f u s i o n protein to i n n e r s e g m e n t s a n d cell bodies.  M i s l o c a l i z a t i o n w a s t h e r e f o r e not a result of high e x p r e s s i o n of  Fig 4 1 . O v e r e x p r e s s i o n of t h e X e n o p u s f u s i o n p r o t e i n s is not t h e c a u s e of d e r e a l i z a t i o n to the rod inner segment or rod degeneration. Transgenic eyes expressing X e n o p u s p e r i p h e r i n - 2 - G F P ( A ) , L185P-peripherin-2-GFP (B), C214S-peripherin-2-GFP (C), and P216L p e r i p h e r i n - 2 - G F P ( D ) w e r e e x c i s e d , f i x e d , a n d s e c t i o n e d in p a r a l l e l . Cryosections from each e y e were consecutively imaged by confocal microscopy. All i m a g e acquisition settings w e r e identical for all four s a m p l e s a n d p o s t m i c r o s c o p y p r o c e s s i n g w a s d o n e in p a r a l l e l t o t h e i m a g e s ( s e e Methods section 5.2.5). G F P (green) and Hoescht 3 3 3 4 2 (blue), o s , rod o u t e r s e g m e n t ; i s , r o d i n n e r s e g m e n t ; n, n u c l e u s . Bar, 5 u m .  Chapter 5 Transgenic Xenopus  164  the mutant fusion proteins. (Fig  41D),  bodies.  fusion  protein  In r o d s e x p r e s s i n g P 2 1 6 L - p e r i p h e r i n - 2 - G F P w a s not  o b s e r v e d in i n n e r s e g m e n t s o r c e l l  O v e r a l l e x p r e s s i o n of this  peripherin-2-GFP observed  in  rods  (compare  to  Fig  expressing the  fusion 41A).  protein  was  Therefore  P216L mutant  was  comparable  the  to  degeneration  not  due  to  high  expression levels.  5.4  DISCUSSION A m o l e c u l a r rationale for h o w mutations  in p e r i p h e r i n - 2 c a u s e R P  h a s b e e n d e v e l o p e d , b u t h a s n o t b e e n t e s t e d in v i v o ( G o l d b e r g ; L o e w e n & Molday, 2001).  1998;  Goldberg &  Molday,  1996a;  L o e w e n , Moritz &  Molday,  T h i s m o d e l s t r e s s e s t h e i m p o r t a n c e o f h a v i n g s u f f i c i e n t l e v e l s of  f u n c t i o n a l o l i g o m e r i c c o m p l e x e s in t h e o u t e r s e g m e n t . r e s u l t in p h o t o r e c e p t o r d e g e n e r a t i o n a n d l o s s o f v i s i o n .  D e c r e a s e d levels It  is u n c l e a r if  the m u t a n t proteins are s i m p l y m i s t a r g e t e d to the i n n e r s e g m e n t t h e r e b y r e s u l t i n g in a n o v e r a l l r e d u c t i o n o f o u t e r s e g m e n t p e r i p h e r i n - 2 , o r if t h e y are targeted to the outer s e g m e n t w h e r e they exert a d o m i n a n t effect on p h o t o r e c e p t o r function. u s i n g t r a n s g e n i c Xenopus  W e h a v e d e v e l o p e d a n in vivo s y s t e m  to e x a m i n e the effects of R P - c a u s i n g  in p e r i p h e r i n - 2 o n r o d p h o t o r e c e p t o r s . t h a t t r a n s g e n i c Xenopus  negative  mutations  W e d e m o n s t r a t e for the first t i m e  can be used a s a m o d e l s y s t e m to study R P -  Chapter 5 Transgenic Xenopus linked  disease  genes  165 and  the  molecular  basis  for  photoreceptor  degeneration. We  show  mistargeting Therefore,  that  defects  core  likely  mutations  was observed  Therefore, interference  in  rods,  RP caused with  dominant-negative Disc  in  to rod inner segments.  result  in d e f e c t i v e  tetramer  with  the  Additionally, a dominant-negative  P216L mutation  in  did not disrupt core or oligomer formation  mistargeting  result  C o r e t e t r a m e r a s s e m b l y is n e c e s s a r y f o r t a r g e t i n g  of p e r i p h e r i n - 2 t o rod o u t e r s e g m e n t s .  mutation  that  formation  h a v e t h e d i s e a s e b e c a u s e of d e c r e a s e d levels of total  p e r i p h e r i n - 2 in R O S .  effect  tetramer  of C 2 1 4 S a n d L 1 8 5 P p e r i p h e r i n - 2  individuals with  formation  in  but  did  result  by mutations  the function  in in  peripherin-2.  a n d did not cause  photoreceptor  peripherin-2  of W T protein  This  degeneration.  also  in o u t e r  results  from  segments  in a  manner.  m o r p h o g e n e s i s in r o d a n d c o n e  photoreceptors  is a  highly  d y n a m i c p r o c e s s t h a t is e s s e n t i a l f o r p h o t o r e c e p t o r v i a b i l i t y a n d v i s i o n . New discs a r e created continuously at a rate of 1 disc e v e r y 7 - 1 0 minutes (Besharse,  Hollyfield & R a y b o r n , 1 9 7 7 ; Y o u n g , 1976).  critical for disc f o r m a t i o n ,  P e r i p h e r i n - 2 is  probably by creating a n d then stabilizing the  disc rim (Arikawa e t a / . , 1992).  Therefore, a steady supply of peripherin-  2 t o t h e o u t e r s e g m e n t s o f p h o t o r e c e p t o r s is r e q u i r e d t o m a i n t a i n  proper  disc morphogenesis.  Xenopus  D i s c m o r p h o g e n e s i s h a s b e e n s t u d i e d in  Chapter 5 Transgenic Xenopus laevis  using  various  166  entrapment  dyes  like  Lucifer  pharmacological compounds  Matsumoto  & Besharse,  determined.  in c o n j u n c t i o n  with  (Hale, Fisher & Matsumoto, 1 9 9 6 ;  but the molecular  basis has not  been  Because photoreceptors are highly polarized cells, they are  also an excellent trafficking  1985),  yellow  model  system  to study  of m e m b r a n e proteins.  protein  targeting,  T h e s i z e o f Xenopus  especially  photoreceptors,  which are 5 - 1 0 times that of their m a m m a l i a n counterparts, m a k e s t h e m easy to analyze ultrastructurally  by microscopy techniques.  W e have  e x p l o i t e d t h e s e a d v a n t a g e s in c o n j u n c t i o n w i t h t h e e a s e a n d s p e e d o f t h e transgenesis technology to study the effects of disease causing in p e r i p h e r i n - 2 o n p r o t e i n t a r g e t i n g a n d p h o t o r e c e p t o r  mutations  morphology.  W e must m a k e certain considerations w h e n analyzing o u r results. First,  transgenesis  complement  present  "knock-in"  m u t a n t s i n Xenopus t h e effects mosaic  2  endogenous  of  procedure  is p r e s e n t  where  a  in t r a n s g e n i c  protein.  dominant  Introduction),  Because  RP where we  feel  does mimic the disease state. of t h e t r a n s g e n e expression  of  alone  the  in  is a l s o  studying  these  It i s m o r e d i f f i c u l t t o  on rod function,  transgene  over  mutations  W T protein that  full rods.  t h e effects of t h e m u t a n t t r a n s g e n e s  cause autosomal  (see Table  Secondly,  gene  studying  of the functional  peripherin-2  isolate  a  of e n d o g e n o u s peripherin-2  We are, therefore, those  is  is  an  however.  unavoidable  c o n s e q u e n c e o f t h e c u r r e n t t r a n s g e n e s i s p r o c e d u r e ( M o r i t z e t al.,  2001).  Chapter 5 Transgenic Xenopus  167  Hence, transgene expression levels will vary from rod to rod and retina to retina.  An advantage of this variability however, is that a rod expressing  the transgene can be compared to a neighbouring rod from the same retina not expressing transgene to determine effects on cell morphology and targeting of endogenous peripherin-2. To compensate for effects due to varying expression levels, relative quantitative confocal microscopy has been performed on all animals so the relative concentrations of fusion proteins can be directly compared.  