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Characterization of two C. elegans molecular chaperone families, CCT (chaperonin containing TCP-1) and… Leroux, Michel Rejean 1997

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Characterization of two C. elegans molecular chaperone families, C C T (chaperonin containing TCP-1) and the small heat shock proteins by Michel Rejean Leroux B. Sc., McGill University, 1991 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy in THE FACULTY OF GRADUATE STUDIES Department of Biochemistry and Molecular Biology  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA June 1997 © Michel Rejean Leroux  in  presenting  degree freely  at  this  the  available  copying  of  department publication  of  in  partial  fulfilment  of  the  University  of  British  Columbia,  I  agree  for  this or  thesis  reference  thesis by  this  for  his thesis  and  scholarly  or for  her  Department The University of British Columbia Vancouver, Canada  DE-6 (2/88)  I further  purposes  gain  shall  that  agree  may  representatives.  financial  permission.  Date  study.  requirements, for  be  It not  that  the  be  by  understood allowed  the  advanced  shall  permission for  granted  is  Library  an  make  extensive  head  that  without  it  of  copying my  my or  written  Abstract  ABSTRACT  * Molecular chaperones belong to a class of proteins whose function is to interact with and stabilize proteins that are partly or totally unfolded, as is the case when proteins are in the process of being synthesized, translocated into an organelle, or damaged by cellular stress. The work presented in this thesis describes the first studies aimed at characterizing some of the structural and functional properties of two molecular chaperone families of the nematode Caenorhabditis  elegans,  CCT (chaperonin containing TCP-1) and the small heat shock proteins  (smHSPs). CCT is involved in folding newly synthesized actin and tubulin, and may play a more general role in protein folding within the eukaryotic cytosol. C. elegans CCT is an ATP-binding complex of about 900 kDa. Sucrose gradient fractionation and ATP-agarose chromatography were used to purify the CCT complex from embryos. Over 7 subunits ranging between 5265 kDa were detectable, of which two were shown to be CCT-1 and CCT-5 by immunoblotting. Native gel electrophoresis of the CCT revealed three distinct species: one contains CCT-1 and CCT-5 and hence represents the CCT complex, another contains HSP60, which has the highest affinity for chemically-denatured actin, and the last species remains unidentified. A 3.7 kb Pstl genomic fragment encoding the 59 kDa CCT-1 protein was cloned and sequenced. The cct-1 transcript undergoes both c/s-splicing of its four introns and frans-splicing to SL1. Three additional cet cDNAs were sequenced (cct-2, cct-4, and cct-5), and the sequence of a fifth cet gene (cct-6) was obtained from the C. elegans sequencing consortium. The C. elegans cet multigene family displays 23-35% sequence identity between members and about 65% identity to the corresponding murine cet homologues. Northern blot analyses show that the five C. elegans cet genes are expressed in all life stages. Transgenic lines carrying a ccf-i-promoterlacZ  construct revealed that cct-1 is expressed in various tissues, including muscles and the  nervous system. The smHSP and a-crystallin genes encode a family of proteins which assemble into large multimeric structures, function as chaperones by preventing protein aggregation, and contain a ii  Abstract conserved region termed the cc-crystallin domain. Studies on wild-type HSP16-2 and five derivatives demonstrate that multimerization and chaperone activity depend on the full-length nonconserved N-terminal region and are not affected by removal of most of the C-terminal extension which follows the cx-crystallin domain. The N-terminal region of HSP16-2 is buried within an oligomeric complex which can accomodate an additional 4 kDa of heterologous protein sequence. It was found that HSP16-2 has an equally high affinity for unfolded actin and tubulin intermediates which form early on the renaturation or aggregation pathway. The structure-function data on HSP16-2 are complemented by studies on HSP12.6, the smallest smHSP to be characterized. In contrast to other smHSPs, the stage-specific HSP12.6 does not multimerize and lacks chaperone activity.  iii  Table of Contents T A B L E OF CONTENTS  ABSTRACT  ii  TABLE OF CONTENTS  iv  LIST OF FIGURES  viii  LIST OF TABLES  ix  LIST OF ABBREVIATIONS  x  ACKNOWLEDGMENTS  xii  CHAPTER I—Studies on the structure, function, and expression of C. elegans C C T  1  I. INTRODUCTION  1  Protein folding  1  Molecular chaperones  2  Chaperonins  5  Chaperonin evolutionary relationships  5  Function of Group I chaperonins  7  Structure-function studies on GroEL and GroES  8  Structure of Group II chaperonins  9  Function of CCT in vitro and in vivo  11  Expression and localization of CCT  12  C. elegans as a model organism  14  The present study  14  II. MATERIALS AND METHODS  16  2.1 Bacterial strains and media  16  2.2 Molecular biological techniques  16  2.2.1 Gel electrophoresis and isolation of DNA fragments  16  2.2.2 Ligations  16  2.2.3 Transformations  17  2.2.4 Purification of plasmid and cosmid DNA  17  2.2.5 Double stranded DNA sequencing  18  2.2.6 Polymerase chain reaction  18  2.2.7 Preparation of DNA, RNA and first strand cDNA  18  2.2.8 Southern and DNA dot blots  19  2.2.9 Northern blots  19 iv  Table of Contents 2.2.10 Preparation of radiolabeled DNA probes  20  2.2.11 Nucleic acid hybridization conditions  20  2.3 General protein and immunological techniques  20  2.3.1 Denaturing and native poly aery lamide gel electrophoresis  20  2.3.2 Polyclonal antibody production  21  2.3.3 Affinity purification of antibodies  21  2.3.4 Western blot analysis  21  2.3.5 Miscellaneous  ;  22  2.4 Purification of Chaperonin Containing TCP-1 (CCT)  22  2.5 Construction and analysis of transgenic nematodes  23  2.5.1 Preparation of constructs for injection  23  2.5.2 Injection of the construct  24  2.5.3 Histochemical detection of P-galactosidase in transgenic lines  24  2.6 Sequence alignments and secondary structure predictions III. RESULTS  24 25  3.1 Sequence analysis of five CCT proteins  25  3.1.1 Sequence determination of five cet cDNAs from a multigene family  25  3.1.2 Primary structure analyses of five CCT proteins  26  3.1.3 Predicted secondary structures of CCTs and other chaperonins  28  3.2 Isolation of genomic cct-1  32  3.3 Physical map positions of five cet genes  37  3.4 Expression of cet genes throughout nematode development  37  3.5 Expression of cct-1 after heat-shock treatment  40  3.6 cct-1 undergoes both czs-splicing of its introns and rrans-splicing to SL1  40  3.7 Expression pattern of ccf-7-promoter-P-galactosidase fusion transgene  43  3.8 Purification of CCT and analysis of protein subunits  45  3.8 Affinity of CCT for denatured actin  48  IV. DISCUSSION  50  A novel eukaryotic cytosolic chaperonin...  50  The cet multigene family of C. elegans  50  Secondary structure analysis of C. elegans CCTs and other chaperonins  53  Denatured actin-binding activities in C. elegans CCT preparations  55  Expression of cet genes in C. elegans and other organisms  57  Future studies  •  v  58  Table of Contents CHAPTER II—Structure-function studies on C. elegans HSP16-2 and HSP12.6 I. INTRODUCTION  60 60  Small HSP family  60  Small HSP family of C. elegans  62  Structure of small HSPs  65  Mechanism of small HSP chaperone function  67  Cellular functions of small HSPs  68  The present study  71  II. MATERIALS AND METHODS  73  2.1 Cloning of hspl2.6 cDNA and preparation of small HSP constructs  73  2.2 Expression and purification of the small HSPs  73  2.3 Molecular weight estimation of the small HSPs  75  2.4 Production and purification of anti-HSP12.6 antibody  75  2.5 Western blot analysis of HSP12.6  76  2.6 Cross-linking of small HSPs  76  2.7 Assay for H6HSP16-2 binding to Ni2+-chelate affinity resin *.  76  2.8 Thermal aggregation studies  77  2.9 Chemical aggregation studies  77  2.10 Native gel analysis of HSP16-2-actin/tubulin binary complexes  77  2.11 Sedimentation velocity and fluorescence measurements  78  2.11.1 Actin and tubulin purification and labeling  78  2.11.2 Fluorescence measurements  79  2.11.3 Sedimentation velocity measurements  79  2.12 Miscellaneous  79  III. RESULTS  81  3.1 Structure and function of HSP16-2  81  3.1.1 Production of HSP 16-2 variants for structure-function studies  81  3.1.2 HSP16-2 quaternary structure and subunit orientation  82  3.1.3 HSP 16-2 interacts with unfolded CS and prevents its aggregation  84  3.1.4 Complex formation between HSP 16-2 and denatured actin and tubulin  89  3.1.5 Chaperone assembly and activity is not affected by C-terminal deletion  93  3.1.6 Correlation between multimerization and chaperone activity  93  3.2 Structure, function and expression of HSP12.6 3.2.1 A novel class of small HSPs from C. elegans vi  96 96  Table of Contents 3.2.2 Cloning, expression, and purification of HSP12.6  99  3.2.3 The expression of HSP12.6 is developmentally regulated  102  3.2.4 The quaternary structure of HSP12.6 differs from that of other smHSPs  102  3.2.5 The in vivo M r of HSP12.6 is identical to that of the recombinant protein...105 3.2.6 HSP12.6 may be functionally distinct from other smHSPs IV. DISCUSSION  :  105 108  Structure of smHSPs  108  Function of smHSPs  112  The data on HSP12.6 complement the HSP16-2 studies  116  Models summarizing HSP16-2 structure and function  118  Future studies  119  REFERENCES  123  APPENDIX  141  I. Bacterial strains used in this study  141  II. List of oligonucleotides used in this study  142  III. Conditions used for polymerase chain reactions  144  IV. Large-scale culturing of C. elegans and isolation of embryos 4.1 Maintenance of C. elegans on plates and in liquid media  145 145  4.2 Preparatory culture used for inoculating 20 litre culture  145  4.3 First 20-litre culture  145  4.4 Second 20-litre culture  147  V . Transgenic lines generated and used in this study VI. C. elegans strain containing a T e l insertion in cct-1 gene  148 149  VII. Antibodies used in this study  150  VIII. Sedimentation velocity analyses of smHSPs  151  vii  List of Figures LIST OF FIGURES Figure 1. Summary of some chaperone functions in the cell  3  Figure 2. Multiple alignment of five C. elegans CCT proteins and TF55  27  Figure 3. Multiple alignment of primary and predicted secondary structures of chaperonins ..30 Figure 4. Southern blot analysis of C. elegans cct-1  33  Figure 5. Isolation of cct-1 from the cosmid T05C12  35  Figure 6. Nucleotide and amino acid sequence of C. elegans genomic cct-1  36  Figure 7. Physical map positions of five C. elegans cet genes  38  Figure 8. Northern blot analysis of five C. elegans cet genes  39  Figure 9. Northern blot analyses of heat-shocked C. elegans RNA  41  Figure 10. The cct-1 primary transcript is trans-spliced to the splice leader-1 (SL1) RNA  42  Figure 11. Localization of cct-1 transgene expression  44  Figure 12. Purification C. elegans CCT and identification of subunits  47  Figure 13. Native gel analysis and affinity of CCT and HSP60 for denatured actin  49  Figure 14. Alignment of C. elegans smHSPs  63  Figure 15. Summary of smHSP constructs, purified proteins, and alignment  83  Figure 16. Elution profile of HSP16-2 and derivatives  85  Figure 17. Affinity of native and denatured H6HSP16-2 for nickel affinity resin  86  Figure 18. Three techniques for measuring the chaperone activity of smHSPs  88  Figure 19. HSP16-2 prevents thermally- and chemically-unfolded CS from aggregating  91  Figure 20. Binary complex formation between HSP16-2 and P-actin and P-tubulin  92  Figure 21. Fluorescence quenching studies  94  Figure 22. HSP16-2 lacking most of its C-terminal extension has chaperone activity  97  Figure 23. Chemical cross-linking of smHSPs  98  Figure 24. Comparison of the C. elegans HSP12 protein family with other smHSPs  100  Figure 25. Overexpression in E. coli and purification of HSP12.6  101  Figure 26. HSP12.6 is present in C. elegans LI larvae  103  Figure 27. Size exclusion chromatography and cross-linking of HSP12.6  104  Figure 28. Size determination of HSP12.6 isolated from C. elegans  106  Figure 29. HSP12.6 lacks molecular chaperone activity  107  Figure 30. Model of in vivo function and oligomeric structure of smHSPs  120  Figure 31. C. elegans strain NL708/PK58 contains a Tel insertion near cct-1  149  Figure 32. Specificities of the polyclonal antibodies against CCT and HSP16-2  150  viii  List of Tables LIST OF TABLES  Table 1. E. coli strains used for plasmid propagation and protein expression  141  Table 2. Oligodeoxyribonucleotides used for sequencing, PCR, and/or subcloning  142  Table 3. Summary of PCR conditions used for amplifying DNA from various sources  144  Table 4. Hydrodynamic parameters of HSP16-2, two derivatives, and HSP12.6  151  ix  LIST OF ABBREVIATIONS  aa  amino acid(s)  ATP  adenosine 5'-triphosphate  bp  base pair(s)  BSA  bovine serum albumin  C. elegans  Caenorhabditis elegans  CCT  chaperonin containing TCP-1 protein  cet  chaperonin containing tcp-1 gene  cDNA  DNA complementary to coding strand  cpm  counts per minute  dATP  deoxyadenosine 5'-triphosphate  dCTP  deoxycytidine 5'-triphosphate  dGTP  deoxyguanosine 5'-triphosphate  dH 0  distilled water  DNA  deoxyribonucleic acid  dNTP  dATP, dCTP, dGTP, dTTP  DTT  dithiothreitol  dTTP  deoxythymidine 5'-triphosphate  E. coli  Escherichia  ECL  enhanced chemiluminescence  EDTA  ethylenediamine tetraacetic acid  GTP  guanosine 5'-triphosphate  HSE  heat shock element  HSP  heat shock protein  hsp  heat shock protein gene  IPTG  isopropyl-p-D-thiogalactopyranoside  kb  kilobase pair(s)  kDa  kilodalton(s)  L1-L4  four C. elegans larval stages  mAb  monoclonal antibody  MDa  megadalton(s)  MES  2- [N-morpholino] ethanesulfonic acid  2  coli  X  List of Abbreviations molecular mass mRNA  messenger RNA  MW  molecular weight  NG  nutrient growth (medium)  NP  50 mM NaCl, 50 mM sodium phosphate  nt  nucleotide  pAb  polyclonal antibody  PAGE  polyacrylamide-gel electrophoresis  PCR  polymerase chain reaction  PMSF  phenylmethylsulfonyl fluoride  PND  25 mM sodium phosphate pH 7.0, 25 mM NaCl, 0.5 mM DTT  PVDF  polyvinylidene difluoride  RNA  ribonucleic acid  RNAse  ribonuclease  SDS  sodium dodecyl sulphate  SL  splice leader  smHSP  small heat shock protein  SSPE  180 mM NaCl, 1 mM EDTA, 10 mM NaH P0 ; pH 7.4  Taq  Thermus aquaticus  TB  terrific broth  TBE  90 mM Tris-borate pH 8.3; 2 mM EDTA  TBS-T  25 mM Tris-HCl, 140 mM NaCl, 0.05% Tween 20; pH 7.4  tcp  r-complex polypeptide gene  TCP-1  ^-complex polypeptide-1 protein  TE  10 mM Tris-HCl, pH 8.0; 1 mM EDTA  TEDKM  50 mM Tris pH 7.4, 0.1 mM EDTA, 1 mM DTT, 50 mM KC1, 10 mM MgCl  TEND  50 mM Tris-HCl, 0.1 mM EDTA, 50 mM NaCl, 1 mM DTT; pH 7.5  TER  10 mM Tris-HCl, pH 8.0; 1 mM EDTA; 1 ug/ml RNAse A  Tris  tris (hydroxymethyl) aminomethane  Tween 20  polyoxyethylene-sorbitan monolaurate  X-GAL  5-bromo-4-chloro-3-indolylgalactosidase  YAC  yeast artificial chromosome  2  4  2  Aknowledgments ACKNOWLEDGMENTS  I would like to give my warmest thanks to my supervisor, Peter Candido, for giving me the opportunity to work on projects that have been very interesting and exciting. Throughout my degree, I have benefited tremendously from many helpful discussions with my lab colleagues, Don, Mei, Dave, Tracy, Bruce, Brian, Sandra, Eve, and Emily. Many of these people have been my friends for many years, and the work environment they created was always productive and enjoyable at the same time. Anyone working with C. elegans knows that researchers from the C. elegans community are very communicative, helpful, and generous. The C. elegans genomic DNA yeast YAC library and cosmids were kind gifts from John Sulston and Alan Coulson (Sanger Centre, Cambridge, UK), and the cet cDNA clones were provided by Robert Waterston (Dept. of Genetics, Washington University, St. Louis). Richard Durbin and Steve Jones (St. Louis) have been helpful in locating and deciphering information from the C. elegans database ACeDB. Research would not be as rewarding if there were no collaborations. I would like to thank Keith Willison (Institute of Cancer Research, Chester Beatty Laboratories, London, UK) for providing samples of his UM1 antibody, as well as Bruce Gordon and Brian Ma (UBC) for helping out with some smHSP purifications, molecular weight determinations, and aggregation assays. Peter also contributed to my research by performing the injections needed to create transgenic strains. A very special thanks to Ronald Melki (Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette, France) for supplying [ S]-labeled actin and 35  tubulin in addition to a sample of purified bovine testis CCT, and for providing me with sedimentation and fluorescence quenching data on some of the smHSPs. Gerard Batelier's (CNRS) expert help with the sedimentation velocity measurements is also appreciated. Most importantly, I would like to thank my wife Tracy for her boundless enthusiasm for my career in health sciences. But above all, merci de m'aimer pour quije suis et de m'avoir montre qu 'il y a plus que la science dans la vie.  xii  Chapter I: Introduction  CHAPTER I— Studies on the structure, function, and expression of C. elegans C C T  Protein folding is one of the most important process in living organisms, as it produces the three-dimensional conformation required by proteins for their specific properties. In this chapter we explore various aspects of a eukaryotic molecular chaperone that has only recently come to light, but whose function appears essential in assisting the proper folding of two of the most abundant cellular proteins, namely the cytoskeletal elements actin and tubulin.  I. INTRODUCTION  Protein folding The process of protein folding, or the, acquisition of tertiary structure, has long been believed to be a spontaneous event, guided strictly by the information contained within the amino acid sequence of a protein. Early work on protein folding by Anfinsen using ribonuclease A demonstrated that if this protein was completely unfolded in denaturant and then diluted into a buffer, it could spontaneously regain full activity (Anfinsen, 1973). Although many purified proteins can refold in vitro under certain conditions, protein misfolding and aggregation is a frequent and major problem which rarely occurs in vivo (Gething and Sambrook, 1992). The reason is that unfolded polypeptides expose core hydrophobic amino acid side chains which tend to associate with each other incorrectly both inter- and intra-molecularly, resulting in the formation of aggregates (Hendrick and Hartl, 1993). Although the extent of aggregation can sometimes be controlled in the test tube by lowering the concentration of the refolding protein or lowering the temperature, the physiological relevance of in vitro refolding experiments is now regarded with caution, for two major reasons. The first reason concerns the environment in which protein folding occurs within the cell. Not only is the concentration of unfolded, nascent chains in the cytoplasm very high (approximately 35 (iM in E. coli, for example; Ellis and Hartl, 1996), but molecular crowding by the bulk of the cellular proteins is 1  Chapter I: Introduction predicted to cause a tremendous increase in molecular association constants over those in dilute solution (Hard, 1996). Secondly, because of the cooperativity of the folding process, the formation of stable tertiary structures requires the presence of a complete polypeptide or at least a complete protein domain (Hartl et al, 1994). Since the polypeptide chain is synthesized de novo in an extended conformation, unproductive associations of the nascent chain with itself or other proteins would be strongly preferred over the correct folding pathway unless a specific machinery were present during most of the folding process to prevent off-pathway reactions. The components of this machinery are termed molecular chaperones.  Molecular chaperones The term 'molecular chaperone' was first used to describe the unique function of the nuclear protein nucleoplasm^ in assisting chromatin assembly (Laskey et al., 1978). This term was later applied more generally to a range of cellular proteins whose role is to promote the folding and assembly of unfolded and partially unfolded forms of other proteins (Ellis, 1987). Chaperones serve many functions that stem from their ability to recognize and modulate the state of folding within cells. The basic, revised definition of a molecular chaperone is "any protein that binds to and stabilizes an otherwise unstable form of another protein, and by controlled binding and release, facilitates its correct fate in vivo, be it folding, oligomeric assembly, transport to a particular subcellular compartment, or disposal by degradation" (Hartl, 1996). Numerous reviews on the subject of molecular chaperones are available (Morimoto et al., 1990; Gething and Sambrook, 1992; Welch, 1993; Georgopoulos and Welch, 1993; Hendrick and Hartl, 1993; Parsell and Lindquist, 1994; Buchner, 1996; Hartl, 1996; Martin and Hartl, 1997). The various roles for some of the well-known chaperones are illustrated in Figure 1. The involvement of molecular chaperones during and following cellular stresses will be discussed in Chapter II, where studies on HSP16-2, a strictly stress-inducible small HSP, are presented.  2  Chapter I: Introduction  Figure 1. Summary of some chaperone functions in the cell. The folding of some nascent polypeptide chains in the eukaryotic cytosol requires a specific machinery consisting of HSP70, its partner HSP40, and CCT. HSP40 and HSP70 (dimer) first interact with the emerging polypeptide, preventing its premature folding and aggregation (A). The partially folded protein is then passed on to CCT for folding to its native state. Proteins destined for the mitochondria require HSP70 and HSP40 to remain in a translocation-competent state (B), and mitochondrial HSP70 (mtHSP70) and HSP60 for proper folding and oligomeric assembly inside the organelle (C). When cells are subjected to stress, some proteins become damaged and unfolded (D), which may result in their aggregation and loss of function (E). Alternatively, these proteins interact with various chaperones such as small HSPs, HSP70, and HSP104 (F). Either through the direct action of these chaperones, or by further intervention of other chaperones, the unfolded proteins may be refolded (G). Alternatively, the damaged proteins may be degraded via the ubiquitinand proteasome-dependent degradation pathway (H). 3  Chapter I: Introduction One of the well-studied functions of chaperones is their ability to assist in the folding of de novo synthesized proteins. A number of proteins have been found to associate with nascent chains in the eukaryotic cytosol, and are believed to be components required to ensure the proper folding of the polypeptide emerging from the ribosome. The first component which is thought to interact with nascent chains is the nascent-chain-associated complex (NAC), which is a heterodimer of 33 and 21 kDa subunits. NAC shields the first -33 residues adjacent to the peptidyl transferase site and prevents the association of the ribosome with the endoplasmic reticulum (ER) membrane unless the polypeptide contains a signal sequence recognized by the signal-recognition particle (SRP) (Wiedmann et al., 1994; Wang et al., 1995). NAC may also be involved in recruiting two chaperones involved in maintaining the nascent polypeptide in a folding-competent conformation, HSP70 and its co-chaperone HSP40. Protein translocation across the ER and mitochondrial membranes also requires the participation of HSP70 and HSP40, which together maintain the polypeptide in a translocation-competent conformation (Hendrick et al, 1993; Hohfeld and Hartl, 1994). HSP70 and HSP40 have been co-purified with ribosome-bound polypeptide chains, and recognize polypeptides less than 85 residues in length (Beckmann et al, 1990; Hendrick et al, 1993; Frydman et al, 1994a). HSP40 is believed to recruit HSP70 onto the nascent chain (Frydman et al, 1994a), where it regulates the ATPase activity of HSP70 (Hohfeld et al, 1995). Yet another factor involved in the HSP70/HSP40 reaction cycle is Hip (HSP70-interacting protein), which itself has chaperone activity and can modulate the activity of HSP70 (Hohfeld et al, 1995). Together, these chaperones maintain the aggregation-sensitive nascent chains in a folding-competent conformation through their co-translational association (Hartl, 1996). After a sufficient length of polypeptide destined for the cytosol has been produced, it is transferred by HSP70/HSP40 to a specialized class of molecular chaperone termed 'chaperonin' for eventual folding to its native state.  4  Chapter I: Introduction  Chaperonins The term 'chaperonin' denotes a family of sequence-related proteins of -60 kDa found in eukaryotic mitochondria and chloroplasts, and in eubacteria such as E. coli (Hernmingsen et al., 1988). These ubiquitous proteins were later shown to exist in the eukaryotic cytosol (Ellis, 1990; Gupta, 1990) and in archaebacteria (Trent et al., 1991). Chaperonins form a distinct class of ATP-dependent molecular chaperones involved in assisting de novo protein folding (Hendrick and Hartl, 1993; Horwich and Willison, 1993; Frydman and Hartl, 1996; Ellis and Hartl, 1996; Hartl, 1996; Martin and Hartl, 1997).  Chaperonin evolutionary relationships Two chaperonin families are known, and can be classified on the basis of evolutionary relationships, structures, and functions. One family (Group I) is represented by eubacterial GroEL, mitochondrial Hsp60, and plastid Rubisco subunit-binding protein (RuBP). The eukaryotic Hsp60 and RuBP chaperonins share a high degree of deduced amino acid sequence identity with GroEL (45-60%), presumably because they are localized within organelles believed to originate from the endosymbiotic capture of a proteobacterium and cyanobacterium, respectively (Gray, 1992; Viale and Arakaki, 1994). In addition to their sequence similarity and shared eubacterial lineage, the so-called classical chaperonins are composed of one or two types of subunits, possess a common quaternary structure consisting of 14 subunits arranged as two stacked toroids with 7-fold rotational symmetry, and usually require an -10 kDa co-chaperonin (termed GroES or chaperonin 10) for function (Hernmingsen et al., 1988; Lubben et al, 1990; Martin et al., 1993; Horwich and Willison, 1993). The other family (Group II) consists of eukaryotic cytosolic and archaebacterial chaperonins. In the eukaryotic cytosol, the chaperonin named CCT (Chaperonin Containing TCP-1) or TRiC (TCP-1 Ring Complex; Frydman et al., 1992) is encoded by 8 members of a Cet multigene family which shares 25-36% pairwise predicted amino acid sequence identity between members (Kubota et al., 1994; Kubota et al, 1995a; Stoldt et al, 1996). An additional testis-specific Cet gene was recently identified (Kubota et al., 1997). TCP-1 (CCTa) was the 5  Chapter I: Introduction first protein from the cytosolic chaperonin to be identified (Lewis et al, 1992; Yaffe et al, 1992). The murine and bovine CCT protein subunits (denoted CCTa, p\ y, 5, e, etc.) assemble to form a hetero-oligomeric double-ring complex with 8- or 9-fold rotational symmetry (Frydman et al, 1992; Gao et al, 1992; Lewis et al, 1992; Marco et al, 1994). Because of the great number of variations in naming the CCT chaperonin genes and subunits, a consistent nomenclature has been proposed (Stoldt et al, 1996), where genes are named cctl, cct2, etc., subunits are designated Cctlp, Cct2p, etc., and the complex is called CCT. However, to be consistent with the accepted C. elegans nomenclature (Hodgkin, 1995), the C. elegans genes are designated as cct-1, cct-2, etc., the proteins they encode as CCT-1, CCT-2, etc., and the complex is referred to as CCT. Remarkably, the subunits of the eukaryotic cytosolic chaperonin are very closely related to a subunit of the archaebacterial chaperonin TF55 (32-39% identity), and are only weakly similar to GroEL (Kubota et al, 1994). The considerable similarity between the eukaryotic cytosolic and archaebacterial chaperonins agrees with phylogenetic analyses which suggest that the eukaryotic cell nucleus evolved from an archaebacterial ancestor (Iwabe et al, 1989; Rivera and Lake, 1992). The evolution of the CCT/TF55 family is also consistent with an alternative chimeric model for the origin of the eukaryotic cell nucleus (Gupta, 1995), which proposes that a fusion between a thermoacidophilic archaebacterium and a Gram-negative bacterium gave rise to a pro-eukaryotic cell (Gupta and Singh, 1994; Irwin, 1994). The quaternary structures of the TF55 chaperonins from the archaebacteria Sulfolobus shibatae and Sulfolobus solfataricus, double toruses with 9 subunits per ring, are also more similar to CCT than to GroEL (Trent et al., 1991; Knapp et al, 1994). An abundant chaperonin isolated from the hyperthermophile Pyrodictium occultum is composed of two types of subunits, like TF55, but seems to possess mostly 8 rather than 9 subunits per ring (Phipps et al, 1991, 1993). Another distinguishing feature of the eukaryotic cytosolic and archaebacterial chaperonins is that they do not appear to associate with a chaperonin-10-like co-chaperonin. Recently, Guagliardi et al. (1994, 1995) have demonstrated that the S. solfataricus chaperonin can prevent the thermal aggregation as well as fold several thermophilic and mesophilic enzymes in vitro. Hence, the eubacteria, 6  Chapter I: Introduction archaebacteria, and eukaryotes all possess one or more evolutionarily related chaperonins whose ubiquitous function is to assist protein folding.  Function of Group I chaperonins Functional studies on chaperonins provided the first evidence that protein folding within cells is a chaperone-dependent process. The E. coli chaperonin GroEL was originally shown to participate in the morphogenesis of bacteriophage X and T4 head structures, suggesting a role in protein assembly (Georgopoulos et al, 1973; Sternberg, 1973). Several years later, the rubisco subunit binding protein (RuBP) was identified as a component associating with newly-translated large subunits of rubisco in the chloroplast, implying a role in protein folding and assembly (Barraclough and Ellis, 1980), and is now thought to have a broader scope of action in this organelle (Lubben et al, 1989). A general role for GroEL in folding newly translated proteins appears likely (Viitanen et al., 1992; Horwich et al., 1993). For example, it was shown that under non-permissive conditions, a number of cytoplasmic proteins including citrate synthase, ketoglutarate dehydrogenase, a maltose-binding protein, and polynucleotide phosphorylase, were translated but failed to reach native form in an E. coli strain producing a temperature-sensitive mutant GroEL (Horwich et al., 1993). Furthermore, a 2D gel analysis of GroEL-deficient cells shows that approximately 30% of the protein species are affected. The folding of polypeptides within the GroEL cavity has been extensively studied. GroEL cooperates with GroES, a single heptameric ring of -10 kDa subunits that binds asymmetrically to GroEL, capping one opening of the cylinder (Saibil, 1996). The substrate interacts with GroEL through multiple ATPase cycles of GroES binding and release (Hayer-Hartl et al., 1995). Folding occurs in iterative steps in which the polypeptide is sequestered in the GroEL cavity, prevented from undergoing aggregation or other off-pathway reactions (Mayhew et al., 1996). ATP binding and hydrolysis triggers the opening of the GroELGroES cage, at which point the folded polypeptide leaves; alternatively, incompletely-folded polypeptide rebinds for a further round of folding and release (Martin et al., 1993; Weissman et  7  Chapter I: Introduction al., 1995; Burston et al., 1996). The most recent reviews on chaperonin-assisted protein folding are those of Hartl (1996) and Martin and Hartl (1997). Eukaryotic mitrochondrial HSP60 performs a function analogous to that of GroEL, and has been implicated in the folding and assembly of many polypeptides—for example, the assembly of newly imported wild-type HSP60 subunits is not observed in intact cells or isolated mitrochondria of the yeast HSP60-defective mutant mif4 (Cheng etal, 1989, 1990). It also was found that the subunits of ornithine transcarbamylase and citrate synthase were found in the mitochondrial matrix compartment at their mature size, but were lacking biological activity: on extraction with nonionic detergents, the proteins were found in the insoluble fractions, suggesting that they had not properly folded, and had instead aggregated upon entry into the matrix. Similarly, Ostermann et al. (1989) found that HSP60 was implicated in the ATPdependent folding of proteins in the mitochondrial matrix. In addition to defective biogenesis of imported proteins in HSP60-deficient mitochondria, the folding of at least one mitochondriallyencoded protein, a ribosomal component, is also affected (Horwich et al, 1992). Unlike the eubacterial and organellar chaperonins, the scope of action of the eukaryotic cytosolic chaperonin CCT may be more restricted, and details concerning its function are discussed in a separate section below.  Structure-function studies on GroEL and GroES The only three-dimensional structure of a chaperonin available is that of E. coli GroEL, which has been solved by X-ray crystallography to a resolution of 2.8 A (Braig et al, 1994). The quaternary structure of the chaperonin consists of a porous cylinder made from two back-to-back heptameric rings. In addition, the structure of GroEL complexed with a non-hydrolyzable form of ATP reveals the location of a novel nucleotide-binding pocket whose primary sequence is conserved among chaperonins (Boisvert et al, 1996). Together with comprehensive mutationfunction studies which have defined numerous critical residues involved in all aspects of chaperonin activity (Braig et al, 1994), the above findings provide significant insight into the mechanism of polypeptide binding and release of this chaperonin. 8  Chapter I: Introduction The bacterial chaperonin monomer is divided into three domains which form a bilobate structure: the amino- and carboxy-terminal portions of the protein make up the equatorial domain, the central region forms the apical domain, and the intermediate region links the two domains. Residues required for inter-subunit contacts and ATPase activity are localized mainly in the equatorial and intermediate domains. As might be expected, residues involved with cochaperonin interaction and polypeptide binding map almost exclusively to the apical domain, which form the ends of the cylinder. Mutations which produce defective release or folding of the bound polypeptide are found in the apical and equatorial domains of GroEL. The crystal structures of two different GroES heptamers are also known (Hunt et al., 1996; Mande et al., 1996). The overall structure of the heptamer is dome-shaped, and it has been shown by electron microscopy to form a cap over the top of one of the two polypeptide binding chambers of GroEL (Martin et al., 1993). GroES is believed to interact in cis with the GroEL ring enclosing the folding polypeptide (Weissman et al., 1995; Burston et al., 1996). Folding is believed to be initiated within the GroEL-substrate-GroES ternary complex, and for some substrates may be completed prior to the release of GroES following ATP hydrolysis (Weissman et al., 1996). The surface of the interior cavity provided by GroES is very hydrophilic. Therefore, the role of GroES, in addition to modulating the cooperativity of ATP hydrolysis by GroEL (Gray and Fersht, 1991), may be to assist the folding of proteins by binding to hydrophilic regions of substrate proteins, while avoiding interactions with hydrophobic patches, which eventually form the interior core of the substrate protein (Mande et al., 1996).  Structure of Group II chaperonins In contrast with GroEL, the crystal structures of the Group II chaperonins have not been determined. However, observations of CCT and TF55 by electron microscopy suggest that their quaternary structures are very similar to each other and superficially similar to that of GroEL. First, the compositions of CCT and archaebacterial chaperonins are more complex, consisting of 7-9 and 2 different subunits, respectively. Second, these chaperonins display 8- or sometimes 9fold rotational symmetry compared with 7-fold for GroEL/HSP60. The most detailed three9  Chapter I: Introduction dimensional view of a Group II chaperonin has been obtained with the TF55-related complex from the archaebacterium Pyrodictium  occultum,  by means of random conical tilt  reconstructions from electron micrographs (Phipps et al., 1993). The chaperonin structure contains two eight-membered rings composed of equal amounts of two polypeptides of 56 and 59 kDa, whose arrangement within the complex is unknown. In contrast, TF55 isolated from Sulfolobus solfataricus by Knapp et al. (1994) and recombinant Thermoplasma  acidophilum  chaperonins (Waldmann et al., 1995c) consist exclusively of nine-membered rings. In the centre of the chaperonin is a large cavity about 6.7 nm in diameter, larger than that of GroEL (4.7 nm). Unlike GroEL, its cavity is partially blocked by a mass which appears to be weakly connected to the main body (Phipps et al., 1991). CCT is an -900 kDa heteromeric complex consisting of an assembly of 7-9 polypeptides of 53-65 kDa (Lewis et al., 1992; Frydman et al., 1992; Rommelaere et al., 1993; Kubota et al., 1994). The subunit composition of CCT varies somewhat depending on its source (Kubota et al., 1994; Hynes et al., 1995). As with the archaebacterial chaperonins, the only data available on the CCT quaternary structure has been obtained by electron microscopy (em). Initially, visual inspection of the electron micrographs of murine CCT by Lewis et al. (1992) suggested that it consisted of 8 or 9 subunits per ring. However, more thorough em analyses of CCT structures reveals that they are likely to consist solely of eight-membered rings (Marco et al., 1994; Waldmann et al., 1995b). Most likely because of the additional subunits in each ring, the pore size of CCT, approximately 6.0 nm, is larger than that of GroEL (Marco et al., 1994). In a review article by Kim et al. (1994), a detailed primary sequence alignment of the seven known members of the CCT protein family (a, p\ y, 5, 8, ^, and r\) and E. coli GroEL was used to assess domain-specific structural similarities between different CCT subunits and GroEL. A high level of conservation among all chaperonin proteins was observed in the region which corresponds to the equatorial, or ATP-binding domain of GroEL. In contrast, little sequence similarity was seen within the region corresponding to the apical domain, or polypeptide-binding region, of GroEL: not only had the seven CCT subunits substantially diverged from GroEL in this region, but they were also surprisingly different from each other, suggesting that each 10  Chapter I: Introduction subunit migh have different structural and/or functional properties. Similarly, on the basis of amino acid sequence alignments and secondary structure predictions, Waldmann et al. (1995a) suggested that the equatorial domain of CCTs and GroEL were likely to be similar, and that the apical domains of these chaperonins may share a common, albeit significantly divergent fold.  Function of CCT in vitro and in vivo A number of reviews describing the structure, function, and expression of CCT have appeared (Horwich and Willison, 1993; Willison and Kubota, 1994; Kim et al, 1994; Kubota et al, 1995b; Stoldt et al, 1996; Lewis et al, 1996; Hartl, 1996). In contrast to the Group I chaperonins, there is considerable evidence that CCT may play a more specialized role in the folding of proteins within the eukarytic cytosol. Two highly abundant cytoskeletal protein families, actins and tubulins, are known to be the major physiological substrates folded by CCT (Yaffe et al, 1992; Sternlicht et al, 1993; Melki and Cowan, 1994; Ursic et al, 1994; Chen et al, 1994). Melki and Cowan (1994) measured the relative affinities of many unfolded proteins for both HSP60 and CCT, and found that CCT appears to be more selective in its choice of target proteins, having greater affinity for actins and tubulins compared to other noncytoskeletal proteins. Interestingly, Tian et al. (1995) showed that HSP60 and GroEL could bind unfolded actin and tubulin, but unlike CCT, could not release them in their properly folded conformation. Mutations in many yeast genes encoding CCT subunits result in exclusively cytoskeletal phenotypes, such as disorganized or disrupted actin and tubulin filaments (Ursic et al, 1991, 1994; Chen et al, 1994; Miklos et al, 1994; Vinh and Drubin, 1994; Schmidt et al, 1996; reviewed in Stoldt et al, 1996). Interestingly, CCT has been shown to be associated with the centrosome, where it is required for mediating the growth of microtubules initiated from this structure (Brown et al, 1996). Overall, these results suggest that the increased complexity of the hetero-oligomeric CCT complex may have evolved to deal with particularly aggregation-prone proteins such as actins and tubulins (Kim et al, 1994). It is also possible that CCT may be involved in folding a broader range of newly synthesized polypeptides. The cytosolic chaperonin, in conjunction with HSP70 and the DnaJ 11  Chapter I: Introduction homolog HSP40, is required for folding firefly luciferase as it emerges from ribosomes (Frydman et al., 1994b, 1996). CCT can refold chemically denatured firefly luciferase in vitro whereas GroEL/GroES can bind the unfolded protein but apparently cannot refold it to its native state (Frydman et al., 1992). An association of CCT with an intermediate in the assembly of hepatitis B virus capsids has also been observed (Lingappa et al., 1994). The relative ability of GroEL, HSP60, and CCT to bind an unfractionated mixture of labeled, unfolded E. coli proteins was recently assessed (Tian et al., 1995). It was found that the pattern of proteins recognized by each chaperonin was very similar, although the overall amount of proteins bound to CCT was approximately 10-fold lower compared with GroEL. It is now evident that the chaperone-mediated protein folding pathway in eubacteria parallels the eukaryotic de novo folding pathway. In a landmark paper by Langer et al. (1992), the sequential action of DnaK, DnaJ, and GroEL was shown to be required for the efficient folding of newly synthesized polypeptides. In the eukaryotic cytosol, the same chaperone system (HSP70, HSP40, and CCT) is involved in folding de novo synthesized firefly luciferase and actin (Nimmesgern and Hartl, 1993; Frydman et al, 1994, 1996). However, in contrast to the CCT-mediated folding of actin (Gao et al, 1992; Melki et al, 1993, 1994), the mechanism by which CCT folds tubulin is substantially more complex. Indeed, four different cofactors (A, C, D, and E) in addition to CCT are required for the correct folding of tubulin in vitro (Gao et al, 1993, 1994; Melki et al, 1996; Tian et al, 1996). As (3-tubulin is synthesized de novo, CCT interacts most strongly with a stretch of -120 amino acids situated in the core (middle) region of tubulin, and the N- and C-terminal regions of p-tubulin are thought to fold back onto the (3tubulin core region (bound to CCT), permitting its release from the chaperonin complex (Dobrzynski et al, 1996).  Expression and localization of CCT Because of the complex subunit composition of CCT, little information regarding the expression of the corresponding genes in higher organisms is available. In general, it appears that the spatial and temporal distribution of CCT subunits corresponds with specific 12  '  Chapter I: Introduction  requirements for folded actin or tubulin. The first subunit to be studied, CCTa (i.e., TCP-1 or CCT-1), is known to be abundant in mouse testis, and is expressed at lower levels in almost all cell types (Willison et al., 1989, 1990). There also exists a murine testis-specific CCT subunit (Kubota et al., 1997). Another process apart from spermatogenesis which requires high rates of tubulin synthesis has been documented for Tetrahymena, where CCTy is upregulated during reciliation (Soares et al, 1994). The same subunit (TRIC-P5, or CCTy) is known to be expressed at the two-cell stage in mouse embryos, suggesting an important function for this protein during the rapid cell division occurring in early development (Sevigny et al., 1995). In a neuronal cell line, CCTa specifically co-localizes with actin at the extreme periphery of growth cones, where the unassembled, globular form of actin and assembly-competent filamentous forms are found. In contrast, other subunits (CCT(3, e, y) are restricted to the perikaryal cytoplasm of these cells, where cytoskeletal proteins are first synthesized (Roobol et al., 1995). These data strongly suggest that CCT subunits do not necessarily assemble into a single type of hetero-oligomeric complex. Indeed, a homo-oligomeric CCTa particle from mammalian brain was reported by Martin etal. (1993). The expression of several cet genes has been described in two vertebrates, a salamander (axolotl) and in Xenopus laevis. In Xenopus, the expression patterns of two CCT subunits are developmentally regulated during embryogenesis, as evidenced by increased expression of CCTa and CCTy in differentiating neural and muscle precursor cells (Dunn and Mercola, 1996). More specifically, the transcripts for CCTa and CCTy are enriched in cranial neural crest cells during embryogenesis, as well as in the developing central nervous system (CNS) and skeletal and cardiac muscle. At least in the CNS and muscles, the need for abundant expression of cet genes is consistent with their potential requirement for actin and tubulin biogenesis. Similarly, temporal and spatial regulation of TCP-1 in axolotl is evident (Sun et al., 1995). At an early stage of development, TCP-1 transcripts accumulate in developing brain and neural tube. At a somewhat later stage, strong expression is seen in the brain, spinal cord, and somites by wholemount in situ hybridization studies and RT-PCR. The authors note that the expression of TCP1 correlates with the expression of actin and myosin during early development. 13  Chapter I: Introduction C. elegans as a model organism The free-living soil nematode Caenorhabditis elegans is an anatomically and genetically simple multicellular organism (Wood, 1988). It is one of the preferred model systems for the study of fundamental biological problems. Because the nematode is self-fertilizing, has a short life cycle of about 3 days at room temperature, and is easily cultured on plates or in liquid media, genetic manipulations using classical Mendelian approaches are possible. C. elegans can also be transformed with cloned DNA to produce stable transgenic lines (Mello et al., 1991). The small size (1 mm length for the adult), small cell number (959 somatic cells in the hermaphrodite), invariant cell lineage and transparency of the organism have allowed the entire cell lineage to be defined (Sulston and Horvitz, 1977; Sultson et al., 1983), and the complete wiring diagram of the nervous system has been determined at the ultrastructural level (White et al, 1986). Targeted gene disruptions, although still not as simple as in S. cerevisiae, are made possible by the use of transposon insertion and imprecise excision (Plasterk and Groenen, 1992). Of particular interest for molecular studies is the existence of a virtually complete physical map of the genome, and the rapid progress on sequencing of the C. elegans genome. At this writing, about two thirds of the genome has been sequenced through a consortium of two laboratories based in St. Louis, MO, and Cambridge, England (Wilson et al., 1994), and completion is projected for late 1998. The sequences of cosmids are deposited in Genbank as they are completed and are therefore available to researchers. In addition, the cosmids, as well as a huge collection of cDNAs isolated in order to identify expressed genes, are distributed freely. The availability of a major part of the genome sequence allows C. elegans researchers to readily study entire protein families, or entire pathways, if desired.  The present study The hetero-oligomeric eukaryotic cytosolic CCT represents the most complex chaperonin discovered to date. By making use of available cDNA sequence tags and the C. elegans genome sequencing project, characterization of five genes that encode subunits of the complex was greatly facilitated. All five genes were shown to be expressed throughout development. One of 14  Chapter I: Introduction the genes, cct-1, was isolated and sequenced. The cct-1 transcript was shown to involve cis- and trans-splicing events to yield the mature 1.9-kb mRNA species. The ability to make transgenic C. elegans carrying promoter-/acZ fusion constructs made it possible to determine the expression pattern of cct-1 within the context of an entire multicellular animal. The CCT complex was purified by sucrose gradient fractionation and ATP-agarose chromatography, and the presence of two of the subunits (CCT-1 and CCT-5) was confirmed by Western blot analysis with polyclonal antibodies generated against C-terminal peptides. The subunit composition of the C. elegans CCT was shown to be very similar to that of bovine CCT, suggesting that the structure of the chaperonin complex is highly conserved in the animal Kingdom. The affinity of CCT for denatured actin appears to be lower than that of a related chaperonin from C. elegans, HSP60.  15  Chapter I: Materials and Methods II. MATERIALS AND METHODS  2.1 Bacterial strains and media The E. coli strains used for propagating plasmids and phage are listed in Appendix I. YT plates were used to grow the various E. coli strains, and Terrific Broth (TB) liquid medium was used for culturing the bacteria for the subsequent isolation of plasmid DNA or overexpressed protein. Both liquid and solid media were supplemented with the appropriate antibiotic(s) and were prepared as reported in Sambrook et al. (1989).  2.2 Molecular biological techniques 2.2.1 Gel electrophoresis and isolation of DNA fragments Restriction enzyme digestion of DNA was usually carried out in a 20 (il reaction mixture containing 0.5-3.0 |ig of DNA, 2 |il of 10X enzyme buffer supplied with the enzyme, and 1-10 units of restriction endonuclease enzyme(s). Restricted DNA fragments were fractionated on an agarose gel (0.4-2.0%) in TBE buffer (45 mM Tris-base, 45 mM boric acid, 2 mM EDTA; pH 8.0). Visualization of DNA bands under UV light was facilitated by the inclusion of 0.25 |lg/ml ethidium bromide in the gel. Restricted DNA fragments were excised from the agarose gel and purified using the Qiaex™ Gel Extraction Kit (Qiagen Inc.).  2.2.2 Ligations For most ligations, about 50 ng (25 fmol) of plasmid vector DNA and a 2-5 fold molar excess of insert DNA were ligated in a 20 |ll reaction mixture containing 50 mM Tris-HCl (pH 7.8), 10 mM MgCl , 10 mM DTT, 1 mM ATP, 25 ug/ml BSA, and 0.25 Weiss units (cohesive2  end ligation) or 2.5 Weiss units (blunt-end ligation) of T4 DNA ligase. Removal of vector DNA 5' phosphoryl groups, when required to prevent self-ligation, was carried out by adding 4 units of calf intestinal alkaline phosphatase to the restriction digest reaction. The phosphatase enzyme was removed using the Qiaex Gel Extraction Kit.  16  Chapter I: Materials and Methods 2.2.3 Transformations Competent DH5a subcloning efficiency cells purchased from BRL were typically used for transformations, although sometimes competent cells were prepared using the method of Hanahan (1983). Competent E. coli cells were transformed with 5 |il of ligation reaction by heat shock. Transformed cells were spread on YT plates containing the appropriate antibiotic(s) and incubated overnight at 37°C for colony growth. When required, X-Gal (50 jul of 20 mg/ml stock) and IPTG (10 (il of 100 mM stock) were spread onto the plates to differentiate recombinants (white) from nonrecombinants (blue), as described by Messing (1983).  2.2.4 Purification of plasmid and cosmid DNA For isolating plasmid DNA suitable for restriction enzyme digests, subcloning, and sequencing, a slightly modified plasmid miniprep protocol from that of Morelle (1989) gave the best and most consistent results. A 2 ml bacterial culture harbouring plasmid or cosmid DNA was grown overnight at 37°C in TB medium supplemented with the appropriate antibiotic (Sambrook et al., 1989). The cells were harvested in a 1.7 ml microcentrifuge tube, and resuspended in 200 ul of a 50 mM glucose, lOmM EDTA, 25 mM Tris-HCl pH 8.0 solution. After a 2 minute incubation, 400 |tl of lysis buffer (0.1 N NaOH and 1% SDS) was added, and the tube mixed gently by inversion. After a 2-minute incubation, 300 pi of 7.5 M ammonium acetate was added, and the tube contents mixed well by inversion and incubated on ice for 10 minutes. Cellular debris, high molecular weight RNA and chromosomal DNA were then pelleted by centrifugation (10000 x g) at room temperature for 8 minutes. To precipitate the DNA, 700 (il of supernatant and 420 |il of isopropanol were mixed together and incubated for 10 minutes. The DNA was pelleted by centrifugation for 10 minutes, washed briefly with 70% ethanol, dried at room temperature for 10 minutes, and then dissolved in 40-60 (tl TER buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA; 1 ug/ml RNAse A).  17  Chapter I: Materials and Methods 2.2.5 Double stranded DNA sequencing For most subcloning and sequencing procedures, restriction digest or PCR D N A fragments were ligated into pBluescript® II KS(+) (Stratagene) and sequenced by the dideoxy chain termination method of Sanger et al. (1977) using reagents from the Sequenase® Version 2.0 sequencing kit (U.S. Biochemical). The double stranded plasmid D N A (15 | i l or at least 5 jig) was denatured for 5 minutes at room temperature by the addition of 2 jil of freshly prepared 2 M NaOH, in the presence of 1 Jill (1 pmol) of sequencing primer. A list of primers used for sequencing and/or P C R is given in Appendix II. The template D N A and primer were precipitated by the addition of lOOixl of 95% ethanol and 8 | i l of 5 M ammonium acetate (pH 7.8), mixing, and incubation at -70°C for 15 minutes. After centrifugation, the pellet was washed in 70% ethanol and air dried. Annealing of the primer to the template D N A was accomplished by resuspending the D N A in 10 ul I X reaction buffer, and incubating at 37°C for 20 minutes. The remaining protocol was performed exactly as described in the Sequenase® Version 2.0 user's manual.  2.2.6 Polymerase chain reaction Selective amplification of various D N A sequences was routinely performed using the polymerase chain reaction (PCR) (Saiki et al, 1988). Typical reaction conditions are outlined in Appendix III. The 50 | i l reactions were overlaid with light mineral oil, and inserted into a thermocycler block (TwinBlock™ System by Ericomp Inc.) preheated to 95°C. The thermal cycling program usually consisted of a 95°C denaturation step (3 minutes) followed by 30 cycles of 95°C for 30 seconds, 48-55°C for 30 seconds, and 72°C for 30-90 seconds. After a final extension step at 72°C for 10 minutes, 20 (il aliquots were analyzed on a 1.5 or 2.0% T B E agarose gel.  2.2.7 Preparation of DNA, RNA and first strand cDNA Genomic D N A was isolated from C. elegans embryos as described by Emmons et al. (1979). Total R N A was prepared from staged nematodes by lysis in guanidine hydrochloride and 18  Chapter I: Materials and Methods separation on a CSC1 gradient (Antonucci, 1985). Alternatively, a mini-scale isolation of RNA (using a few hundred nematodes) was carried out (Mei Zhen, Ph. D. thesis, University of British Columbia, 1995). First strand cDNA was prepared from 10-20 p,g of total RNA according to Jones and Candido (1993).  2.2.8 Southern and DNA dot blots Aliquots of C. elegans genomic DNA (10 |ig) or cosmid DNA (500 ng) were digested overnight with one or more restriction enzyme(s) and electrophoresed on a 0.7% TBE agarose gel. Prior to transfer, gels were soaked in 0.25 N HC1 for 10 minutes, denaturation solution (1.0 M NaCl, 0.5 M NaOH) for 2 x 15 minutes, and neutralization solution (1.5 M NaCl, 0.5 M TrisHCl, pH 7.4) for 2 x 15 minutes. Denatured DNA was then transferred to a Hybond™-N nylon membrane (Amersham) by capillary action using 10X SSPE (1.8 M NaCl, 10 mM EDTA, 100 mM  NaH2PC<4; pH 7.4).  For dot blots, plasmid DNA (about 50 ng) or cosmid DNA (about  150 ng) was denatured with 0.1 volume of 3 M NaOH for 30 minutes at 65°C, and then neutralized by the addition of SSPE to a final concentration of 5X. The entire denatured DNA solution was spotted in 3 |il aliquots on a Hybond™-N nylon membrane previously soaked in 5X SSPE, air dried, and placed on top of 2 sheets of dry filter paper.For both methods, DNA was crosslinked to the nylon membranes by UV crosslinking (approximate exposure: 120 mJ/cm ), 2  or by baking the membrane at 80°C for 30 minutes.  2.2.9 Northern blots The fractionation of RNA through an agarose gel containing formaldehyde was performed as described by Sambrook et al. (1989). Total cellular RNA samples (20 jig) were fractionated on a 1.2% formaldehyde gel alongside 5 p.g of 0.24-9.5 kb RNA ladder molecular weight markers (BRL). After electrophoresis, the gel was rinsed in dH20 for 15 minutes and the RNA was then transferred to a Hybond™-N nylon membrane by capillary action using 20X SSPE. The RNA was immobilized on the nylon membrane by UV crosslinking. RNA molecular weight markers were visualized by staining the membrane section containing the markers with 0.02% 19  Chapter I: Materials and Methods methylene blue dye in 0.3 M NaOAc pH 5.5 (Herrin and Schmidt, 1988). If removal of the radiolabeled probe was required, the membrane was stripped in 0.1X SSPE, 0.1% SDS at 100°C for 5 minutes.  2.2.10 Preparation of radiolabeled DNA probes DNA (25-50 ng) was uniformly labeled with [a- P]dATP or [a- P]dCTP using random 32  32  hexadeoxyribonucleotides (Pharmacia) and Klenow enzyme (Pharmacia) as described by Feinberg and Vogelstein (1983). Alternatively, probes were random-primer labeled with [oc32  P]dCTP using the QuickPrime™ kit from Pharmacia. The radiolabeled probes were purified T7  by chromatography on 1-ml Sephadex G50 spun columns to remove unincorporated dNTPs.  2.2.11 Nucleic acid hybridization conditions Southern and Northern blots were pre-hybridized at 42°C for 1 hour in hybridization solution (final composition for 10 ml: 50% (v/v) deionized formamide, 5X SSPE, 0.5% (w/v) SDS, 5X Denhardt's reagent (diluted from a 50X stock consisting of 10 |Xg/ml each of Ficoll, polyvinylpyrrolidone, and BSA), and 0.5 mg/ml heparin). Denatured probe (0.1 ml; 1-6 X 10  7  cpm) was then added to the fresh hybridization solution and allowed to hybridize to the target DNA or RNA overnight at 42°C. Membranes were washed twice in 250 ml of 0.1X SSPE/0.1% SDS at room temperature for 15-30 minutes. For more stringent washes, membranes were washed twice in 250 ml of 0.1 X SSPE/0.1% SDS at 65°C for 15 minutes. Membranes were exposed to X-OMAT™ AR film (Kodak) with an intensifying screen for autoradiography.  2.3 General protein and immunological techniques 2.3.1 Denaturing and native polyacrylamide gel electrophoresis Denaturing, discontinuous SDS-PAGE of protein samples (Laemmli, 1970) was carried out on a Bio-Rad Mini-PROTEAN II gel. Gels (7.5%-15% acrylamide) and protein samples were prepared as described by Laemmli (1970). Native gels (4.5% acrylamide) were made with 80 mM MES pH 6.8 and 1 mM EGTA, and run on a Bio-Rad mini gel apparatus in the same 20  Chapter I: Materials and Methods buffer at a constant power of 5 W for 1.5 hours. The running buffer in the upper and lower tank chambers was mixed every 30 minutes. For actin folding reactions, gels were supplemented with 1 mM ATP/ 1 mM MgC^, and the running buffer contained 0.1 mM ATP and 1 mM MgC^. Samples were supplemented with glycerol to 20% before loading.  2.3.2 Polyclonal antibody production Peptides whose sequences match those of the 15 last residues of C. elegans CCT-1 ( r C L D K Q E P L G G D D C H A - C O O H ) and CCT-5 (KIDDVRVPDDERMGY-COOH), respectively, were obtained from API (Alberta Peptide Institute). Each peptide came conjugated to KLH (for immunization) and BSA (for affinity purification). For each antigen, one rabbit was immunized with 0.5 mg of the KLH-conjugated peptide in Freund's complete adjuvant. New Zealand White rabbits were then boosted with 0.5 mg of the KLH-conjugated peptide in Freund's incomplete adjuvant after two weeks, and then once more after a further 3 weeks before sacrificing (bleeding) the rabbit two weeks later. Antiserum was prepared by allowing the blood to clot overnight at 37°C, precipitating the IgG with 45% ammonium sulfate, and dialyzing the resuspended salt-IgG pellet against TBS (25 mM Tris-HCl, 140 mM NaCl; pH 7.4).  2.3.3 Affinity purification of antibodies The polyclonal antibodies (pAbs) were affinity purified on an Affi-Gel 10 resin coupled with the appropriate BSA-conjugated peptide according to the manufacturer's instructions. Antibodies were eluted with 0.1 M glycine pH 2.5, and immediately neutralized with 15 p:l IM Tris base per ml of eluant. Fractions containing antibody (monitored by A280) were pooled and dialyzed against TBS.  2.3.4 Western blot analysis Approximately 10 (Xg of total C. elegans protein extract (solubilized in Laemmli sample buffer), or <1 |ig of partly purified or purified chaperonin was separated by SDS-PAGE, soaked in transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol) for 10 minutes, and transferred 21  Chapter I: Materials and Methods onto an Immobilon™-P PVDF membrane (Millipore) in transfer buffer for 1 hour at 250 mA or overnight at 50 mA. Following transfer, the membrane was blocked for 1 hour in TBS-T (TBS supplemented with 0.05% Tween 20) supplemented with 10% skim milk powder (Carnation). The membrane was briefly washed with TBS-T, and incubated with primary antibody (usually 1:3000 to 1:10000 dilution of purified and unpurified anti-CCT-1 or anti-CCT-5 antibodies, respectively) for 1 hour. After 3 washes with TBS-T (5 minutes each), the membrane was incubated with a 1:10000 dilution of Amersham peroxidase-labeled anti-rabbit antibody for 1 hour, and washed as before. Immunodetection was carried out using Amersham's enhanced chemiluminescence (ECL) kit.  2.3.5 Miscellaneous Protein concentrations were determined with the Bio-Rad protein assay kit using bovine IgG as standard.  2.4 Purification of Chaperonin Containing TCP-1 (CCT) Large-scale isolation of C. elegans embryos was performed as described in Appendix IV. C. elegans CCT was purified essentially as described by Lewis et al. (1992). A post-nuclear supernatant of frozen C. elegans embryos (50% v/v in 250mM sucrose, lOmM Tris-HCl, pH 8.0, lOmM MgCh) was prepared by homogenizing thawed embryos using a Potter Elvejhem homogenizer, and pelleting the cell debris at 12000 x g. Protease inhibitors (100 p:g PMSF, 2 (ig antipain, 2 \ig aprotinin, and 1 |ig pepstatin A, all per ml; 1 mM EDTA) were added to the thawed embryos before homogenization. Approximately 2.5-ml aliquots of the supernatant were loaded onto 36.5-ml 10-40% (w/w) sucrose gradients (made in 50 mM KC1, 50 mM Tris-HCl pH 7.4, 1 mM DTT, 0.5 mM EDTA) and centrifuged in a SW28 rotor at 26000 rpm for 18 hrs at 4°C. A total of 19 2-ml fractions were collected from the bottom of the polyallomer centrifuge tubes (Beckman), and 15 jLLl aliquots from each fraction was assayed for CCT-1 by Western blotting. Positive fractions were dialyzed against TEDKM buffer (50 mM Tris-HCl pH 7.4, 50 mM KC1, 1 mM DTT, 0.1 mM EDTA, 10 mM MgCh) and loaded onto a column containing 22  Chapter I: Materials and Methods ATP-agarose resin (Sigma) equilibrated with the same buffer. After washing the column with 3 column volumes of TEDKM/ 100 mM NaCl buffer, ATP-binding proteins were eluted with three column volumes of TEDKM/ 100 mM NaCl/ 10 mM ATP/ 10 mM MgC^. The second and third fractions were dialyzed against TEDKM buffer diluted 10-fold with dH^O, and 200 |il aliquots were concentrated approximately 10-fold by vacuum centrifugation.  2.5 Construction and analysis of transgenic nematodes 2.5.1 Preparation of construct for injection To make a ccr-i-promoter-/acZ expression construct, the -3.7 kb Hindlll restriction fragment upstream of the internal cct-1 /f/ndlll site was cloned from the cosmid T05C12 (pBScct-1 [Hindlll-HindTLl] clone). A nematode expression vector containing the P-galactosidase gene (pPD 16.43 vector; Fire et al, 1990) was used to fuse, in frame, the 5'-end (promoter region) of the cct-1 gene fused to the P-galactosidase coding region, and to add the 3'untranslated region of cct-1, as described below. First, the pPD 16.43 vector was cut with Eagl and Stul (16.43.ES). The 3'-end of cct-1 was restricted with BgH and Psil, and subcloned into pBluescript cut with the same enzymes. From the latter vector, a HincWEagl  fragment was  isolated, and subcloned into 16.43.ES, creating pPD16.43.3'. A fragment containing the 5'-end of cct-1 was then made by amplifying the pBS-cct-1 [Hindlll-Hindlll]  clone with the cct-1-  specific oligo MIC5 (see Appendix II) and the pBS vector primer T3. The amplified product and 16.43.3' were then cut with Hindlll and Sail and ligated together, forming pPD16.43.5'.3'. This construct therefore contained 2.7 kb of upstream noncoding region, the first intron in the cct-1 gene, followed by 32 amino acids of the cct-1 coding region fused in-frame to the nuclear localization signal (NLS) and (3-galactosidase gene, as well as approximately 1000 bp of cct-1 3'-noncoding region (including the polyadenylation signal). This vector was tested for expression in transgenic strains (below).  23  Chapter I: Materials and Methods 2.5.2 Injection of the construct The construct was prepared by the regular plasmid DNA isolation protocol, and was resuspended in distilled water at a concentration of 200 ng/jil. The plasmid marker pRF4 (Mello et al., 1991) was co-injected at the same concentration into the gonad of the Bristol N2 strain. Transformed progeny, identified by their rolling phenotype caused by the mutant collagen encoded by pRF4, were transferred to separate plates to determine if the extrachromosomal array was passed on to subsequent generations. Transient lines were frozen in liquid nitrogen as described in Lewis and Fleming (1995), and are listed in Appendix V.  2.5.3 Histochemical detection of (3-gaIactosidase in transgenic lines Strains containing the cet-1 -promoter-lacZ construct were stained according to Fire et al. (1992). The animals were dried in vacuo on a microscope slide, permeabilized with acetone, and incubated with 75 jil of staining solution overnight at 37°C in a moist chamber. The stained animals were mounted in 80% glycerol, 20 mM Tris (pH 8.0), and 200 mM sodium azide, and then examined by Nomarski microscopy. The cell types were identified on the basis of their position (relative to the animal and other cells), size, and shape, based on published C. elegans anatomy and cell lineage (Wood, 1988).  2.6 Sequence alignments and secondary structure predictions Amino acid sequences were deduced using the Macintosh program DNA Strider 1.2 (Christian Marck). Multiple amino acid sequence alignments were assisted by the program MacDNASIS Pro v3.2 (Hitachi Software Engineering Co.), and refined manually to minimize the number of gaps/insertions within the alignment, and to take into account predicted secondary structures. Secondary structure predictions for the chaperonin proteins were made with the program PHD (1995 version). PHD scans the SWISSPROT database for sequences similar to the input sequence, and generates a multiple sequence alignment which is then used by a three-level neural network algorithm for making predictions at better than 70% accuracy (Rost and Sander, 1993, 1994). 24  Chapter I: Results III. RESULTS  3.1 Sequence analysis of five CCT proteins 3.1.1 Sequence determination of five cet cDNAs from a multigene family As an adjunct to the genomic sequencing of C. elegans, a complementary project has provided investigators with a large collection of sequence tags from a sorted cDNA library (Waterston et al, 1992). For each cDNA clone, a single sequence read from the 5' end is made available through ACeDB (the genome database of C. elegans, accessible through NCBI, EMBL, and GenBank), and the clones themselves are freely available. Four different X.SHLX2 phage harbouring the C. elegans cet cDNAs encoding CCT-1, CCT-2, CCT-4, and CCT-5 were obtained from this library; these clones correspond to cm08gl0, cml7cl, cml5el 1, and cml3e8, respectively. The BLASTX scores (Altschul et al, 1990) for the predicted protein sequences encoded by the cDNAs showed them to be significantly similar to murine CCTa (Kubota et al, 1992) and CCT(3, CCTy, and CCTe (Kubota et al, 1994). The pRatll plasmids containing the cet cDNAs (pRatII-ccr-7, pRatII-ccf-2, pRatII-ccr-4, and pRatII-cct-5) were excised from the phage DNA using the 'popout' E. coli strain BM25.8 (Palazzolo et al, 1990). The plasmid DNAs were isolated and used to transform competent DH5cc E. coli for efficient propagation of the plasmids. NotVApal fragments containing the fulllength cDNAs were isolated from the pRatll plasmids and subcloned into pBluescript (pBS) vectors cut with NotVApal, yielding pBS-cct-1, pBS-ccr-2, pBS-cct-4, and pBS-cct-5. The pBS vectors containing the cet cDNAs were digested with multiple restriction enzymes, and the fragments generated were subcloned into pBS for sequencing using the primers T3 and T7, as well as some gene-specific primers. The entire cDNAs (approximately 1.9 kb each) were sequenced in this manner. Sequence coverage included all of both strands for cct-1, and both strands for the other cet cDNAs except in some regions where the sequence was clearly unambiguous. The deduced amino acid sequence of C. elegans CCT-6 was obtained by translating the open reading frames of the cct-6 gene sequence determined by the C. elegans genome sequencing project (Wilson et al, 1994). 25  Chapter I: Results 3.1.2 Primary structure analyses of five CCT proteins The open reading frames of the cct-1, cct-2, cct-4, cct-5, and cct-6 genes encode proteins of 58.8 kDa (549 aa), 57.0 kDa (529 aa), 58.4 kDa (540 aa), 59.4 kDa (542 aa), and 58.9 kDa (539 aa), respectively. Kubota et al. (1994) reported that members of the murine CCT protein family are as closely related to the archaebacterial protein TF55 as they are to other members of the CCT family. Sequence analysis of the five C. elegans CCT proteins and TF55 corroborate this observation. An amino acid sequence alignment of the C. elegans CCT proteins with TF55 (Figure 2A) reveals that members of the C. elegans CCT protein family share 23-35% pairwise predicted amino acid sequence identity between each other and 31-35% sequence identity to TF55 (Figure 2B). The C. elegans proteins CCT-1, 2, 4, 5, and 6 share 66%, 66%, 63%, 68%, and 67% amino acid sequence identity with the orthologous mouse C C T a , P, 8, £, and C, subunits, respectively. Similarly, various CCT proteins from humans, yeast, and Drosophila share between 62-68% amino acid sequence identity with their C. elegans orthologs. The strong similarity between the CCT orthologs spans the entire protein sequence, and the calculated MWs of the orthologs are nearly identical. Some sequence motifs that are conserved in nearly all chaperonins, including the classical chaperonins (Lewis et al., 1992) and members of the CCT/TF55 protein family (Kim et al., 1994; Kubota et al., 1994), are also conserved in the C. elegans CCT protein family (Figure 2A). One highly conserved sequence is GDGTT, centered on position 110 of the C. elegans CCT and TF55 alignment. This sequence was proposed to form part of a putative ATP-binding domain based on limited sequence homology to the cAMP-dependent protein kinase family (Lewis et al., 1992; Kubota et al., 1994). Mutational analysis of the aspartic acid residue present within this sequence confirms that it is required for ATP hydrolysis and polypeptide release (Fenton et al., 1994).  