Thirdly, proteins are expressed with  GFP fused to their C-terminus to facilitate rapid and easy screening for transgenic animals, and for fusion protein localization by fluorescence microscopy (Fig 28). It is possible that GFP may interfere with the function of peripherin2 . This may occur by ( 1 ) interfering with targeting to outer segment discs; ( 2 ) disrupting the putative C-terminal fusion domain; ( 3 ) disrupting interactions with other proteins; or ( 4 ) disrupting core and oligomer formation.  Our  results  indicate  that  GFP does  not  interfere  with  peripherin-2 function since peripherin-2-GFP ( 1 ) targets to incisures and rims of rod outer segment discs similarly to endogenous peripherin-2; ( 2 ) is found in phagosomes of RPE cells suggesting that proper fusion and shedding occurs; ( 3 ) does not cause rod degeneration; and ( 4 ) forms core and oligomeric complexes similarly to WT bovine peripherin-2.  Chapter 5 Transgenic Xenopus  168  W e h a v e c h a r a c t e r i z e d r o d s e x p r e s s i n g Xenopus b y c o n f o c a l a n d e l e c t r o n m i c r o s c o p y (Fig fusion protein  31  & 32).  peripherin-2-GFP L o c a l i z a t i o n of t h e  to o u t e r s e g m e n t s c o n f i r m s t h a t G F P d o e s not  with outer segment targeting.  interfere  V e r y l i t t l e f u s i o n p r o t e i n w a s o b s e r v e d in  i n n e r s e g m e n t s . T h i s is i n d i c a t i v e o f a v e r y f a s t r a t e o f t r a f f i c k i n g t o t h e o u t e r s e g m e n t ( M o r i t z e t al.,  2001).  rims  proper formation  and  incisures confirms  T a r g e t i n g of f u s i o n protein to disc of t h e s e structures.  regions of o u t e r s e g m e n t s e x p r e s s i n g v e r y high levels of fusion  In  protein,  l o c a l i z a t i o n is n o t r e s t r i c t e d t o r i m s a n d i n c i s u r e s , b u t is o b s e r v e d in t h e l a m e l l a r r e g i o n s o f d i s c s o r o t h e r m e m b r a n e s t r u c t u r e s (Fig  32A).  This  is l i k e l y d u e t o a n o v e r f l o w e f f e c t c a u s e d b y e x c e s s f u s i o n p r o t e i n non-rim regions rather than a GFP-related mistargeting.  It is n o t  into  known  w h a t e f f e c t s t h e f u s i o n p r o t e i n h a s o n t h e s e r e g i o n s , b u t in m a n y r o d s , s m a l l e r d i s c s a n d s m a l l e r o u t e r s e g m e n t d i a m e t e r a r e o b s e r v e d in r e g i o n s o f h i g h e x p r e s s i o n . It is p o s s i b l e t h a t t h e i n c r e a s e d l e v e l s o f p e r i p h e r i n - 2 result  in  increased  rates  of  rim  formation  and  hence  smaller  a s s u m i n g the rate of m e m b r a n e e v a g i n a t i o n r e m a i n s t h e s a m e .  discs, If  the  fusion protein inhibited d i s c f o r m a t i o n , fusion protein a c c u m u l a t i o n at the b a s e of t h e o u t e r s e g m e n t p r o x i m a l to t h e c o n n e c t i n g c i l i u m w o u l d expected.  be  N e i t h e r t h i s n o r a p h e n o t y p e s i m i l a r t o w h a t is o b s e r v e d in t h e  rds/+ heterozygous mouse was observed. o c c u r in a n y Xenopus  Retinal d e g e n e r a t i o n did  peripherin-2-GFP transgenic animals.  not  T h e r e f o r e , in  Chapter 5 Transgenic Xenopus the  full  range  rhodopsin  of  169  expression  levels and  higher  levels,  than  which  may  endogenous  for  determining  the  effects  of  comparable  peripherin-2,  protein did not c a u s e photoreceptor d e g e n e r a t i o n . control  be  the  T h i s is a n  disease-causing  to  fusion  important  mutations  on  photoreceptor morphology and survival. Expression  of  bovine  peripherin-2-GFP  r e s u l t e d in a s i m i l a r l o c a l i z a t i o n a s t h e Xenopus  in  transgenic  o r t h o l o g (Fig  33).  r e s u l t s a r e s i g n i f i c a n t b e c a u s e it s h o w s t h a t t h e p r o t e i n s o r t i n g has been conserved through mammalian  genes  in  this  evolution system.  d e s i g n e d in t r a n s g e n i c Xenopus  bovine  Therefore,  experiments  rod  outer  conjunction assembles  segments with our  was  possible  disc  molecular  incisures data  peripherin-2  (Fig  to  33).  showing (Fig  that  29)  t h e s e p r o t e i n s a r e in c o m p l e x in t r a n s g e n i c r o d s . from  rods  Colocalized peripherin-2  expressing  extremely  mistargeting to  inner  can  be  By  examine  the  peripherin-2.  co-localized with endogenous peripherin-2  and  w i t h Xenopus  study  o r m a m m a l i a n o r t h o l o g s of g e n e s .  p e r i p h e r i n - 2 - G F P , it  protein  machinery  a n d t h a t it is p o s s i b l e t o  effects of f u s i o n protein o n t h e localization of e n d o g e n o u s The bovine fusion  These  to elucidate the signals a n d m e c h a n i s m s  o f p r o t e i n t a r g e t i n g u s i n g Xenopus expressing  Xenopus  of  high  both  segments  the  levels of fusion  was observed  These  bovine  strongly  data  to in  peripherin-2 suggest  that  Further evidence comes bovine  protein (data  fusion  and not  protein.  endogenous shown).  We  Chapter 5 Transgenic Xenopus believe that this transgene  170  mistargeting  expression  is d u e t o t h e  (much  higher  abnormally  than  high  observed  levels  for  of  Xenopus  p e r i p h e r i n - 2 - G F P ) a n d is n o t a r e s u l t o f a n i n h e r e n t m i s t a r g e t i n g  property  of t h e b o v i n e f u s i o n p r o t e i n . O t h e r w i s e m i s t a r g e t i n g w o u l d be o b s e r v e d at moderate These  to  low  results  fusion  do  protein  confirm  that  levels similarly the  fusion  to  the  protein  is  C214S in  mutant.  complex  with  endogenous peripherin-2. W e h a v e e x a m i n e d the effects of the R P - c a u s i n g C 2 1 4 S m u t a t i o n p e r i p h e r i n - 2 b y e x p r e s s i o n i n t r a n s g e n i c Xenopus  (Fig  34).  in  An obvious  t a r g e t i n g d e f e c t is p r e s e n t in t h i s p r o t e i n w h i c h p r e v e n t s m o s t o f it f r o m reaching  the  membranes higher  outer  segment.  of the  inner segment  levels, fusion  segment.  Mutant  Electron  protein  fusion  a n d cell b o d y .  accumulates  microscopy  protein  (Fig  35)  In  near the  accumulates rods  expressing  b a s e of the  revealed that  in  fusion  outer protein  a c c u m u l a t e d a t t h e c o n n e c t i n g c i l i u m in t h e i n n e r s e g m e n t a n d a l o n g t h e e d g e of the  outer  s e g m e n t proximal to the  connecting cilium.  protein incorporation into disc incisures w a s not o b s e r v e d . indicate  that  fusion  protein  that  reaches  incorporated effectively into disc rims.  the  outer  T h e s e results  segment  endogenous  peripherin-2  to  inner  is  not  T h e m u t a n t fusion protein did not  d i s r u p t d i s c s t r u c t u r e , w h i c h s u g g e s t s t h a t it is n o t i n t e r f e r i n g f u n c t i o n of e n d o g e n o u s p e r i p h e r i n - 2 .  Fusion  with  the  W e did not o b s e r v e m i s t a r g e t i n g of segments  or  cell  bodies  in  these  Chapter 5 Transgenic Xenopus  171  a n i m a l s (Fig 34).  T h i s is f u r t h e r e v i d e n c e t h a t t h e m u t a n t f u s i o n  protein  does  w i t h W T in  by o u r  not interact  vivo.  This finding  is s u p p o r t e d  previous studies that show that the C 2 1 4 S mutant does not interact with rom-1 (Goldberg, Loewen & Molday, 1998).  It is l i k e l y t h a t  photoreceptor  d e g e n e r a t i o n d i d n o t o c c u r in t h e s e a n i m a l s b e c a u s e t h e m u t a n t  protein  did not interfere with the targeting or function of e n d o g e n o u s peripherin2.  In h u m a n s , t h e C 2 1 4 S m u t a t i o n likely p r e v e n t s t a r g e t i n g to t h e o u t e r  s e g m e n t a n d interaction with endogenous W T peripherin-2. decreased  total  levels  of  peripherin-2  in  rod outer  This leads to  segments  and a  disease phenotype.  R e t i n a l d e g e n e r a t i o n is o b s e r v e d i n t h e h e t e r o z y g o u s  rds/+  results from  mouse which  ( C h e n g etal.,  a 5 0 % reduction  in t o t a l  1997; Sanyal & Jansen, 1981).  To understand the molecular basis for mistargeting m u t a n t i n t r a n s g e n i c Xenopus, is  capable  of  oligomerization tetramers sizes  core (Fig  tetramer  36).  for  membranes  & Molday, disc  formation  W T peripherin-2  2000).  morphogenesis  and creating  disc rims  possibly  disulfide-mediated  forms  core  prevents  core  tetramer  by  noncovalent  oligomers of various are thought  binding  opposing  (Loewen & Molday, 2000; 2000).  formation  protein  and  These oligomers  M o r i t z & M o l d a y , 2 0 0 1 ; W r i g l e y e r al., mutation  of the C 2 1 4 S  w e h a v e d e t e r m i n e d if t h e m u t a n t  t h a t a s s e m b l e into large disulfide-linked  (Loewen  important  peripherin-2  to be disc  Loewen,  W e find that t h e C 2 1 4 S and  disulfide-mediated  Chapter 5 Transgenic Xenopus oligomerization  and  172  r e s u l t s in  non-specific aggregation  of the  protein.  T h e inability of C 2 1 4 S p e r i p h e r i n - 2 to f o r m t h e s e n a t i v e c o m p l e x e s m u s t be  the  cause  of  the  mistargeting  observed  in  transgenic  Xenopus.  M i s t a r g e t i n g c o u l d r e s u l t f r o m r e t e n t i o n in t h e E R a n d G o l g i m e m b r a n e s by m o l e c u l a r c h a p e r o n e s b e c a u s e of t h e m i s f o l d e d n a t u r e of t h e  protein,  o r b e c a u s e a f u n c t i o n a l o u t e r s e g m e n t s o r t i n g s i g n a l is n o l o n g e r p r e s e n t . Accumulation  of fusion  m e m b r a n e s (Fig  35)  protein  at  the  connecting  cilium  in  post-Golgi  a n d l o w l e v e l s o f f u s i o n p r o t e i n in t h e E R ( d a t a  not  s h o w n ) a r g u e f o r t h e l a t t e r in w h i c h m i s t a r g e t i n g l i k e l y r e s u l t s f r o m  the  disruption of a n o u t e r s e g m e n t sorting s i g n a l .  C o r e t e t r a m e r f o r m a t i o n is  l i k e l y r e q u i r e d f o r s i g n a l l i n g to t h e o u t e r s e g m e n t a n d will b e d i s c u s s e d in greater detail below. Recently,  the  molecular  in  that  cannot  oligomerize  disulfide-linked complexes unless W T peripherin-2 or rom-1 Therefore,  the  core  tetramer  is  required  for  &  in  T h e L 1 8 5 P c o r e c o m p l e x is a n o n c o v a l e n t d i m e r t h a t c a n d i s u l f i d e It  (Loewen, Moritz  result  2001).  a covalent tetramer.  detail  defects  RP have  form  examined  and  digenic  bond to  been  interactions  Molday,  into  larger  is p r e s e n t .  disulfide-mediated  oligomerization.  T h e m o l e c u l a r r e s c u e o f L 1 8 5 P b y W T p e r i p h e r i n - 2 is n o t  100%  however,  efficient,  remains.  and  a  fraction  of  noncovalent  heterodimer  It h a s b e e n p r o p o s e d t h a t d e c r e a s e d l e v e l s o f o l i g o m e r s in t h e  o u t e r s e g m e n t r e s u l t s in p h o t o r e c e p t o r d e g e n e r a t i o n ( L o e w e n , M o r i t z  &  Chapter 5 Transgenic Xenopus Molday, 2001). Xenopus  173  W e have expressed the L185P mutation  a n d found that a fraction of the fusion protein mistargets to the  inner segment while the majority likely  in t r a n s g e n i c  that the mutant  fusion  form  a  is l o c a t e d i n t h e o u t e r s e g m e n t .  protein  mixture  assembles with  endogenous WT  peripherin-2  to  noncovalent  dimers  experiments.  T h e oligomers probably target to the outer s e g m e n t where  similar  to  of  It is  disulfide-linked  what  is  observed  oligomers in  and  C O S - 1 cell  they m a y function n o r m a l l y , a n d t h e n o n - t e t r a m e r i c core c o m p l e x e s likely m i s t a r g e t t o t h e i n n e r s e g m e n t in a f a s h i o n s i m i l a r t o t h a t o b s e r v e d f o r the C 2 1 4 S Rod  mutant. degeneration  w a s not  observed  p r o b a b l y b e c a u s e t h e ratio of m u t a n t  in  these  animals.  This  is  fusion protein to endogenous W T  peripherin-2 did not reach a degenerative threshold level.  In h u m a n R P ,  w h e r e t h e r a t i o o f m u t a n t t o W T is p r e d i c t e d t o b e 1 : 1 , m i s t a r g e t i n g o f dimeric  complex  to  the  inner  segment  would  d e c r e a s e in t o t a l o u t e r s e g m e n t p e r i p h e r i n - 2 . mutation,  we  predict  that  the  L185P  result  in  a  significant  T h e r e f o r e , like t h e C 2 1 4 S  mutation  causes  disease  by  d e c r e a s i n g t h e t o t a l level o f p e r i p h e r i n - 2 in t h e o u t e r s e g m e n t - a r e s u l t of m i s t a r g e t i n g of m u t a n t d i m e r s to t h e inner s e g m e n t . O n l y o n e R P - c a u s i n g m u t a t i o n , P 2 1 6 L , h a s b e e n e x a m i n e d in vivo in transgenic  m i c e ( K e d z i e r s k i et al.,  1996).  In t h e r d s / +  heterozygous  background, which mimics the human disease state, the P216L  mutation  Chapter 5 Transgenic Xenopus caused  photoreceptor  174  degeneration  and  cell  death.  Because  the  t r a n s g e n e w a s i n d i s t i n g u i s h a b l e f r o m e n d o g e n o u s p e r i p h e r i n - 2 , it w a s n o t p o s s i b l e t o d e t e r m i n e its s u b c e l l u l a r l o c a l i z a t i o n a n d h e n c e a n y defects d u e to the m u t a t i o n .  targeting  W e h a v e s h o w n in t r a n s g e n i c Xenopus  that  the P 2 1 6 L m u t a n t t a r g e t s to o u t e r s e g m e n t disc r i m s a n d i n c i s u r e s , but causes photoreceptor degeneration.  It d o e s n o t m i s t a r g e t t o r o d  inner  s e g m e n t s o r cell b o d i e s .  D e g e n e r a t i o n w a s c o n f i n e d to t h e r o d s of t h e  central  s e g m e n t s of t h e s e cells w e r e  highly  retina. The outer  d i s o r g a n i z e d c o n s i s t i n g of  unaffected at this s t a g e .  whorls  of  shortened  membranes.  Cones  and were  B e c a u s e p h o t o r e c e p t o r s of the central retina are  considerably older than peripheral rods (Hollyfield, 1971) we reason that a  time-dependent  degeneration  p r o t e i n in t h e o u t e r s e g m e n t s .  results  from  the  p r e s e n c e of  This t y p e of central to p e r i p h e r y directed  d e g e n e r a t i o n h a s b e e n o b s e r v e d in t r a n s g e n i c Xenopus rab8 mutants  (O. Moritz, personal communication).  