26  Chapter I: Results  A 10  CCT-1 CCT-2 CCT-4 CCT-5 CCT-6 TF55  . 2 0 . 3 0 . 4 0 . 5 0 . 6 0 . 7 0 . 8 0 . 9 0 100 110 . 120 IIiasagdsilaltgkRtTgggiRSONVtAAvalANIVKSSlGFVGLDKMLVDD vGDVivTNDGATII^qLEVEHPAgKVI.vELAqLQDeEVGIX3TTSVVIvAAE mlPVqllKdnAqEFi^EsARLSSFvGAIMGDLVKSTIiGPKGMDKILiSGn —mppavpaaaAtarqsaSgrernfkDkDKPesvRnSNIVAAKAVADAvRTSliCSPrGMEKMIQsG nGDVTITNDGATILnQMsViHPtAkMLVELSKAQDIEAGDGTTtVVvinAGa ITiaqSsaqLlFDEsGqPFivmreQenqkRitGvEAvKSHIlAArAVANTlRTSIjGPrGLDKMlVsp DGDVTIlWDGATImekMDVqHhvAKLJ^VELSKSQDhEIGDGTTGWVLBGA mssiqcLNPKAElARhaAALelNISgARGLQDVinRsNLGPKGTlKMLVSG AGDIKLTKIXSNVLIjHEMalQHPTASlTLrAKasTAQDDvTGDGTTStVLlIGE MATATVATTPEGIPVIIIJOZXjSSRTYGKEAIjRANIAAVKAIEEAljKSTyGPRGMDKMFVDS l^DITITHIXSATILDKMDUJHPTGKLLVQIAKGQDEETADGTKTAVILAGE  CCT-1 CCT-2 CCT-4 CCT-5 CCT-6 TF55  . 130 . 140 150 . 160 . 170 . . 180 . 190 . 200 210 . 220 230 240 LLKrADELVKQKvHPTtilnGyPiACKEAVkYIsENisf tsDsiGP^SVvNaAKTS^ LLkFAEkLvnqrlHPQTIISGyRrAlgiAqEsLkkSsiesGdn-irdD^^ LdAIqilKKLGGSmneSYIjDEGFLLeKlp LI^aqnllsKGIHPTtISESFQsAaaeaekII/2eMSsPVdL£-ndalLnkmAT^ T.T.F.EAeklidRGIHPIkIADGfdlAckkAletLDsI5DK£pVe-HrE^LveTAqTsIjGSKivMrslRQ£AEIAVdAVLsVADiEs—kDVnFEmlKmEGKVGGRLECTiLvKGiviDKtm LLKQAeslvlEGLHPRIvTEGFEwAntKtLelLEkfKkeapve—RdlLveVcRTaLRTKlHqkLADhiTEcWDavLAIRrdgE epDLhMVEkMEMhHdSdmETtliVRGLVLDHGA IJiKKAmiiYKEIHPTIIVSGYKKJ^EIAIJCriQDIAOPVSIN-ETDVIiRKVALT  CCT-1 CCT-2 CCT-4 CCT-5 CCT-6 TF55  250 260 270 . 280 . 290 . 300 . aSQaMPlRvqNAKIACIJ3FSIlIlKaKMhL-GisVWedPaKI^aIForeE£DITKrRiaKILkaG GnifQ-PrRvEkAKILIAWIpMDTDKvKvrcSRVRVDgvAKVAElEaAEKlKMKEKVdklLaHn mgrGapTRiEKAKIGLIQFqiSpPKTOM-eNQviitDYAQMDRaLkEERqYlLeicKQIKaaG SHPQMPKelknAKvAlLTCPFEPPKPKT-KHKLDitStEDEKALrdYErEtFEtMIrQvKEsG FMPE^rhVkdAYILTCNVSLEYEKTEV-NSGlFYKUtfcERE^laAEReFI^^ VHPGMPKRIEHAKIAT ,T .nASLEVEKPEL-DAEIRIMDPTQMHKFI .EEEEMILKEKVDKIAATG  CCT-1 CCT-2 CCT-4 CCT-5 CCT-6 TF55  370 380 . 390 . 400 . 410 . 420 430 . 440 . ltvsMtLEGdE--aFdaslLGhAdEiVQERIsDDELILIKgpKsRTasSIIIJlGAm^ IvSTFDsPqtaq fGSCdLIEEiMIG EDrLlrFSGVkIX5EACs\rt/IJ<GATC<2ILDEsE^LHIlAI^^ ilGcrPVAsvDhFnADal^AdLvEEiptg-GdGKviKvTGvqnPG^ IVPRFSELskEK I^t^GlVrEItFXS-aaKDrMLsIEqCpNnkAVTIFvRQGNKMIIdEAKRalJIDA AvNSvDDLtPed LGwAGLVYEhsLG EFJ<yTFIEeCraPkSVTLLiKGPNKHTiTQIKDAIhIXjIiRAVfNtIvD^ VISNIDELTSQD LGYAALVEERKVG EDKMVFVH3AKNPKSVSILIRGGLFjWVDETERALRDALG^  CCT-1 CCT-2 CCT-4 CCT-5 CCT-6 TF55  . 490 . 500 . 510 . 520 . 530 . 540 . 550 . 560 . 570 . 580 I i i l P k v I A s N A A r D S T D L V t f LRAyHskAQliPqlqhIJ(WaGLDI^eGtiRDNKe^ LaqLPTIIcDNAGlDSAeLVtrLRAeHanG rlwGiDiekGevaDvtto3viESynVKlcmvsSAAEAtEqILRVDdIIKAAPRaRaqDrirPC lElIPyTLAENAGLsPIhTVTELRNnHAnG nssyGvNVRKGyvtdlwEEdWOPLLVtaSAikqAsEcVRSILKIDDiVhiavr LEsIPMALaENSGlaPIdalsdlkAkQiet gkssLGIDavfaGtNIMkeQkVIETLlsKreQISLATQvVRMILKlDDvRvPdderiT^y LLvIPKtLAvKgGyDaQETLVKlieEktaa GpdiavGlDLeTGgavep—qgiWDNvtVKKns isSaTVlAcNlLLVDEvMRAGHtnLKQpqpe IEGLIHILAENAGLDPIDKLMQLRSLHENE TNKWYGLNLFTGNPEDMWKI/^IEPALVKMNAIKAATEAVTLVIiRID^  B CCT-1 CCT-2 CCT-4 CCT-5 CCT-6 TF55  CCT-1  549 34 29 30 25 38  CCT-2 8(271  52V 30 29 29 36  CCT-4 5(13) 6.18)  540 30 22 39  CCT-5 5(14) 6(13) 3(7)  542 28 38  310  .  320 330 . 340 . 350 360 ANVvLTTGGIDDlcLKqFVEsGAMAVRRckKsDLKriAKAtGAt cWvFINRQLIYNYPEQLFadAkVMAIEHADFeGiERLALVlGGE QJVLLIQKSILRDAvneLALHFLaKMKrMciKDiEREDIEFysr AtLAICQWGFDDEANHLLqaNdLPAVRWVGGPEIELlAIATnaR  ANWICQKGIDEVAQHYLAKKGIIAVRRAKKSDLEKLARATGGR  CCT-6 7(32) 8(271 6(25) 6(20)  5.1V 33  450  460  470  .  . 480  590  TF55 4(14) 5(13) 4(9) 2(4) 5(20)  552  Figure 2. Multiple alignment of five C. elegans CCT proteins and TF55. A. Single-letter amino acid designations for each C. elegans CCT protein are shown in uppercase if they are identical to the orthologous mouse CCT protein at the same respective position (the TF55 sequence is shown entirely in uppercase). Amino acids which are identical at five of six positions in the C. elegans CCT and TF55 proteins are shown in bold to denote highly conserved positions. Dashes indicate gaps introduced to maximize the number of identical or conserved residues aligned between the CCT and TF55 amino acid sequences. The numeric scale represents relative amino acid positions in the alignment. B. A summary of the percentage amino acid identities of the C. elegans CCT proteins and TF55 is shown below the diagonal. The total number of gaps required for the pairwise alignments, and the total number of spaces within the gaps (in parentheses) are shown above the diagonal. The size of each protein in amino acids is shown along the diagonal. The sequences of the nucleotide and deduced amino acid sequences of the C. elegans cet genes are available in Genbank, under the following accession numbers: CCT-1 (U07941), CCT-2 (U25632), CCT-4 (U25697), CCT-5 (U25698), and CCT-6 (U13070). 27  Chapter I: Results The sequence (I/V)T(K7N)DG (positions 75-79), conserved in all chaperonins, is also located near the ATP-binding region in GroEL. Another conserved region, LGP(K/R)G, is centered on position 55 and is the site of a known temperature-sensitive mutation in yeast TCPIB (Miklos et al, 1994). Lastly, a highly conserved V(P/A/Y)GGG motif (positions 443447), is present in the C.  elegans  CCT proteins  in the  somewhat  modified  (V7L)(P/A/Y)G(G/A)(G/A) form. The triple-glycine motif is part of a tubulin-binding domain present in the microtubule-associated MAP2 and tau proteins, and is the site of a temperaturesensitive mutation in a yeast mutant (tcpl-1) which is synthetic-lethal in combination with the tubulin tub2-402 mutation (Lewis etal, 1988; Ursic etal, 1994).  3.1.3 Predicted secondary structures of CCTs and other chaperonins The structural and functional information available on GroEL facilitates a more thorough analysis of the CCT/TF55 chaperonin family structure and function. Kim et al (1994) demonstrated that the primary amino acid sequence of the CCT proteins and GroEL are much more conserved in their equatorial (ATP-binding) domains than in their apical (polypeptide binding) domains. The fact that residues within the apical region of the CCT subunits were poorly conserved suggested that the polypeptide binding domains of the CCT subunits had diverged in response to the eukaryote's increasing variety of substrates to fold, or to specialize in the folding of specific sets of proteins, such as tubulins and actins. Important questions regarding the structure and function of CCT/TF55 proteins remain (Kim et al, 1994). Do individual CCT/TF55 members share the same fold or have they all diverged from one another, especially in their polypeptide binding domain? Do the classical and CCT/TF55 chaperonins share evolutionarily conserved secondary and tertiary structures? Lastly, are differences in substrate affinities, folding properties, and requirement for a co-chaperonin in the GroEL/Hsp60 and CCT chaperonins due to changes in secondary and/or tertiary structures of the presumed polypeptide binding domain? To provide some insight into these questions, alignments of the primary and predicted secondary structures of the five C. elegans CCT members along with two murine CCTs, TF55, 28  Chapter I: Results GroEL, Hsp60 and RuBP were performed, and rooted to the known GroEL secondary structure (Figure 3). The defined secondary structure of GroEL provided an important internal standard with which to compare the predicted structures of the two chaperonin families. Secondary structure predictions were performed with the program PHD, which uses information from homologous amino acid sequences to make predictions with an overall accuracy of greater than 70% (Rost and Sander, 1993, 1994). Since the GroEL/Hsp60/RuBP chaperonin family is highly conserved (44-55% amino acid sequence identity), it was expected that the predicted secondary structures of the members would be very similar. Indeed, the alignment of the Group I chaperonins clearly shows that their predicted secondary structures are almost identical. However, while PHD predicts almost identical secondary structures for GroEL, Hsp60, and RuBP, the predicted secondary structure of GroEL is very similar, but not identical to the known secondary structure of this chaperonin. PHD correctly predicted 11 out of 19 (3-sheets and 16 out of 18 helices; the low prediction rate for (3-sheets seems to be due to the relative inability of the algorithm to correctly predict (3-sheet structures which are immediately adjacent to a helix (only 1 out of 5 such (3-sheets was predicted). Therefore, although the secondary structure predictions are only an approximation of the true secondary structure, they are nonetheless useful for detecting conserved secondary structures within related proteins. The alignment of the CCT and TF55 proteins suggests that the secondary structures of these chaperonins are very closely related. Except for a few minor differences, the predicted secondary structures of these chaperonins are virtually identical. Moreover, the predicted secondary structures of the C. elegans and mouse orthologues are completely superimposable (not shown). On this basis, the structural backbone of the CCT/TF55 chaperonin family appears to have been evolutionarily conserved, despite the divergence in primary amino acid sequence of each member.  29  Chapter  70 . 8 0 . 9 0 . 100 . 110 . 120 30 . 4 0 . 5 0 . 6 0 20 10 -maakdvk fGndarvkrnJ^GvnvIJUDaVTCv^ - —fGaptlTklX^SVareleLecakfei^^ -mir s s w r sra 11 r p l l r rays shke Ikf Gvegras lLkGve t LAEaVaaTLGPKGmvLIecrp-- - f (^pkITltf)GvTVaks IvLkcTkf gadake ia f dqksraalqaGvekTJtaaVgvTWPRGrnWLde— —yGnpkVvNIX3vTIaraleLanpmenaGA^ PAGkVLVEDaqlQDEEvGDGTTSwI _ mas agds i1a1tgkr t tGqg i r s qnV tAava lAniVKSSLGPvG lDKMLVdd- - - vGdviVTMXSaTILkqLeVeH -irdpvqilkdnaqeer<^sarlssfvGaiaIGDlVI^TT^ PAAkVXVEMsrntQDhEvGDGTTSvtV —mppavpaaaa t arqsasgrern f kclkdUqpesvrnsnlvAakaVADaVRTSLGPRGrnDKMIqsg nGdvt ITOJDGaTILnqMsViH P tAkMLVELs kaQDiEaGDGTTTvvV irfiqssaqllfdesgcjpfivnrreqenqkxitGv^ dGdvtlTNDGaTIMekMdVqH HvAklJlvTLsksQDhEiGDGTTgvvV mssiqclnpkaelarhaaalelnlsGargliqDvMRSnljGPKGtlKMLVsg aGdikLTkDGnvLLheMalqH PtAsMIakastaQDDvtGDGTTStvL mmghrpvl v l s qn t kr e sGrkvqsgn InAak t IAD i IRTcLGPKsnTnKMLLdp rnGgivreTNDGnalLrelqVqH PAAksMIE Isrt QDEEvGDGTTSv i I rr^tpvillkegtdssqGipqlvsnlsAcqv^ rGka t ISHDGaTILklLdVvH PAAktLVDIaksQDaEvGDGTTSvtL rnatatvattpegipviilkegssrtyGkealranlaAvkaleEat^TyGPRGnCiI^fvds lGdi t ITNDGaTI LdkMdLqH PtGkLLVqlakgQDEEtADGTkTavI  GroEL Hsp60 RuBP CCT-1 CCT-2 CCT-4 CCT-5 CCT-6 CCTY CCTT)  TF55  . EEEEHHHHHHHHHHHHHHHHHHH.  GroEL°  CCT-2 CCT-4 CCT-5  CCT-6 CCTY CCTT) TF55  HHHHHEEE...HHHHHHHHHHHHHHHHHHH....HHHHHH  EEE  .EEEEEEE  .HHHHHHHHHHHHHHHHHHHHEE. .HHHHHHHHHH..HHHHHHHHHHHHHHHHHHHHEE. HHHHHHHHHHHHHHHHHHHHEE EEEEE. HHHHHHHHHHHHHHHHHHHH HHHHHHEE. . .EEEE HHHHHHHHHHHHHHHHHHHH. . . .HHHHHHHE HHHHHHHHHHHHHHHHHHH HHHHHHH. . . EEEE HHHHHHHHHHHHHHHHHHH HHHHHHE. . HHHHHHHHHHHHHHHHHHHHHHHHH EEEEE. . . EEEEEE HHHHHHHHHHHHHHHHHHH HHHHE. . ... EE HHHHHHHHHHHHHHHHHHH.... HHHHHHHE. . . EEEEEE HHHHHHHHHHHHHHHHHHHH HHHHHHHH.  GroEL Hsp60 RuBP CCT-1  EEE. EEE. EEE. EEE. EEE. EEE. EEE. EEE.  HHHHHHHHHHHHHHHHH EEEEEEEE. HHHHHHHHHHHHHHHHH EEEEEEEE. HHHHHHHHHHHHHHHHH HHHHHHHHHHHHHH HHHHHHHHH.. HHHHHHHHHHH HHHHHHHHHHHHHH HHHHHHHHHH. . HHHHHHHHHHHHH HHHHHHHHHHH HHHHHHHHHHHHHH .HHHHHHHHHH . HHHHHHHHHHHH .HHHHHHHHH. HHHHHHHHHHHHH HHHHHHHHHH. .HHHHHHHHHHHHH .HHHHHHHHH. HHHHHHHHHHHHHH  HHHHH HHHHHH HHHHHH HHHHHH HHHHHH HHHHHH HHHHHH HHHHH HHHHH HHHHHH HHHHH  . 130 . 140 . 150 . 160 . 170 . 180 . 190 . 200 210 . 220 . 230 . 240 i^allteGlkaVaaGMriPirdjJcrGidkAvtaaV skaiaciygtisansdetVgkliaeairrikVGkEGV^ IjGralf tesvknVaaGcnPrrdl^rGsc^ seeiaqVatisangclShVgkllasamekvT^kJX^tlregrtledelevtegMrfdRgf ispyf iTdpks IJUrEIIklGilsVtsGanPvslJdcGidktvqglleeLerkarpvkg sgdikaVasisagndellgairdadaidkVGpDGVLsIessssf ettvdveegMeldRgyispqfvTnLek V^AEIjLkxAdelVkqkVHPttlinGYrlAckeaVkylsenisf tsd sigrqsWriaakTsMsSKIIgpdadf fgeLvVnAaeaVrvenn-gk^typlnaVriVlKahGksarESvLVk LAAETJ.keAeklVnqrlHPqtlisGYrrAlgiaqesLkkssiesgd nirddLLkiarTcLgSKILscihJceh^ LdalqliKklGgsmnESyLde MAGaTrTidaAqnlLskGIHPttlsesFqsAaaeaekiLdemsspvdl snda 1 Lnkma tTsLnSKWs qhsw 11 apMAVnAVkk I ins en—dsnvnLkmlkliKkrrGdtveESeLIe I^al^eAekllidrGIHPikladGFdlAckkal-etljdsisdkfpv enrerLVetaqTsLgSKIVnrslrqf aelAVTlAVLsVadies kdvnfemlkMeglwGgrleDTiLVk L xG ELLkqAe s 1V l eGLHPr i Vt eGFewAn t k tLe ltekfkkeapv erdlLVevcrTaLrTKLhqkladhi tecvVDAVLalrrdge epdliimVekmernhhds dmDT tLVr I^EMLsvAehfl^qc^lHPtWisAYrr^^ rmreir^siinSsItTKVlsrwsslacnlJUJ^ LAAEfLkqvkpyVeeGIjHPqil^ LkmlglkKvqGgaleESqLVa LAGELakJcAedlLykelHPtilvsGYldcAeeialiktlcjdiacrpvsi ndtcrvl^kvalTsJ^SKaVagareyladLvVkAV^^  GroEL Hsp60  RuBP CCT-1 CCT-2 CCT-4 CCT-5  CCT-6 CCTy CCTTl  TF55 GroEL  I: Results  0  GroEL Hsp60 RuBP CCT-1 CCT-2 CCT-4 CCT-5  CCT-6 eery CCTT] TF55  GroEL Hsp60 RuBP CCT-1 CCT-2 CCT-4 CCT-5  CCT-6 CCTy CCTT)  TF55 GroEL" GroEL Hsp60 RuBP  CCT-1 CCT-2 CCT-4 CCT-5  CCT-6 CCT/ CCTT|  TF55  —HHHHHHHHHHH  HHHHHHHHHHH.  .EEEEEE.EEEE.  HHHHHHHHHHHHH  HHHHHHHHHHHHHHHHHHHH.  HHHHHHHHHHHHHH HHHHHHHHHHHHHH HHHHHHHHHHHHHH HHHHHHHHHHHHHHH HHHHHHHHHHHHHHH HHHHHHHHHHHHHHH HHHHHHHHHHHHHHHH HHHHHHHHHHHHHHH HHHHHHHHHHHHHHH HHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHH  EEEEEE EEEEEE. .EE. . . .E..EE. HHHHHHHHHHHHHHHHHHHHH .HHHHHHEEE HHHHHHHHHHHHHH .EEEEEE. .EE EE HHHHH.HHHHHHHHHHHHHHH HHHHHHH. .EE. . . .HHHHHHHHHHHHHH .EEEEEE. ..EEEEE..EE....EE....E HHHHH. HHHHHHHHHHHHHHH . HHHHHHEEE HHHHHHHHHHHHHH EEEE. HHHHHHHHHHHHHHHHHHHH . . .HHHHHHHHHH HHHHHHHHHHHHHHHHHH . . .EE. . .EEEEE . . EEEEEHHH EE. . . HHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHH HHHHHHHHHHHHHHHH. . EEEE EEE. HHHHHHHHHHHHHHHHHHHH . .HHHHHHHHH HHHHHHHHHHHHHHHHHH. HHHHHHHHHHHHHHHHHHH . .HHHHHHHHHHH HHHHHHHHHHHHHHHHHHHH EHHHHHHHH EE EHHHHHHHHHHHHHHHHHHH . . HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH. EEEEE EEEEE EEHHHHHHHHHHHHHHHHHHHHE. . . . . .HHHHHHHH E HHHHHHHHHHHHHHHH. . EEEEEE EEE. HHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHH HHHHHHHHHHHHHHHHH. . . .E EEEE HHH. . HHHHHHHHHHHHHHHHHHHH . .HHHHHHHHHHH E HHHHHHHHHHHHHHHHHH  . 250 260 270 . 280 290 300 310 . GaVelesp f illadKKls—nlreMlpvLEavakag-kplLilaedvegeAI^tavvntlrgiVkvaavkA skVefekp llllseKKIs—slqdllpaLEisnqsr-rplLilaecrvTigeAI^ac^ siVefena r v l i t d q K I t — slkeliplrjEqttqlr-cplfiVaeditgeAI^tlwnkLrgilnvaaikA GyaLnctv asqamPlF^qnaKIacI^fsUrkaKnihl-gis GfLLeklp gmf qPRRVe kaK 11 i an tpMDtdKvkv f g s rVrVdgva kvaeLeaaEkl kMke kvdk 11 ahn GaLldqkc n^rgaPtRIekaKIgllqfqlsppKtdm-enqV^ GiVTdktm shpqituPKeLknaKVaiLtcpfEppKpkt-klikLclItst GlVLdhga rhpdrru^FJiV^daylltcnvsIjEye^ GvMInkdv thprmrRylkripRIvlLdssLEykKges-qtdleltreedf trllqmEeeylhqlcedliqlk Gva £ kk t f syag femqPKKyknpK I a iLnve LElkaekd-nael rVh t vecryqAI vdaEwn i LydkLek IhqsG GiWdkev vhpgmPKRlenaKIallx3asl^eKpel-daeI^ .EEEE..- -EEEEEE. . EEEE. . . .EEE. . . . .EEEE'. . . EEEEEE. . EEEEEE.. EEEEEE.. EEEEE... EEEE.... EEEEEE.. EEEEEEE . EEEEE..  .HHHHHHHHHH.  EEEEEE --HHHHHHHHHHHHHHH EEEEE --HHHHHHHHHHHHHHH FPRRR —HHHHHHHHHHHHHHH HHHH.HHHHHEHHHHHHH HHHEEE. . HHHHHHEEEEEE. HHHH. . EEEEEE. . HHHHHHHEEEEEE. EEEEEEE. E.EEE. . E. EEEE . HHHH  . EEEEE  HHHHHHHHH  EEEEEEE -  320 330 340 . 350 . 360 pgfgdrrkandp^IatltG^tviseeigmelekatLedLgqak pgfgdnrkntigdlavltGgCvf ceeldlkpeqctlenLgscD psfgerrkavlqdlaivtGaeylakdlgllvenatVdqLgtar anWLttgGIddlclkqfvesgamaVrrckksD cnVf Inrqllynypeqlf adakVmalehadf eg cnVLLiqksIlrdavnelalhf LakMkil-lcikD atLalcqwGfddeanhllqandLpaVrWVggpE pdVVItekGIsdlaqhyliriranVCalrrVrktD akVILsklpIgdvatqyfadrdMf cagrVpeeD anWlcqkGIdevaqhylakkgllaVrrakksD . .HHHHHHHHHHHHHH  EEEE.  . EEEEE. . . HHHHHHHHHHHHHH. . . EEEEEE. HHHHHHHHHH .H.HH.EEE . HHHHH . EEEEE....HHHHHHHHHHHHH...EEEEEE. ....HHHHHHHHHHHHHH. . . EEE E HHHH.... . EEEEE. . .HHHHHHHHHHHHHH. . .EEEEEE. . . . .HHHHHHHHH .H.HH .HEEE . HHHH .... .EEEE. . .HHHHHHHHHHHHHHHHHHHHHHHH. .EEEEE....HHHHHHHHHHHHHHHHHHHHHHH .EEEE. .HHHHHHHHHHHHHHHHHHHHHHHH. . .EEEEE....HHHHHHHHHHHHHHHHHHHHHHH . . .HHHHHHHHHHHHHHHHHHHHHH .EEEE. . .HHHHHHHHHHHHHHHHHHHHHHHH. .EEEEEE. . .HH . EEEE. . . HHHHHHHHHHHHHHHHHHHHHHH. . . EEEEE. .HHHHHHHHHHHHHHHHHHHHH . . .HHHHHHHHHH. .HHHHHHHHHH . EEEE. . HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH EEEEEE. HHHHHHHHHHHHHHHHHHHHHHHH .EEEE. . .HHHHHHHHHHHHHHHHHHHHHHHH. .EEEEE... .HHH.HHHHHHHHHHHHHHHHHHHHH . EEEE. . . HHHHHHHHHHHHHHHHHHHHHHH. . . EEEEE. . .H.HHHHHHHHHHHHHHHHHHHHHH . EEEE . . HHHHHHHHHHHHHHHHHHHHHHHH .. . EEEEE.  3. Multiple alignment of the deduced amino acid sequences and predicted secondary structures of the GroEL/Hsp60/RuBP and CCT/TF55 chaperonin families. The C. elegans CCT family (CCT-1, 2, 4, 5, and 6) and two murine CCTs (CCTy and rj) are shown in the alignment. GroEL, Hsp60, and RuBP, as well as TF55 are also included. Amino acids in uppercase or bold uppercase indicate conserved amino acids present in 7 or 9 of the 11 chaperonins, respectively. Conserved amino acids were placed in the following groups: A, G; M , I, L, V; D, E; K, R; S, T; N, Q; F, W, Y. Predicted secondary structure alignments are shown below the amino acid sequences. The known GroEL secondary structure (GroEL ) is shown for comparison. Protein conformations are indicated as follows: dots represent coils, turns, or bends; H, a helix; E, P-sheet. Dashes indicate gaps introduced in the protein primary and predicted secondary structures. Numbers in brackets represent the number of truncated amino acids from the ends of some sequences. Chaperonin sequences (with accession numbers) used in this alignment are as follows: GroEL [X07850] and RuBP [X07851] (Hernmingsen etal, 1988); Hsp60 [M33301] (Reading et al, 1989); murine CCTy [Z31556] andt] [Z31399] (Kubota^a/., 1994); TF55 [X63834] (Trent et al., 1991). Figure  0  30  Chapter I: Results GroEL Hsp60 RuBP CCT-1 CCT-2 CCT-4 CCT-5 CCT-6 CCTy CCTn TF55  • 370 . 380 . 390 . 400 . 410 . 420 . 430 . 440 . 450 . 460 . 470 . 480 r w l n k d t t t i l d gVgeEaaiqgrvaqirqqiEeats-dya^EKLqervAklaggvavIkVgAAteveMkEkkarVenAlhatraaVeEg-vVaGGGvalIrVaskI.adlrgq—n sitvtkedtviLn gsgpkeaiqerieqikgsiDitttnsyekEKIxgerlAklsggvavIrVgGAseveVgEkkdrydnftLnatraaVeEg-ILpGGGtalvkasrvLdevvvd—n kitlhqttttlla daaskDe-iqarvaqlkkelsetds-iydsEKLaeriAklsggvavIkVgActeteLEDrqlrlenAknatfaaleEg-iypGGGAayVhLstYVpaiketied LkRIakatgAtLtvslatLegDe—afdasllghadEivqe-risdDeLilikGpksrtasSIIl^GAn6SmiIJ}EmeRsVhDsLcvVrrvLeskkLVaGGGAvEtsLslfLetyaqt-ls IeRLalvlgGelvstfdspqtaq fg s c d l i e E i m i g eDRLlrf sGvklgeacSVvLrGAtqqiLDEseRsLhDALcvLvthVkEsktVaGAGAsEIlMssalaveaqk-va IeRediefysrllgcrpvasvDhfnadalgyadlveEiptg—gdgkvIkvtgvqnpghavSIIJ^GsnklvLEEao^sIhDALcvIrclVkkkaLIipGGGApEMelavkLrnlaqt-qh IelLaiatnArlvprfseLskEk l g t a g l v r E i t f g—aakDRMlsieqcpimkavTIfVrGGnkmiIDEakRaLhnALcvIrnlVrDsrIVyGGGsaEDaaaiqVakeadr-id MeRLqlavgGeavnsvddLtpEd lgwaglvyEhslg eEKytf ieecrapksvTLLIkGpnkhtltqikdalhDGLraVfntlvDkaVLpGAAAf ElaayvmLkkdven-lk nnRIaracgArlvsrpeeLreDd v g t g a g l l e i k k - - i g d E y f t f itdckdpkacTILLrGAskeiLsEveRnLqDAMqvcrnvLlDpqLVpGGGAsEMaVahaLtekska-nit LkRtmmacgGsIqtsvnaLvpDv lghcqvfEetqi ggDRynf f t G c p k a k t c T I I L r G G a e q f M E E t e R s L h D A I n d V r r a l k n d s W a G G G A i E M e L s k y L r d y s r t - i p LeKLaratgGrVisnideLtsqd lgyaalvEerkv geDKMvfveGaknpksvSILIrGGlervVDEteRaLrDALgtVadvIrDgraVaGGGAvEIelakrLrkyapq-vg . . . .HHHHHHHHHHHHHHH. . . . -  .HHHHHHHHHHHHHHHHHHHHH. . -  HHHHHHHHH.  GroEL  . EEEE. . EEEEEE  GroEL Hsp60 RuBP CCT-1 CCT-2 CCT-4 CCT-5 CCT-6 CCTy CCTH TF55  EEEEEE...EEEE ....HHHHHHHHHHHHHHHHH...• •HHHHHHHHHHHHHHH. . .EEEEE. HHHHHHHHHHHHHHH. . .EEEEE. EEEEEE. . .EEEE . . . .HHHHHHHHHHHHHHH EEEEEE. . .EEEE HHHHHHHHHHHHHHHHH. . .•-HHHHHHHHHHHHHHH...EEEEE. EEEEEE EEEEE. HHHHHHH. . .HHHHHHHH — E E . HHHHHHHHHHHHH •E . .EEEEEEE EEEEE. HHHHHHHH . . . . E E . . . HHHHHHHE. . • EEEEE. HHHHHHHHHHHH. . . . E E E . .HHH. HHHHHHHHH.. — . .HHHHEEE EEEEEE. HHHHHHHEE. . — . . . . EEEEEEE HHHHHHH. . .HHHHHHHH. . ...EEEEEE.. EEEEE. . . .EEEEEE. HHHHHHH.. .EEEE HHHHHH. — . . . .EEEEEE.' EEEEE. HHHHHHH. . .EEEE HHHHHHHH. HHHHHHHHH. . . . .HHHHHHHHH. -— . . .EEEEEE EEEEE. HHHHHHH. . HHHHHHHHHHHH. .HHHHHHHHH. •— . . .EEEEEE EEEEE.  HHHHHHH.  .HHHHHHHHHHHHHHHHHHHHHHHHH. HHHHHHHHHHHHHHH. . . .HHHHHHHHHHHHHHHHHHHHHHHH. HHHHHHHHHHHHHHH. . . . HHHHHHHHHHHHHHHHHHHHHHHHH-HH. . . HHHHHHHHHHHHHHHH. . . .HHHHHHHHHHHHHHHHHHHHHHH. . . . E . . . HHHHHHHHHHHHHHHHH. .HHHHHHHHHHHHHHHHHHHHHHH. . . . E . . . . HHHHHHHHHHHHHHHH. .HHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHH. .HHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHH. . .HHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHH. .HHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHH. .HHHHHHHHHHHHHHHHHHHHHHH. . . . E . . . HHHHHHHHHHHHHHHHH. .HHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHH -  . 490 . 500 . 510 . 520 . 530 . 540 . 550 . 560 . 570 . 580 . 590 . 600 GroEL edqNVGikvalrAMeaplrqlvlNcGeEpsvVantVKggdgn yGynaateeygnMidmGILDPtkVtrsaIjqyAasvagLMIttEcMVtdlpk-ndaadlgaag(12) Hsp60 fdqkLGvdiirkAItrpakqlieNAGeEgsvIIgkLideygd dfakGydaskseytdMlatGIIDPfkWrsgLvdASgvasLLattEvalvdape—ppaaagagg{12) RuBP hderLGadiiqkALqapaslIanNAGvEgevVIeklKesewe inGynamtdkyenLiesGVIDPakvtrcaLqnAasvsgMVLttqaIWekpkpkpkvaepaegq{3} CCT-1 sreQI^vaeFAsALlilPkvLasNAarDstdLVtfLRayhskaqlipqlqhlkwaGLdleeGtirdnkeaGILEPalsKvksLkfATeaaitlLrlDDLIkldkqeplggddcha CCT-2 GkesLAveAFGrALaqLPtilcdNAGlDsaeLVtrLRaehan grhnmGIdiekGevadVtklGVIEsynVklcmVssAaeateqILrVDDIIkaapraraqdnrpc CCT-4 GatQycwrAFAdALellPytLaeNAGlspihtvteLRnnhan gnssyGVnvrkGyvtdMveedWqPllvtasalkqASecvrsILklDDIVtaavr CCT-5 GieQyAfrAFAdALesIPniaLaeNsGlapidaLsdLKakqie tgksslGIdavfAgtndMkeqkVIEtllsKreqlslATqwrMILklDDVrvpddernigy CCT-6 GrakLGaeAFAqALlvIPktLavNGGyDaqetLvkLieekta agpdiavGLdletGgavep—qGIwDnvtVKknsIssATvlacnLLlVDEVMragmtnlkqpqpe CCTy GveQwpyrAvAqALevIPrtLiqNcGastirLLtsLRakhtq escetwGVngetGtlvdMkelGIwEPlaVKlqtyktAvetavLLLrIDDIVsghkkkgddqnrqtgap{ 5} CCTn, GkqQLligAYAkALeilPrqLcdNAGfDatnlLnkljRarhaq gginwyGVdinneniadnfqafvwEPamVRinaLtaASeaacLIVsVDEtlkn-prstvdppapsagr {8} TF55 GkeQLAieAYAnAIegLimiLaeNAGlDpidkLmqLRslhen etnkwyGLnlf tGnpedMwklGVIEPalVKitinaIkaATeavtLVLrIDDIVaagkkggsepggkkeke{6} GroEL° GroEL Hsp60 RuBP CCT-1 CCT-2 CCT-4 CCT-5 CCT-6 CCTy CCTT| TF55  . .HHHHHHHHHHHHHHHHHHHHHHH. . .HHHHHHHHH. . . . . . HHHHHHHHHHHHHHHHHHHHHH. .. . EEEEEEE HHHHHHHHHHHHHHHHHHHHHH.... . .EEEEEEE HHHHHHHHHHHHHHHHHHH. .. .EEEEEEE .HHHHHHHHHHHHHHHHHHHHHHH.. . HHHHHHHHHHHHHH .HHHHHHHHHHHHHHHHHHHHHHH. ..HHHHHHHHHHHHH. .HHHHHHHHHHHHHHHHHHHHHHH. ..HHHHHHHHHHHHHH .HHHHHHHHHHHHHHHHHHHHHHH.. .HHHHHHHHHHHHHH .HHHHHHHHHHHHHHHHHHHHHHH. ..HHHHHHHHHHHHHH .HHHHHHHHHHHHHHHHHHHHHHH.. . HHHHHHHHHHHHHH .HHHHHHHHHHHHHHHHHHHHHHH. ..HHHHHHHHHHHHHH .HHHHHHHHHHHHHHHHHHHHHHH. .. HHHHHHHHHHHHHH  r GroEL/Hsp60/RuBP GroEL (determined) CCT/TF55  EEEEE. .EEEEHHHHHEEEE. .HHHHHHHHHHHHHHHHHHH. .EEEE HHHHHHHH HHHHHHHHH HHHHHHHHHH  E EEE . . . .EEEEE . . . . EEEEEE  HHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHH. .HHH HHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHH. .HHHH - - . . EEEHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHH.HHH HHHHHHHHHHHHHHHHHHHHHHHHHH  600 300 400 500 200 n—i—i—i—i—i—i—i—i—i—i—r T—r —i—i—i—r • jDx-helix E3 I I1E3 E3 TS] LU • III I II S I E l HI DH E l QIETJ • E 3 CIO I 13 EDEZ3 GZ3 E3 II I I 0 I E I E 2 III £3 • • EE Q EIZIQ IDE0I | ^3-sheet • g / p mix I E 3 3IE3E] E 3 E 3 H E3 I • I I EH) I E 3D • I I E Z 3 H 100  "i  i  i  i  i  i  r  •same idiffeient  CCT/TF55= GroEL? GroEL N ^ domains  '-^LJLS  Equatorial  Apical  Intermediate  /  /  /  S /  /€.  Intermediate  Equatorial  Figure 3 cont'd. A summary of chaperonin predicted secondary structures is depicted below the alignments. Secondary structures (helix is shown dotted, (3-sheet in black, and combination of helix/p-sheet in white) predicted in all members of the GroEL/Hsp60/RuBP family, or in five or more members of the C C T / T F 5 5 family are shown aligned with the known G r o E L secondary structure. Below, regions where the C C T / T F 5 5 family are different from the determined G r o E L secondary structure are highlighted in gray, identical regions in white. Also shown are the relative positions of the three domains found in GroEL. A l l of the secondary structures, the C C T / T F 5 5 - G r o E L structure comparison, and the G r o E L domains are aligned against a scale (1-600) which corresponds to the amino acid sequence positions in the sequence alignment.  31  Chapter I: Results  Although the two chaperonin families (Group I and II) are evolutionarily related, basic differences in their primary amino acid sequence, quaternary structure, substrate specificity, and requirements for a co-chaperonin suggest that some alterations in their secondary structures would be required. The results obtained indicate that important similarities, as well as differences, exist between the predicted secondary structures of the two families. Comparison of the predicted secondary structures of the CCT/TF55 family members with the determined GroEL secondary structure (Figure 3) reveals significant correspondence in their structures. The entire equatorial domain (two halves), the C-terminal intermediate domain, and the left half of the N-terminal intermediate domain are very similar in the CCT/TF55 family and GroEL. These regions are rich in helical structures, and comprise residues required for ATP binding and hydrolysis, as well as for making inter-subunit contacts. Significant differences between the predicted secondary structures of the CCT/TF55 family and the known GroEL secondary structure are present in the second half of the N-terminal intermediate domain and most but not all of the apical domain. These two regions encompass a cluster of (3-sheet structures (positions 199-373) which contains 10 of the 19 P-sheet structures of GroEL. This region is also predicted to be rich in p-sheets in the CCT/TF55 family, but the positions of 4 of its 6 P-sheets do not coincide with the P-sheets of GroEL. In contrast, 8 out of 10 P-sheets are correctly predicted in the GroEL/Hsp60/RuBP proteins. Also, the region where a P-sheet and an extended helical structure is predicted in CCT/TF55 (positions 329-367) consists of a helix, an extended loop and two P-sheets in GroEL.  3.2 Isolation of genomic cct-1 Southern blot analysis of C. elegans genomic DNA reveals that cct-1 is a single copy gene (Figure 4). In order to isolate genomic cct-1, the cDNA was used to screen a polytene membrane containing ordered yeast artificial chromosome (YAC) clones representing approximately 90% of the C. elegans genome (Waterston et al, 1992). Two overlapping YAC clones (Y50H5 and Y50D9) mapping to chromosome II hybridized to the cct-1 probe (Figure 5A). 32  Chapter I: Results  kb  1 2 3 45  23.1  0.56 -  Figure 4. Southern blot analysis of C. elegans cct-1. Genomic DNA was digested with Pstl (1), EcoRl (2), HMIII (3), Dral (4), and Xhol (5), and probed with P-labeled cct-1 cDNA under stringent hybridization conditions. 32  33  Chapter I: Results Cosmids spanning the overlapping YAC clones were obtained and screened by dot blot analysis using the same probe; one of the cosmids, T05C12, was found to contain the gene of interest (Figure 5B). A Southern blot of the T05C12 cosmid DNA digested with Pstl, Pstl/Xhol, EcoRl, and Hindlll was hybridized to a cct-1 probe (Figure 5C). The restriction map patterns of the cosmid and genomic cct-1 gene were found to be identical. Consequently, a 3.7 kb Pstl restriction fragment was isolated from the cosmid DNA, subcloned into pBluescript, and sequenced. The open reading frame sequence of the cct-1 gene was found to be identical to that of the cDNA. As shown in Figure 5D, the cct-1 gene locus was mapped to cosmid T05C12 on the central (generich) region of chromosome II, between fer-15 and rol-6. The C. elegans cct-1 gene sequence is shown in Figure 6. Sequence analysis of cct-1 shows that the coding region is divided into 5 exons and 4 introns. The region upstream from the translation start site contains a distinctive degenerate sequence repeated five times: NAAANATTTTTATTTTTA. The repeats contain a core sequence with nucleotides (nt) present in 4 or 5 of the five repeats (underlined), and more variable nt present in 3 of the repeats (not underlined; N means any nt). Three of these repeats are separated by 5 and 7 nt and are proximal to the trans-splice site (see section 3.6). The repeats do not match any known transcription factor consensus binding site. Also present upstream from the start codon are two potential TATA boxes and a CCAAT box (Jones et ai, 1987). No HSE consensus sequences, which are involved in the transcriptional regulation of HSP genes (Sorger, 1991), are present. The 3'untranslated region of the gene contains a putative polyadenylation signal, AATGAA, present 13 nt upstream from the polyadenylation site defined by the cDNA sequence. Lastly, a potential transcription stop signal (Vandenbergh etal., 1991) consisting of an imperfect 13 bp inverted repeat is present 26 nt downstream from the polyadenylation site.  34  Chapter I: Results  B 1 6  2 , 7  12  3 8  13  4_ 9  14  5  10 15  1 1 16  IY50H5 fY50D9  kb  23.1  1  2  cct-1  ^_  9.4 66 -  2.3 _ 2.0  rol-6.  3 lin-5. unc-105  • •  LGII Cluster  §  fer-15  let-23.  "i—i—r 0.50  I  I  1.00  0.75  0.05 units  C30D12  M110  1  0 56 —  F58D3  T05C12  S  F32C7  C01E2  Figure 5. Isolation of cct-1 from the cosmid T05C12. A. Detection of two cross-hybridizing, overlapping YACs (Y50H5 and Y50D9) in an ordered YAC filter (Waterston et al, 1992) using P-labeled cct-1 cDNA as a probe. B. Cosmid DNA samples spanning the two YACs were probed in a dot blot using the same probe. Listed in order, the cosmids were: DH11, C09D2, C08B11, C04H4, B0223, C24E1, C30D12, C30G9, ZK656, ZK658, T05C12. T05H10, F10E8, ZK660, T07C3, and F49C2 (the positive cosmid, number 11, is underlined). C. Southern blots of T05C12 cosmid DNA digested with Pstl (1), Pstl-Xhol (2), EcoRI (3), and Hindlll (4) were probed with P-labeled cct-1 cDNA. D. Map position of cct-1 relative to cosmids surrounding T05C12 as well as other genetic markers on chromosome II. 32  32  35  Chapter I: Results  P.StI 20 . 40 « 6 0 • ctgcagCctgotgtgcatggcggcCccataaatgttagaaatctgaattcaatatCCtaaatCaCageJc^  80  •  100  •  120  •  140 -317  CtCaatttatcggcaataaatcgcgggtgggaacaatgttgcaaatacgccctagtgacagctggtgttgggtctcggcacgatCacccttctagcatgcatgaagtacgatgcgctctgagaatattctacaagaaCtttaattttgat -167 tcagaattcaagggatccccgattttctggaagcatttttatttttactgaaatattttcatttctacg|cata!tttttatttttgagtgcttattcaaagcccctttttgactcaatatcgaatgtcctactctattgctaagtcaatac -17 ggttgtgtttcaggtaATGGCATCAGCTGGAGATTCCATTCTTGCCCTCACCGGTAAAAGAA k M A S A G D S I L A L T G K R T T  134 T A A V 30 Xhol GCG ATCG C C AATATTGTG AA G TC A TC TC TTGGC CC TGTC GGAC TTGATAAAATGC TTG TCG ATGATGTTGGAGATGTC ATTGTC AC AAATGA CGG AG CCA C AATTCTG AAAC AACTC GAGGTTGAGCA TC CGGC TGGAAAAGTG CTTGTA 284 A I A N I V K S S L G P V G L D K M L V D D V G D V I V T N D G A T 1 L K Q L E V E H P A G K V L V 50 G  Q  G  I  R  S  Q  N  V  G AA CTTG C AC AGC TG C AAG A CGAGGAGG TCGG A G A TGG AAC TA CTTCTGTCGTTATTG TGGC GGC TG AGCTCTTG AAGAG AGC C G A TG AG C TTGTGAAACAAAAAG TTC A TC CG AC GAC TATTATC AA TGGTTA C C GTCTCG CG TG C AAG 43 4 E L A O L Q D E E V G D G T T S V V I V A A E L L K R A D E L V K Q K V H P T T I I N G Y R I i A C K UO GAAGCCGTCAAGTACATTAGTGAAAACATCTCATTCACTTCCGACTCGATTGGTAGACA^ E A V K Y T S E N I S F T S D S I G R Q S  V  V  N  A  A  K  T  S  M  S  S  K  I  584 167  I G P  C c agAG ACG C CG ATTTCTTCGG AG AGC TGGTTG TTG ATGC CGCGG AAG CTGTTC GTGTGG AAAATAAC GGG AAAGTC ACTTATC CTATC AATG C AGTC AATCTTC TGAAGG CC C ACGG AAAGAGC GCTC GCG AA TC AGTTTTGGTG AAAG 734 D A D F F G E L V V D A A E A V R V E N N G K V ' T Y P I N A V N V L K A H G K S A R E S V L V K G 216 Hindlll G ATATG CAC TC AATTGC AC AG TTGCC AG TC AGG CC ATGC CAC TTC G TG TTC AAAATGC C AAG ATCGC ATGTCTCG ATTTC TC TTTG ATGAAGG C TAAG ATGC AC C TC GGT ATTTC AGTCGTTGTTG AAG ATC CAG CC AAGCTTGAGGCTA 6 84 Y A L N C T V A S Q A M P L R V Q N A K I A C L D F S L M K A K M H L G I S V V V E D P A K L E A I 266 EcuRI Clal TTCGCAGAGAgtgagttgaaactattcgtttctttttaagctatggaattttcagAOAATTCGATATTACC^^ 1034 R R E E F D I T K R R I D K I L K A G A N V V L T T G G I D D L C L K 301 Hindni AGC AATTTG TCGAATC TGG AG C TATGG CTGTTC GTCGATGC AAGAAATC AG ACTTG AAGAGAATTG CC AAAGCTACTGGAG C C AC ATTGAC TGTTTCC TTGG C TACTTTG GAAGGAGATGAAGCTTTC G ATG CC TXT G CTTC TTGGACATG 1184 Q F V E S G A M A V R R C K K S D L K R I A K A T G A T L T V S L A T L E G D E A F D A S L L G H A 351 C CG ATG AAATTG TTC AAG AAAG AATTAGTG ACG ACGAGC TC ATTC TC ATC AAGGG AC CGAAATC TCGT A CTGC C AGCAGCATTATC C TC CG TGG AG CG AAC G ATGTGATG C TC GATGAAATGGAGAGATCGG TTC ACG AC TC ACTC TGTG 1334 D E I V Q E R I S D D E L I L I K G P K S R T A S S I I L R G A N D V M L D E M E R S V H D S L C V 401 EcoRl TTGTTCGT AG AGTTC TGG AAAGC AAG AAACTTGTGGC TGGAGG AG GTGC TGTTG AG ACTTC TCTC AGTCTTTTC C TTG AAAC TT ATGC AC AAAC C TTGTCTTC TCG CG AG C AGCTTG C TGTTGCTQAATTCGCTTC AG CGC TTCTC ATC A 1484 V R R V L E S K K L V A G G G A V E T S L S L F L E T Y A Q T L S S R E Q L A V A E F A S A L L I I 451 TTC CGAAGGTTTTGGCAAGCAATGCTGC AAGAG ATTC TACTGATTTAGTGACTITTC P K V L A S N A A R D S T D L V T F L R  Dru.1  A  Y  H  S  K  A  Q  L  I  P  Q  L  Q  H  L  K  W  1634 488  atgttagettaatgtaataaaattaaaataatttatttcaaaaaatttcgttttgtgcttagaaaaagcgtctaattcatgttttctgaatttgagtcagtttattcactctttttttagGGCTGGTTTGGATCTCGAAGAAGGCACGAT 1784 A G L D L E E G T I 498 C CG C GATAAC AAGGAGG C TGG AATTTTGG AGCC AGC TC TTAG TAAGGTC AAGTC TC TG AAGTTCGC C ACTG AGGC AGCC A TTACG ATATTG CGTATTG ATG AC C TC ATC AAAC TTG AC AAG C AAG AGC CAC TTG GAGGAG ATG ATTG CCA 1934 R D N K E A G I L E P A L S K V K S L K F A T E A A 1 T I L R I D D L I K I j D K Q E P L G G D D C H 548 CGCTTAAatttecccgttcaccccgtttatatatccctgttttccgcgtgcttctcacataattccgaCctgctgctccttatcccaaattctcatgttcagcttCtgttttcttcttttgatgatactttattgaacgaaatgttgtaa 2084 A * , 549 Hindlll gttttaatgttttgatttcaaaqttgtttgtattcgtttttcattattcaaacaatgaagaagctttgccacatttagttgtattactcatcgccgcccagtgatgtgaaatcgcgatttttcggatcactgttagtacgacggcgCagg 2234  Figure 6. Nucleotide and amino acid sequence of C. elegans genomic cct-1. Part of the 3.7-kb Pstl restriction fragment isolated from the cosmid T05C12 is shown here. The nucleotide (nt) and amino acid numbering are indicated on the right side of the sequence; the first nt (+1) begins at the start codon (A in A T G ) , and negative numbering begins upstream of the start codon. Exons are in uppercase type, and have the corresponding aa residue indicated for each codon; introns are shown in lower case type. Five repeats within the non-coding promoter region are singly underlined. Putative C C A A T and T A T A consensus sequences are shown boxed. The SL1 frarcs-splicing site is indicated by an arrow pointing up. The likely polyadenylation signal ( A A T G A A ) is double underlined, and the respective polyadenylation site is shown by an arrow pointing down. A n inverted repeat downstream of the polyadenylation site is denoted by two horizontal arrows. The accession number for C. elegans cct-1 is U07941.  36  Chapter I: Results 3.3 Physical map positions of five cet genes Many C. elegans genes are transcribed as polycistronic mRNAs, much like bacterial operons (Krause and Hirsh, 1987). Processing of the individual transcripts requires transsplicing to splice leader RNAs (SL1 and SL2; reviewed in Blumenthal, 1995). In theory, operons may serve to coordinate the expression of genes with related functions or which assemble into an oligomeric complex, as is the case with CCT. Both cct-1 and cct-2 were mapped by hybridization to a filter containing overlapping ordered yeast artificial chomosome (YAC) clones representing about 90% of the C. elegans genome (Waterston et al., 1992), and cct-4, cct5, and cct-6 were identified in the genome sequencing project. The five C. elegans cet genes are located on chromosomes II and III (Figure 7). Three genes, cct-1, cct-2, and cct-4 are between 50 and 300 kb apart on chromosome II, and thus are unlikely to be part of a single transcriptional unit. Likewise, the two genes on chromosome III, cct-5 and cct-6, are widely separated from each other.  