rods expressing F u r t h e r a n a l y s i s of  t r a n s g e n i c a n i m a l s at v a r i o u s later s t a g e s will be r e q u i r e d to define  the  time  mutant  c o u r s e of d e g e n e r a t i o n  and  to  s e e if  rigorously  peripheral  rods  eventually degenerate. T h e m o l e c u l a r d e f e c t l e a d i n g t o t h i s d e g e n e r a t i o n is c u r r e n t l y known.  Biochemical analysis  of  bovine  r e v e a l e d a n y d e f e c t s in c o r e t e t r a m e r o l i g o m e r i z a t i o n (Fig  36).  P216L  formation  peripherin-2 or  has  not not  disulfide-mediated  However, these results are consistent with the  Chapter 5 Transgenic Xenopus  175  l o c a l i z a t i o n o b s e r v e d in r o d s p r i o r t o d e g e n e r a t i o n .  S i n c e t h e m u t a n t is  n o t d e f e c t i v e s t r u c t u r a l l y , it t a r g e t s p r o p e r l y t o d i s c i n c i s u r e s a n d r i m s i n the outer segment. of  the  mutant  degeneration.  A defective molecular event d o w n s t r e a m of a s s e m b l y  into  discs  must  Close examination  result  in  destabilization  of the degenerating  reveals that organized disc structure  outer  and  rod  segments  is n o l o n g e r p r e s e n t a n d  mutant  p r o t e i n is l o c a t e d t h r o u g h o u t w h o r l s of m e m b r a n e s , s i m i l a r l y t o t h e r d s / + heterozygous mouse. mutant  protein  interactions  mutants,  in discs destabilizes their  with  organization.  It is p o s s i b l e t h a t o v e r t i m e t h e p r e s e n c e o f t h e  other  factors  that  regulate  or disrupts disc  W e have not yet e x a m i n e d the stability  and  interaction  of  peripherin-2  p r o t e i n s is c u r r e n t l y u n d e r i n v e s t i g a t i o n . f o r p e r i p h e r i n - 2 in v i s u a l t r a n s d u c t i o n . rims  structure  and incisures (Molday,  1998).  with  other  critical  stability of  and  peripherin-2  outer  segment  T h e r e is n o e v i d e n c e f o r a r o l e However, A B C R localizes to disc  Its activity  might  be affected  by  malformed disc rims. Rod  photoreceptors  sorting/trafficking specifically  must  possess  multiple  p a t h w a y s to a c c o m p l i s h the task of targeting  to the various  compartments  of the outer  protein proteins  segment.  The  N a / C a - K exchanger a n d the cGMP-gated channel for e x a m p l e are only located in t h e p l a s m a m e m b r a n e w h e r e a s p e r i p h e r i n - 2 , r o m - 1 a n d A B C R a r e o n l y f o u n d in t h e r i m r e g i o n o f d i s c s ( M o l d a y , 1 9 9 8 ) .  Rhodopsin on  Chapter 5 Transgenic Xenopus  176  t h e o t h e r h a n d is e x c l u d e d f r o m t h e r i m s , b u t i s l o c a t e d i n t h e l a m e l l a r r e g i o n o f d i s c s a n d in t h e p l a s m a m e m b r a n e  (Molday,  1998).  Sorting  s i g n a t u r e s m u s t t h e r e f o r e e x i s t in t h e s e p r o t e i n s t o t a r g e t t h e m t o t h e i r correct locations. similar signals.  Proteins targeted to t h e s a m e location likely  possess  Recently, the C-terminal eight a m i n o acids of rhodopsin  have been shown to comprise an outer s e g m e n t sorting/retention u s i n g t r a n s g e n i c Xenopus  ( T a r n etal.,  signal  2000).  W e h a v e identified a c o m p o n e n t of this s i g n a l in p e r i p h e r i n - 2 42).  Both the C214S and the L185P mutations  mistargeting Xenopus.  of Both  formation. formation  fusion  protein  of these  The P216L  to  rod  mutations mutation  inner  result  does  r o d s e x p r e s s i n g Xenopus  r e s u l t in a n y m i s t a r g e t i n g mutation  prevents  in d e f e c t i v e  in core  core  cause  transgenic tetramer  or  oligomer  in t r a n s g e n i c r o d s .  Analysis  C 1 5 0 S - p e r i p h e r i n - 2 - G F P did not  of fusion protein to t h e inner s e g m e n t .  disulfide-mediated  oligomerization,  tetramer formation (Loewen & Molday, 2000). the core tetramer,  segments  not disrupt  a n d does not cause mistargeting  of t r a n s g e n i c  in peripherin-2  (Fig  but  not  T h e s e results indicate that  It is l i k e l y t h a t t h e s o r t i n g s i g n a l i n p e r i p h e r i n - 2 is  c o n t a i n e d in its c y t o p l a s m i c C - t e r m i n u s a n d r e q u i r e s t e t r a m e r i z a t i o n by  core  n o t t h e d i s u l f i d e - l i n k e d o l i g o m e r , is r e q u i r e d f o r o u t e r  segment localization.  recognition  This  the  sorting  machinery.  Experiments  are  for  currently  177  Species  Complex  Targeting  WT  SwwSwCCtiwr v.v.v.v.v.vXv  P216L  • ••••• •• •• •• •• •• •• •• •• •• •• • r« • ••••••••••  C150S  WwXvX"X*X»>  L185P  X»X»X»X»X*X«X'  C214S Fig 4 2 . M o l e c u l a r r a t i o n a l e for t a r g e t i n g of p e r i p h e r i n - 2 to R O S . W T peripherin-2 forms core tetramers and disulfide-linked oligomers and t a r g e t s s p e c i f i c a l l y to R O S . T h e P 2 1 6 L m u t a t i o n d o e s not d i s r u p t c o r e or o l i g o m e r f o r m a t i o n a n d d o e s not disrupt targeting to R O S . T h e C 1 5 0 S m u t a t i o n p r e v e n t s d i s u l f i d e - l i n k e d o l i g o m e r i z a t i o n b u t d o e s n o t r e s u l t in m i s t a r g e t i n g t o R I S . T h e r e f o r e , d i s u l f i d e - m e d i a t e d o l i g o m e r i z a t i o n is n o t required for R O S t a r g e t i n g . T h e L 1 8 5 P m u t a t i o n p r e v e n t s c o r e t e t r a m e r and o l i g o m e r f o r m a t i o n in t h e a b s e n c e o f W T a n d r e s u l t s i n m i s t a r g e t i n g t o R I S . A p o r t i o n of L 1 8 5 P p e r i p h e r i n - 2 f o r m s c o r e t e t r a m e r s a n d o l i g o m e r s w i t h W T a n d t a r g e t s to R O S . T h e C 2 1 4 S m u t a t i o n p r e v e n t s c o r e t e t r a m e r a n d o l i g o m e r f o r m a t i o n a n d d o e s not interact w i t h WT. C 2 1 4 S peripherin-2 m i s t a r g e t s t o R I S . T h e r e f o r e , t h e c o r e t e t r a m e r is r e q u i r e d f o r t a r g e t i n g o f p e r i p h e r i n - 2 to R O S . R P - c a u s i n g m u t a t i o n s r e s u l t in d i s e a s e b y ( 1 ) d e c r e a s i n g t h e l e v e l o f p e r i p h e r i n - 2 in R O S b e c a u s e o f m i s t a r g e t i n g , ( 2 ) e x e r t i n g a d o m i n a n t n e g a t i v e e f f e c t in R O S o n r o d f u n c t i o n .  Chapter 5 Transgenic Xenopus  178  u n d e r w a y to identify this s e q u e n c e . Restoration  of functional  photoreceptor  outer  segments  by  gene  t h e r a p y in t h e r d s m o u s e h a s r e c e n t l y b e e n a c c o m p l i s h e d b y a d d i n g b a c k W T p e r i p h e r i n - 2 (Ali e t a / . , 2 0 0 0 ) .  This technique m a y prove very useful  in t r e a t i n g o r c u r i n g b l i n d n e s s i n p e o p l e w i t h d e f e c t s i n t h e g e n e e n c o d i n g peripherin-2.  H o w e v e r , it i s e s s e n t i a l t o d e t e r m i n e t h e m o l e c u l a r b a s i s  for h o w mutations  i n p e r i p h e r i n - 2 c a u s e p h o t o r e c e p t o r d e g e n e r a t i o n in  order to develop effective gene therapy treatment  strategies.  shown that,  (Sung & Tai, 2000),  mutations  in  like  RP-causing  peripherin-2  rhodopsin  cause  protein to the inner segment. total  levels of peripherin-2  mutations  RP through  mistargeting  W e have  of  mutant  Therefore, RP that results from decreased  in t h e o u t e r  segment  (like t h e C 2 1 4 S a n d  L185P mutations, and probably the C 1 6 5 S mutation) by i n c r e a s i n g t h e level of W t p e r i p h e r i n - 2 .  should be treatable  RP resulting from the P216L  mutation and other dominant negative mutations, m a y require strategies to inactivate o r d e c r e a s e t h e level of t h e m u t a n t  additional  protein.  Chapter 6 Conclusion CHAPTER 6 6.1  179  CONCLUSION  SUMMARY The results of this investigation have provided valuable n e w insight  into t h e structural  features of p e r i p h e r i n - 2  and rom-1 complexes, and  w h a t f u n c t i o n s t h e y s e r v e in p h o t o r e c e p t o r o u t e r s e g m e n t d e v e l o p m e n t  -  a p r o c e s s t h a t is a b s o l u t e l y r e q u i r e d f o r p h o t o r e c e p t o r v i a b i l i t y a n d f o r vision.  Without  this  intricate  architecture,  we would  find  no use for  i n v e n t i o n s like t h e light bulb o r t h e printing p r e s s , a n d w e w o u l d t a k e no p l e a s u r e in t h e s i g h t s of p a i n t i n g s , p h o t o g r a p h s , a s p r i n g f l o w e r , a s n o w capped m o u n t a i n , or of those dearest to us. A s s u c h , peripherin-2 plays a dominant  role in p h o t o r e c e p t o r d e v e l o p m e n t a n d c r e a t e s t h e s c a f f o l d  through which we see the world. Disulfide peripherin-2  bond  is critical  tetrameric complex. region  formation  are likely  within  for folding  the  intradiscal  and subunit  loop  assembly  of the  of  core  S i x of t h e s e v e n c o n s e r v e d c y s t e i n e s within t h e loop  involved  in  forming  intramolecular  although these have y e t to be d e m o n s t r a t e d .  disulfide  t h a t instead of f o r m i n g  bonds,  One unique cysteine,  is s o l e l y r e s p o n s i b l e f o r i n t e r m o l e c u l a r d i s u l f i d e b o n d f o r m a t i o n . demonstrated  region  disulfide dimers  C150,  W e have  within the core  c o m p l e x , C 1 5 0 r e s i d u e s actually link t e t r a m e r s into o c t a m e r s a n d o t h e r higher-order formation.  oligomeric  forms  through  intermolecular  disulfide  bond  T h i s d i s u l f i d e b o n d is r e a d i l y r e d u c e d w h e n s u b j e c t e d t o o n l y  Chapter 6 Conclusion mildly  180  reducing conditions a n d facilitates dissociation of oligomers  into  core tetrameric c o m p l e x e s . This result a n d t h e fact that noncovalent core t e t r a m e r s a r e a l s o p r e s e n t in R O S , i m p l i e s t h a t a l e v e l o f p l a s t i c i t y e x i s t s in o l i g o m e r f o r m a t i o n , s i z e a n d s t a b i l i t y , a n d s u g g e s t s a d y n a m i c r o l e f o r t h e s e c o m p l e x e s in t h e r i m s of d i s c s . formation  W e have shown that core tetramer  is r e q u i r e d f o r o l i g o m e r i z a t i o n  and that formation  of h i g h e r - o r d e r  but not disulfide  oligomers, therefore,  covalent (disulfide bonds) a n d noncovalent interactions. the  core  dimers.  tetramer  has two-fold  symmetry  E x p e r i m e n t s in t r a n s g e n i c f r o g s  involves  both  W e propose that  and exists have  dimerization,  as a  dimer-of-  revealed that the core  t e t r a m e r is r e q u i r e d f o r t a r g e t i n g t o p h o t o r e c e p t o r o u t e r s e g m e n t s . W e h a v e d e m o n s t r a t e d a role f o r r o m - 1 in i n h i b i t i o n o r r e g u l a t i o n of higher-order oligomer formation.  Peripherin-2 complexes from R O S that  c o n t a i n r o m - 1 a r e i n c o m p l e t e l y d i s u l f i d e b o n d e d a n d a r e l i m i t e d in s i z e to octamers.  R o m - 1 in t h e a b s e n c e o f p e r i p h e r i n - 2 h a s a d e c r e a s e d ability  to  disulfide-linked  form  peripherin-2  in a h e t e r o l o g o u s  c o m p l e x e s to o c t a m e r s . prevents oligomers  octamers,  disulfide  when  s y s t e m , limits  reconstituted  the size  of  with  peripherin-2  T h e r e f o r e , it is l i k e l y t h a t a s s o c i a t i o n w i t h r o m - 1  the conformational upon  and  change  linking  required  to  produce  of C 1 5 0 residues.  higher-order  T h e s e results are  c o n s i s t e n t w i t h t h e d o m i n a n t role f o r p e r i p h e r i n - 2 in d i s c m o r p h o g e n e s i s a s d e m o n s t r a t e d b y t h e d r a s t i c p h e n o t y p e a s s o c i a t e d w i t h t h e rds  mouse  Chapter 6 Conclusion and  a  181  regulatory  role  for r o m - 1 as demonstrated  by the very  mild  phenotype of the r o m - 1 knockout m o u s e . By  examining  peripherin-2  on  the  its  effects  oligomeric  of  disease-causing  structure  and  mutations  targeting  in  in  rod  photoreceptors, w e have developed a molecular rationale describing how misfolding  and  mistargetting  oligomeric  complexes  in  result  in  functional P216L,  targets  dominant  oligomerization  significance to  of t h e s e  negative  the others.  structural  provides  oligomers  further in  effect  on photoreceptor  L185P)  morphology  inherent  complexity  the  mutant,  but exerts a n d results  lead  suggests that either to  photoreceptor  subtle  a in  and  heterogeneity  undetected  degeneration,  a n o t h e r critical function or interaction h a s b e e n d i s r u p t e d . the  for  Another  normal,  The  prevent  evidence  ROS.  of  T h i s m u t a n t is c l e a r l y b e h a v i n g d i f f e r e n t l y  This finding  abnormalities  levels  degeneration.  (C165Y, C214S,  R O S a n d appears structurally  photoreceptor degeneration. than  decreased  R O S and photoreceptor  important finding that s o m e mutations disulfide-mediated  overall  of  or  that  In a n y c a s e ,  disease  phenotypes  a s s o c i a t e d w i t h m u t a t i o n s in p e r i p h e r i n - 2 h a s b e e n d e m o n s t r a t e d . Out  of  necessity,  through  the  course  of  this  work  we  have  developed n e w methods to study the structure a n d function of m e m b r a n e proteins. sedimentation  The  2-dimensional  and  non-reducing  method  which  SDS-PAGE  for  combines  velocity  determining  the  Chapter 6 Conclusion contribution structures  of  182 intermolecular  is a p p l i c a b l e t o  disulfide  other  bonds  membrane  to  oligomeric  proteins.  We  have  d e v e l o p e d an S D S - d e n a t u r a t i o n - i m m u n o p u r i f i c a t i o n m e t h o d to the  absolute amount  of a g i v e n  protein  subunit  from  t h e f i r s t t i m e , t h a t t r a n s g e n i c Xenopus  also  quantitate  a tissue.  t e c h n i q u e is c u r r e n t l y b e i n g u s e d t o d e t e r m i n e t h e s u b u n i t of the c G M P - g a t e d c h a n n e l c o m p l e x .  protein  This  stoichiometry  Lastly, we have demonstrated laevis  can be used as a  for  model  s y s t e m t o s t u d y t h e e f f e c t s o f d i s e a s e - c a u s i n g m u t a t i o n s in p h o t o r e c e p t o r genes on protein trafficking, photoreceptor m o r p h o l o g y , and viability.  