3.4 Expression of cet genes throughout nematode development The expression levels of the five C. elegans cet genes throughout development were examined by Northern blot analysis. Total RNA (approximately 20 fxg) prepared from the nematode's six major life cycle stages was separated on a 2.2 M formaldehyde-1.2% agarose gel and transferred to a Hybond-N membrane. The membrane was then sequentially hybridized to individual cet cDNAs, autoradiographed, and then stripped before the next hybridization. As a control for RNA loading, the membrane was also hybridized to a P-labeled actin-1 probe 32  which hybridizes to at least four actin genes in C. elegans, that, overall, are expressed at the same approximate level during development (Krause and Hirsh, 1986). Northern blot signal intensities were quantified by densitometry, and the expression levels of the cet genes were then normalized to the actin control. Figure 8 summarizes the results from the Northern blot analysis. Each C. elegans cet gene is expressed at a similar level throughout development. An mRNA transcript of approximately 1.9 kb, which closely matches the sizes of the cDNAs (1.8-2.0 kb), is present for each gene during all life stages. :  37  Chapter I: Results  A -30 -20 -10 I I I I I I I I I I I 1 map unit = 1 cM  I  1  I  I  I  1  I  1  I  1  I  1  I  1  I  gene  cosmid(s)  YACs  chromosome  determined  cct-1  T05C12  Y50H5, Y50D9  II  this study  cct-2  T21B10  Y55H12, Y50B6  II  this study  cct-4  K01C8  II  genome seq.  cct-5  F25F2/C07G2  III  genome seq.  cct-6  F01F1, W04D12  III  genome seq.  Figure 7. Physical map positions of five C. elegans cetgenes. A. The cet genes, marked in bold and shown with some genomic landmarks, are distributed on two of the five C. elegans autosomes. Precise locations of the genes on the physical map can be found in the C. elegans ACeDB database (Richard Durbin and Jean Thierry-Mieg). Each map unit corresponds to more than 1.5 Mb in the gene-rich, central chromosomal clusters of C. elegans (Sulston et al., 1992). B. Summary of cet gene mapping to cosmids and/or YACs. "genome seq." refers to genes already mapped and sequenced by the C elegans sequencing consortium.  38  Chapter I: Results  Figure 8. Northern blot analysis of five C. elegans cet genes. RNA from each major developmental stage (embryo, L1-L4 larvae, and adult) was separated on a 1.2% denaturing agarose gel and transferred to a Hybond-N membrane. The membrane was then sequentially hybridized to five cet cDNAs and a control actin-1 probe. After each hybridization and autoradiography, probes were stripped from the membrane. The bar graph shows relative mRNA levels of each cet gene during the developmental stages. Expression levels are normalized with respect to the actin-1 control probe. Below the graph are representative Northern blot autoradiograms of each gene at the L4 larval stage, with the sizes of the RNA molecular weight markers indicated on the left.  39  Chapter I: Results 3.5 Expression of cct-1 after heat-shock treatment A characteristic difference between yeast cct-1 and other chaperonin genes is that the level of RNA transcript is not upregulated following heat-shock treatment; rather, there appears to be a decrease in the amount of transcript produced during heat-shock (Ursic and Culbertson, 1992). A Nothern blot containing RNA samples isolated from heat-shocked (2 hours at 30°C and 30 minute recovery at room temperature) and control nematode embryos was successively hybridized to P-labeled cct-1 cDNA, actin-1, and hspl6-48 probes (Figure 9). The relative 32  signal intensity from the cct-1 (Figure 9A) compared with the actin-1 (Figure 9B) probing of the control and heat-shocked RNAs is somewhat weaker, indicating that C. elegans cct-1 expression is not heat-inducible and might be slightly lower. The actin-1 probe serves as a loading control, as it is expressed at a similar level during heat-shock; a control probing (Figure 9C) shows that the RNA from the heat-shocked nematodes but not the control nematodes hybridizes strongly to the strictly heat-inducible hsp!6-48 gene (Jones et al., 1989).  3.6 cct-1 undergoes both m-splicing of its introns and trans-splicing to SL1 Inspection of the cet-1 cDNA sequence immediately upstream of the methionine start codon revealed part of the SL1 rrans-splice RNA leader sequence, which is spliced onto many C. elegans gene transcripts (Krause and Hirsh, 1987). To determine if cct-1 is trans-spliced to the SL1 or SL2 RNA leaders, PCR was performed on a C. elegans embryo cDNA library using an SL1 and SL2 primers in conjunction with an internal cct-1 primer (MIC3, complementary to nt 242-227 on the genomic DNA sequence). A DNA fragment amplified with these primers was identified as trans-spliced cct-1 by sequencing (Figure 10). Therefore, the beginning of the fully processed mRNA sequence is 5' -GGTTT A ATT ACCC A AGTTTG AGGT AA TG. where the underlined nt are derived from the SL1 RNA leader, which istarns-splicedat the splicing consensus signal (TTTCAG), and ATG is the start codon. In addition, the four introns in cct-1 are absent from the cDNA sequence, indicating that these introns are c/s-spliced from the RNA transcript.  40  Chapter I: Results  Figure 9. Northern blot analyses of heat-shocked C. elegans RNA. A Northern blot prepared with embryonic total RNA from control (C) or heat-shocked (HS) nematodes was sequentially hybridized to P-labeled cct-1 (A), actin-1 (B), and hsp 16-48 (C) probes. After each hybridization and autoradiography, probes were stripped from the membrane. 32  41  Chapter I: Results  Figure 10. The cct-1 primary transcript is taws-spliced to the splice leader-1 (SL1) R N A . A . Three amplified fragments were obtained when C. elegans embryo first strand c D N A was used in a PCR reaction containing a primer with the SL1 sequence and an internal cct-1 primer (MIC 3). One major product was obtained when the PCR was carried out with an SL2 primer and the same cct-1 internal primer. Shown here are the individual products re-amplified from the SL1 reaction (lanes 1-3) and the SL2 reaction (lane 4), and analyzed on a 1.5% agarose gel. Upon isolation, subcloning, and sequencing of all four fragments, it was found that only one of them (lane 2) was trans-spliced cct-1 cDNA. B. Sequence of cct-1 near the trans-splicing site. The consensus splice site is boxed, and the initiating methionine and two additonal amino acids are shown below the D N A sequence. C . Sequence of the 5'-end of cct-1 c D N A , with the SL1 sequence underlined.  42  Chapter I: Results 3.7 Expression pattern of ccM-promoter-P-galactosidase fusion transgene In order to determine the spatial expression pattern of the cct-1 gene, an additional -2 kb of upstream sequence from the Pstl site was cloned (see Materials and Methods). An expression construct harbouring the entire 5'-noncoding region (-2.7 kb) and -1 kb of 3'-noncoding region (including the polyadenylation signal), which is likely to contain the promoter and/or regulatory elements of the cct-1 gene, was created (Figure 11A). To monitor expression from the cct-1 promoter, the first 32 amino acids of the CCT-1 protein was fused in-frame with the SV40 nuclear localization signal (NLS) and P-galactosidase as a marker. The NLS is used to restrict the localization of the marker enzyme to the nucleus, thereby assisting in the identification of cell types. This construct was then injected into C. elegans, and four stable lines carrying extrachromosomal arrays of the construct were isolated (see Appendix V). The expression of the cc?-i-promoter-P-galactosidase fusion transgene was monitored by permeabilizing the nematodes with acetone and staining with X-GAL. The staining pattern of selected nematodes is shown in Figure 11B. In the transgenic strains analyzed, expression of the marker enzyme occurs throughout development, from embryos to adults. This result was expected given that cct-1 transcripts are detectable during all life stages. Note that because the constructs are propagated as extrachromosomal arrays, not all cells or tissues will contain the vector construct and therefore staining will differ from animal to animal. However, specific staining patterns in embryos and larvae/adults were reproducibly obtained in the four independent strains. Late-stage embryos expressed the transgene, often in pairs of closely-associated cells, or sometimes in a greater number of cells. Expression in larvae was particularly strong in the head region near the pharynx, where numerous muscle and neuronal cells are present. Similarly, tail expression was prominent near the anal sphincter, a site also rich in the same cell types. Many cells from the ventral nerve cord and nerve ring stained prominently. Occasionally, expression of the Pgalactosidase marker was visible in hypodermal in larvae and in some vulval cells of adults.  43  Chapter I: Results  Figure 11. Localization of cct-1 transgene expression, a. Schematic of the construct used to monitor cct-1 promoter-driven expression of (3-galactosidase. The construct contains -2.7 kb of 5'-noncoding region and -1 kb of 3'-noncoding region, and the first intron of cct-1. The vector encodes a translational fusion of the first 32 aa of the CCT-1 protein and the SV40 nuclear localization signal (NLS) and [3-galactosidase coding region. Transgenic nematodes carrying extrachromosomal arrays of the above construct were dessicated, permeabilized with acetone, and stained with a solution containing X - G A L . b. L4 hermaphrodite larva showing staining in pharyngeal (PH) and head region, in the nerve cord (NC), and in the tail (T). c. L4 male staining in the head region (left), NC, and reproductive organ in tail, copulatory bursa (CB). d. Young larva showing staining in hypodermal (HYP) cells, e. L 2 or L3 showing intense staining in PH. f. embryos showing staining.  44  Chapter I: Results 3.8 Purification of CCT and analysis of protein subunits In order to obtain sufficient quantities of starting material for purifications, over 100 g of embryos were isolated from 2 separate 20-liter nematode fermentor cultures (see Appendix IV). Embryos rather than adult nematodes were used because of the gut protease contamination which occurs when adults are homogenized. A purification of CCT from adults resulted in a substantial amount of proteolytically-degraded CCT protein (not shown), which was avoided when embryos were used. Interestingly, a similar 30 kDa proteolytic fragment of CCTe is observed in some preparations of mouse testis CCT (Hynes et al., 1995), perhaps indicating the presence of a common proteolytically-sensitive region in the CCT subunits. To help in the detection of CCT subunits during and after the purification of the chaperonin complex, polyclonal antibodies against peptides corresponding to the last 15 amino acids of C. elegans CCT-1 and CCT-5 were produced. Both antibodies specifically recognize proteins of approximately 59 kDa in whole nematode extracts (Appendix VII). The CCT complex appears to be largely cytosolic. Preparations of cytosolic and nuclear fractions from embryos showed that the majority of the CCT is in the cytosol, although there may be a small amount in the nucleus (not shown), as has been previously observed (Joly et al., 1994). Isolation of CCT from embryos involved a two-step purification, the first being a size separation on 10-40% sucrose gradients, followed by affinity purification on an ATP-agarose column. Sucrose gradients (2-8) were poured and a 2.5 g aliquot of cleared embryonic protein extract was applied to each gradient and centrifuged at 26000 rpm for 18 hours. Nineteen 2-ml fractions were collected from the bottom of each gradient and assayed for the presence of CCT-1 by Western blotting (Figure 12A). Positive fractions were dialyzed and applied to a column containing ATP-agarose resin. After extensive washing, the ATP-bound proteins were eluted using 10 mM ATP. The fractions containing CCT were dialyzed against a low salt buffer and concentrated by vacuum centrifugation. The CCT complex was also purified by a combination of sucrose gradient fractionation, ion-exchange chromatography, and ATP-agarose chromatography, but this yielded a substantially lower amount of CCT without greatly  45  Chapter I: Results increasing the purity as judged by silver staining (data not shown; CCT binds to DEAE Sephacel resin and is eluted at 450-550 mM NaCl concentrations). The purified CCT complex contains at least 7 visible polypeptides when separated on a single-dimension 7.5% SDS-gel (Figure 12B). These range in size from 52-65 kDa, and appear to be present in approximately equimolar amounts. When C. elegans and bovine testis CCT (kind gift from R. Melki) preparations are analyzed in parallel on an SDS-gel, the number and molecular weights of the polypeptides present appear essentially identical, suggesting that the subunit composition and stoichiometry of the CCT complexes from evolutionarily distant organisms are very similar. Even the contaminants from both preparations are similar. Both contain HSP70, which is known to associate specifically with CCT (Lewis et al., 1992). Actin, a known substrate for CCT, is likely to be present in both preparations. HSP60, whose size and ATP-binding activity are almost identical to those of CCT, represents another contaminant. The presence of HSP70 and HSP60 in the C. elegans CCT purified by both of the above described methods was confirmed by Western blotting (not shown; HSP70 was detected with a mAb against HSP70/HSC70, and HSP60 was detected with a pAb against moth HSP60; both are from StressGen). Two CCT subunits, CCT-1 and CCT-5, were detected in the purified CCT preparation by Western blot analysis with the purified anti-CCT-1 and anti-CCT-5 pAbs (Figure 12C). These two proteins were previously shown to be present in both murine and bovine CCT (Kubota et al., 1994). In addition, several CCT-related subunits within the complex were identified by Western blot analysis with the UM1 pAb (kindly provided by Keith Willison), which crossreacts with all murine CCT subunits (Hynes et al., 1995; also see Appendix VII). The reactivity of the C. elegans CCT complex subunits toward these three antibodies further suggests that the CCTs from different organisms possess similar subunit species.  46  Chapter I: Results  Figure 12. Purification C. elegans CCT and identification of subunits. A. SDS-PAGE of 10 (il aliquots from 2 ml sucrose gradient fractions (left to right corresponding to decreasing sucrose concentrations). Aligned below the gel are the corresponding sucrose gradient fractions (6-15) probed with the anti-CCT-1 antibody. Fractions containing the most CCT-1 (9-12) were isolated and dialyzed against TEDKM buffer (see Materials and Methods) before loading onto an ATPagarose column equilibrated in the same buffer. ATP-binding proteins were then eluted with ATP, and concentrated by vacuum centrifugation. B. A fraction of the C. elegans CCT preparation (10 ul) was analyzed on a 9% SDS-gel in parallel with partially purified bovine testis CCT (obtained from Ronald Melki). Protein MW markers are indicated on the left of the gel. The 2 prominent polypeptides not associated with the bovine CCT complex are HSP70 (-70 kDa) and actin (-45 kDa) (R. Melki, personal communication). C. This panel shows the cross-reactivity of the C. elegans CCT complex with the C. elegans anti-CCT-1 and anti-CCT-5 pAbs, as well as the pAb which recognizes a conserved region in CCTs, UM1 (supplied by Keith Willison and described in Hynes et al, 1995).  47  Chapter I: Results 3.9 Affinity of CCT for denatured actin The fact that only very small amounts of C. elegans CCT could be purified greatly limited the functional assays which could be carried out. Therefore, an assay involving the binding of the chaperonin to [ S]-labeled, denatured actin was chosen because of its convenience and 35  sensitivity. First, the purified C. elegans CCT was separated by electrophoresis on a 4.5% native gel (Figure 13A). Rather than a single migrating species, three Coomassie-stainable bands are observable (Bands A, B, and C). Bands A and B are approximately of equal intensity, and band C is approximately one-fifth the intensity of the others. By Western blotting, Band A was shown to contain CCT-1 and CCT-5, and therefore likely represents the CCT complex. The mobilities of C. elegans and bovine testis CCTs on the native gel are identical, further confirming the identity of Band A. The third band cross-reacts with an anti-HSP60 antibody, and therefore is likely to contain the co-purified HSP60 contaminant. The identity of Band B is unknown, although it is apparent that the bovine testis CCT preparation contains a small amount of material with the same mobility on the native gel. Bovine and C. elegans CCTs were incubated with [ S]-labeled, urea-denatured actin (a 35  kind gift from R. Melki) and separated on a 4.5% native gel (Figure 13B, lanes 1 and 2). After autoradiography (lanes 4 and 5), it is apparent that an actin-binding activity is associated specifically with bovine CCT (lane 4). In contrast, an actin-binding activity is found almost exclusively with the HSP60-containing Band C in the C. elegans CCT preparation (lane 5). A substantially lower amount of radiolabeled actin is also found associated with the CCTcontaining Band A. In order to diminish the actin-binding activity associated with the HSP60 contaminant, the CCT preparation was immunodepleted of HSP60 using an excess of StressGen's anti-moth HSP60 (lane 3). Indeed, the immunodepleted reaction mixture had little labeled actin associated with Band C (which contains HSP60), and had a correspondingly (slightly) larger amount of actin associated with the CCT-containing Band A (lane 6). It should be noted that the bovine CCT preparation refolded a portion of the denatured actin in the presence of ATP (lane 4; arrow shows native actin species). Neither the untreated or immunodepleted C. elegans preparation yielded native actin (lanes 5 and 6). 48  Chapter I: Results  Coomassie stain CCT prep.  Immunoblots CCT-1  CCT-5  UM1  HSP60  Hand A [ Band B [ Band I [  Coomassie stain  B bovine CCT prep.  autoradiograph  C. elegans C. elegans CCT CCT prep, prep. (- HSP60)  C. elegans bovine C. elegans CCT CCT CCT prep, prep. prep. (- HSP60)  Band A Band B £ BandC  Figure 13. Native gel analysis and affinity of C C T and HSP60 for denatured actin. A . Separation of -10 [Xg C. elegans C C T preparation on a 4.5% native gel. Three species can be detected (Bands A , B , and C) by Coomassie staining. Immunoblot analysis of the same preparation with antibodies against CCT-1, CCT-5, U M 1 , and HSP60 are shown aligned with the Coomassie-stained gel. B . Binding/refolding assays using [ S]-labelled denatured actin. Bovine C C T (lanes 1 and 4) or C. elegans C C T (lanes 2, 3, 5, and 6) preparations were incubated in an ATP-containing buffer with identical amounts of labelled, unfolded actin and separated on 4.5% native gels. The C. elegans C C T preparation in lanes 3 and 6 was first immunodepleted of HSP60. Lanes 1-3 show the Coomassie-stained gel, and lanes 4-6 show the autoradiograms of lanes 1-3, respectively. The arrow points to native actin (lane 4). 35  49  Chapter I: Discussion IV. DISCUSSION  A novel eukaryotic cytosolic chaperonin Our understanding of the mechanisms by which chaperonins facilitate the folding of newly synthesized proteins, as well as proteins translocated across biological membranes, has dramatically increased since the identification by Hemmingsen et al. (1988) of the homologous genes encoding GroEL/ES and RuBP, proteins which could chaperone protein folding and assembly in vivo. Moreover, the recent identification (Lewis et al., 1992; Gao et al., 1992; Frydman et al., 1992) of a eukaryotic cytosolic chaperonin structurally and functionally similar to the Group I chaperonins has generated tremendous interest in the field of protein folding (Ellis, 1992). The unique multimeric nature (Frydman et al., 1992; Lewis et al., 1992) and possible heterogeneous subunit composition (Creutz et al, 1994; Roobol etal,  1995) of the  CCT complex make it a particularly interesting and challenging chaperonin model system to study.  The cet multigene family ofC. elegans The genes encoding CCT subunits are common to all eukaryotes, including yeast (Ursic and Culbertson, 1991), Drosophila (Ursic and Ganetzky, 1988), vertebrates (human, mouse, bovine, Xenopus, axolotl, etc.; reviewed in Willison and Kubota., 1994; Kubota et al., 1995b), plants (Ehmann et al., 1993), and protozoa (Soares et al., 1994; Maercker and Lipps, 1994). So far, the full complement of cet genes (8 different genes) have been sequenced in mouse (Kubota et al., 1994, 1995a) and yeast (reviewed in Stoldt et ai, 1996). A testis-specific mouse chaperonin gene is also known (Kubota et ai, 1997). In this study, five members of the C. elegans cet multigene family were characterized. The C. elegans cet genes studied thus far (cct-1, cct-2, cct-4, cct-5, and cct-6) are well conserved across species, displaying 62-69% predicted amino acid sequence identity with mouse, human and yeast orthologs. Also, sequence motifs and residues conserved in all chaperonins are conserved in the C. elegans CCT family, including those known to be involved in the ATPase 50  Chapter I: Discussion function of GroEL. The C. elegans CCT subunits are 23-35% identical to each other, and 31-35% identical to one of the two archaebacterial chaperonin subunits, TF55. From an evolutionary perspective, phylogenetic analyses of CCT proteins and TF55 point to an early divergence of the gene family—as early as two billion years ago, or approximately as old as the origin of eukaryotes (Willison et al, 1994; Kubota et al, 1994, 1995b). It has been remarked that the amino acid substitution rate of TCP-1 is nearly constant and that it evolves as slowly as the highly conserved protein cytochrome c (Kubota et al, 1995b). Genomic mapping data on several human and mouse Cet genes (reviewed in Kubota et al, 1995b) is also consistent with an early divergence of this gene family. No clusters of CCT subunit genes are known in any organism including yeasts and mammals. Although it is interesting that three of the five C. elegans cet genes are somewhat closely associated on chromosome II (see Figure 7), the distance between the cet genes on the two different chromosomes argues against the possibility that they are transcribed as polycistronic messages, as a few known genes with related functions are in C. elegans (Blumenthal, 1995). Taken together, these findings imply that the cet genes diverged from one or perhaps two ancestral genes common to both eukaryotes and archaebacteria to encode proteins with conserved, specialized functions. Indeed, attempts to rescue yeast strains deficient in a particular Cet gene by overexpression of a yeast Cet homolog have revealed that each Cet gene examined has an irreplaceable function(s) in this organism (Chen et al, 1994). Moreover, in the present study, attempts at rescuing a yeast cct-1 mutant (DUY4; Ursic and Culbertson, 1991) with the C. elegans cct-1 gene were not successful (not shown). These results suggest that not only are different CCT subunits functionally distinct but that homologous genes from different organisms, although closely related (61% amino acid sequence identity between yeast and C. elegans CCT-1), require compensatory mutations in other subunits for proper assembly or activity of the CCT complex. There is presently no evidence that the different subunits carry out different functions on their own; instead, a single functional CCT complex containing the eight different subunits is likely to be required for carrying out its chaperone activity.  51  Chapter I: Discussion Structure of C. elegans CCT complex The quaternary structures of the mammalian CCT complexes are known to be well conserved, most likely consisting of 8 or 9 subunits per ring (Lewis et al, 1992; Frydman et al., 1992; Marco et al, 1994). The subunit composition of different mammalian CCT complexes has been extensively investigated. Two-dimensional polyacrylamide gel electrophoretic analysis of mouse and bovine CCTs shows that each complex contains nine subunit species (Kubota et al, 1994). In contrast, eight subunits of rabbit reticulocyte lysate CCT were identified using an HPLC purification method (Rommelaere et al, 1993). Human HEp-2 and mouse F9 cells were shown to contain only seven subunit species (Lewis et al, 1992; Kubota et al, 1994), and Roobol and Carden (1993) also noted differences in the number of subunits present in rodent testis and brain CCT preparations. These data suggest that the subunit composition of CCT may vary between different cell or tissue types. In contrast, the structure of the CCTs from different organisms has received much less attention. Thus far, only cucumber (Ahnert et al, 1996) and S. cerevisiae (Miklos et al, 1994) CCT complexes have been isolated. Both were shown to contain TCP-1 (CCT-1) and the yeast chaperonin contained CCTp (CCT-2) in addition. Although it is claimed that the CCT complexes isolated in the above studies are comparable to those of mammalian CCTs, no direct (side-by-side) comparisons were made. To assess whether the subunit composition of CCT is similar between highly diverged organisms, it was of interest to determine the makeup of the C. elegans CCT complex and compare it with a mammalian counterpart. A large-scale preparation of C. elegans embryos provided enough starting material for the two-step (sucrose gradient fractionation and ATPagarose chromatography) purification of CCT, as first described by Lewis et al. (1992) to isolate murine testis CCT. The much lower concentration of CCT in C. elegans embryos compared with testis made it very difficult and impractical to add an additional purification step. Consequently, the C. elegans CCT was not purified to near-homogeneity, and several contaminants were shown to be present, including HSP60 and HSP70 by immunoblotting, and a 45 kDa protein (most likely actin). More importantly however, at least seven polypeptides between 53-65 kDa are distinguishable in the C. elegans preparation, which is precisely the range of subunit sizes 52  Chapter I: Discussion observed by SDS-PAGE for both mouse and bovine CCT (Gao et al, 1992; Lewis et al, 1992; Frydman et al, 1992; Kubota et al, 1994). A side-by-side comparison of bovine and C. elegans CCTs shows the subunit composition of these evolutionarily distant CCT complexes to be remarkably similar. Interestingly, similar contaminants were present in the partially-purified bovine CCT complex and C. elegans CCT (HSP70 and actin; R. Melki, personal communication). The C. elegans CCT preparation also contains some HSP60 (see below). The presence of HSP70 'contaminants' in CCT preparations may be biologically relevant because it cooperates with CCT during protein folding (Frydman et al, 1994, 1996). Indeed, HSP70 is specifically immunoprecipitated with anti-TCP-1 antibodies (Lewis etal, 1992). Surprisingly, native gel analysis of the CCT preparation revealed three distinct species, of which the slowest migrating is most likely CCT, as it corresponded in mobility to bovine CCT and contained CCT-1 and CCT-5 (detected by specific antibodies against the C-termini of the two corresponding C. elegans chaperonin subunits) as well as polypeptides recognized by the UM1 antibody, which recognizes a region conserved in CCT subunits (Hynes et al, 1995). Interestingly, the second fastest migrating species is approximately equal in abundance to the first, but it is not recognized by any of the CCT-specific antibodies, suggesting that it is unrelated to CCT. The identity of this species therefore remains unknown. The fastest migrating species reacted with an anti-moth HSP60 antibody, and therefore is likely to be (or to contain) HSP60.  Secondary structure analysis of C. elegans CCTs and other chaperonins The unique hetero-oligomeric structure of CCT and divergent primary structure of each CCT subunit suggest that Group II chaperonins may be significantly different structurally from each other and from Group I chaperonins. Perhaps contrary to expectations, analysis of the C. elegans and murine CCT proteins revealed that all the members have nearly identical predicted secondary structural folds, despite displaying limited pairwise sequence identity between members (23-35%). This observation suggests that the divergence in the primary structures of the CCT subunits has not resulted in major alterations in their secondary or tertiary 53  Chapter I: Discussion structures, but has allowed for significant variability in amino acid composition, perhaps resulting in diversification of function. Most of the highly conserved regions within chaperonins are probably involved in ATP hydrolysis (Fenton et ai, 1994; Kim et al., 1994; Lorimer, 1995). The analysis presented here provides evidence that the secondary structures within the equatorial and part of the intermediate domains of the Group I and II chaperonins are very similar and therefore likely to have been evolutionarily conserved. This finding suggests that the ATPase function of the two chaperonin families is maintained through the conservation of secondary and perhaps also tertiary structures, as well as residues critical for ATP binding and hydrolysis. Interestingly, a number of residues known to affect GroEL ATPase activity (e.g., residues at positions 180-182 in Figure 3) are not conserved in the CCT/TF55 family but are present within regions predicted to have equivalent secondary structures. On the other hand, the predicted secondary structures of the CCT proteins in the region encompassing part of the N-terminal intermediate domain and most of the apical domain are substantially different from the known GroEL secondary structure. Therefore, the differences predicted for the polypeptide binding domain of the CCT subunits and GroEL may account for the different range of target polypeptides which can be bound to and folded by the two chaperonins. Also, differences in primary structure are likely to account for some of the specificity. For example, hydrophobic residues within the apical domain which are known to be involved in polypeptide binding are not all conserved between the CCT subunits and GroEL (Kim et al., 1994; this study). However, a few important secondary structures required for polypeptide binding may be conserved between the two groups of chaperonins (e.g., the helix centered on position 298). Furthermore, the three P-sheet structures predicted in the apical domain of the CCT proteins (beween positions 220-270) may form a structural backbone analogous to the GroEL p-sheets present between positions 205-260 (see Figure 3 alignment). In GroEL, these P-sheets are located on the outside surface of the apical domain and do not appear to contain residues critical for polypeptide binding (Fenton et al., 1994); hence, their role  54  Chapter I: Discussion may be strictly structural in character, and could be performed in the CCT proteins by a proximal but different, non-conserved region of the protein. The predicted secondary structure of TF55 closely matches that of the CCT members, which along with their shared primary and quaternary structures, further demonstrates that CCT proteins and TF55 constitute a family of closely related chaperonins. However, the scope of action of the two chaperonins may be different. Whether CCT folds mainly tubulins and actins in vivo is still unclear, but the heat-inducible TF55 chaperonin probably folds a large number of substrates, and prevents the denaturation of many proteins during cellular stress (Phipps et al., 1991; Guagliardi et al., 1994). Since the predicted secondary structures of TF55 and the CCT subunits are nearly identical, it is likely that their tertiary structures are also very similar, and that it is the differences in exposed amino acids within their polypeptide binding domains which are responsible for their (apparently) different substrate specificities. The lack of requirement for a co-chaperonin such as GroES by CCT and TF55 may be due to differences in primary structures within the apical domain, since the residues required for polypeptide binding are also required for GroES interaction with GroEL (Fenton et al., 1994). However, it is also possible that the differences between the predicted CCT/TF55 and known GroEL secondary structures may be functionally significant. An intriguing possibility is that the CCT/TF55 chaperonins incorporate a chaperonin- 10-like functionality within their apical domains. Through random conical tilt reconstruction of the Pyrodictium  occultum  archaebacterial chaperonin complex from electron micrographs, Phipps et al. (1993) were able to detect a solid mass partially occluding the central channel, in a location analogous to where the co-chaperonin binds (Saibil, 1996).  Denatured actin-binding activities in C. elegans CCT preparations The eukaryotic cytosolic chaperonin CCT is known to interact with unfolded actin and promote its folding in the presence of ATP in vitro (Gao et al., 1992; Rommelaere et al., 1993; Melki and Cowan, 1994), and in vivo (Sternlicht et al., 1993). The ability of C. elegans CCT to  55  Chapter I: Discussion interact with this target protein was assessed by diluting [ S]-labelled, urea-denatured actin into 35  the CCT preparation, and monitoring binary complex formation after separation on native gels. Unexpectedly, a relatively 'minor' component of the CCT preparation (the fastest migrating of three species) was clearly the most active in binding to the denatured actin. Interestingly, this species is likely to be HSP60, a mitochondrial chaperonin which co-purified with CCT. While it cannot be discounted that the CCT in the preparation was damaged during the purification procedure, or was lacking the proper conditions for binding to the denatured substrate, two publications aimed at comparing the differences in the affinities and specificities of HSP60 and CCT provide evidence that the data obtained in the above experiment supports what is currently known about the two chaperonins. In a study by Melki and Cowan (1994), it was found that the cytoplasmic chaperonin had a higher relative affinity for actin folding intermediates which formed within a few minutes of dilution from denaturant, whereas HSP60 had a correspondingly higher affinity than CCT for actin conformers which are present immediately after dilution of the denatured actin. Given this observation, it should therefore be expected that HSP60 whould have a higher affinity than CCT in the actin-binding experiment performed with the C. elegans CCT preparation, which was the case. Furthermore, the above authors also reported that the relative affinity of CCT compared to HSP60 for denatured (3-actin (the same substrate used in the present studies) was slightly less than one-half. Tian et al. (1995) suggest that the ability of CCT to bind various unfolded substrates is lower by a factor of approximately 10-fold compared with HSP60. Interestingly, HSP60 is unable to fold actin or tubulin to their native state, unlike CCT, and this observation is consistent with the fact that no folded actin was generated by the C. elegans CCT preparation whereas bovine CCT was able to refold some of the actin (Figure 13B). Overall, the above data suggest that HSP60 has a higher affinity for unfolded substrate, and therefore is likely to outcompete CCT in substrate binding in vitro. Whether this is biologically important in the function of CCT and HSP60 in vivo is unknown, but the fact that CCT but not HSP60 nor GroEL can refold actin and tubulin may indicate true functional differences between Group I and Group II chaperonins. 56  Chapter I: Discussion Expression of cet genes in C. elegans and other organisms The expression of C. elegans cet genes at all stages suggests that each gene encodes a protein whose function(s) may be required throughout nematode development. Since CCT is involved in cytoskeletal protein folding, it is possible that the expression of the C. elegans cet genes roughly parallels the expression of the major cytoskeletal genes tubulin and actin. In support of this hypothesis, actin genes (Krause and Hirsh, 1986) and at least one tubulin gene (Fukushige et al., 1993) are indeed known to be expressed constitutively throughout C. elegans development. A direct correlation between the expression of Tetrahymena pyriformis TpCCTy and tubulin genes during ciliogenesis has been documented (Soares et al., 1994). In addition, constitutive expression of the five C. elegans cet genes may also signify that all are required to form a functional CCT complex. The finding that the C. elegans cct-1 promoter drives the expression of a reporter gene in muscle and neuronal cells is entirely consistent with the expression pattern of CCT subunit genes in Xenopus laevis (Dunn and Mercola, 1996) and in the axolotl Ambystoma mexicanum, a salamander (Sun et al., 1995). These data are likely to reflect the increased need for folding actin and tubulin by CCT in these tissues (Roobol et ah, 1995; Dunn and Mercola, 1996). Interestingly, the Cctg gene of Xenopus is expressed in all tissues examined but is particularly abundant in the ovary (Walkley et al., 1996). No P-galactosidase staining in the gonad of the adult transgenic C. elegans can be detected. On the one hand, this finding is unexpected since developing oocytes would be expected to require the presence of CCT for the folding of cytoskeletal proteins, which would be needed in great quantity given the tremendous amount of cell divisions occuring. On the other hand, the lack of expression in C. elegans germ cells is not surprising given that the activity of a germline specific factor (PIE-1) blocks new gene expression in the early embryonic germ lineage (Seydoux et al., 1996; Mello et al., 1996). Thus, CCT-1 may be present (or even abundant) in germ cells but would be detectable only by whole-mount immunostaining with the anti-CCT-1 antibody. Northern blot analysis of RNA samples from control and heat shocked C. elegans reveals that cct-1 expression is not increased and may be slightly decreased in heat-shocked nematodes. 57  Chapter I: Discussion It was also noted by Ursic and Culbertson (1992) that yeast TCP-1 expression was slighly repressed following a heat shock treatment, implying that this chaperonin was not a member of the HSPs like most other molecular chaperones. Sun et al. (1995) showed that the transcription of TCP-1 was also repressed in heat-shocked axolotl embryos. Similarly, the Tetrahymena homolog of Cctg is not heat-inducible (Soares etal., 1994). Despite these findings, it is possible that CCT may play a role in protecting certain proteins from unfolding during biological stresses, as has been shown to be the case with HSP60. Although only induced approximately two-fold during stress conditions, in vivo experiments demonstrated that HSP60 was required to prevent the thermal inactivation of a protein (dihydrofolate reductase) imported into mitochondria (Martin et al., 1992). Also, chaperonins are the main proteins expressed under conditions of heat shock in thermophilic Archaea, which suggests that they play an important role in the thermotolerance of these organisms (Conway de Macario and Macario, 1994). Remarkably, the Pyrodictium occultum archaebacterial chaperonin constitutes approximately 73% of the total soluble protein at 108°C, approximately 10°C above its optimal growth temperature (Phipps et al., 1991).  Future studies One of the primary goals of studying CCT is to understand its function in vivo. C. elegans is an excellent model organism in which to study the role(s) that this chaperonin play(s) within a complex, multicellular organism. In collaboration with Ronald Plasterk, a strain containing a Tel transposon insertion near cct-1 was uncovered. The site of this Tel insertion was determined to be between the stop codon and polyadenylation signal (see Appendix VI). Using a well-established technique of screening for imprecise excisions of Tel (Plasterk and Groenen, 1992), a cct-1 knockout could be generated using this strain. The resulting phenotype might itself be very interesting, and disruption of cct-1 would also provide an avenue for attempting rescues with mutant forms of cct-1 (some of which may be temperature sensitive, or have specific defects and therefore display a range of phenotypes). Alternatively, or in parallel with such experiments, lethals mapping near cct-6 (isolated by David Baillie, Simon Fraser 58  Chapter I: Discussion University) could be investigated by injecting wild-type cct-6 into these strains in an attempt to rescue the lethal phenotype and identify mutations in this gene. Similarly, other cet genes mapping near known lethals could be possible targets for rescue experiments. To date, chaperonin mutants have been uncovered only in yeast and prokaryotes. Therefore, the isolation and characterization of a C. elegans strain carrying a mutant cet gene might yield the first significant insight into the function of this molecular chaperone in higher eukaryotes. One of the persisting mysteries concerning CCT is its complex subunit composition compared with the Group I and archaebacterial chaperonins. It is possible that some cet genes are differentially regulated and that the subunit composition of CCT may differ depending on the stage of development or localization within different tissues. Consequently, CCT variants may have different affinities for its substrates. A thorough analysis of the spatial and temporal expression of all of the cet genes within C. elegans would be possible as soon the entire genome sequence becomes available, and would likely provide useful information concerning this possibility. Meanwhile, the promoters of five cet genes are available. By making cet promoterlacZ fusion constructs comparable to the cct-1 promoter-/acZ construct analyzed in this study, a detailed comparison of the expression patterns of all cet genes is possible. Furthermore, in situ immunostaining studies on staged nematodes would provide complementary data on the localization of CCT proteins in the organism; this could be done starting with the anti-CCT-1 and anti-CCT-5 polyclonal antibodies developed in this study.  59  Chapter II: Introduction CHAPTER II— Structure-function studies on C. elegans HSP16-2 and HSP12.6  Organisms are frequently subjected to environmental stresses which must be dealt with in order to prevent cellular damage. Protection at the molecular level requires a specific set ofproteins termed stress or heat shock proteins. In the present chapter, studies on the structure and function of two small heat shock proteins provide insight on how this family of chaperones assembles into large multimeric complexes which can interact with structurally compromised proteins.  I. INTRODUCTION  Small HSP family The small HSPs (smHSPs) and oc-crystallins form a highly divergent protein family with members present in Eukarya, Archaea, and Bacteria (Caspers et al., 1995; Bult et al., 1996). a-crystallin is a highly abundant protein in eye lenses, and is usually found as large aggregates consisting of two types of related subunits, a A and ocB (Groenen et al., 1994). It is also present to a lesser extent in various tissues outside the lens (Bhat and Nagineni, 1989; Kato et al., 1991). The evolutionary relatedness between a-crystallins and smHSPs was first noted by Ingolia and Craig (1982), and is restricted to a conserved ~85-amino acid a-crystallin domain (Ingolia and Craig, 1982; Russnak et al., 1983; Wistow, 1985). Among the prominent molecular chaperone families (including HSP 104, HSP90, HSP70, HSP60, and HSP40), the smHSP family is the most structurally diverse, ranging in size from 17-26 kDa in plants, 20-27 kDa in vertebrates, 2024 kDa in Drosophila, 24-43 kDa in yeast, and 16-21 kDa in bacteria (Ingolia and Craig, 1982; Bossier et al, 1989; Caspers et al., 1995; Waters et al., 1996; Wotton et al., 1996). The first archaebacterial smHSP, an 18-kDa protein most closely related to plant cytosolic smHSPs, was recently uncovered by the sequencing of the Methanococcus jannaschii genome (Bult et al., 1996). The N-terminal domains and C-terminal extensions of smHSPs, which flank the  60  Chapter II: Introduction evolutionarily conserved a-crystallin domains, differ substantially in length and amino acid sequence and account for most of the structural diversity between different members. All organisms cope with changes in their environment, including exposure to elevated temperatures and environmental pollutants such as heavy metals and toxins, by increasing their levels of stress proteins (Lindquist and Craig, 1988; Morimoto et al, 1990). Although many smHSPs are present during development under physiological conditions, smHSPs are among the most highly inducible HSPs during heat shock or other stresses (Arrigo and Landry, 1994). In plants, some smHSPs are induced more than 200-fold under stress conditions, and the accumulation of the proteins is proportional to the temperature and duration of the stress (Waters et al, 1996). Following the stress event, smHSPs persist for long time periods, having half-lives of 30-50 hours (Chen et al, 1990; DeRocher et al, 1991). This suggests that their function is probably critical during the recovery period (Waters et al, 1996). In mammalian cells, induction of smHSPs is typically 10-20-fold, and in the lens, HSP27 and aB-crystallin represent about 0.2-1% and 2-10% of total proteins, respectively (Arrigo and Landry, 1994). In the yeast Saccharomyces cerevisiae, both HSP26 and HSP42 accumulate to high levels during heat shock, although HSP42 is present at lower levels during physiological conditions (Wotton etal, 1996) Transcriptional activation of smHSP genes is thought to be the major regulatory mechanism by which smHSPs preferentially accumulate after stresses in Drosophila, C. elegans, yeast, plants, and Xenopus (Arrigo and Landry, 1994). The induction of smHSPs during heat shock or other stresses is mediated by the binding of the heat shock transcription factor (HSF) to repeats of the regulatory DNA sequence termed heat shock element (HSE) (Morimoto et al, 1990). HSE repeats are present upstream of all known stress-induced smHSP genes. It is likely that in many cases, there are other regulatory elements influencing smHSP gene expression. It is now well established that smHSPs are regulated not only by environmental stressors, but also by a variety of other environmental and developmental cues (Arrigo and Landry, 1994; Waters et al, 1996). Thus, the expression of plant smHSPs is regulated, in the absence of heat stress, in developing embryo (Coca et al, 1994; Vierling and Sun, 1989), during germination (Vierling and Sun, 61  Chapter II: Introduction 1989), pollen development (Bouchard, 1990), fruit maturation (Fray et al., 1990), and cold storage (Van Berkel et al., 1994). In Drosophila, the spatial and temporal expression patterns of four of the known smHSP genes (Ingolia and Craig, 1982) have been extensively characterized (Arrigo and Landry, 1994). Not only are some smHSPs produced during different life stages (embryos, larvae, pupae, and adults), but the localization of these proteins differs during development. In constrast, the genes are expressed in almost all tissues following heat shock (Arrigo and Tanguay, 1991).  Small HSP family of C. elegans  During the early and mid 1980's, two cDNAs encoding small HSPs (Russnak et ah, 1983) and four genes encoding two different classes of smHSPs (Russnak and Candido, 1985; Jones et al, 1986) were isolated. These genes (hspl6-l, hspl6-2, hsp!6-41, hspl6-4S) were shown to contain a region highly homologous to a conserved sequence in the a-crystallin and Drosophila small hsp genes. Until recently, no additional small hsp genes had been isolated, but the C. elegans genome sequencing project has now uncovered 11 novel genes which are clearly related to the small hsp gene family (Figure 14). The smHSPs can be subdivided into three classes based on size alone. Class I smHSPs may be the most abundant and range from 16-18 kDa, which roughly parallels the size of eubacterial, most plant, and archaebacterial smHSPs (Caspers et al, 1995; Bult et al, 1996). Interestingly, the size distribution of the Class I smHSPs is quite narrow (145-159 amino acids), especially in light of the fact that other smHSPs are more than 200 amino acids in length. The C. elegans Class II smHSPs are the smallest known members, ranging from 12.2-12.6 kDa. They are so far unique to nematodes, as they have not yet been observed in other organisms. A comparison of their sizes (there are two pairs, of 109 and 110 aa in length) and similar sequence (43-73% overall aa sequence identity between members) suggests that they form a highly conserved, novel family of smHSPs. Class III smHSPs are the largest in size, ranging from 20-30 kDa. In terms of size alone, these are most closely related to higher eukaryote smHSPs (e.g., murine HSP25, Drosophila and human HSP27, etc.). 62  Chapter II: Introduction  HSP16-1 HSP16-2 F08H9.3 F08H9.4 HSP16-41 HSP16-48 F43D9 F52E1.7 F38E11.1 HSP13 C14B9.1 ZK1128.7  HSP16-1 HSP16-2 F08H9.3 F08H9.4 HSP16-41 HSP16-48 F43D9 F52E1.7 F38E11.1 HSP13 C14B9.1 ZK1128.7  HSP16-1 HSP16-2 F08H9.3 F08H9.4 HSP16-41 HSP16-48 F43D9 F52E1.7 F38E11.1 HSP13 C14B9.1 ZK112 8.7  N-terminal domani MSLYH MSXiYH'  IRDMAQMERHFTPVCRGSPSESS  g  iRDMALMERMFAPVCRISPSESS  5  IMVFGDNGRI^JPEYVPISENNDDLS JR-DMGGMQRRLMPISf T F N P M T  DCRN " DDS '  [ALDHFLDELTGSVMFP' MSVNP DgNVLDHFLDEITGSVWFP' M S MLMLRSgFS VSP gRPTGLFRDFEgMMP' I LMMSLSRLSCI H Y T f?, 1 - M'D R R J J~[3FS--PFFNHGRRFFDDVDFDRHMIRP PJ MSVAIDr  M - ( 4 0 aa)  -  IHNSFN  FSDNIGj  -JADHNSFN FSDNIG j .QRHSMLNNFNNIVPQQLN| IQTMLTGH RVGDAID •WPFQKGDG W K  GTKjJJDWPLQKGDG WN :SVP' iSAIEVTJ STjJJDWPLQHNDG WK -PRSSHSNMIYGYPINYKETVFPTHRGPPDQSYDSJEYTKITESRPRSPGPVAGAGS  a-crystalin domani DQ^SAX  amino  C-etrmn ius  acids 145 145 147 147 143 143 159 149 109 110 110  B - E A I QGJ^JJJQQAPVEQKTSE SMDHHHIE gj- EAVOGBJpraioOAIVEEKSAE 'AKH V-EAKKTNFFGFLSKFRCMPESQAKH [TSAGHAVTQKPSSTTTTGKHIVPKRN r  SK—WLWlEnLONTYSRVLVKDGVROASOAITQQLNETWQEV  218 1  Class I Class II CalSS  III  Figure 14. Alignment of C. elegans smHSPs. The two smHSPs characterized in this study (HSP16-2 and HSP12.6) are aligned to other members of the C. elegans smHSP multigene family. The family can be divided into three classes based on size; Class I smHSPs are between 16-18 kDa (similar to plant and bacterial homologues), Class II smHSPs are the smallest known members of any organism at 12-13 kDa, and Class III smHSPs are 25-30 kDa in size, which is similar to higher-eukaryote smHSPs. The three basic regions of small HSPs, the nonconserved N-terminal domain, the conserved a-crystallin domain, and the C-terminal extension are grouped into separate blocks. The Genbank accession numbers for the C. elegans smHSPs are: HSP16-1 and HSP16-48 (K03273), HSP16-2 and HSP16-41 (M14334), F08H9.3 and F08H9.4 (Z77657), SEC-1 (Z35640), F52E1.7 (U41109), HSP12.6 (also known as F38E11.2; Z68342 and U92044) and HSP12.3 (also known as F38E11.1; Z68342), ZK1128.7 (Z47357), C14F11.5 (U39645), and C09B8.6 (U29612). The asterisk at the end of the C14F11.5 coding region indicates that the sequence is predicted to continue by the program GeneFinder, but may end at the stop signal present immediately following the putative intron at this location.  63  Chapter II: Introduction A few of the other known nematode proteins also belong to class III smHSPs; for example, there exists immunologically-related 27-kDa smHSPs (p27s) in D. immitis, B. malayi, O. volvulus, L. carinii, A. viteae, T. spiralis, T. canis, T. leonino, as well as in C. elegans (Lillibridge et al., 1996). In D. immitis, the hypodermally-localized p27 is developmentallyregulated, but is not upregulated by increased temperatures. A different, constitutively-expressed and heat-inducible 22-24 kDa smHSP from B. pahangi was identified by Jecock et al. (1992). Yet another nematode smHSP, a 20-kDa protein from the intestinal nematode N. brasiliensis, has been found to be developmentally regulated and heat-induced (Tweedie et al., 1993). Homologs of the class II smHSPs have also been uncovered as part of a tag sequenching approach to identify genes expressed in the human filarial nematode parasites B. malayi and O. volvulus (Blaxter etal, 1996). Whereas most HSPs perform necessary functions in non-stressed cells and are also induced following cellular insults (Gething and Sambrook, 1992; Parsell and Lindquist, 1994; Arrigo and Landry, 1994), the C. elegans HSP16-class smHSPs are produced only under stress conditions, in a tissue-general manner (Dixon et al., 1990; Stringham et al., 1992; Jones et al., 1986; Candido et al., 1989). More significantly, their expression correlates specifically with the presence of agents which induce protein damage (Jones et al., 1996). A series of compounds closely related to Captan, a commonly used agricultural fungicide, were tested for the induction of p-galactosidase production in a transgenic C. elegans harbouring an integrated hspl6promoter-/acZ construct. It was found that only compounds possessing N-(trichloromethylthio) or N-(tetrachloroethylthio) groups, which denature proteins by reacting with amino and thiol groups, induced transgene expression. These data on the C. elegans HSP 16 family strongly suggest that these proteins are produced only under conditions which induce the stress response, which has been shown to be strongly correlated with the presence of proteotoxic agents within the cell (Hightower, 1980; Ananthan et al, 1986)  64  Chapter II: Introduction Structure of small HSPs Wistow (1985) proposed that the overall structure of a-crystallin consists of two major domains, a globular N-terminal domain and a somewhat larger C-terminal domain, with an exposed C-terminal arm. There is considerable genetic and experimental evidence to support such a model. First, some smHSP genes contain an intron which delineates the N-terminal and a-crystallin domains (King and Piatigorsky, 1983; Russnak and Candido, 1985). Primary structure sequence alignments of smHSPs show the boundary between the nonconserved N-terminal domain and the highly conserved a-crystallin domain to be highly conspicuous, which is suggestive of two separate folding domains. Second, folding-unfolding and NMR studies also support a two-domain structure (van den Oetelaar and Hoenders, 1989; Carver et al., 1993). A computer-modelling study by Groth-Vasselli et al. (1995) predicts the a-crystallin monomer to have an elongated, dumbbell-like structure. Based on molecular modelling studies and partial primary sequence similarities, the structure of E. coli IbpB, a smHSP, was suggested to resemble the middle region of CaflM from Yersinia pestis, a protein related to the periplasmic molecular chaperone PapD of E. coli (Zav'yalov et al., 1995). The crystal structure of PapD is known and consists mostly of p-sheets (Holmgren and Branden, 1989). Overall, a-crystallin and other smHSPs appear to be composed of >90% p-sheets on the basis of CD spectra (Siezen and Argos, 1983; Merck etal, 1993a). A common feature of smHSPs is their formation of large oligomeric complexes. The simplest and best characterized smHSP quaternary structure is the 150-kDa trimer of trimers formed by M. tuberculosis HSP16.3 (Chang et al, 1996). Plant smHSPs assemble into circular or triangular complexes of 200-300 kDa, or 12 subunits (Lee et al, 1995; Waters et al, 1996), and the typical oligomeric size of a-crystallins and smHSPs from yeast and mammals is between 400 and 800 kDa (Bentley et al, 1992; Groenen et al, 1994). A detailed analysis of recombinant murine HSP25 by hydrodynamic and electron microscopic techniques showed that it consisted of circular, elliptical, triangular, or polygonal 15-18 nm particles of about 730 kDa (32 monomers) (Behlke et al, 1991). Curiously, cardiac a-crystallin appears to possess a central cavity (like chaperonins) (Longoni et al, 1990), a feature not present in other smHSPs studied. The smHSP . 65  Chapter II: Introduction quaternary structures are therefore highly diverse. Furthermore, the oligomeric state of mammalian smHSPs depends on various factors, such as the state of phosphorylation and temperature (Siezen et al., 1980b; Van den Oetelaar and Hoender, 1989; Kato et al, 1994). The C-terminal extensions of smHSPs appear relatively unstructured (Carver et al, 1992, 1995a) and are known to undergo numerous modifications, including truncations (reviewed in Groenen et al, 1994). They are therefore likely to be exposed on the surface of the oligomers, and could contribute significantly to maintaining the solubility of the complex (Smulders et al, 1996). Several contrasting models have been proposed to account for the arrangement of subunits and diversity of a-crystallin aggregates. The review by Groenen et al. (1994) addresses these issues in detail, and suggests that although there is as yet no single consistent model for smHSP quaternary structure, it is likely that the smHSP N-terminal domain is buried in the aggregate whereas the C-terminal domain is exposed at the surface, and that all subunits are in equivalent positions within a roughly spherical aggregate. The first quaternary structure models proposed for the a-crystallin aggregate were tetrahedral-based three-layer models which consisted of a core of 13 or 12 subunits, a second layer of 14 or 6 subunits, and a third layer of 16 or 24 subunits, for a total of 43 (Bindels et al, 1979) or 42 (Tardieu et al, 1986) subunits, respectively. These similar models were based on the observation that the approximately 800-kDa a-crystallin (~ 40 subunits) can be dissociated reversibly at various temperatures into particles of smaller sizes (Groenen et al, 1994). Variations of this model have been suggested (Walsh et al, 1991). A micelle-like structure in which the relatively hydrophobic N-terminal domains were oriented toward the interior, providing the driving force for aggregation, was also suggested based on several experimental results (Augusteyn and Koretz, 1987). A later model based on tetrameric building blocks was proposed to explain the observation by Merck et al. (1992) that N-terminally-deleted a-crystallin dissociated into dimers/tetramers (Wistow, 1993). Finally, a chaperonin-like structure has been proposed for cardiac a-crystallin (Carver et al, 1994a).  66  Chapter II: Introduction Mechanism of small HSP chaperone function The first demonstration that smHSPs could function as molecular chaperones came from Horwitz (1992), who showed that ccA- and aB-crystallin were highly effective in suppressing the thermally-induced aggregation of (3- and y-crystallins as well as alcohol dehydrogenase. Subsequently, numerous in vitro studies have documented the ability of smHSPs to prevent the aggregation of various test proteins at elevated temperatures (Jakob et al., 1993; Merck et al., 1993a; Lee et al., 1995; Chang et al., 1996; Rajaraman et al., 1996). In addition, many (Jakob et al, 1993; Lee et al, 1995) but not all (Horwitz, 1992; Chang et al, 1996) smHSPs appear to promote the refolding of denatured proteins. The mechanism by which smHSPs recognize and capture aggregation-prone polypeptides is largely unknown. As with other molecular chaperones (e.g., HSP60; Horowitz et al, 1995), there is some evidence that hydrophobic patches in a-crystallin are required for interaction with the hydrophobic regions exposed by unfolded proteins (Raman and Rao, 1994). It has been observed that a-crystallin undergoes a conformational transition at elevated temperatures which results in a marked increase in surface hydrophobicity, and a concomitant increase in chaperone activity (Das and Surewicz, 1995). Therefore, it is likely that the masking and stabilizing of hydrophobic core regions of unfolded proteins by smHSPs is a key aspect of their ability to suppress protein aggregation. It is apparent, however, that electrostatic interactions are also likely to be critical in substrate binding. For example, an Asp ->-Ser aA-crystallin mutant was 69  69  shown to have a substantially reduced protective ability despite its formation of wild-type-like oligomeric complexes (Smulders etal, 1995). Another informative study, on the interaction of a-crystallin with spin-labeled peptides, revealed that the side chains of the peptides were immobilized within a polar environment (Farahbakhsh et al, 1995). Studies aimed at investigating the structural conformations of polypeptides bound to smHSPs provide inconsistent data on this important aspect of chaperone activity. Carver etal. (1995b) demonstrated that a-crystallin does not interact with unfolded, hydrophobic but stable proteins (e.g., reduced and carboxymethylated a-lactalbumin and acasein), but does complex with grossly unfolded and unstable proteins which are about to 67  Chapter II: Introduction precipitate out of solution. Similarly, Das et al. (1995, 1996) suggested that unlike other chaperones, a-crystallin has very low affinity for folding intermediates formed during protein refolding reactions in vitro (i.e., protein conformers which are nearly fully denatured), but has a high affinity for aggregation-prone intermediates which are characterized by a very low degree of unfolding. Another study showed that a-crystallin bound to a molten-globule form of carbonic anhydrase, which displays a significant amount of secondary structure but little permanent tertiary structure (Rajaraman etal., 1996). Unlike some other well characterized molecular chaperones such as HSP60 and HSP70 (Hartl, 1996), little experimental data are available on the site of interaction of the unfolded proteins with smHSPs. In one of the aforementioned studies by Farahbakhsh et al. (1995), two spin-labeled peptides (insulin B chain and melittin) were used to demonstrate that these small, largely unstructured polypeptides bound not on the surface or in an interior cavity of the complex, but instead were found to be largely separated in distinctly polar environments. In one of the few studies on a smHSP other than a-crystallin, Lee et al. (1997) demonstrated that when a hydrophobic probe (bis-ANS) was incubated with the dodecameric pea HSP 18.1 complex, it could be cross-linked specifically to a hydrophobic region within the first half of the a-crystallin domain. Interestingly, a stretch of hydrophobic residues near the end of the a-crystallin domain (GVLTVTV) which is highly conserved among smHSPs was not labeled with bis-ANS, suggesting that this region might be solvent-inaccessible.  Cellular functions of small HSPs Unlike the HSP60 molecular chaperone family, the smHSPs are unlikely to play a critical role in the folding of nascent polypeptides. One reason is that smHSPs are not encoded in either of  the  two  fully  sequenced  bacterial  genomes  (Haemophilus influenzae and  Mycoplasma genitalium), while all of the other classes of chaperones found in prokaryotes and eukaryotes (DnaK/HSP70, DnaJ/HSP40, GroEL/HSP60, GroES/HSPIO, and GrpE) are present (Fraser et al., 1995; Fleischmann et al., 1995). Most of these ubiquitous chaperones are involved in folding nascent polypeptide chains. Thus, the function of smHSPs may be restricted to the 68  Chapter II: Introduction protection of the organism from cellular stress by preventing the aggregation of stress-damaged proteins. Consistent with this argument is the fact that one 18-kDa smHSP is indeed found in Methanococcus jannaschii, a thermophilic organism whose genome was recently sequenced (Bult et al., 1996). Interestingly, the only other major chaperone present in M. Jannaschii is a chaperonin related to other archaebacterial and eukaryotic cytosolic chaperonins. Chaperonins are not only required for protein folding, but have also been shown to play a protective role during stress conditions (Martin etal, 1992). Given that smHSPs are highly induced under stress conditions, it is not surprising that they are likely to play an important role in induced thermotolerance. This increased resistance of organisms to severe stresses following mild pre-exposures to a similar or different stress is observed in virtually every organism studied (Parsell and Lindquist, 1994). As with other chaperones, including inducible HSP70s (Feder et al., 1996) and HSP104 (Sanchez and Lindquist, 1990; Sanchez et al., 1992), it is well documented that smHSPs play important roles in protecting organisms from stress and in the establishment of thermotolerance (Landry et al., 1989; Lavoie et al, 1993a; Yeh et al, 1994; Arrigo and Landry, 1994). The repertoire of proteins protected by smHSPs during stress conditions is presently unknown. It is possible that smHSPs nonspecifically recognize and stabilize the bulk of cellular proteins susceptible to unfolding. On the other hand, they may be more specialized, acting on a limited number of target proteins. In the cell, cytoskeletal proteins, which are particularly sensitive to cellular stresses (Welch and Suhan, 1985), appear to be targets for smHSPs. Specific binding of smHSPs to actin and the intermediate filaments desmin, vimentin, and GFAP has been demonstrated (Chiesi et al, 1990; Bennardini et al, 1992; Gopalakrishnan and Takemoto, 1992; Nicholl and Quinlan, 1994). HSP27 has been implicated in regulating the dynamics of actin filaments (Lavoie et al, 1993b; Landry and Huot, 1995), and a-crystallin participates in intermediate filament assembly (Nicholl and Quinlan, 1994). Furthermore, HSP27 and acrystallin can enhance the survival of cells subjected to heat shock or oxidative stresses by conferring increased stability to actin fibers (Lavoie et al, 1993a, 1995; Huot et al, 1996; Wang and Spector, 1996). 69  Chapter II: Introduction The finding that smHSPs can modulate cytoskeleton dynamics is consistent with the fact that many smHSPs can inhibit actin polymerization (Miron et al., 1991; Benndorf et ai, 1994; Rahman et al., 1995). Many factors influence the equilibrium between actin monomers and filaments in vitro, including ATP, divalent cations, and proteins that bind to actin (Vandekerckhove, 1990; Cooper, 1992). It has been proposed by Rahman et al. (1995) that a possible site of interaction between smHSPs and actin lies within a region in actin thought to be important for the polymerization of G-actin into F-actin, and for the binding of actin to DNAse I. This hydrophobic sequence motif, G-[V/I]-L-T-[X]3-P, is conserved in actin at residues 61-69, and in oc-crystallins and many smHSPs near the end of the a-crystallin domain. Interestingly, labeled bis-ANS (a hydrophobic probe) was not photoincorporated into the equivalent region of pea HSP 18.1, suggesting that this region might not be the site of interaction with hydrophobic regions of unfolded polypeptides (Lee etal, 1997). Thus, the conserved motif in smHSPs may have a specialized role in binding to the corresponding sequence in actin, thereby influencing the polymerization of the cytoskeletal protein. In contrast to the above function of smHSPs, Wang and Spector (1996) have recently shown that a-crystallin and its individual subunits ( a A and a B )  are able to prevent  cytochalasin D-induced ^polymerization of actin. Furthermore, it was shown that a-crystallin stabilizes actin from dilution-induced depolymerization and enhances polymerization at higher actin concentrations. It was suggested that there is no likely interaction between a-crystallin and actin monomers, as the effect of a-crystallin in enhancing actin polymerization is not apparent before some polymerization has occurred. The reason for the apparently disparate roles of a-crystallin and other smHSPs in influencing actin polymerization is unknown, but as the authors suggest, a-crystallin, like other smHSPs, is clearly able to influence actin dynamics. It should be noted that no link has been made between the above function of smHSPs and their chaperone activity; while it is conceivable that the two activities are functionally related, only the chaperone activity has thus far been shown to be a ubiquitous feature of smHSPs. One interesting aspect of smHSP chaperone activity is its ATP-independence (Horwitz, 1992; Jakob et al., 1993; Merck et al., 1993a; Lee et al., 1995). During heat shock conditions, 70  Chapter II: Introduction significant changes in energy metabolism occur, including decreases in the cellular levels of ATP (Findly et al., 1983). As a consequence, the recruitment of the smHSPs to protect the cell against the accumulation of unfolding proteins may be advantageous. Since the heat-induced interaction of smHSPs with unfolded proteins is ATP-independent and stable in vitro, it is possible that the productive release of the bound protein may occur later (perhaps after the stress condition), and require the assistance of other ATP-dependent molecular chaperones. Recently published studies are shedding light into the fate of the smHSP-bound proteins. Ehrnsperger et al. (1997) demonstrated that HSP70, which is present at high concentrations after heat shock, is able to trigger the release of citrate synthase from HSP25 and promote refolding in an ATP-dependent manner. However, the percentage of citrate synthase reactivated with HSP70 was only about 13%, whereas without HSP70, the level of spontaneous refolding was approximately 2%. As the authors suggest, it is likely that co-chaperones (for example, HSP40 and Hip), would participate in this process in vivo, increasing the efficiency of the refolding reaction. Indeed, Lee et al. (1997) showed that pea HSP18.1-bound firefly luciferase could be folded more efficiently (to 40% of total activity) when incubated with rabbit reticulocyte lysate. Essentially no refolding occurred in the presence of apyrase, which causes a depletion of ATP. Although it is unclear what components are involved in initiating the refolding of the bound protein, the rabbit reticulocyte lysate is known to contain all of the necessary chaperone machinery to fold firefly luciferase synthesized de novo and from the chemically-denatured state (Frydman et ai, 1994b; Frydman and Hartl, 1996).  The present study Our laboratory has extensively examined the induction of two classes of C. elegans smHSPs by various stresses, both at the transcription and protein levels. Conditions which induce expression invariably promote protein denaturation, and range from heat stress to exposure to many kinds of chemicals, including alcohols, heavy metals, and pesticides. Although it is likely that the smHSPs have a protective function in vivo, the details concerning their assembly and mechanism of action is largely unknown. In an effort to understand the structure and function of 71  Chapter II: Introduction smHSPs in general, the C. elegans HSP16-2 wild-type protein and various deletion variants were expressed in E. coli and purified. HSP16-2 is an ideal small HSP to study for two main reasons. Firstly, a great deal is already known about its expression during cellular stresses. Secondly, it reflects the minimal functional unit for a small HSP, as the minimum size of functional smHSPs is very close to 16 kDa. In brief, structure-function studies on HSP 16-2 revealed that there is a strict requirement for an intact N-terminal region for the formation of high-MW complexes which are functional as molecular chaperones. The C-terminal extension is not required for oligomerization or chaperone activity, but appears to be involved in maintaining the solubility of the complex. HSP16-2 forms stable binary complexes with unfolded actin and tubulin, and preferentially interacts with early unfolded intermediates which form along the renaturation or aggregation pathway. The findings of this study are likely to be applicable to other smHSPs, and provide insight into our understanding of the protective function of these chaperones in vivo. Lastly, characterization of a unique, stage-specific 12.6-kDa smHSP from C. elegans provided supporting evidence for the structure-function model proposed.  