6.2 DISC  O R G A N I Z A T I O N O F P E R I P H E R I N - 2 C O M P L E X E S IN  THE  RIM W e have proposed two m o d e l s for how peripherin-2 oligomers are  s i t u a t e d in t h e d i s c r i m b a s e d o n w h e t h e r d i s u l f i d e b o n d i n g o f t e t r a m e r s occurs laterally within one m e m b r a n e  (Fig  m a n n e r a c r o s s t w o m e m b r a n e s (Fig 43B). of t h e c o r e t e t r a m e r  43A)  head-to-head  W e a s s u m e that the subunits  e x i s t in a s i n g l e m e m b r a n e  c o m p l e x w e c a n i s o l a t e f r o m b o v i n e R O S is t h e T h e shell  o r in a  because the  smallest  tetramer.  model predicts that laterally disulfide-bonded  tetramers  will m a k e a n o l i g o m e r i c ring or coil d e p e n d i n g o n w h e t h e r t h e  tetramers  are perfectly aligned or off-set w h e n they are linked.  T h e s e rings  m a y associate noncovalently and circumscribe the disc rim.  then  Peripherin-2  183  Fig 4 3 . O r g a n i z a t i o n of p e r i p h e r i n - 2 in t h e d i s c r i m . T w o p o s s i b l e m o d e l s are s h o w n for h o w peripherin-2 oligomers create a n d stabilize t h e disc rim. For details, see the text.  Chapter 6 Conclusion  184  oligomers that do not contain r o m - 1 need to be perfectly disulfide-linked, whereas  those  Tetramers potential  containing  that  rom-1  d o not align  do  not  properly  role of t h e intermediate  comes  tetramers  in  from the  evidence  Molday,  2000).  linked.  The  o l i g o m e r o r o c t a m e r is n o t a s c l e a r l y  of  heterologous  &  are not disulfide  d e f i n e d i n t h i s m o d e l a s i n t h e zippering model  (Loewen  model.  Support for the  disulfide-mediated C O S - 1 cell  system,  shell  oligomerization which  can  of  likely  a c c o m m o d a t e linking of t e t r a m e r s within t h e s a m e m e m b r a n e ( L o e w e n & Molday,  However,  2000).  a  direct  role  for oligomers  in  binding  of  a d j a c e n t m e m b r a n e s is n o t a s o b v i o u s i n t h i s m o d e l a n d d o e s n o t a g r e e as well  with  microsomal oligomers  recent  studies showing  membranes  could  (Wrigley  et  peripherin-2-induced al.,  2000).  flattening  Although,  coils  possibly induce " k i n k i n g " of t h e v e s i c l e s w h i c h  of of  might  result in t h e o b s e r v e d v e s i c l e m o r p h o l o g y . T h e zippering membranes  at  the  model  predicts that core tetramers  lamellar-rim  border  octamers which bind the m e m b r a n e s . order  oligomer  formation  formation  which  align  from  opposing  head-to-head  Octamerization facilitates  proceeds around  the disc  to  form  higher-  driving  rim  a n d c i r c u m s c r i b i n g it.  Tetramers that do not align with the  correct geometry are not linked.  This model emphasizes the octamer as  a  discrete  structural  and functional  element  a n d is s u p p o r t e d  by the  e x i s t e n c e o f t h e s e c o m p l e x e s in b o t h R O S a n d t r a n s f e c t e d C O S - 1 cells.  Chapter 6 Conclusion  185  T h e ability o f p e r i p h e r i n - 2 c o m p l e x e s t o f l a t t e n m i c r o s o m a l v e s i c l e s in a disulfide-dependent protein  Po  m a n n e r , a n d the existence of the myelin  which  binds  opposing  e x t r a c e l l u l a r s p a c e ( S h a p i r o e t al., potential  problem  for  the  myelin  membranes  stabilizing  across  the  1996) further support this model.  zippering  model  comes  from  A  C O S - 1 cell  expression studies where disulfide-linked oligomerization does occur.  If  t e t r a m e r s a r e l i n k i n g a c r o s s m e m b r a n e s in C O S - 1 c e l l s , t h e n p i n c h i n g o r flattening of these m e m b r a n e s must be occuring there also. if t h e s e  types  of m e m b r a n e  a n d vesicle morphologies  It is u n c l e a r  are present  in  transfected C O S - 1 cells.  6.3  CONSIDERATIONS  DISC  FOR THE MOLECULAR  BASIS  FOR  MORPHOGENESIS A model for disc m o r p h o g e n e s i s involving the action of peripherin-2  o l i g o m e r s is s h o w n i n Fig 44. mechanism  of  disc  A l t h o u g h t h e role for p e r i p h e r i n - 2 a n d t h e  morphogenesis  has  yet  to  be  unequivocally  d e t e r m i n e d , e n o u g h e v i d e n c e h a s a c c u m u l a t e d that w e c a n begin to build a simple molecular model for h o w this results expressing peripherin-2  process occurs.  and peripherin-2  mutants  Based on our in t r a n s g e n i c  frogs, a n d t h e results of others e x a m i n i n g t h e e x p r e s s i o n of mutants  in t r a n s g e n i c p h o t o r e c e p t o r s  ( G r e e n e t al.,  rhodopsin  2 0 0 0 ; Moritz et  2 0 0 1 ; S u n g e t a / . , 1 9 9 4 ) , it is c l e a r t h a t p r o t e i n s d e s t i n e d f o r t h e r o d  al.,  Chapter 6 Conclusion F i g 4 4 . Z i p p e r i n g m o d e l f o r disc m o r p h o g e n e s i s in r o d s .  186 In n o r m a l  d i s c m o r p h o g e n e s i s , ( 1 ) c o r e t e t r a m e r s a s s e m b l e d in t h e E R a n d G o l g i are p a c k a g e d into transport vesicles a n d migrate to t h e connecting cilium (2) w h e r e t h e y e n c o u n t e r a c h e c k p o i n t . (3) V e s i c l e s c o n t a i n i n g properly assembled tetramers are actively targeted to the disc rim forming region w h e r e t h e y fuse with m e m b r a n e s of t h e g r o w i n g r i m . (4) N o n - c o v a l e n t tetramers in opposing membranes are linked together through intermolecular disulfide bonds by PDI to form disulfide-linked octamers which bind a n d stabilize the adjacent m e m b r a n e s . (5) Disulfide bonding of t e t r a m e r s facilitates a c o n f o r m a t i o n a l c h a n g e t h a t a l l o w s a s s o c i a t i o n of adjacent o c t a m e r s into higher-order oligomeric c o m p l e x e s which (6) drives elongation of the disc rim. (7) M e m b r a n e fusion catalyzed by the C - t e r m i n a l r e g i o n o f p e r i p h e r i n r e s u l t s in c l o s e d i n t a c t d i s c s w h i c h a r e separate from the plasma membrane. ( 8 ) F u s i o n a c t i v i t y is c a p p e d b y association with another protein (GARP) a n d mature discs migrate axially t o w a r d s t h e distal tip of t h e r o d . (9) D i s s o c i a t i o n of G A R P in t h e distal tip region allows fusion between disc and plasma m e m b r a n e s and p h a g o c y t o s i s b y t h e R P E . A b n o r m a l c o r e t e t r a m e r f o r m a t i o n ( a s in R P ) r e s u l t s i n m i s t a r g e t i n g o f p e r i p h e r i n - 2 t o t h e i n n e r s e g m e n t , (i) M u t a n t d i m e r s a s s e m b l e d in t h e E R a n d G o l g i a r e p a c k a g e d into t r a n s p o r t vesicles a n d migrate to the connecting cilium where they encounter the c h e c k p o i n t (ii) a n d a r e r e f u s e d e n t r y into t h e O S . (iii) V e s i c l e s c o n t a i n i n g mutant proteins are targeted for degradation and may affect photoreceptor fitness.  187  Golgi  Chapter 6 Conclusion  188  outer s e g m e n t are actively transported a n d must pass a checkpoint at the connecting  cilium.  In  the case  of  rhodopsin,  mutations  that  either  i n t e r f e r e w i t h t h e C - t e r m i n a l O S t a r g e t i n g s i g n a l o r t h o s e t h a t r e s u l t in a misfolded protein segment.  