72  Chapter II: Materials and Methods II. MATERIALS AND METHODS  2.1 Cloning of hspl2.6 cDNA and preparation of small HSP constructs The hspl6-2 coding region had previously been subcloned into the pRSET A expression vector (Invitrogen) at the BamRl-EcoRl  site (Jones et al, 1996) for producing H6HSP16-2 (i.e.,  wild-type HSP 16-2 fused to an N-terminal ~4 kDa polyhistidine-containing fusion tag). A construct designed to express wild-type HSP 16-2 was made by excising the Ndel-BamUl fragment containing the polyhistidine tag, blunting, and religating. To make the vector encoding H6A1-15 HSP16-2 (a tagged HSP16-2 lacking the first 15 amino acids), the /^76-2-containing clone was amplified with the primers OH2-A and OBM2 (see Appendix II), and the XholiscoRI-restricted PCR product was subcloned into pRSET B (Xhol-EcoRT). The vector encoding H6A130-145 HSP16-2 (like H6HSP16-2 but missing the last 16 amino acids) was generated by subcloning the PCR product amplified with the primers OBM1 and OH2-C into pRSET A (BamHl-EcoRT). The constructs designed to encode H6A1-32 and H6A1-44 HSP 16-2 were made by subcloning the Pstl-EcoRl fragment of the pH6HSP16-2 vector into pRSET B (Pstl-EcoRI), and the Hpal-Hindlll fragment into pRSET B (PvMlI-//wdIII), respectively. The hsp!2.6 coding region was amplified from mixed-stage first-strand cDNA (prepared according to Jones and Candido, 1993) with the primers F38A and F38B and subcloned into ZJaraHI-rYmdlll restricted pRSET A vector after restriction with the same enzymes. This expression vector encodes H6HSP12.6 (HSP12.6 fused at the N-terminus to the pRSET vector polyhistidine-containing tag). To create a vector expressing wild-type HSP 12.6, the BamRlHindlll cut PCR product was subcloned into the Bglll-Hindlll  site of a modified pRSET A  vector lacking the polyhistidine-containing Ndel-BamRI fragment. The sequence of the cloned cDNA matches the coding region predicted from the genomic DNA.  2.2 Expression and purification of the small HSPs All expression constructs were transformed into the BL21(DE3) E. coli strain (Studier et al, 1990), and 0.5-2.0-liter cultures were grown in TB/Amp medium to an O D 6 0 0 between 0.8-1.0, 73  Chapter II: Materials and Methods induced with 1 mM IPTG, and harvested 3 hours later. Cells were frozen and thawed four times and insoluble inclusion bodies were isolated and washed once in TEND buffer (50 mM TrisHCl, 0.1 mM EDTA, 50 mM NaCl, 1 mM DTT; pH 7.5). For the purification of the HSP16-2 protein, the inclusion bodies were solubilized in TEND buffer supplemented with 4 M urea, and 2.5 ml of the cleared supernatant was applied to an ~40-ml Sephacryl S-100 column equilibrated with TEND/4 M urea buffer. The peak fractions were then collected and dialyzed overnight in TEND buffer. A 2-ml sample was applied to an ~70-ml Sephacryl S-300 column equilibrated in TEND buffer, and the peak fractions were collected, snap-frozen in a dry-ice-ethanol bath, and kept at -70°C. To purify the polyhistidine-containing proteins, the inclusion bodies were solubilized in UNT buffer (8 M urea, 50 mM NaCl, 50 mM Tris; pH 8.0) and centrifuged at 10000 x g. The cleared supernatant was applied to a column containing 0.5-2.0-ml Ni -chelate 2+  affinity resin (Qiagen) equilibrated in UNT buffer. The column was extensively washed with UNT (pH 8.0) followed by UNT (pH 6.3) buffers, and the protein was eluted with UNT (pH 4.3) buffer. Purified proteins were then dialyzed against TEND buffer and used in assays immediately. The wild-type and polyhistidine-tagged HSP 12.6 recombinant proteins were also produced in E. coli BL21(DE3). The cells were resuspended in PND buffer (25 mM sodium phosphate pH 7.0, 25 mM NaCl, 0.5 mM DTT) supplemented with protease inhibitors (2 mM PMSF, 10 (ig/ml aprotinin, 10 p.g/ml pepstatin A, 10 u.g/m.1 leupeptin, 5 mM EDTA), disrupted by sonication, and the insoluble fraction pelleted by centrifugation at 12000 x g. The soluble HSP12.6 was first fractionated by size exclusion chromatography on Sephacryl S200-HR in PND buffer. Peak fractions containing HSP 12.6 were collected, then loaded on a BIO GEL® HTP hydroxylapatite column (Bio-Rad). The column was washed with PND buffer, and the bound HSP12.6 was eluted using a 10-300 mM sodium phosphate pH 7.0 gradient made in 25 mM NaCl and 0.5 mM DTT. Positive fractions were then dialyzed extensively against PND buffer and used immediately. N-terminal sequencing of the purified HSP 12.6 confirmed its identity and revealed some microheterogeneity at the N-terminus, which was probably due to processing (cleaving) of one or both of the two methionine residues present at the start of the 74  Chapter II: Materials and Methods coding region. The native H6HSP12.6 protein was purified on a Ni -chelate affinity column 2+  using PND buffer adjusted to pH 6.3 for washes, and pH 4.3 for elution.  2.3 Molecular weight estimation of the small HSPs To estimate the sizes of the native smHSP complexes, approximately 5 mg of purified protein was subjected to size exclusion chromatography over a 1.5 x 80 cm Sephacryl S-300HR. The column was first calibrated with the following molecular weight standards: thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and RNAse A (14 kDa). The column was run in TEND buffer at room temperature. Samples were separated on 12.5 % SDS-gels, and the elution volume of each was used to estimate the molecular weight. The native MW of recombinant HSP 12.6 was estimated by size exclusion chromatography on a 1.5 x 100 cm Sephacryl S-200HR column in PND buffer at 4°C. Fractions were collected, separated on 13.5% SDS-gels, and relative amounts of HSP12.6 were determined by densitometry. To estimate the in vivo M of HSP12.6, a soluble protein extract was prepared by T  Dounce-homogenizing and sonicating ~1 ml of packed LI-stage larvae in 1.5 ml PND buffer supplemented with protease inhibitiors (2 mM PMSF, 2 mM EDTA, and 10 p-g/ml each of aprotinin, pepstatin A, and leupeptin). The entire clarified protein extract was chromatographed on a 1 x 50 cm S-200HR column in PND buffer at 4°C. Fractions were analyzed by Western blotting as described below.  2.4 Production and purification of anti-HSP12.6 antibody Purified HgHSP12.6 was dialyzed against TBS and used to immunize rabbits for pAb production. Rabbits were injected with 0.5 mg of antigen in Freund's complete adjuvant, then boosted three times at two-week intervals using the same amount of H6HSP12.6 in Freund's incomplete adjuvant. Antiserum was affinity purified over H6HSP12.6 affixed to an Affi-Gel 10 column (Bio-Rad) according to the manufacturer's instructions.  75  Chapter II: Materials and Methods 2.5 Western blot analysis of HSP12.6 Synchronous populations of C. elegans were obtained as described (Emmons et al., 1979), and nematodes were cultured in liquid medium at 15°C. Protein extracts were prepared by boiling the nematodes in Laemmli IX sample buffer for 20 minutes. For Western blot analysis, 20 u\g of protein extract was separated by electrophoresis on a 13.5% gel, transferred to an Immobilon-P membrane (Amersham), and probed with a 1:500 dilution of the anti-HSP12.6 pAb. Immunoblots were developed using Amersham's enhanced chemiluminescence system.  2.6 Cross-linking of small HSPs Cross-linking reactions consisted of 1.5 pM HSP16-2 and its variants (based on monomeric size), 15 pM BSA (to minimize spurious cross-linking), and 2 mM of freshly-prepared bis (sulfosuccinimidyl) suberate (BS ; from Pierce). Protein samples to be cross-linked were first 3  dialyzed in cross-linking buffer (25 mM MES pH 7.5, 25 mM NaCl, 0.5 mM DTT), and reactions were carried out in the same buffer for 30 minutes. Aliquots (5-15 ill) were then electrophoresed on 12% SDS-gels, transferred to a PVDF membrane, and probed with a polyclonal antibody against HSP16-2 (Jones et al., 1996; see also Appendix VII). Cross-linking reactions with HSP 12.6 were performed under the same conditions with the exception that 0.25 or 1 pM protein was used, and the Western blot was developed using the anti-HSP12.6 pAb.  2.7 Assay for H HSP16-2 binding to Ni -chelate affinity resin 2+  6  H6HSP16-2 was purified by nickel affinity chromatography under denaturing (8M urea) conditions, and dialyzed against a 50 mM NaCl and 50 mM sodium phosphate buffer (pH 8.0) (NP buffer). Two 150 (i,l samples containing 175 \ig of H6HSP16-2 were prepared, one in NP buffer (Sample 'N' for 'Native') and the other in NP buffer containing 6M urea (Sample 'D' for 'Denatured'). A small amount of Qiagen nickel affinity resin (30 pi) was added to samples N and D. The samples were mixed gently at room temperature for 15 minutes, then washed twice with 500 pi NP buffer (without urea for sample N and with 6M urea for sample D, both adjusted to pH 6.3), and any bound protein was eluted by resuspending the nickel resin pellet with two 76  Chapter II: Materials and Methods 50 ui washes of 100 mM EDTA, which chelates the nickel bound to the resin and therefore elutes all the adsorbed proteins. Aliquots (15 u.1) of the EDTA wash of sample N and the pH 6.3 and EDTA washes of sample D were separated on a 12% SDS-gel, and stained with Coomassie Blue.  2.8 Thermal aggregation studies The effect of small HSPs on the thermally-induced aggregation of a 150 nM solution of porcine heart citrate synthase (Sigma) was monitored by light scattering at 320 nm in a Cary 210 Varian spectrophotometer equipped with a thermostated cell compartment pre-heated to 45°C. The appropriate dilutions of citrate synthase and smHSP were made with 50 mM NaP04 (pH 8.0) buffer. The relative amount of CS aggregation was normalized to the 100% aggregation value obtained when CS was incubated alone.  2.9 Chemical aggregation studies The effect of HSP 16-2 on the chemically-induced aggregation of CS was measured in an SPF-500C™ stirred-cell spectrofluorometer with excitation and emission wavelengths set to 500 nm and a bandpass of 2 nm. CS was denatured in 6 M guanidine hydrochloride for at least 45 minutes and diluted 100-fold in a total volume of 2 ml to give a final monomeric concentration of 200 nM. All aggregation experiments were carried out in 50 mM NaP04 buffer (pH 7.5), and were normalized as described above.  2.10 Native gel analysis of HSP16-2-actin/tubulin binary complexes The kinetics of HSP16-2-actin/tubulin binary complex formation was measured by diluting [ S]-labeled 7.5 M urea-denatured actin or tubulin 100-fold (final actin/tubulin concentration, 35  approximately 225 nM) into a solution containing 0.5 mg/ml (1.6 |iM) HSP16-2 and immediately mixing well by pipetting up and down. Appropriate dilutions of HSP 16-2 were made using 'refolding buffer' (25 mM MES pH 7.5, 25 mM NaCI, and 0.5 mM DTT). At each time point, 20 fil of the reaction mix was mixed in with 5 JJ.1 glycerol, and snap-frozen in an 77  Chapter II: Materials and Methods ethanol dry-ice bath. Samples were then separated on native gels (prepared and run as described in the Materials and Methods of Chapter I), and subjected to autoradiography. To measure the binding of HSP 16-2 to progressively more aggregated forms of actin and tubulin, the following experiments were done. Labeled, denatured actin or tubulin was diluted into 'refolding buffer' for various lengths of time, and an aliquot was added to a tube containing HSP 16-2 such that the final volume was 20 pi, and the HSP 16-2 concentration was 1.6 pM and that of actin or tubulin approximately 225 nM. The binding was allowed to proceed for 20 minutes at room temperature, and the reaction was snap-frozen after the addition of glycerol to 20%. Samples were then separated on native gels as before.  2.11 Sedimentation velocity and fluorescence measurements The experiments in this section were performed by Ronald Melki and Gerard B atelier as part of a collaboration, and the methodologies are included here for reference only.  2.11.1 Actin and tubulin purification and labeling Actin was purified from rabbit muscle acetone powder (Spudich and Watt, 1971; Eisenberg and Kielley, 1974) and isolated as CaATP-G-actin by chromatography through Sephadex G-200 (McLean-Fletcher and Pollard, 1980) at 4°C equilibrated in 5 mM Tris-HCl, pH 7.8, 0.2 mM DTT, 0.2 mM ATP, 0.1 mM CaCl , 0.01% NaN . G-actin (50 pM) was stored on ice and used 2  3  within two weeks. Pyrenyl-labeled actin and NBD-labeled actin were prepared according to Kouyama and Mihashi (1981) and Detmers et al. (1981), respectively. Pure tubulin was prepared from fresh pig brain by three assembly-disassembly cycles (Shelanski et ai, 1973) followed by phosphocellulose (Whatman P l l ) chromatography (Weingarten et al., 1975). Tubulin (100-150 uM) in 0.1 M PIPES, pH 6.9, 1 mM EGTA, 0.5 mM MgCl and 1 mM GTP was flash 2  frozen in liquid nitrogen and stored at -80°C. DTAF-labeled tubulin was prepared as described by Mejillano and Himes (1989). Native Pyrenyl- or NBD-actin and DTAF-tubulin were denatured by addition of urea to a final concentration of 7.5 M.  78  Chapter II: Materials and Methods 2.11.2 Fluorescence measurements All measurements were made at 20°C in a Spex fluorolog 2 spectrofluorometer. The excitation monochromator was set at 345, 468, and 492 nm and the emissions recorded at 386, 535, and 517 nm for pyrenyl-actin, NBD-actin and DTAF-tubulin, respectively.  2.11.3 Sedimentation velocity measurements Sedimentation velocity experiments were carried out with a Beckman Optima X L - A analytical ultracentrifuge equipped with an AN 60Ti four-hole rotor and cells with two-channel 12 mm path length centerpieces. Sample volumes of 400 fll were centrifuged at 60 000 rpm. Radial scans of absorbance at 278 nm were taken at 10-minute intervals. Data were analyzed to provide the apparent distribution of sedimentation coefficients using the programs DCDT (Stafford, 1992) and SVEDBERG (Philo 1994). The partial specific volumes, v, at 20°C, were calculated from the amino acid compositions and the solvent density was 1.00 g/cm . The degrees of hydration of the totally unfolded proteins were estimated based on the amino acid compositions by the method of Kuntz (1971) according to Laue et al. (1992). The degrees of hydration used for all calculations, 0.316, 0.314, and 0.337 g of H2O per g of protein, for wildtype HSP16-2, H A130-145 HSP16-2 and H Al-44 HSP16-2, respectively, were the result of 6  6  correcting the calculated degrees of hydration by a factor of 0.7, obtained by comparing degrees of hydration for several proteins in their folded state to that based on their amino acid composition (Lin et al, 1991).  2.12 Miscellaneous SmHSP protein concentrations were determined using the Bio-Rad Bradford assay kit and IgG as a standard. Citrate synthase concentrations were determined by absorbance at 280 nm using an extinction coefficient of 1.55 X 10 M -5  _ 1  cm" (Singh et al., 1970). A heat-shocked C. elegans 1  embryo extract was prepared by homogenizing 1 g of embryos (incubated at 30°C for 2 hours and recovered at 15°C for 30 minutes) in a Dounce homogenizer in the presence of 2 ml TEND buffer supplemented with protease inhibitors (2 mM PMSF, 2 |ig/ml Leupeptin, 1 |ig/ml 79  Chapter II: Materials and Methods Aprotinin, 1 pg/ml Pepstatin A), and clearing the cellular debris by centrifugation at 12000 x g. The concentrations of HSP16-2 and H.6A130-145 complexes were calculated according to the average of the two molecular masses obtained by sedimentation velocity measurements (317 kDa and 421 kDa, respectively). All other smHSP concentrations were based on the theoretical, monomeric molecular weights.  80  Chapter II: Results III. RESULTS  3.1 Structure and function of HSP16-2 3.1.1 Production of HSP16-2 variants for structure-function studies Although the sizes of smHSPs vary substantially, a detailed sequence comparison of known members suggests that the minimal functional unit consists of a core region of about 85 amino acids (the a-crystallin domain) which is flanked by an N-terminal region of at least 39 residues (in E. coli IbpA), and a C-terminal extension of at least 12 residues (in N. crassa HSP30). Accordingly, the most compact smHSP characterized to date is E. coli IbpA, which has a very short C-terminus of 14 residues and totals 137 amino acids (15.8 kDa). The nematode HSP12 family represents the only known exception to the apparent minimum size restriction of smHSPs. C. elegans HSP16-2 (145 amino acids) is only slightly larger than IbpA, and is therefore a good model smHSP for delineating the structural requirements for smHSP oligomerization and chaperone activity in these proteins. Expression constructs harbouring wild-type HSP16-2 and six derivatives were created (Figure 15A). The cDNA encoding HSP16-2 was initially isolated and subcloned into pRSET for the purpose of producing and purifying a polyhistidine-containing fusion protein (H6HSP16-2), which was used to make anti-HSP16-2 polyclonal antibodies (Jones et al., 1996). An HSP 16-2 construct lacking the polyhistidine fusion leader was created for the expression of wild-type HSP 16-2. Three additional constructs designed to express polyhistidine-containing Nterminal truncations of HSP16-2 were made. These encode H6A1-15, HgAl-32, and H6A1-44 HSP16-2, which lack the first 15, 32, and 44 amino acids of HSP16-2, respectively, but contain a 4-kDa N-terminal polyhistidine-containing region to facilitate purification. Finally, a construct designed to express a polyhistidine-containing variant of HSP 16-2 lacking the last 16 amino acids (HgA130-145 HSP16-2) was created. All smHSPs were produced in the E. coli BL21(DE3) strain (Studier et al., 1990) and purified from inclusion bodies to >90% homogeneity (Figure 15B). Wild-type HSP 16-2 was purified by a combination of denaturing and nondenaturing size exclusion chromatography, while the polyhistidine-containing smHSPs 81  Chapter II: Results were purified by binding to Ni -chelate affinity resin and eluting with a low pH buffer. The 2+  smHSPs were then dialyzed in an appropriate buffer before use. An alignment of C. elegans HSP 16-2 and HSP 12.6, as well as other smHSP sequences from various organisms (Figure 15C) illustrates the three distinct regions found in smHSPs, and delineates the regions of the HSP 16-2 derivatives which are truncated relative to wild-type HSP16-2, HSP12.6, and other smHSPs. The cDNA encoding HSP 16-48, a member of another class of 16-kDa smHSPs in C. elegans (Russnak and Candido, 1985) was amplified by PCR from first strand cDNA prepared from total (heat-shocked) RNA, and subcloned into a pRSET vector. The wild-type HSP 16-48 protein was produced in E. coli, and its size and cross-reactivity toward the antiHSP16-2 antibody strongly suggests that it represents (probably along with its highly identical homologue, HSP 16-41) the lower-MW protein seen in Western blots of stressed nematode extracts probed with the same antibody (see Appendix VII). No additional studies were attempted with this protein.  3.1.2 HSP16-2 quaternary structure and subunit orientation The size of recombinant wild-type HSP 16-2 was estimated by gel permeation chromatography on a calibrated Sephacryl S-300HR column. The majority of HSP16-2 elutes as a single peak between the MW markers thyroglobulin (669 kDa) and ferritin (440 kDa), and has an apparent molecular mass of 550 kDa (Figure 16A and 16B). The estimated size of the complex is essentially identical to that of HSP16-2 isolated from a heat-shocked nematode extract (Hockertz et al, 1991). Interestingly, the H6HSP16-2 protein also assembles into large oligomeric complexes of somewhat larger size (680 kDa), as judged by size exclusion chromatography (Figure 16B). These data suggest that HSP 16-2 and H6HSP16-2 contain approximately 34 and 37 subunits, respectively. While gel permeation chromatography provides estimated sizes based on the size and shape (Stake's radius) of any protein or protein complex, significantly better size estimates can be obtained by sedimentation velocity measurements. 82  Chapter II: Results  A  r  r  nr.  kDa 20068 43 -  QpOtyHjS tag |  H, IM' i,-:  |  ii,, \i-i5 nspit>-:  QcMtll I  29-  J Hj,AI-.12 l l s l ' K . - :  18-  I H , \l-44 I I S I ' K . - : (  14-  II,, M -0-145 I I S P U . - :  5.5 -  1SPI2.G  2.8-  N-terminal domain HSP16-2 -MSLYHYFRPAQRSVFGDLMRDMALMERQFAQVCQISPSES s HSP12.6 MMSVPVMADEGTKWDWPLQKGD GW HSP16-48 MLMLRS PrSD SNVLDHFLDEITGSVQ DHNSFNFSD NIG SEC-1 MSSLCPYTGRPTGLFRDFEDI BRHSMLNNFNNI -VPQQLN uA-cryst. MDIAIQHPWFKRTLGPFYPSRLFDQFFGEGLFEYDLLPFLSSTI S{ 2SLFRTV LDSGIS HSP25 MTEPJ(VPFSUJ«PSWEPETffiWYPAHSRIJIK2AFGVPPiPDEWS0WFSAAGWPGYVRPLPAATAEGPAAVT: rSRALNRQ LSSGVS HSP27 PLI^LJIP^IJDHDYRTDWGHLL^X)DFGFGVHAHDLFHPRRLLLPNTLGLGRRP.YS£ BjSHGKHNQMSRRASGGPNAL HSP17 . 6 MSLIPSIFGGPRSNVFDPFSLDMWDPFKD rSSVSAENSA FVNTRV HSP16 . 3 _ -MATTLPVQRHFRSLFPEFSELF. SFAGLRPTFD TRLMRL M  HSP16-2 HSP12.6 HSP16-48 SEC-1 c,A-cryot. HSP25 HSP27 HSP17.6 HSP16.3  S  I  I  C-teiminal extension KEAVQG iSlsngiDOAI - -VEEJ^SAEMH KTNSS FVAKH TGSNTTVTSAGHAVTGSPSSTTTTGKH IPSGVDAGHS] —REE^PSSAPSS PKAVTQSAE FEARAQIGGPEAGKSEQSGAK PPSKEQAKSE-glW TGPAHLSVFJAPAPEAGDGKAENGSGEKMETSK EEVKKSDV G SEGKPTESTK  N  a  25 45  it 62 90 81 49 42  (i 32 83 82 82 83 83 83 90 66  ( 22 2 16 33 28 36 39 15 16  lota 145 110 143 159 173 209 2 13 154 144  Figure 15. Summary of smHSP constructs, purified proteins, and alignment. A. Schematic of all smHSPs used in this study, drawn approximately to scale. The two-exon structure (exon 1, light grey and exon 2, dark grey) applies to hsp!6-2 and hsp!2.6, as both have a single intron in the same relative position. The polyhistidine-containing pRSET tag is shown in white. B. Purified smHSPs separated on a 15% SDS-gel. Lanes 1-7 represent the constructs shown in (B) from top to bottom, respectively. C. Amino acid sequence alignment of smHSPs (with Genbank accession numbers): C. elegans HSP16-2 (M14334), HSP12.6 (also identified as F38E11.2; Z68342 and U92044), HSP16-48 (K03273), SEC-1 (Z35640); bovine aA-crystallin (M26142); murine HSP25 (L07577); Drosophila HSP27 (J01101); Soybean HSP17.6 (Ml 1317); M. tuberculosis HSP16.3 (M76712). The first block of aligned sequences corresponds to the Nterminal domain, the second block corresponds to the a-crystallin domain, and the third block shows the C-terminal extension. Identical amino acids present in at least 4 of the 9 sequences are highlighted in black, and structurally similar amino acids (at least 4 of 9) are shaded. 83  Chapter II: Results In collaboration with Ronald Melki and Gerard Batelier (Laboratoire d' Enzymologie et Biochimie Structurales, CNRS, France), such studies were carried out on HSP16-2, H6A130145, H6A1-44, and HSP12.6 (summarized in Appendix VIII). Sedimentation data confirmed that HSP 16-2 forms large oligomeric complexes. The data fitted relatively well to a twocomponent system involving 10.5 S and 14.7 S species with proportions of about 58% and 42%, respectively (Appendix VIII). These correspond to apparent molecular masses of 239 and 395 kDa for the two species, consistent with the behaviour of wild-type HSP 16-2 as an oligomer of approximately 14 and 24 subunits, respectively. Interestingly, the frictional ratio values if I /o) suggest that HSP16-2 is somewhat asymmetrical. Waldmann et al. (1995c) previously reported difficulty in purifying an archaebacterial chaperonin complex carrying a polyhistidine extension fused to its C-terminus, due to poor accessibility of this region to the Ni -chelate affinity resin. Since H6HSP16-2 also forms an 2+  aggregate, a test to determine whether its polyhistidine-containing tag was exposed or not was carried out. Aliquots of native or urea-denatured H6HSP16-2 were incubated with nickel-chelate affinity resin, and after washing the resin to remove non-specifically adsorbed proteins, specifically bound protein was eluted with 100 mM EDTA. No detectable binding of the native H6HSP16-2 complex to the nickel resin was seen, whereas the unfolded HgHSP16-2, with its exposed H6 tag, bound efficiently (Figure 17). It is therefore likely that the N-terminal region of wild-type HSP 16-2 is sequestered within the interior of the native complex.  3.1.3 HSP16-2 interacts with unfolded CS and prevents its aggregation The various methods employed to assay for molecular chaperone activity all make use of the affinity of chaperones for unfolded proteins. The three assays used in this study to measure the interaction of smHSPs with unfolded proteins are summarized in Figure 18. The first method involves the protection of a given protein from aggregating at an elevated temperature. Similarly, the second assay measures the protection of a substrate from aggregation after dilution from a chemical denaturant. The third assay involves the detection of binary complexes formed between the chaperone and a denatured protein by native gel electrophoresis. 84  Chapter II: Results  30  32 34 36 38 40 elution volume (ml)  42  I • • •• i •• • • i • • • i • " 1 " " i • • " i • " i " • • I 1  30  35  1  40 45 50 55 60 elution volume (ml)  1  65  70  Figure 16. Elution profile of HSP 16-2 and derivatives from a Sephacryl S-300HR size exclusion column. A . HSP 16-2 elutes as a single peak corresponding to a species of approximately 550 kDa. B. Peak elution volumes of the smHSPs (O) relative to a series of molecular weight protein standards (•), thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), BSA (67 kDa), ovalbumin (43 kDa), and RNAse A (14 kDa). The estimated sizes of the eluting species, shown in parentheses, were derived from the log(MW) versus elution volume (ml) standard curve.  85  Chapter II: Results  Native H HSP16-2  Denatured H HSP16-2  6  6  kDa 29 18 14 -  1  2  3  4  5  Figure 17. Affinity of native and denatured H.6HSP16-2 for nickel affinity resin. Native HSP 162 complex and 6 M-urea denatured complex were incubated with separate aliquots of N i chelate affinity resin equilibrated in buffer without or with 6 M urea, respectively. The resins were then washed multiple times with a pH 6.3 buffer without or with 6 M urea, respectively, and the first two washes without urea were combined and analyzed on a 12% SDS-gel (lane 1). Following the washes, any protein bound to the resin was eluted with 100 m M E D T A buffer. The E D T A washes without urea are shown in lanes 2 and 3, and the washes with urea are shown in lanes 4 and 5. Note that only the denatured HSP 16-2 bound to the resin and therefore was eluted in the E D T A washes. 2 +  86  Chapter II: Results The molecular chaperone activity of HSP 16-2 was investigated using citrate synthase (CS), a dimer of two identical 49-kDa subunits. The denaturation and renaturation of CS has been well studied (Zhi et al, 1992), and CS has been used in chaperone studies with HSP90 and associated proteins (Wiech et al, 1992; Jacob et al, 1995; Bose et al, 1996), GroEL (Buchner et al, 1991), and smHSPs (Jakob et al, 1993; Lee et al, 1995; Chang er al, 1996). When CS is incubated alone at 40-45°C it aggregates irreversibly, as detected by light scattering at 320nm, and loses activity. However, when CS is incubated together with increasing amounts of HSP162, there is a concomitant decrease in CS aggregation (Figure 19A). IgG has no effect on CS aggregation under these conditions, demonstrating the specificity of the chaperone-mediated inhibition of aggregation. The ability of HSP16-2 to prevent CS aggregation over a range of concentrations is summarized in Figure 19B. At molar ratios of HSP 16-2 complex to CS monomer of 1:1 or higher, aggregation of CS is almost completely inhibited. The H6HSP16-2 variant is also equally effective in preventing CS aggregation (see Figure 29). To determine if HSP 16-2 can re-solubilize protein aggregates, a 3.5-fold molar excess of HSP16-2 was added to a solution of CS which had aggregated for 15 minutes (Figure 19C). The addition of HSP 16-2 prevented further aggregation, but did not reduce the light scattering caused by the preformed CS aggregates. This behavior of HSP 16-2 parallels that of bovine a-crystallin and M. tuberculosis HSP16.3 (Wang and Spector, 1994; Chang et al, 1996). The aggregation of proteins at high temperatures may involve unfolded intermediates which are different from those generated when the proteins are diluted into buffer from chaotropes such as urea or guanidine hydrochloride. We therefore examined whether HSP 16-2 can prevent the aggregation of 6 M guanidine hydrochloride-denatured CS which occurs after 100-fold dilution of the chaotrope. HSP 16-2 interacts with and stabilizes CS diluted from denaturant essentially as efficiently as it prevents the thermally-induced aggregation of CS (Figure 19D).  87  Chapter II: Results  Functional assays used for small HSPs A  thermal aggregation assay  > 3  o N  a 3  a  citrate synthase to 150 nM +/small HSP in 0.9 ml  B  3 .•£!  chemical aggregation assay +/- small HSP final volume with CS  (added later) is 2 ml  c  native gel denatured protein binding assay combine small HSP + 7.5 M ureadenatured [ S]-labeled actin/tubulin (diluted lOOx) ^in20(d 35  insert cuvette in water-jacketed spectrophotometer set to 320 nm and 45°C  measure: increasing amounts of small HSP  insert cuvette in spectrofluorometer (with stirrer) set parameters to: excitation: 500 nm ernrnission: 500 nm bandpasses: 2 nm HV 800 fluorescence; Gain 100 HV 305 reference; Gain 10 Time base: 50 sec/inch add CS denatured in 6M GuHCl to ,200 nM  time measure:  increasing amounts of small HSP  incubate at room temp, for required 'time add 4 ul glycerol and load  4.5% native ge run at 5W for 1.5 hours  i  dry gel and expose to screen  detect and quantitate binary complexes  time  Figure 18. Three techniques for measuring the chaperone activity of smHSPs. A . Thermal aggregation assay using citrate synthase as substrate. B. Chemical aggregation assay using citrate synthase as substrate. C . Measuring binary complexes between smHSPs and labeled, denatured actin or tubulin on a native polyacrylamide gel.  88  Chapter II: Results 3.1.4 Complex formation between HSP16-2 and denatured actin and tubulin Although smHSPs have been shown to bind to and stabilize actin filaments (Bennardini etal., 1992; Lavoie etal., 1993b; Wang and Spector, 1996), detailed studies on the nature of the interaction between smHSPs and actin have not been reported. To further explore this property of smHSPs, we first investigated the formation of binary complexes between HSP 16-2 and unfolded p-actin using a gel mobility assay. Figure 20A shows reactions in which urea-denatured [ S]-labeled actin was diluted 100-fold into a solution with (lanes 1-4) or 35  without (lanes 5 and 6) HSP16-2, or with HSP16-2 and a 2-fold molar excess of native BSA (lane 7), and separated on a non-denaturing gel. The denatured actin in reaction 4 was first incubated for 150 minutes before it was mixed with HSP16-2. The top panel of Figure 20A shows the Coomassie Blue-stained native gel (with the positions of the HSP 16-2 complex and BSA indicated), and the bottom panel represents an autoradiogram of the same gel. In lanes 1-3, the binary (HSP16-2-actin) complex is visible, whereas if the actin is allowed to aggregate for 150 minutes before HSP 16-2 is added, essentially no binary complex is detectable (lane 4). Reactions in which actin was incubated in the absence of HSP 16-2 show only a trace of actin aggregates (lanes 5 and 6). The presence of a two-fold molar excess of BSA has no effect on binary complex formation between HSP16-2 and denatured actin (lane 7), indicating that HSP 16-2 shows selective binding to unfolded polypeptide but not to native proteins. The time course of binary complex formation between HSP 16-2 and unfolded actin (Figure 20B) reveals that approximately 70% of the binary complexes are formed within the first minute of incubation. Interestingly, identical kinetic experiments performed with HSP 16-2 and labeled, denatured (3-tubulin gave very similar results (Figure 20C). This demonstrates that HSP 16-2 has similar affinities for unfolded actin and tubulin, despite the fact that these proteins share no sequence homology. Melki and Cowan (1994) also showed that binary complexes between denatured actin and GroEL or HSP60 also formed very quickly (within 15 seconds). Although the binding of HSP 16-2 to denatured actin is initially very rapid, it then proceeds with observable kinetics until a plateau is reached after about 20 minutes. The slower phase of binary  89  Chapter II: Results  complex formation may indicate that the actin intermediates formed at later times are less well recognized by the smHSP or that the available binding sites become progressively saturated. In light of the above results, the ability of HSP 16-2 to interact with actin intermediates formed along the aggregation pathway was investigated. A constant amount of HSP 16-2 was added to a reaction mixture containing actin which had been, incubated alone in buffer for varying lengths of time. The binding reactions were allowed to proceed for 20 minutes, and then analyzed by native PAGE (Figure 20D). Under these conditions, the yield of binary complex formed decreased to almost zero over time. After a brief 2-minute pre-incubation of the actin alone, a significant proportion (about 25%) of the actin fails to form a complex with HSP16-2, and after a 5-minute pre-incubation, only approximately 50% of the actin is found in a binary complex, relative to the amount of actin bound to HSP 16-2 in the absence of a pre-incubation. The same experiment with HSP16-2 and (3-tubulin gave almost identical results (Figure 20E). It is noteworthy that the chaperonins HSP60 and CCT also have a decreasing ability to recognize actin intermediates which form during aggregation over the same time period (Melki and Cowan, 1994). Overall, the actin and tubulin binding studies suggest that HSP 16-2 has a distinctly greater affinity for early refolding or aggregating intermediates but displays little protein specificity. To complement the HSP 16-2 actin and tubulin binding studies performed above, the affinity of HSP 16-2 for actin and tubulin was further examined using fluorescently-labeled derivatives. These studies, presented in Figure 21, were carried out by Ronald Melki. Addition of increasing amounts of HSP 16-2 to denatured pyrenyl-actin and DTAF-tubulin resulted in a quenching of the fluorescence of these proteins (Figure 21A and B). These data show that HSP 16-2 binds to labeled, denatured actin and tubulin with a single association constant of 2.8 x 10 M" . The -6  1  stoichiometry of binding appears to be 1:1. In contrast, addition of up to a 5-fold molar excess of HSP 16-2 to the native pyrenyl- or NBD-labeled actin and DTAF-tubulin did not result in a quenching of the fluorescence of the labeled proteins. This demonstrates that HSP 16-2 has an equally high affinity for unfolded actin and tubulin, but not for the native proteins.  90  Chapter II: Results  Figure 19. HSP16-2 prevents thermally- and chemically-unfolded CS from aggregating. A. Influence of various concentrations of the HSP16-2 oligomeric complex (O, 260 nM; • ,130 nM; • , 85 nM; • , 44 nM; • , 0 nM) and IgG (A, 300 nM) on the thermally-induced aggregation of CS (monomer concentration of 150 nM) at 45°C. B. Summary of data from the first panel, showing the ratio of HSP 16-2 complex to CS monomers versus the % of CS aggregated after 40 min. C. Effect of no additions (•) or addition of HSP16-2 (to a final concentration of 525 nM, 0) to a solution of CS (150 nM) which had aggregated for 15 minutes at 45°C (denoted by arrow). D. summary of the ability of various concentrations of HSP 16-2 to prevent guanidine hydrochloride-denatured CS (200 nM) from aggregating when diluted into buffer at room temperature. 