a r e d e t a i n e d a n d a c c u m u l a t e in v e s i c l e s o f t h e i n n e r  W e h a v e f o u n d w i t h p e r i p h e r i n - 2 , t h a t if c o r e t e t r a m e r s a r e n o t  assembled correctly, they also are turned  back at the connecting  cilium  a n d a c c u m u l a t e in v e s i c l e s in t h e i n n e r s e g m e n t . From complexes measure.  the  rod cell's  from  entering  It m u s t  perspective, the  outer  preventing  segment  nontetrameric  must  be  a  core  protective  be advantageous to have slightly decreased overall  l e v e l s o f p e r i p h e r i n - 2 in R O S t h a n t o a l l o w d e f e c t i v e c o r e c o m p l e x e s t o interfere with the d y n a m i c a n d sensitive process of disc m o r p h o g e n e s i s . T h i s s e n s i t i v i t y is d e m o n s t r a t e d in f r o g s e x p r e s s i n g P 2 1 6 L p e r i p h e r i n - 2 GFP.  T h i s m u t a t i o n c a u s e s n o d e t e c t a b l e d e f e c t i n o l i g o m e r i z a t i o n a n d is  presumably  not caught  by the checkpoint,  but  its  presence  in R O S  i n t e r f e r e s w i t h s o m e a s p e c t o f d i s c s t a b i l i t y t h a t r e s u l t s in p h o t o r e c e p t o r degeneration.  It  is  possible  that  if  the  photoreceptor  could  have  r e c o g n i z e d a defect in this protein a n d p r e v e n t e d trafficking to t h e o u t e r s e g m e n t , it c o u l d h a v e l e s s e n e d t h e d a m a g i n g e f f e c t s a n d i n c r e a s e d i t s chances for survival. T h e m o d e l i n Fig 4 4 p r e d i c t s d i s u l f i d e - m e d i a t e d o l i g o m e r i z a t i o n t o occur at the disc rim-forming  r e g i o n i n t h e o u t e r s e g m e n t a n d n o t in t h e  Chapter 6 Conclusion ER of the cell. protein  189 W e have evidence that the ER proteins calnexin and  d i s u l f i d e i s o m e r a s e ( P D I ) a r e p r e s e n t in R O S d i s c s ( C . L o e w e n  a n d R. M o l d a y , u n p u b l i s h e d r e s u l t s ) . and  disulfide  calnexin  bond  formation  or another  core tetramer  This suggests that protein  c a n o c c u r in d i s c s .  photoreceptor  specific protein  It  folding  is p o s s i b l e  that  associates with  the  i n t h e E R a n d p r e v e n t s it f r o m d i s u l f i d e b o n d i n g u n t i l it  reaches the rim forming region, where PDI then catalyzes disulfide-linking of  tetramers  and  rim  formation.  Alternatively,  the  geometric  r e q u i r e m e n t s f o r p r o p e r d i s u l f i d e - l i n k i n g o f t e t r a m e r s m a y n o t b e m e t in the  ER thereby  preventing  oligomerization.  A  unique  feature  of  p e r i p h e r i n - 2 o l i g o m e r i c c o m p l e x e s i s o l a t e d f r o m R O S m e m b r a n e s is t h e presence  of  oligomers. thought  to  noncovalent This  is v e r y  tetramers unusual  be an all-or-none  in  addition  since disulfide  to  disulfide-linked  bonding  process as the tertiary  and  is  generally quaternary  s t r u c t u r e o f t h e p r o t e i n c r i t i c a l l y d e p e n d s o n it. Further evidence for disulfide-mediated oligomerization at the disc rim  comes  variant.  from  analysis  of  transgenic  frogs  expressing  the  C150S  In t h e s e f r o g s , d e f e c t i v e d i s u l f i d e - m e d i a t e d o l i g o m e r i z a t i o n d o e s  not r e s u l t in m i s l o c a l i z a t i o n of C 1 5 0 S p e r i p h e r i n - 2 - G F P c o m p l e x e s to t h e inner segment.  A s i m p l e e x p l a n a t i o n f o r t h i s o b s e r v a t i o n is t h a t n o n -  disulfide  complexes are not detained  bonded  at the connecting  cilium  Chapter 6 Conclusion checkpoint  190  because disulfide-mediated  oligomerization  normally  occurs  a f t e r it a t t h e r i m f o r m i n g r e g i o n . It  is  possible  o c c u r s in t h e E R .  however,  that  disulfide-mediated  oligomerization  H e t e r o l o g o u s e x p r e s s i o n s t u d i e s in C O S - 1 c e l l s s h o w  t h a t d i s u l f i d e - m e d i a t e d oligomerization of p e r i p h e r i n - 2 o c c u r s in a similar manner  as  observed  in  ROS.  In  these  cells,  both  peripherin-2  noncovalent tetramers and disulfide-linked oligomers are observed.  It is  possible that the geometric requirements for complete disulfide-mediated o l i g o m e r i z a t i o n a r e n o t m e t in e i t h e r t h e E R o f C O S - 1 c e l l s o r t h e E R of photoreceptors. in i n c o m p l e t e  I m p r o p e r a l i g n m e n t of t e t r a m e r s in t h e E R w o u l d  disulfide-mediated  oligomerization  result  and could explain the  p r e s e n c e of n o n c o v a l e n t t e t r a m e r s in R O S . A n alternative e x p l a n a t i o n of the  results  observed  in  C O S - 1 cells  is  that  disulfide-mediated  o l i g o m e r i z a t i o n o c c u r s o u t s i d e of t h e E R in i n t r a c e l l u l a r v e s i c l e s s i m i l a r l y to p h o t o r e c e p t o r s .  T h e s e vesicles w o u l d m i m i c t h e r i m f o r m i n g region of  photoreceptors and m a y contain PDI and calnexin. Regulation of disc formation rom-1.  Incorporation  likely involves t h e inhibitory action of  of r o m - 1 with peripherin-2 oligomers at the r i m -  f o r m i n g r e g i o n will d e c r e a s e t h e e x t e n t of disulfide b o n d i n g a n d r e d u c e the level a n d size of higher order oligomers.  This likely m o d u l a t e s the  stability  of t h e rim region a n d also affects t h e rate  Because  rim formation  occurs independently  of rim  of m e m b r a n e  formation. evagination  Chapter 6 Conclusion  (Arikawa  er  191  al.,  1992; Steinberg,  imperative that the photoreceptor knockout mice, slightly  Fisher  &  Anderson,  1980),  regulate these processes.  of  diameter.  more  l a r g e r d i s c s a r e o b s e r v e d ( C l a r k e e r al.,  stable  disc  rims  that  allow  for even  I n f a c t , t h e p h o t o r e c e p t o r d i s c s o f Xenopus  2000).  or to the larger  laevis  is  In r o m - 1  This m a y be d u e either to increased rates of rim formation production  it  disc  c a n be ten  t i m e s l a r g e r in d i a m e t e r t h a n m a m m a l i a n d i s c s a n d c o n t a i n u p to s i x t e e n incisures which increase the rim surface area even greater. do  not appear to contain  Interestingly, rods  a rom-1 ortholog  Xenopus  ( K e d z i e r s k i et  w e do not observe proper incisure formation  expressing  C150S  analogous to what  peripherin-2-GFP.  This  is o b s e r v e d in m a m m a l i a n  al.,  rods 1996).  in t r a n s g e n i c  phenotype  rods which  may  be  express the  rom-1 g e n e a n d have f e w , or often only a single incisure. Analysis  of the photoreceptors  of h e t e r o z y g o u s  rds  mice  which  should contain only half t h e a m o u n t of p e r i p h e r i n - 2 a s W T reveals that disc  rim  formation  membranes Jansen,  incomplete  are present  1981).  peripherin-2  is  It  adjacent  appears,  and  large  whorls  to the connecting  therefore,  that  evagination.  cilium  insufficient  r e s u l t in t h e l a c k o f r i m f o r m a t i o n ,  process of m e m b r a n e  of  evaginating (Sanyal amounts  but d o not affect  This provides more  evidence  & of  the that  peripherin-2 creates the disc rim a n d that r o m - 1 cannot compensate for its a c t i v i t y .  Chapter 6 Conclusion  192  A m e m b r a n e f u s i o n e v e n t is r e q u i r e d u p o n m e e t i n g o f t h e t w o s i d e s of t h e g r o w i n g disc rim to seal t h e disc a n d create t h e p l a s m a m e m b r a n e . A role f o r t h e C - t e r m i n a l r e g i o n of p e r i p h e r i n - 2 in m e m b r a n e f u s i o n h a s b e e n p r o p o s e d , b u t h a s n o t b e e n c o n f i r m e d in v i v o ( B o e s z e - B a t t a g l i a e r al.,  1998).  If p e r i p h e r i n - 2 c a t a l y z e s t h i s f u s i o n e v e n t i n r o d s , t h e n i t s  fusion activity  must  be inhibited  in t h e m a t u r e  distally t o w a r d s t h e tip of t h e r o d . has  disc while  it  A g l u t a m i c acid rich protein  migrates (GARP),  been found to interact with the cytoplasmic region of only disulfide-  linked  peripherin-2  complexes  unpublished results). binding  to  formation disc  (A.  as disulfide-mediated  are occurring. and  rods  Poetsch  and  R.  Molday,  G A R P m a y function to c a p the fusion activity  peripherin-2  rims  in  between  by  oligomerization  and rim  G A R P m a y also mediate interactions  between  rims  and  the  plasma  membrane  interactions between peripherin-2 and the p-subunit  through  of t h e c G M P  gated  tip of t h e r o d , a signaling  event  channel. As initiated GARP again.  the discs  reach the distal  by the R P E (or possibly another  from  the  rim a n d allow  photoreceptor)  peripherin-2-catalyzed  m a y release  fusion  to  occur  T h i s w o u l d r e s u l t in f u s i o n b e t w e e n t h e d i s c r i m a n d t h e p l a s m a  m e m b r a n e a n d r e l e a s e of a p a c k e t of d i s c s ( s e e Fig 5 in t h e i n t r o d u c t i o n ) . Penetration  of  the  outer  segment  by  pseudopodia  of  the  RPE and  Chapter 6 Conclusion ingestion  193  m a y increase  the  local  reducing  potential  a n d initiate  the  process of rim a n d disc collapse. The  differences  observed  between  discs of rod a n d cone  outer  s e g m e n t s m i g h t also be d u e to differential regulation of o l i g o m e r i z a t i o n of peripherin-2.  It h a s b e e n p r o p o s e d t h a t r i m f o r m a t i o n  in c o n e s o c c u r s  m o r e s l o w l y a n d t h e r e f o r e is n o t o f t e n c o m p l e t e u n t i l t h e d i s c s n e a r t h e d i s t a l t i p o f t h e p h o t o r e c e p t o r ( A r i k a w a e r al., the  majority  of  cone  discs  remain  open  1992).  to  the  T h e r e s u l t is t h a t  extracellular  space.  Peripherin-2 h a s been found to extend along the discs only a s far a s the r i m in c o n e s .  If c o n e s s y n t h e s i z e l e s s t o t a l p e r i p h e r i n - 2 , o r if t h e r a t i o o f  p e r i p h e r i n - 2 t o r o m - 1 is d e c r e a s e d c o m p a r e d t o r o d s ( w h e r e (Loewen & Molday, 2000)), oligomers of peripherin-2 formation  the size a n d a b u n d a n c e of disulfide-linked  will  m a y result - this  it i s 2 : 1  be decreased and a slower  rate  of rim  being s o m e h o w a d v a n t a g e o u s to the cone  cell.  6.4  FUTURE DIRECTIONS Many  questions  remain  unanswered  functionally.  The importance  of  photoreceptor  c e l l s is o b v i o u s .  It  -  peripherin-2  both in  structurally  maintaining  plays an intriguing  role in  and  healthy modeling  m e m b r a n e s a n d has a n u m b e r of unique structural features that imply a m e c h a n i s m f o r its a c t i o n .  A c r y s t a l s t r u c t u r e o f t h e c o m p l e x is a t t h e t o p  Chapter 6 Conclusion  194  of t h e list o f p r i o r i t i e s . technology, possible  W i t h c u r r e n t a d v a n c e s in X - r a y c r y s t a l l o g r a p h y  crystallization  (Moffat,  1997;  of  whole  membrane  Pebay-Peyroula  er  protein  al.,  complexes  1997).  The  is  high  a b u n d a n c e o f p e r i p h e r i n - 2 in b o v i n e p h o t o r e c e p t o r s a n d t h e a v a i l a b i l i t y of l a r g e a m o u n t s o f b o v i n e r e t i n a l t i s s u e m a k e it p o s s i b l e t o p u r i f y e n o u g h c o m p l e x for t h e s e t y p e s of studies.  Also, expression of the intradiscal  l o o p r e g i o n in a b a c t e r i a l s y s t e m a n d s t r u c t u r a l a n a l y s i s i n c l u d i n g X - r a y crystallography and N M R techniques would structural  studies  include:  electron  be very  informative.  microscopic analysis of  More  oligomeric  c o m p l e x e s in t h e d e t e r g e n t s o l u b i l i z e d a n d r e c o n s t i t u t e d s t a t e s ; effect of reductant on the s h a p e a n d stability of photoreceptor discs; isolation a n d characterization of peripherin-2-containing vesicles f r o m transfected cells by i m m u n o - E M ; m a p p i n g of the proposed intramolecular disulfide bonds within the loop region by proteolysis a n d HPLC analysis. E x p e r i m e n t s c a n be d e s i g n e d also to a d d r e s s t h e role of disulfidemediated  oligomerization  impermeable sulfhydryl  in d i s c f o r m a t i o n .  T h e u s e of  r e a g e n t s in c o n j u n c t i o n  membrane-  with labeling dyes  like  L u c i f e r y e l l o w t o s t u d y d i s c f o r m a t i o n in p h o t o r e c e p t o r s in e y e c u p s s h o u l d d e t e r m i n e t h e i m p o r t a n c e of disulfide b o n d i n g at t h e d i s c - f o r m i n g and  identify  revealed  the  proteins  involved.  t h e p r e s e n c e of proteins  with  Preliminary free  experiments  sulfhydryl  r e g i o n ( C . L o e w e n a n d R. M o l d a y , u n p u b l i s h e d ) .  region  groups  have in  this  Antibodies generated to  Chapter 6 Conclusion  195  t h e i n t r a d i s c a l r e g i o n o f p e r i p h e r i n - 2 m a y a l s o b e u s e f u l in t h i s t y p e study.  of  S i n c e a n a t u r a l k n o c k o u t is a v a i l a b l e , t r a n s g e n i c s t u d i e s in m i c e  adding-back C 1 5 0 S peripherin-2 or other chimeras would informative  in  determining  the  mechanisms  of  disc  be  extremely  morphogenesis.  Certain research g r o u p s are also using inducible p r o m o t e r s to begin  to  study this role for p e r i p h e r i n - 2 . Studies  in  transgenic  Xenopus  examining  outer s e g m e n t targeting are currently underway. u s e d to  test the  role of the  f u s i o n in v i v o .  Interestingly,  morphogenesis  due  another  has found  group  peripherin-2 2001). frog  with  a  to  the  to  a  for  T h i s s y s t e m c a n also be in  membrane  w e d i d n o t f i n d a n y s e r i o u s d e f e c t s in d i s c p r e s e n c e of  no  truncated  obtain  requirements  C-terminus of peripherin-2  serious  G F P at  problems  C-terminus  (Lee,  Also, more disease causing mutations  system  the  more  in  C-terminus,  frogs  that  Burnside &  and  express Flannery,  c a n b e e x a m i n e d in  comprehensive  molecular basis for retinal dystrophies.  the  understanding  of  the the  Lastly, the role of G A R P a n d the  i n t e r a c t i o n of p e r i p h e r i n - 2 w i t h o t h e r p r o t e i n s , p o s s i b l y c y t o s k e l e t a l , will tell us m o r e a b o u t t h e o r g a n i z a t i o n of p h o t o r e c e p t o r o u t e r s e g m e n t s .  196  References  REFERENCES A M , R. R . , S a r r a , G . 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