91  Chapter II: Results  ACTIN  denatured 35S-actin pre-incubation time HSP16-2 native B S A incubation time  + + 1  + 150  20  + -  1  150  1  5  0  10  y  20 30 4 0 T i m e (mm)  50  60 0  10  20 30 40 T i m e (min)  50  60  TUBULIN  ACTIN  0 2 5 10 30 60 120  HSP16-2/actin binary c£> complex  2 5 10 20 30 60  0000111  HSP16-2C>  BSA C >  1  2 5 10 20 30 60  0 2 5 10 30 60140  tf 0  20  40 60 80 Time (min)  100 120 0  20 4 0 6 0 8 0 100 120 140 T i m e (mm)  Figure 20. Binary complex formation between HSP16-2 and P-actin and P-tubulin. A . [ S]35  labelled, chemically-unfolded p-actin was diluted 100-fold into reactions containing HSP 16-2 (lanes 1-3), no smHSP (lanes 5 and 6), or HSP16-2 and a two-fold molar excess of BSA (lane 7). Reactions were incubated for the times shown, and analyzed on a native gel. The upper panel shows the Coomassie-stained gel and the bottom panel is an autoradiogram of the same gel. Lane 4 shows the result of pre-incubating the unfolded actin before the addition of HSP16-2. The native HSP16-2 oligomeric species is indicated, as is native BSA. Binary complexes are shown in the autoradiogram. The kinetics of binary complex formation between HSP 16-2 and Pactin (B) or P-tubulin (C) were measured by diluting the labelled, unfolded target proteins into reactions containing a fixed amount of HSP 16-2, incubating for the times shown, and analyzing the reactions on native gels as in (A). The relative yields of binary complexes were quantified by phosphorimager. The binding of HSP 16-2 to P-actin (D) or P-tubulin (E) intermediates formed on the aggregation pathway was measured by diluting the labelled, unfolded target protein into buffer alone, incubating for the times shown, and then adding a fixed amount of HSP 16-2. Binding reactions were allowed to proceed for 20 minutes before analysis on native gels as before.  92  Chapter II: Results 3.1.5 Chaperone assembly and activity is not affected by C-terminal deletion The C-terminal extensions of smHSPs are variable in size and sequence, but often contain a region of limited similarity having the motif [R/K]-X-[I7V]-X-[17V] (see Figure 14 and 15C). It was therefore of interest to determine whether the C-terminal extension of HSP 16-2 (including the conserved motif RSIPI) plays a structural or functional role by studying the C-terminallytruncated H A130-145 HSP 16-2. Gel permeation chromatography of H A130-145 HSP 16-2 6  6  reveals that like HgHSP16-2, the N-terminally-tagged variant forms a somewhat larger aggregate than wild-type HSP 16-2, with an estimated M of 675 000 (Figure 16B). T  Sedimentation velocity measurements of H6A130-145 HSP16-2 were also carried out (by R. Melki and G. Batelier), and the data agree with this smHSP variant having a large oligomeric size. The best theoretical fit of the sedimentation data was obtained assuming a two-component system involving a 12.8 S and a 17.6 S species with proportions of about 49% and 51%, respectively. The apparent molecular masses obtained for the two species are 322 and 520 kDa, which is consistent with oligomeric complexes consisting of approximately 17 and 28 subunits (Appendix VIII). The ability of H6A130-145 HSP 16-2 to protect CS from aggregation at an elevated temperature was then tested (Figure 22). Surprisingly, removal of the C-terminal extension has little or no influence on the chaperone function of HSP 16-2: H6A130-145 is as effective as the wild-type smHSP in suppressing CS aggregation (compare with Figure 19B). In light of this data, it was expected that H6A130-145 HSP 16-2 would have an affinity for denatured actin or tubulin. Indeed, results similar to those with wild-type HSP 16-2 were obtained when H6A130-145 was added to pyrenyl-labeled actin (Figure 21C), demonstrating that the C-terminally-truncated smHSP interacts with unfolded polypeptides.  3.1.6 Correlation between multimerization and chaperone activity Perhaps the most conspicuous feature of smHSPs is the presence of a nonconserved N-terminal domain which is highly variable in size. Three N-terminal truncations of HSP 16-2, Al-15, Al-32, and Al-44 were used to probe the function of this domain. 93  Chapter II: Results  Figure 2 1 . Fluorescence quenching studies. The quenching of pyrenyl, NBD and DTAF fluorescence upon binding of HSP16-2; H A130-145 HSP16-2, H Al-44 HSP16-2 and HSP12.6 to actin and tubulin in their native or denatured states was measured. A. increasing amounts of HSP 16-2 were added to solutions of native (O) or urea-denatured (•) pyrenyl-actin and native (•) or denatured (•) NBD-actin in 5 mM Tris-HCl, pH 7.8, 0.2 mM DTT, 0.2 mM ATP, 0.1 mM CaCh. B. increasing amounts of HSP 16-2 were added to solutions of native ( T ) or ureadenatured ( A ) DTAF-tubulin in 0.1 M PIPES, pH 6.9, 1 mM EGTA, 0.5 mM MgCl and 1 mM GTP. C. increasing amounts of H6A130-145 HSP16-2 were added to a solution of native (O) or urea-denatured (•) pyrenyl-actin. (D) increasing amounts of H6A1-44HSP16-2 (•) or HSP12.6 (O) were added to a solution of urea-denatured pyrenyl-actin. The concentration of actin and tubulin solutions was 1 pM. The fluorescence was recorded after each addition as described in Materials and Methods. (Figure kindly provided by Ronald Melki). 6  6  2  94  Chapter II: Results  To examine the quaternary structure of these N-terminal truncations as well as of other HSP 16-2 derivatives, the purified proteins were subjected to chemical cross-linking using the homobifunctional cross-linker bis (sulfosuccinimidyl) suberate (BS ) (Staros, 1982). To 3  minimize nonspecific intermolecular cross-linking of non-oligomerized smHSP subunits, crosslinking reactions were carried out in the presence of low protein (1.5 |iM monomer) and B S  3  (2 mM) concentrations, and a 10-fold molar excess of BSA (15 |J,M). Reactions were analyzed by Western blotting with an anti-HSP16-2 polyclonal antibody (Figure 23). All of the proteins yielded discernible monomeric, dimeric, and trimeric species. From the large number of products present in the high-MW range (above 100 kDa and at the origin of the separating gel), it can be concluded that the HSP16-2, H HSP16-2, and H6A130-145 proteins also form higher-order 6  oligomeric structures which are too large to be well resolved on the 12% SDS-gel. These finding are consistent with the behaviour of these proteins as high-MW oligomers as judged by size exclusion chromatography and sedimentation velocity measurements. In contrast, the H6A1-15, H6A1-32, and H6A1-44 HSP16-2 proteins yielded essentially no high-MW cross-linked products but could be cross-linked to trimers (and possibly tetramers), suggesting that they exist as small aggregates. The presence of the N-terminal H6 tag in each of these truncated proteins is unlikely to have resulted in disruption of the multimers since both H6HSP16-2 and H6A130-145 HSP 16-2 are able to form large oligomeric complexes. The sizes of the N-terminally-truncated smHSPs were estimated by gel permeation chromatography on a Sephacryl S-300HR column. These smHSP derivatives elute as particles of 45-115 kDa (Figure 16B), and therefore appear to form small aggregates of trimers-heptamers. These results resemble those obtained on the C-terminal domains of ocA-, aB-crystallin, and HSP25 . Removal of the N-terminal domains of these smHSPs resulted in dimers, tetramers, and higher-order multimers, based on size exclusion and cross-linking data (Merck et al., 1992, 1993b). In order to determine the true subunit stoichiometry of one of the truncated smHSPs, H6A1-44 HSP 16-2, sedimentation velocity measurements were performed (by R. Melki and  95  Chapter II: Results  G. Batelier). The sedimentation velocity data are best fitted by a two-component system involving 1.66 S and 4.0 S species. The apparent molecular masses obtained from the data are 15 and 56 kDa, consistent with the behaviour of H6A1-44 HSP 16-2 as a monomer (95%) with a small proportion (5%) of tetramers (Appendix VIII) Since the N-terminally-truncated smHSPs did not form wild-type-like oligomeric complexes, it was of interest to determine whether they still possessed molecular chaperone activity. The three derivatives were therefore tested for the ability to prevent thermally- or chemically-induced aggregation of CS. None of these smHSPs was effective in suppressing CS aggregation, even at a molar excess of 65 molecules of smHSP to 1 CS monomer (not shown). Furthermore, H6A1-44 HSP 16-2 did not quench the fluorescence of denatured forms of pyrenylactin (Figure 21D).  3.2 Structure, function and expression of HSP12.6 3.2.1 A novel class of small HSPs from C. elegans The advent of whole genome sequencing projects has resulted in a tremendous amount of information becoming rapidly available, which facilitates comparative studies of large multigene families. Caspers et al. (Caspers et al., 1995) first reported the existence of an unusually diminutive smHSP which was uncovered by the C. elegans genome sequencing consortium (Wilson et al., 1994). More recently, three additional closely related members in the genome sequence were identified. These four smHSP genes encode proteins of 12.2-12.6 kDa (109 and 110 amino acids) which are highly similar to each other throughout their entire length (42-67% amino acid sequence identity), as shown in Figure 24A. The alignment also outlines the similarity between the a-crystallin domain of the C. elegans HSP 12s and several other smHSPs. Compared with all other known smHSPs, the HSP12 proteins have the shortest N - and C-terminal regions, and represent a novel class of smHSPs. Previously, the smallest known member of the smHSP family was E. coli IbpA, at 15.8 kDa.  96  Chapter II: Results  0  0.8  1.6  2.4  3.2  ratio A130-145:CS monomer  Figure 22.  H S P 16-2 lacking most of its C-terminal extension has chaperone activity. Summary of the H6A130-145 HSP16-2 concentration-dependent suppression of aggregation of a 150 n M (monomer) C S solution incubated at 4 5 ° C . Aggregation assays were carried out for 40 minutes, and are normalized with respect to a control incubation with C S alone (100% o f total C S aggregation).  97  Chapter II: Results  r<^  kDa  2009868 —  •a  ^  &  &  ^  t>  & v>  ^  "•  -origin Utetramers Itrimers  43dimers 2918 —  monomers  14 —  Figure 23. Chemical cross-linking of smHSPs. Samples of HSP 16-2 and five derivatives were cross-linked with the cross-linking agent BS , electrophoresed on a 12% SDS-gel, transferred to a PVDF membrane, and probed with an anti-HSP16-2 antibody. Monomers and cross-linked products corresponding to dimers, trimers, and tetramers are indicated. The separating gel origin is also shown. 3  98  Chapter II: Results The predicted secondary structures of the a-crystallin domain of the C. elegans HSP 12 and HSP16-2 smHSPs, and murine aB-crystallin are nearly identical, consisting almost exclusively of (3-sheets (Figure 24B); this observation is consistent with the >90% p-sheet structure predicted for various smHSPs by circular dichroism studies (Merck et ai, 1993a, 1993b). The notion that the HSP 12 proteins are evolutionarily related to other smHSPs is further supported by the presence of introns at conserved positions (Figure 24C). In particular, the intron which delineates the N-terminal region from the a-crystallin domain is found in three of the HSP 12 genes, as well as in murine aB-crystallin, and many other C. elegans smHSPs, including HSP16-2 (Figure 24C). The hsp!2 multigene family can be inferred to have arisen by gene duplications and gains/losses of introns. For example, the genes encoding HSP 12.6 and HSP 12.3 are more similar to each other (67% amino acid sequence identity) than to either C14B9.1 or T22A3.2 (42-48% pairwise identity), and are found duplicated approximately 1000 bp apart in a head-to-tail orientation on chromosome IV. Similarly, the genes encoding T22A3.2 and C14B9.1, which are present on different chromosomes (I and III, respectively), are also more closely related to each other (63% identity), and share an intron near a location corresponding to the second intron of the murine aB-crystallin gene.  3.2.2 Cloning, expression, and purification of HSP12.6 To characterize the structural and functional properties of the HSP 12 class of smHSPs from C. elegans, HSP 12.6 was cloned from first-strand cDNA by PCR. Both wild-type and polyhistidine-tagged HSP12.6 (H6HSP12.6) were overexpressed in E. coli from a T7 promoter, producing soluble protein in each case. HSP12.6 was purified by size exclusion and hydroxylapatite chromatography, and HgHSP12.6 was purified by Ni -chelate affinity 2+  chromatography (Figure 25). The gene encoding HSP12.3 (67% identity to HSP12.6) was also cloned by PCR (using the primers HSP13B.5 and HSP13B.3; see Appendix II), overexpressed in E. coli, and used to assess the specificity of the anti-HSP12.6 pAb; it was found that recombinant HSP12.6 and HSP12.3 are detected with similar efficiency in Western blots (see Appendix VII). 99  Chapter II: Results  Figure 24. Comparison of the C. elegans HSP12 protein family with other smHSPs. A . an alignment of the four C. elegans HSP12 proteins and other selected smHSPs (Genbank accession numbers in parentheses): C. elegans HSP12.6, also referred to as F38E11.2 (Z68342 and U92044), HSP12.3, also referred to as F38E11.1 (Z68342), C14B9.1 (L15181), T22A3.2 (Z81125); E. coli IbpA (M94104); C. elegans HSP16-2 (M14334); murine aB-crystallin (M73741); Drosophila HSP27 (J01101). Conserved residues present in at least four of the smHSPs are highlighted. The a-crystallin domain residues are numbered for reference. The total number of amino acids in each smHSP is indicated at the end of the sequences, and numbers in parentheses represent smHSP residues not shown in the alignment. B. secondary structures of the four HSP12 proteins, aB-crystallin, and HSP16-2, as predicted by the program PHD; arrows represent (3-sheets and striped bars a-helices. The scale parallels the a-crystallin domain numbering scheme. C. the intron positions of the genes shown in B are indicated by an arrow and follow the codon corresponding to the numbered a-crystallin domain amino acid residues. 100  Chapter II: Results  kDa 2009 7 6 7 -  43-  1814-  Figure 25. Overexpression in E. coli and purification of HSP 12.6. A. Polyacrylamide gel electrophoresis of various protein samples on a 13.5% SDS-gel. Lane 1, uninduced BL21(DE3) soluble protein extract; lane 2, soluble fraction of BL21(DE3) expressing recombinant wild-type HSP12.6; lane 3, HSP12.6 purified by size exclusion and hydroxylapatite chromatography; lane 4, recombinant HSP 12.6 containing N-terminal polyhistidine tag (H6HSP12.6) purified by Ni -chelate affinity chromatography, and used for antibody production. 2+  101  Chapter II: Results 3.2.3 The expression of HSP12.6 is developmentally regulated The developmental expression pattern of hsp 12.6 in C. elegans was examined at the protein level using polyclonal antibodies raised against the purified H6HSP12.6 protein. Western blots of total protein extracts from the major C. elegans developmental stages (embryo, larval, and adult stages) were probed with the anti-HSP12.6 pAb. Surprisingly, a cross-reacting polypeptide of approximately 13 kDa was detected only in the first larval (LI) stage (Figure 26). It is possible that the closely-related HSP12.3 smHSP is also recognized by the pAb and is likewise present only in the LI-stage, as a slightly smaller cross-reacting species of the same size as recombinant HSP12.3 is sometimes visible (see Figure 28). Interestingly, the production of HSP 12.6 is not appreciably augmented when C. elegans is exposed to a variety of stressors known to induce the expression of hspl6 genes, including heat shock, heavy metals, and alcohols (Brian Ma, personal communication).  3.2.4 The quaternary structure of HSP12.6 differs from that of other smHSPs As noted above, smHSPs thus far examined form large oligomeric assemblies. The native size of HSP12.6 was therefore examined by size exclusion chromatography. Interestingly, HSP 12.6 eluted from a Sephacryl S-200HR column near chymotrypsinogen A (25 kDa), with an estimated MW of 23 kDa (Figure 27A). Given a calculated MW of 12.6 kDa, this experiment suggested that HSP12.6 exists as a dimer of -25 kDa. The oligomeric nature of HSP12.6 was further probed using the homobifunctional crosslinker BS , in the absence or presence of BSA competitor. At a relatively high concentration of 3  HSP 12.6 (1 pM) without competitor protein, a dimer and a few higher-MW cross-linked products were detected by Western blotting (Figure 27B, lane 1). In the presence of BSA (lane 2), however, a significally smaller amount of dimer product was formed, suggesting that non-specific cross-linking of HSP 12.6 occurs at this concentration in the absence of competitor When a lower concentration of HSP12.6 was used (0.25 pM), little or no dimer product was seen with or without competitor (lanes 3 and 4), suggesting that HSP 12.6 is monomeric, and that its behavior on the size exclusion matrix may be due to a non-globular conformation. 102  Chapter II: Results  V  Figure 26. HSP12.6 is present in C. elegans LI larvae. Total protein extracts (20 pg) from C. elegans embryos, first larval stage (LI), a mixture of second and third larval stages (L2-L3), and mixed fourth larval and adult stages (L4-adult) were separated on a 13.5% SDS-gel, transferred to a PVDF membrane, and probed with the antibody against HSP12.6. The antibody cross-reacts strongly with HSP12.6 in the LI stage (prominent band near the 14 kDa marker).  103  Chapter II: Results  67  40  44  43  48  25  52  56  60  14  64  68  72  Elution volume (ml)  B HSP12.6(uM) 1 cross-linker + BSA  1 + +  0.25 0.25 + + +  1 0.25  976743291814kDa  ••  Figure 27. Size exclusion chromatography and cross-linking of HSP 12.6. A . the native size of HSP 12.6 was estimated by size exclusion chromatography on a Sephacryl S-200HR column. The elution volumes of the M W standards B S A (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and ribonuclease A (14 kDa) are indicated above the graph. B . HSP12.6 was incubated with 2 m M B S cross-linker for 30 minutes in the presence or absence of 15 p M B S A competitor (lanes 1-4). Control reactions without cross-linker were also carried out (lanes 5 and 6). A l l reactions were then analyzed by Western blotting with the antibody against HSP 12.6. 3  104  Chapter II: Results To determine whether the elution behavior of HSP12.6 from a size exclusion column is due to dimerization or to an extended, non-globular conformation, sedimentation velocity experiments were carried out (by R. Melki and G. Batelier). The sedimentation data best fits a monomer model involving a 1.43 S species, which corresponds to a molecular mass of 12.2 kDa. The frictional ratio values (f/fo) suggest that HSP12.6 is asymmetrical (Appendix VIII).  3.2.5 The in vivo M of HSP12.6 is identical to that of the recombinant protein r  The size of HSP12.6 isolated from C. elegans L I larvae was estimated by size exclusion chromatography of a protein extract on an S-200HR column under the same conditions used for recombinant HSP12.6. It was found that the endogenous HSP12.6 elutes as a single peak corresponding to a molecular mass of approximately 25 kDa (Figure 28). Based on the behavior of the recombinant protein on a sizing column, it is clear that the oligomeric structures of the natural and recombinant proteins are identical.  3.2.6 HSP12.6 may be functionally distinct from other smHSPs Although the exact role of oligomeric assembly in the function of smHSPs is unclear, it is likely to be important given its ubiquitous nature. Since HSP12.6 is likely to be monomeric, it was of interest to determine whether it might still be functional as a molecular chaperone. HSP 12.6 was therefore tested for the ability to prevent the aggregation of thermally-unfolded citrate synthase (CS). Contrary to other smHSPs and molecular chaperones in general, HSP12.6 has no significant effect on temperature-induced CS aggregation, even when present at a 225fold molar excess over CS (Figure 29). However, the kinetics of CS aggregation were somewhat slowed in the presence of the large excess of HSP 12.6, probably due to nonspecific stabilization of the denaturing CS by the additional protein, as shown by Zhi et al. (1992). To ensure that the assay conditions were optimal, a parallel experiment was performed using H6HSP16-2. Addition of this protein effectively suppressed CS aggregation.  105  Chapter II: Results  void volume T  67  25  T  T  kDa 432918-  0  14611  13  15  17 19  -  21  23  25  Fraction number  Figure 28. Size determination of HSP 12.6 isolated from C. elegans. A protein extract from C. elegans first-stage larvae was prepared and chromatographed on a Sephacryl S-200HR column calibrated with the protein standards BSA (67 kDa) and chymotrypsinogen A (25 kDa). Fractions eluting after the void volume were Western-blotted using the anti-HSP12.6 pAb. Note the presence of two cross-reacting species which have slightly different elution profiles and therefore are partially resolved in certain fractions: the upper band is the same size as recombinant HSP 12.6 and is visible in fraction 21; the lower band corresponds to the same size as recombinant HSP12.3 (see text) and is distinguishable as a single species in lane 13 (lane 15, in comparison, shows both upper and lower cross-reacting polypeptides). See Appendix VII for pAb specificity against HSP12.6 and HSP12.3.  106  Chapter II: Results  i  0  1  1  1  1  1  1  i  i  r  5 10 15 20 25 30 35 40 45 Time (min)  Figure 29. HSP12.6 lacks molecular chaperone activity. The effect of HSP12.6 and H HSP16-2 on the aggregation of a 150 nM solution of citrate synthase (CS) incubated at 45°C. The aggregation curves shown are of CS incubated alone (•), CS incubated with a 40-fold molar excess of HSP12.6 (O), CS incubated with a 225-fold molar excess of HSP12.6 (•), and CS incubated with a 60-fold molar excess of H6HSP16-2 (•) as a positive control. HSP12.6, H6HSP16-2, and CS concentrations refer to monomers. An experiment with a 1:1 ratio of CS to HSP12.6 gave results identical to that of CS incubated alone. 6  107  Chapter II: Discussion IV. DISCUSSION The discovery by Ingolia and Craig in 1982 that four small Drosophila HSPs were related in sequence to the well-studied but poorly understood a-crystallins found in eye lenses hinted at the importance and ubiquitous nature of this class of proteins. Later studies (Caspers et al, 1995) and recent whole-genome sequencing projects (Bult et al, 1996) have identified smHSPs in the three Kingdoms of life, and have demonstrated their presence in a wide variety of tissues in multicellular eukaryotes in the presence and absence of stress (Stringham et al., 1992; Arrigo and Landry, 1994; Groenen et al., 1994; Caspers et al, 1995; Waters et al, 1996). The smHSPs are likely to play key roles during both normal development and particularly when organisms are subjected to environmental stressors. The C. elegans HSP16s are among the smallest members of the a-crystallin family of HSPs, and it is reported here that they possess the multimeric structure and chaperone activities typical of these proteins. Very few mutational studies have been carried out on smHSPs, and these have concentrated mainly on mammalian a-crystallins (Merck et al, 1992, 1993b; Smulders etal. 1995, 1996; Plater et al, 1996). The present studies on HSP16-2 represent the most extensive structure-function analysis of a stress-inducible smHSP reported to date. The small size of the HSP 16s is an advantage in such studies, and the combination of in vitro mutagenesis with the functional chaperone assays and substrate binding measurements reported here provide a number of important insights into the structure and function of smHSPs.  Structure of smHSPs Many molecular chaperones are active as homo- or hetero-oligomers, requiring multiple subunit contacts with the unfolded polypeptide for activity. Chaperonins such as GroEL, HSP60, and CCT have characteristic bitoroidal structures containing 7-9 identical or related subunits per ring (reviewed in Hartl, 1996). The eukaryotic HSP104 and bacterial Clp homologues assemble into similar multimeric ring complexes (reviewed in Schirmer et al, 1996). SmHSPs also form large oligomeric assemblies of one or two types of subunits, but their quaternary structures are remarkably variable. Thus, cardiac a-crystallin has been reported to exist as a torus resembling 108  Chapter II: Discussion  that of chaperonins (Longoni et al., 1990) whereas other vertebrate a-crystallins and smHSPs assemble into roughly globular particles of various sizes and shapes and contain 32-42 subunits (Behlke et al., 1991; Groenen et al., 1994); M. tuberculosis HSP16.3 forms a trimer of trimers particle (Chang et al., 1996); lastly, two plant smHSPs were shown to form globular, or mixed globular and triangular structures of 12 subunits (Lee et al., 1995). The analyses of wild-type HSP 16-2 and various derivatives demonstrate that the N-terminal domain of smHSPs is required for subunit assembly and is buried within an aggregate which is highly accomodating for N-terminal regions of different sizes and compositions. Recombinant wild-type HSP 16-2 readily assembles into large oligomeric assemblies resembling those of the natural protein, and of -550 kDa as estimated by size exclusion chromatography. Sedimentation velocity measurements reveal that HSP 16-2  forms  heterogeneous mixtures of two particles of 238 and 412 kDa, suggesting that the number of subunits per complex is approximately 14 and 24, respectively. The reason for the size discrepancy between the two techniques is unclear; it is possible that the HSP 16-2 aggregates are loosely packed, or that they are non-globular. Interestingly, a similar apparent dichotomy exists for mammalian HSP27, which by size exclusion behaves as an -800 kDa oligomer, whereas by native gel electrophoresis migrates as a doublet of approximately 200 and 250 kDa (Lavoie et al., 1995). In sucrose gradients, HSP27 sediments as two distinct forms, one of 300-400 kDa and another of <70 kDa, depending on the phosphorylation state (Kato et al., 1994). It is possible that the various smHSP species are in slow equilibrium with each other. The H6HSP16-2 variant, which forms active oligomeric complexes  essentially  indistinguishable from wild-type HSP 16-2, was used to determine the orientation of the HSP 16-2 monomers within the complex. It was found that the 4-kDa N-terminal H6 tag was inaccessible for binding to a Ni -chelate affinity matrix. Thus either the tag is collaterally 2+  positioned in the complex and masked by the HSP 16-2 protein and neighbouring subunits (which would very likely have a detrimental effect on the assembly of the complex), or, more likely, the tag is accommodated within a central cavity in the complex. It is therefore very likely  109  Chapter II: Discussion  that the N-terminal domain of HSP 16-2 is oriented toward the interior of the complex. Also, the fact that HSP 16-2 can accomodate an additional 4 kDa (36 residues) at its N-terminus, making this domain larger than those of a-crystallins and Drosophila HSP27, and only 8 residues shorter than that of murine HSP25 (refer to the alignment in Figure 15C), suggests that the arrangement of subunits within smHSP complexes provides considerable freedom for N-terminal regions of different lengths and amino acid sequence. Indeed, the alternatively-spliced aA -crystallin ins  variant which contains a 23-residue insertion near the N-terminal/a-crystallin domain boundary also has structural properties comparable to that of wild-type aA-crystallin, although its chaperone activity is somewhat reduced (Smulders et al, 1995). Merck et al. (1992) reported that the isolated N-terminal domain of a-crystallin formed large aggregates, while the removal of the entire N-terminal domain from a-crystallin largely abrogated multimerization, suggesting that this domain is important for subunit assembly. Interestingly, it was also shown that the C-terminal region (a-crystallin domain and C-terminal extension) of aA-, aB-crystallin, and HSP25 independently formed dimers, tetramers, or higher oligomers (Merck et al., 1992, 1993b). The present work shows that deletion of only approximately one-third (15 residues) of the N-terminal domain of HSP 16-2 results in a dramatic reduction in the size of the complex as judged by size exclusion chromatography and crosslinking analysis, suggesting that the region involved in multimerization may be distally located in the N-terminal domain. It is therefore conceivable that the HSP 16-2 monomer has an extended conformation with the N-terminal region oriented toward the interior of the complex and provides the necessary contacts for subunit aggregation. In support of this hypothesis, HSP12.6, which has 16 fewer residues than HSP16-2 in the N-terminal region, is monomeric and predicted to be asymmetrical by sedimentation velocity analysis. The studies by Merck et al. (1992, 1993b) suggest the existence of at least two major sites of interaction between smHSP monomers: one within the N-terminal domain, which permits the assembly of the complex, and the other in the C-terminal domain, which appears to have an inherent ability to form smaller oligomers. Chang et al. (1996) found that mycobacterial  110  Chapter II: Discussion  HSP16.3 nonamers could be disassembled into trimers by treatment with 4 M urea or 1 M guanidine hydrochloride, also suggesting two different types of subunit interactions—one ;  involved in trimer formation and another involved in trimer oligomerization. The data presented here lend further support to this notion, as removal of all or part of the HSP 16-2 N-terminal domain prevents the formation of the native complex, but apparently permits the assembly of smaller oligomers: the estimated sizes of the truncated smHSPs are 45-115 kDa, and all crosslink to at least trimers. On the other hand, the sedimentation velocity measurements of H6A1-44 HSP16-2 and HSP12.6, which possess complete a-crystallin domains, reveal that these proteins are monomeric, with the exception that a small percentage of the H6A1-44 HSP16-2 (5%) assembles into tetramers. It is therefore possible that all N-terminally-truncated smHSPs are monomeric and are in equilibrium with higher-order oligomeric species. Sedimentation velocity and cross-linking analyses of HSP 16-2 and other smHSP derivatives containing only the acrystallin domain and C-terminal extension would help to determine whether this region has an intrinsic ability to oligomerize. The removal of the last 16 residues of HSP 16-2 has no effect on multimerization, but decreases the solubility of the complex significantly. Over 90% of H6A130-145 HSP16-2 precipitates from solution after freeze-thawing, while wild-type HSP16-2 and H6HSP16-2 remain completely soluble following the same treatment (not shown). Similarly, a role for the C-terminal extension in the solubilization of a-crystallin particles has recently been documented (Smulders et al., 1996). It has also been shown by U NMR studies that the last 8-10 and 18 l  C-terminal residues of a-crystallins and HSP25, respectively, form flexible extensions (Carver etal., 1992, 1995a). Accordingly, it seems likely that the C-terminal extension in HSP 16-2 is on the surface of the complex and exposed to solvent, where it imparts increased solubility to the complex. Augusteyn and Koretz (1987) proposed that a-crystallin forms micelle-like structures, with the hydrophobic N-terminal domain buried inside and providing the driving force for subunit aggregation. The structural studies presented here on HSP 16-2 are entirely consistent with the  111  Chapter II: Discussion  micellar-like aggregate model (Tardieu etal, 1986; Wistow, 1993), and further demonstrate the inherent heterogeneity in the quaternary structures of many smHSPs.  Function  of  smHSPs  Under stress conditions, heat shock proteins such as HSP60, HSP70, HSP90, and smHSPs likely prevent partially unfolded proteins from aggregating and becoming insoluble (Martin et al, 1992; Horwitz, 1992; Jakob et al, 1993, 1995; Freeman and Morimoto, 1996). Whereas most HSPs perform necessary functions in non-stressed cells and are also induced following cellular insults (Gething and Sambrook, 1992; Parsell and Lindquist, 1994; Arrigo and Landry, 1994), the C. elegans HSP16-class smHSPs are produced exclusively when nematodes are exposed to a heat shock or to proteotoxic agents (Jones et al, 1986, 1996). The present studies on HSP 16-2 confirm the ability of this class of smHSPs to interact with and stabilize proteins which become unfolded following heat or chemical treatment. The HSP 16s therefore likely constitute specialized smHSP chaperones that protect cells by stabilizing proteins which become structurally compromised during stress conditions. It is noteworthy that in eukaryotes, cytoskeletal proteins are one of the most sensitive targets of cellular stresses, and there is increasing evidence that smHSPs specifically interact with cytoskeletal elements, modulating their dynamic nature, and protecting them during stress conditions (Welch and Suhan, 1985; Chiesi et al, 1990; Bennardini et al, 1992; Lavoie et al, 1993a, 1993b, 1995; Nicholl and Quinlan, 1994). It was shown that the affinity of HSP16-2 for native actin and tubulin is negligible, whereas it is very high for the same thermally- or chemically-unfolded proteins. Similarly, the interaction of cardiac aB-crystallin with actin and desmin filaments was shown to be enhanced during ischemic conditions, where a decrease in the pH of the cytosol is known to affect the stability of cytoskeletal elements (Bennardini et al, 1992). Interestingly, HSP16-2 has nearly identical affinities for actin and tubulin, and can also prevent the aggregation of citrate synthase, suggesting that this chaperone may have a general affinity for unfolded proteins in vivo. Other smHSPs also display little protein specificity,  112  Chapter II: Discussion  preventing the aggregation of numerous proteins. Thus, whether smHSP chaperones have specialized roles in protecting the cellular cytoskeleton network during stresses remains to be seen. Linder et al. (1996) reported on a novel 18-kDa C. elegans protein highly similar to the HSP 16 smHSPs and named SEC-1 (for Small Embryonic Chaperone-1) (see Figure 15C alignment for sequence). SEC-1 is detectable only during early embryogenesis, where it performs an essential but unknown function. Interestingly, SEC-1 confers thermotolerance to E. coli, even though its expression is not augmented by environmental stresses. Therefore, although the physiological functions of this smHSP are not redundant with those of the stressinducible HSP16s, it is likely that both possess similar structural and functional characteristics as molecular chaperones. Recent data suggest that a-crystallin preferentially interacts with thermally-generated early unfolding intermediates which have exposed hydrophobic regions and are committed to the aggregation pathway (Carver et al., 1995b; Das et al, 1996; Rajaraman et al., 1996). In agreement with this notion, a-crystallin does not interact with largely unfolded but stable proteins (e.g., reduced and carboxymethylated a-lactalbumin and a-casein) (Carver et al., 1995b), or prevent the aggregation of chemically-denatured rhodanese which occurs following dilution into buffer, even at a three-fold molar excess of oligomer over unfolded substrate (Das and Surewicz, 1995). This unusual characteristic of a-crystallin is intriguing, since other molecular chaperones (e.g., HSP90, HSP70, HSP60, and HSP40) can interact with and stabilize such unfolded intermediates (Hartl, 1996; Wiech et al., 1992). The present study demonstrates that HSP 16-2 is equally capable of preventing the aggregation of thermally- and chemicallyunfolded citrate synthase. Furthermore, it is shown that HSP 16-2 has a substantially higher affinity for actin and tubulin unfolded intermediates with occur early on the renaturation or aggregation pathway, and is incapable of resolubilizing citrate synthase aggregates. Taken together, these data present strong evidence that HSP 16-2 and perhaps other smHSPs can  113  Chapter II: Discussion  interact with and stabilize proteins in the early stages of unfolding, or which are grossly unfolded, before the onset of extensive aggregation. The mechanism by which smHSPs interact with and prevent unfolded polypeptides from aggregating is unknown. It is generally agreed that hydrophobic regions in smHSPs are important for polypeptide binding (Carver et al, 1994b; Plater et al, 1996), but hydrophilic interactions may be necessary as well (Farahbakhsh et al, 1995; Smulders et al, 1995). Although C-terminal extensions are likely to be flexible and exposed on the surface of smHSPs (Carver et al, 1992, 1995a), there is some evidence that unfolded polypeptides are not necessarily involved in specific interactions with these structures (Carver et al, 1994b, 1995b). In accord with these observations, it was found that deletion of the last 16 C-terminal residues has little effect on HSP 16-2 structure or chaperone activity. The C-terminal extension seems to play a role in maintaining the solubility of the oligomeric complex (presumably by virtue of its exposure to solvent), and this may be the case with other smHSPs as well (Smulders et al, 1996). In contrast, studies involving deletions or mutations of the aA-crystallin C-terminal extension suggest a role for this region in chaperone activity (Takemoto et al, 1993; Andley, et al, 1996; Smulders et al, 1996). The reason for this discrepancy is unclear, and will require the characterization of C-terminal regions from other smHSPs for clarification. Since the C-terminal extension of HSP16-2 appears to be largely dispensable for polypeptide binding and chaperone activity, the interaction between unfolded polypeptides and the smHSP may not take place at the surface, but instead may occur in a region between subunits, in contact with the a-crystallin domain. In agreement with this, studies with unstructured spin-labeled peptides have suggested that these were not bound on the surface or clustered in an interior cavity of the a-crystallin complex, but rather were strongly immobilized at least 25 A apart in polar environments (Farahbakhsh et al, 1995). A detailed computergenerated model of the interaction between partially unfolded y-crystallin and a-crystallin which predicted the site of polypeptide interaction to be within clefts in the a-crystallin complex (Singh et al, 1995) is also consistent with the results presented here.  114  Chapter II: Discussion  Merck et al. (1992, 1993b) showed that the chaperone activity of a-crystallins and HSP25 lacking their entire N-terminal region was lost. The present studies on HSP 16-2 show that removal of only one-third of the N-terminal domain disrupts multimerization and abrogates chaperone activity. Therefore, it seems unlikely that the N-terminal domain per se is required for chaperone activity—it is probably the assembly of subunits which is important for this function. In addition, smHSPs not present in a complex (i.e., the Al-44 HSP16-2 and HSP12.6 proteins) are incapable of interacting with unfolded proteins. The structure-function studies on HSP 16-2 and HSP12.6 strongly suggest that smHSP multimerization may depend on a limited region of the N-terminal domain, and is a prerequisite both for the interaction of the chaperone with unfolded protein and for chaperone activity. In conclusion, it was demonstrated that HSP 16-2 forms large oligomeric complexes which have a high affinity for unfolded actin and tubulin and prevent the thermally- or chemicallyinduced aggregation of citrate synthase. Despite considerable effort, conditions were not found under which HSP 16-2 could refold denatured test proteins in vitro. The function of HSP 16-2 in vivo may be to interact with and stabilize a large variety of unfolding proteins, effectively buffering the cell against the deleterious effects caused by misfolded and aggregated proteins. The fate(s) of the unfolded proteins bound to smHSPs is poorly understood. However, it is now established that many molecular chaperones cooperate in refolding denatured proteins (Langer et al., 1992; Freeman and Morimoto, 1996; Freeman et al., 1996; Melki et al., 1996), and are involved in targeting proteins for degradation (Straus et al., 1988; also reviewed in Hayes and Dice, 1996). Recently, HSP25 and HSP70 were shown to act in concert in vitro in the refolding of citrate synthase (Ehrnsperger et al., 1997). It is therefore possible that in vivo, the smHSPbound unfolded proteins are folded by molecular chaperones such as those involved in de novo protein folding (HSP40/HSP70, HSP60; Hartl et al, 1996), or are degraded by the cellular proteolytic system.  115  Chapter II: Discussion The data on HSP12.6 complement the HSP16-2 studies The studies on C. elegans HSP12.6, a member of a novel class of smHSP, provide new insights into the structure and function of smHSPs, and reinforce the notion that two distinct features of smHSPs, namely their assembly into variable quaternary structures and molecular chaperone activity, may be strictly dependent on sequences present in their structurally divergent N-terminal regions. Although HSP12.6 possesses only 16 and 17 fewer N-terminal residues than C. elegans HSP16-2 or M. tuberculosis HSP16.3, respectively, it does not form high-MW complexes. It is doubtful that this inability to multimerize could be attributed to the shorter C-terminal extension of HSP12.6 since this region in smHSPs appears unnecessary for aggregate formation (Siezen et al., 1979, and the present work). Rather, it is likely that the N-terminal domains of smHSPs have strict minimum structural requirements for multimerization, and that any alteration in their size and perhaps composition can influence the assembly and overall quaternary structures of these proteins. The short N-terminus and monomeric nature of HSP 12.6 suggest that the oligomerization domain of smHSPs may be located in the distal end of the N-terminal region. There is also evidence that smHSPs may assemble cooperatively from smaller aggregates such as dimers/tetramers or trimers (Chang et al., 1996, Merck et al., 1993b). However, the data on HSP12.6 provide evidence that beyond N-terminal-dependent multimerization, smHSPs have no intrinsic ability to self-associate. In support of these results, bifunctional cross-linking experiments have suggested that a-crystallin aggregates are not likely to be built up of smaller clusters such as trimers or tetramers (Bindels et al, 1979; Siezen et al., 1980a). Another recent study also suggests that the minimal cooperative unit of a-crystallin unit is the monomer (Gesierich and Pfeil, 1996). Since HSP12.6 is clearly related to other smHSPs, it is both intriguing and informative that it is not functional as a molecular chaperone. While it is possible that HSP 12.6 lacks chaperone activity because of its unusually short C-terminal extension, it should be noted that N-terminallydeleted aA-, aB-crystallin, and HSP25, which fail to form native-like complexes, are also ineffective in preventing thermally-induced protein aggregation (Merck et al, 1993b). These results imply that the conserved a-crystallin domain of smHSPs, which is presumed to be the site 116  Chapter II: Discussion of interaction with unfolded protein, is insufficient in itself for molecular chaperone activity but is functional in the context of an oligomeric assembly. Computer-modeling studies indicate that the interaction between an unfolded polypeptide and a-crystallin takes place within clefts formed by adjoining subunits, implying that smHSP multimerization may be a prerequisite for chaperone activity (Singh etal., 1995). Again, the work by Farahbakhsh et al. (1995) on spinlabeled lends further support to this observation. Despite its apparent lack of chaperone activity in vitro, HSP12.6 presumably carries out specific function(s) in vivo, as it is one of a family of similar proteins found in C. elegans. One possible clue to the function of HSP12 proteins comes from the fact that there is a precedent for a monomeric smHSP in vivo. Murine HSP25 from Ehrlich ascites tumor cells can be recovered as two species, one mostly monomeric and the other multimeric (Benndorf et al., 1994). The presence of HSP25 monomers is surprising given that recombinant HSP25 readily forms 730kDa particles (Behlke et al., 1991). Nevertheless, non-phosphorylated HSP25 monomers were shown to be effective in inhibiting actin polymerization, a property shared by other smHSPs (Miron etal., 1991; Rahman et al., 1995). Whether HSP12.6 is active in preventing actin polymerization remains to be tested experimentally; it will be interesting to see whether the chaperone activity of HSP 12.6 has been lost and uncoupled from an ability to influence actin polymerization. It is noteworthy that a region thought to be involved in actin polymerization and binding to DNAse I, and suggested to be required for the inhibition of actin polymerization activity of smHSPs (Rahman et al., 1995), is partly conserved in the HSP 12 family. The motif, G-[V/I]-L-T-[X ]-P, is present in full in C14B9.1 and T22A3.2 with the exception of the proline 3  residue (see Figure 14; perhaps non-coincidentally, this residue is found in nearly all the C. elegans Class I and III smHSPs). Alternatively, HSP 12.6 may regulate the function of other smHSPs by preventing their oligomerization, or act as a co-chaperone with smHSPs or other molecular chaperones. It is becoming increasingly apparent that some smHSPs perform necessary functions during specific stages of development, and are not necessarily induced under physiological stresses. For example, expression of the 20-kDa smHSP from the gastrointestinal nematode Nippostrongylus 117  Chapter II: Discussion brasiliensis is developmentally regulated, but is not increased by stress conditions (Tweedie etal, 1993). In plants, specific subsets of smHSPs are produced during many developmental stages, although usually in response to heat stress (Waters et al, 1996). The developmentally-regulated expression pattern of HSP12.6 is reminiscent of the expression of C. elegans SEC-1, an HSP16-like smHSP produced only during embryogenesis, where it serves an unknown but essential function (Linder et al, 1996). Like HSP12.6, SEC-1 is not induced by stress conditions, which suggests that these smHSPs may have fundamentally different roles from other stress-inducible smHSPs. It has been suggested by Linder et al. (1996) that SEC-1 may be required to facilitate the folding of the large number of nascent polypeptides produced, or to regulate the assembly and disassembly of cytoskeletal structures during early embryonic development. HSP12.6 may play similar role(s) during the first larval stage, a period when a significant number of somatic cell divisions occur (Wood, 1988). Although hspl2 genes from other species have not been uncovered, the gene family is likely to be ubiquitous in nematodes. Numerous cDNAs homologous to C. elegans C14B9.1 from two other nematodes, Brugia malayi and Onchocerca volvulus, were recently isolated as part of a pilot project designed to identify genes expressed in these human filarial nematode parasites (Blaxter et al, 1996). The B. malayi HSP 12 cDNA encodes a protein of 113 amino acids which displays 60% identity to its C. elegans homolog over its entire length. Interestingly, the cDNAs were isolated from third- and fourth-stage infective larvae. It is possible that the difference in expression patterns of the hspl2 genes in the free-living and parasitic nematodes may be due to different temporal requirements for the smHSP.  Models summarizing HSP16-2 structure and function The present work on C. elegans HSP 16-2 and HSP 12.6 provides insights into the structure and function of HSP16-2 and other smHSPs in general. The functional data obtained on HSP 16-2 are in agreement with an ability of this protein to interact with and stabilize proteins which become structurally compromised during cellular insults. Specifically, HSP 16-2 recognizes protein conformers which are in the early stages of the aggregation pathway, and 118  Chapter II: Discussion prevents their aggregation (Figure 30A). It is likely that the mechanism of action of other smHSPs is very similar, although the HSP 16s may have more specialized roles compared with some other smHSPs, since they are produced only during stress conditions. Of all the structural models proposed to date for a-crystallin, the micellar-like aggregate and variations on this structure have received the most support (Tardieu et al., 1986; Walsh et al, 1991; Wistow, 1993; Groth-Vasselli et al., 1995; Singh et al, 1995), although much controversy remains. The structural studies presented here on two minimalist members of the smHSP family are entirely consistent with such a model. Although this model is presently rather crude, it is likely that its basic premises, including N-terminal-dependent oligomerization, the presence of a central cavity which can accomodate N-terminal domains of various sizes and sequences, an exposed C-terminal extension which imparts aggregate solubility, and the necessity for multiple subunit interactions for binding to the unfolded substrate, are all valid for smHSPs in general (Figure 30B).  Future studies Major advances in our understanding of chaperone function have resulted from the structural determination of a limited number of chaperones, including bacterial GroEL/GroES (Braig et al, 1994; Hunt et al, 1996), HSP70 ATPase and DnaK polypeptide-binding domains (Flaherty et al, 1990; Zhu et al, 1996), and PapD (Holmgren and Branden, 1989). In contrast, the multimeric and heterogeneous nature of smHSPs have represented major obstacles to determining their structure by crystallization or high-resolution 2D NMR techniques. Consequently, progress on determining the function of small HSPs has been hampered by a lack of structural information. The simple structure of HSP12.6 may make it amenable to structure determination, and such studies are now underway in collaboration with Andrzej Joachimiak, a crystallographer who was involved in solving the GroEL structure (Braig et al, 1994).  119  Chapter II: Discussion  Figure 30. Model of in vivo function and oligomeric structure of smHSPs. A. Proposed model for the in vivo function of stress-inducible smHSPs. SmHSPs interact with structurallycompromised polypeptides which expose hydrophobic regions, and prevents them from forming insoluble aggregates. The smHSP therefore acts as a reservoir for unfolded proteins which may then be refolded in the presence or absence of other molecular chaperones, or may be degraded by the cellular proteolytic machinery. B. Model for small HSP oligomeric structure and interaction with unfolded polypeptides. This schematic depicts a hypothetical cross-section through a smHSP complex with an arbitrary number of subunits and quaternary structure. SmHSP monomers are shown as having a two-domain structure consisting of the N-terminal domain and C-terminal domain (which includes the a-crystallin domain and C-terminal extension). Double-headed arrows indicate possible sites of interaction between different smHSP monomers and between smHSP subunits and unfolded substrates, a. A proposed feature of smHSPs is their central cavity, which can accomodate N-terminal domains of varying lengths and sequences, b. N-terminal domain interactions are necessary for subunit assembly and are likely to modulate the arrangement of subunits within the smHSP quaternary structure. The multimerization domain may be restricted to a small section (e.g., the distal end) of the Nterminal region, c. the chaperone activity of smHSPs relies on their ability to interact with and stabilize unfolded polypeptides; this interaction is likely to be mediated through multiple contacts with a-crystallin domains in clefts formed by neighboring subunits. d. although the role of the C-terminal extension in the interaction of the smHSP with unfolded polypeptides is unclear, it is probable that this region is largely responsible for maintaining the solubility of the smHSP complex, e. there is evidence that smHSPs assemble cooperatively from small oligomers, but the exact region of interaction between subunits, and whether this is a general feature of smHSPs, is unknown. 120  Chapter II: Discussion If successful, this project would constitute a significant breakthrough in the study of these proteins. Since HSP12.6 contains all of the core a-crystallin domain in addition to a significant amount of N-terminal domain region, insights into the assembly of subunits in complexes might be obtained by modeling studies. Furthermore, information regarding the mechanisms of polypeptide binding and chaperone activity could probably be inferred from the threedimensional structure. Additional studies on HSP 16-2, which represents a good model system for studying smHSPs in general, could further our understanding of the oligomeric assembly and function of these chaperones. While the present study revealed a strict correlation between multimerization and chaperone activity, the amino acids required for multimerization were not precisely defined. Removal of fewer than 16 amino acids (one third of the N-terminal domain), and site directed mutagenesis of one or more residues may pinpoint more accurately the regions involved in the formation of oligomeric complexes. A domain-swapping experiment in which the N-terminal domains of HSP 16-2 and HSP 16-48 are interchanged could reveal whether the N- and C-terminal domains of smHSPs are relatively independent of each other in the native complex. This experiment would determine whether compensating changes between the two domains have evolved in the two closely-related smHSPs in order to maintain function. Also, further truncations of the C-terminal extension (beyond the A130-145 truncation) could provide additional insight with regard to which regions within the smHSP are required for chaperone activity in the context of a normal oligomeric assembly. An intriguing question raised in this study concerns the function of HSP 12.6 in vivo. First, the stage-specific expression of HSP 12.6 during normal physiological conditions and the fact that it does not prevent thermally-induced protein aggregation in vitro may suggest a specialized function in the cell. It may, for example, be able to bind unfolded protein and stabilize it temporarily until it can be assisted by other molecular chaperones, much in the way that HSP40 requires the presence of HSP70 for assisting in the renaturation of unfolded polypeptides (Langer et al., 1992; Rassow et al, 1995). Alternatively, HSP12s may have an inhibitory effect on the function of other smHSPs. The presence of HSP 12s under non-stress conditions could 121  Chapter II: Discussion serve to regulate the activity of various stress-inducible smHSPs by binding to and preventing the oligomeric assembly of the newly synthesized smHSPs. Such a possible role for HSP12.6 could be demonstrated by determining whether HSP 16-2 complex formation is suppressed in the presence of a comparable or sub-stoichiometric amount of HSP 12.6. The function of HSP12s might also be ascertained by testing to see whether they bind certain C. elegans proteins—even native proteins cannot be excluded. 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(1994) Plant low-molecularmass heat-shock proteins: their relationship to the acquisition of thermotolerance in plants. Biotech, and Appl. Biochem., 19, 41-49. Zav'yalov, V. P., Zav'yalova, G. A., Denesyuk, A. I., Gaestel, M., and Korpela, T. (1995) Structural and functional homology between periplasmic bacterial molecular chaperones and small heat shock proteins. FEMS Immunol. Med. Microbiol. 11, 265-272. Zhen, M . (1995) Cloning, characterization and functional analysis of ubc-2, a gene encoding a ubiquiting-conjugating enzyme in the nematode, Caenorhabditis elegans. Ph.D. thesis, University of British Columbia, Vancouver, B. C , Canada. Zhi, W., Landry, S. J., Gierasch, L. M., and Srere, P. A. (1992) Renaturation of citrate synthase: influence of denaturant and folding assistants. Protein Sci. 1, 522-529. Zhu, X., Zhao, X., Burkholder, W. F., Gragerov, A., Ogata, C. M., Gottesman, M . E., and Hendrickson, W. A. (1996) Structural analysis of substrate binding by the molecular chaperone DnaK. Science 272, 1606-1614.  140  Appendix APPENDIX  I. Bacterial strains used in this study Table 1. E. coli strains used for plasmid propagation and protein expression. Strain DH5a  Genotype  Use  supEAA Mac U169 (080 lacZAMl5)  a  hsdRll Propagation of plasmids for  recAX endAX gyrA96 thi-l relAX JM109  subcloning and sequencing  recAl supEAA endAl hsdRll gyrA96 relAl thi Electrotransformation  b  A(lac-proAB)  F'[traD36  proAB+  l a d with plasmid DNA  /acZAM15] BM25.8  recA+ F T c Ximm434kan PlCmRrnr s  C  Conversion  of ?iSHLX2  clones to pRatll plasmids BL21(DE3)  d  hsdS gal (kcltsSSl indl Sam7 ninS /acUV5-T7 Propagation of bacterial gene 1  expression vectors  a. Hanahan, 1983.  c. Palazollo et al, 1990.  b. Yanisch-Perron et al, 1985.  d. Studier and Moffatt, 1986.  141  Appendix II. List of oligonucleotides used in this study Table 2. Oligodeoxyribonucleotides used for sequencing, PCR, and/or subcloning. Name  Sequence (5'->3')  Position spanned  MIC1 MIC2 MIC3 MIC4 MIC5  primers for CCT studies AGTCTGATGTGCATGGCGGCTC GCATCAGCTGGAGATTC CAGAATTGTGGCTCCG ACGGATCGAATTGCAGCACCAG GCAGTCGACGCGATCGCAACTGCCGCGGT Sail  462^-441 (cct-1 gene) 4-»20 (cct-1 gene) 242^227 (cct-1 gene) 2530->2508 (cct-1 gene) 142->123 (cct-1 gene)  MIC6 MIC7 MIC8 MIC9 MIC 10 MIC 11 MIC 12 MIC 13 MIC 14 OZM12 OZM13 SL1 SL2 oligodT  OH2-A OH2-C OBM1 OBM2  a  CTTGCTCAATTGCCAAC TGGACACTGACAAAGTG ACTTCACATTGTAAGAC CCAGAAATGATCGTCTG TGAGGATCCATGCGCGAGATCGTTC BamUl T A AT ACGGTTGTGTTTC AGG TATTACCAAACGCCGCATTG AGAAC A ACGTTGGCTCCGGC ATATAAACGGGGTAAACGGG TCACAAGCTGATCGACTCGATGCCACGTCG GATTTTGTGAACACTGTGGTGAAG GGTTTAATTACCCAAGTTTGAG GTTTTAACCCAGTTACTCAA CGAGC ATGCGTCG AC AGGC A(T) 1 7  primers for small HSP studies GACTCGAGGTGATCTTATGAG Xhol  b  1308->1325 (cct-2 cDNA) 713->729 (cct-2 cDNA) 1483-^1467 (cct-2 cDNA) 377->361 (cct-2 cDNA) 1 1 6 (mec-7 cDNA) -23->-3 (cct-1 gene) 949^968 (cct-1 gene) 1002-»983 (cct-1 gene) 1974->1955 (cct-1 gene) binds Tc 1 sequence binds Tel sequence binds splice leader 1 binds splice leader 2 binds polyA  46->59 (hspl6-2 cDNA)  GCTGAATTCTTATCCTTGAACCGCTTCTTTC 387->369 (hspl6-2 cDNA) EcoRl GCGTGGATCCATGTCACTTTACCACTATTTC 1—>21 (hsp!6-2 cDNA) BamUl CGATGAATTCGTTATTCAGCAGATTTCTCTTCGAC 438-^415 (hsp!6-2 cDNA) EcoRl  142  Appendix  Table 2 cont'd. Oligodeoxyribonucleotides used for sequencing, PCR, and/or subcloning. Name  Sequence (5'H>3 )  Position spanned  OH48-A  CTACATATGCTCATGCTCCGTTC Ndel CTAGGATCCAAGATTAATGTTTTGCAAC BamWl ATGGGATCCATGATGAGCGTTCCAGTG BamRl ATGAAGCTTTTAATGCATTTTTCTTGCTTC Hindll!  1—>17 (hsp 16-48 cDNA)  OH48-B F38A F38B HSP13B.5  , a  AGTCATATGTCTGTTGCTATTGATCAC  432^414 (hsp 16-48 cDNA) 1—»18 (hsp 12.6 cDNA) 333—>313 (hspl2.6 cDNA) 1—»21 (hsp 12.3 cDNA)  Ndel HSP13B.3  GCTC^ATCCTTACTTTTTCTTGTTTCCGGAGATGTG  330^304 (hspl2.3 cDNA)  BamHI Vector-specific primers RG02 GTGTGGAATTGTGAGCGGAT 860->841 (pBSH KS, MCS) RG05 AGGGTTTTCCCAGTCACGAC 577-4596 (pBSII KS, MCS) KS CGAGGTCGACGGTATCG 743-4727 (pBSII KS, MCS) SK TCTAGAACTAGTGGATC 677->693 (pBSIIKS, MCS) T3 ATTAACCCTCACTAAAG 790->774 (pBSII KS, MCS) T7 AATACGACTCACTATAG 627-4643 (pBSII KS, MCS) pRAT II MCS (5' of cDNA) SP6-5' ATAGAATATGCATCAAGCTGAG pRAT II MCS (3' of cDNA) T7-3' ACTATAGGGAGCTAAGCTTGG a. Some PCR primers contain engineered restriction sites at their 5'-ends (underlined). These sites are for subcloning purposes only and are not complementary to the target sequence. b. Numbering for the cet genes follows the convention of increasingly positive numbers beginning with the first nucleotide in the start codon (ATG, where A is +1), and increasingly negative numbers 5' of the start codon (-1, -2, etc.). Numbering for cDNAs begins from the start codon (ATG, where A is +1). c. The numbering used here parallels that of the pBluescript plasmid restriction map in the Stratagene® catalogue. MCS, Multiple Cloning Site. C  143  Appendix  I I I . Conditions used for polymerase chain reactions Table 3. Summary of PCR conditions used for amplifying DNA from various sources. Sample  Plasmid DNA  cDNA  Genomic DNA  Cosmid DNA  Concentration  1 ng/pi  -300 ng/u.1  500 ng/ul  ~5 ng/ul  Sample DNA  1.0 |il  2.0  1.0  1.0  Oligo 1 (50 pmol/ul)  1.0 u.1  1.0  1.0  1.0  Oligo 2 (50 pmol/ui)  1.0 u.1  1.0  1.0  1.0  10X PCR buffer  5.0 u i  5.0  5.0  5.0  MgCl (25 mM)b  3.0 u.1  3.0  3.0  3.0  dNTPs  1.0 |il  1.0  1.0  1.0  37.75 ul  36.75  37.75  37.75  0.25 u.1  0.25  0.25  0.25  50  50  50 |ll  50 |ll  3  2  c  dH 0 2  Taq DNA pol. (5 U/|ll) total volume  d  ui  ul  ,  a. Promega 10X PCR buffer: 500 mM KC1, 100 mM Tris-HCl (pH 9.0), 1.0% Triton X-100 b. The final MgCl concentration used (1.5 mM) was sometimes adjusted to 0.5 or 1.0 mM. 2  c. 5 mM of each deoxyribonucleotide (dATP, dCTP, dGTP, and dTTP) in 20 mM MgCl  2  d. Taq DNA polymerase enzyme was purchased from Promega or Pharmacia; Pfu DNA polymerase (Stratagene) was sometimes used because of its proof-reading function.  144  Appendix IV. Large-scale culturing of C. elegans and isolation of embryos  4.1 Maintenance of C. elegans on plates and in liquid media The C. elegans Bristol (N2) strain was maintained on NG plates (0.3% NaCl, 0.25% tryptone, 1 mM CaCl , 1 mM MgS0 , 10 pg/ml cholesterol, 25 mM K H P 0 , and 1.7% 2  4  2  4  agarose) spread with a lawn of E. coli OP50 (Brenner, 1974). Nematodes were grown in a vigorously aerated liquid growth medium consisting of 50 mM KH2P04/K2HPC>4 buffer (pH 6.0), 2 mM potassium citrate (pH 6.0), 100 mM NaCl, 0.3 mM CaCl , 0.3 mM MgS0 , and 2  4  trace metals (0.5 ml of a 2.5 mM FeS0 , 1 mM MnCl , 1 mM ZnS0 , and 0.1 mM CuS0 4  2  4  4  solution per litre). Frozen E. coli (K12, log phase, from U. of Alabama Fermentation Facility) was added periodically as a food source. Synchronous nematode populations were obtained by isolating embryos from gravid adults and allowing the embryos to hatch completely in the absense of food, thereby causing them to arrest at the LI stage.  4.2 Preparatory culture used for inoculating 20 litre culture Two 250-ml cultures were grown and the embryos harvested and floated on a sucrose cushion essentially according to Sulston and Brenner (1974). Following this, two one-litre cultures were each inoculated with approximately 6 million of the embryos. The embryos were allowed to hatch in the absence of food for 15 hours at 15°C, and 15 g of frozen E. coli was then added to each culture. Over the course of 6 days, an additional 35-40 g of frozen E. coli was added to each culture. The embryos were then harvested and floated. This gave 7.5 g of packed, live embryos (i.e., embryos not staining blue upon incubation with a methylene blue solution). These were placed in 200 ml of liquid medium without food and allowed to hatch completely overnight at 15°C.  4.3 First 20-litre culture 18.5 litres of water and one litre of 20X Basal S (117 g NaCl in 1 M potassium phosphate pH 6.0) were autoclaved in situ in a 35-litre fermenter. The following solutions were filter145  Appendix sterilized into the medium after cooling to room temperature: 12 ml CaCl (1 M), 12 ml MgS04 2  (1 M), 40 ml potassium citrate pH 5.75 (1 M), 10 ml trace metals, 40 ml cholesterol (5 mg/ml in EtOH), and 25 ml antifoam C (Sigma). The LI larvae were then added via an ethanol-rinsed funnel, as was 500 g of frozen E. coli (thawed completely in 300 ml of Basal S at 50°C for about 3 hours). The air flow was adjusted from the beginning to 20 litres/min, and the stirring rate was set to 250 rpm to maintain proper aeration. The rpm was increased to 300, then 420 after the first and second days following inoculation. Antifoam C was added periodically to keep frothing to a minimum; about 40-50 ml of the antifoam was added in total. 45 hours after inoculation, an extra 250 g of thawed E. coli in 150 ml of Basal S was added to the culture (concomitantly with 10 ml of antifoam). The temperature of the culture was maintained at 20°C except for the final 15 hours, where it was increased to 20.7°C to allow the adults to become fully gravid (i.e., contain at least 10 embryos per nematode). After 72 hours, the nematodes were harvested. The 20-litre culture was subdivided equally into five-10 litre buckets, which were placed on a large tray filled with ice. The buckets were stored in a cold room at 4°C for 3 hours, allowing the nematodes to settle completely. Approximately 4.2 litres of the top layer from the 5-litre cultures were removed. This left a total of four litres of culture, which was centrifuged in 50 ml Falcon tubes (2000 x g for 2 minutes), and divided such that each tube had between 5-6 ml of packed nematodes. These were kept on ice. Isolation of embryos was carried out using an alkaline hypochlorite treatment as described previously (Emmons et al, 1979). This was done most efficiently using these guidelines: 1. pack 5-6 ml of nematodes and remove all supernatant 2. add 50 ml of bleaching solution (88 ml dH 0, 12 ml bleach, 2 g NaOH per 100 ml) 2  3. shake vigorously for 2 min and then centrifuge at 2000 rpm in a benchtop centrifuge 4. remove supernatant completely, and add 45-50 ml of fresh bleaching solution 5. shake vigorously until the total elapsed time from the first addition of bleach is 9 min 6. centrifuge, remove supernatant, then wash twice with 0.14 M NaCI 7. remove supernatant, combine embryos into one 50-ml tube, and add NaCI to 50 ml 8. keep on ice until ready-check for viability and presence of undissolved nematodes 146  Appendix The yield for the 20-litre culture was 73 g of unfloated embryos, although 18 g were accidentally lost during the procedure. The preparation was sufficiently clean to avoid sucrose flotation, and viability of the embryos, as judged by methylene blue exclusion was greater than 90%. 50 g of embryos was made into a 50% slurry by adding 50 ml of homogenization buffer (250 mM sucrose, 10 mM Tris pH 8.0, 10 mM MgCl2). The slurry was frozen dropwise in liquid nitrogen, and stored in 4 separate vials. The remaining 15 g of unfloated embryos were added to 200 ml liquid medium and allowed to starve-hatch overnight at 15°C, and then for about 8 hours at room temperature to ensure complete hatching.  4.4 Second 20-litre culture The starve-hatched nematodes were used to inoculate a 20-litre culture identical to the first. 500 g of thawed E. coli (in 250 ml of Basal S) was added to the embryos, as well as 25 ml of Antifoam C. The rpm level was kept at 215 initially, raised to 275 after 15 hours of inoculation, and was maintained at approximately 420 after about 36 hours. After 48 hours, an additional 250 g of thawed E. coli were added (in 150 ml Basal S). The rpm was increased to a final value of 490. During this run, the pH of the culture increased above the optimal 6.0, so to keep the pH below this level, glacial acetic acid, diluted three-fold in distilled water, was added periodically. After growing for 3 days at 20°C and overnight at 14°C, the nematodes were harvested as before. These were packed to approximately 5 ml and bleached as before except that the spin after the second bleach was initiated after only 7.5 min, and not 9 min as before. The reason for this is that the nematodes were harvested at a slightly later stage than in the first isolation, and less bacteria was present. This resulted in more efficient bleaching. The eggs were floated on a sucrose cushion as there was a greater amount of undissolved debris compared to the first isolation. Flotation was performed by packing eggs to a volume of about 8 ml, resuspending in 0.14 M NaCl to a volume of 20 ml, adding 20 ml of 60% (w/v) sucrose, briefly mixing, and centrifuging at 2000 rpm. The floated eggs were then washed twice in the salt solution, centrifuged, and frozen in liquid nitrogen as a 50% slurry. The final yield of floated eggs was 61 g, so 111 g were frozen and stored in 6 separate vials.  147  Appendix V. Transgenic lines generated and used in this study The following C. elegans transient or integrated transgenic line stocks are available frozen in liquid nitrogen in the containers shown:  Strain  Integrated?  Can#  Cane#  Description  NL708/PK58  No  OR#5  Ml  cct-lv.Tcl  PC146 UbExl32 (#8)  Maybe  OR#5  PI 00  cct-1 promoter-tacZ (pPD16.43.5'.3')  PC147 UbExl33(#9)  No  OR#5  P101  cct-1 promoter-tocZ  PC148UbExl34(#ll)  No  OR#5  P102  cct-1 promoter-/«cZ  PC149UbExl35 (#16)  No  OR#5  P103  cct-1 promoter-/acZ  insertion strain  Note: all strains except the first are in an N2 background contain the pRF4 selection marker.  148  Appendix VI. C. elegans strain containing a Tel insertion in cct-1 gene The C. elegans strain NL708/PK58 (cct-1 ::Tcl) contains a Tel insertion between the stop codon and polyadenylation signal (Figure 31). This location corresponds to nt 2145 in the cet genomic sequence. The strain has not been outcrossed to wild-type N2s to remove other possible disruptions caused by multiple Tel insertions.  C  H  A  *  TGCCACGCTTAAattttcccgtttaccccgtttatatatccctgttttccgcgtgcttctcacata<Tcl>attccg atctgctgctccttatcccaaattctcatgttcagcttttgttttcttcttttgatgatactttattgaacgaaatg ttgtaagttttaatgttttgatttcaaagttgtttgtattcgtttttcattattcaaacaatgaagaagctttgcca  Figure 31. C. elegans strain NL708/PK58 contains a Tel insertion near cct-1. Two sets of nested PCR primers (MIC1/MIC2 at the 5'-end and MIC3/MIC4 at the 3'-end) flanking cct-1 were designed and used to isolate a strain from a Tel-insertion bank containing a Tel insertion near cct-1 (this strain was isolated by Ronald Plasterk). The region containing the Tel insertion site was then amplified by nested PCR using MIC1/OZM12 and then MIC2/OZM13 (which yields an ~2 kb frament). This fragment was then subcloned into pBS and sequenced. Shown here are the last three codons of cct-1 (including amino acid above), the location of the Tel insertion (labeled <Tcl>), and the polyadenylation signal (shown in bold).  149  Appendix  VII. Antibodies used in this study Various polyclonal antibodies were produced and used for detection of specific proteins in Western blots. The specificity of each antibody for one or more proteins is shown in Figure 32.  UM-l  CCT-1 CCT-2 CCT-4 CCT-5 CCT-6  B  C C T - 1  KIDDVRVPDDERMGY  kDa  kDa  KLDKQEPLGGDDCHA CCT-5  CCT-1  CCT-5  ^  200 97 68 43 29 18 14  HSP12.6 HSP12.3  Figure 32. Specificities of the polyclonal antibodies against CCT and HSP16-2. A. A peptide with the sequence shown (UM-l) was used to generate polyclonal antibodies against a conserved region in all CCT subunits (provided by Keith Willison; Hynes et al, 1995). An alignment with the five known C. elegans CCT subunits is shown for comparison. B. Peptides corresponding to the last 15 amino acids of C. elegans CCT-1 and CCT-5 were used to produce polyclonal antibodies against the respective CCT subunits. The antibodies (anti-CCT-1 and anti-CCT-5) cross-react specifically with approximately 59 kDa proteins in a total C. elegans extract. C. Polyclonal antibodies raised against HgHSP16-2 (Jones et al., 1996) do not cross-react with any protein in a control, total C. elegans extract (lane 1), but recognize two proteins of approximately 16.5 and 15.5 kDa in a heat-shocked nematode extract (lane 2). The sizes of these two proteins on the 18% SDS-gel correspond precisely to the sizes of the bacterially-produced wild-type HSP 16-2 (lane 3) and HSP 16-48 (lane 4), which were separated on the same gel and Western blotted with the same antibody. D. Polyclonal antibodies raised against H6HSP12.6 cross-react with recombinant HSP 12.6 and HSP 12.3 from bacterial extracts with similar efficiency (black arrow); three different amounts of each recombinant protein were Western blotted with a 1:500 dilution of the anti-HSP12.6 pAb. 150  Appendix VIII. Sedimentation velocity analyses of smHSPs Sedimentation velocity measurements on smHSPs were performed by Ronald Melki and Gerard Batelier. The sedimentation data were fitted by nonlinear least-squares procedures as described by Philo (1994) (see Material and Methods). Results are presented in Table 4 below.  Table 4. Hydrodynamic parameters of HSP16-2, two derivatives, and HSP12.6  a  maoslecu(laD ra)V (cmg")' m 3  theoretical  HSP162 H SP161 2 fo o m r fm r 2  16232 '  fifes  0.735  -  -  0.735 0.735  10.48 14.47  1.10 1.08  relative a b undance R* (m) n-mer (%)  -  .  experimental 239470 394920  4.82 5.65  x x  10" 10"  9  14-15 24-25  58 42  -  -  17-18 28  49 51  -  -  1 3-4  95 5  9  theoretical A130-145  18580  0.723  -  -  322395 520385  0.723 0.723  12.8 17.6  1.16 1.16  -  experimental A130-145  fm r 21 o fo rm  5.29 6.20  x x  10" 10"  9  9  theoretical A1-44  15227  0.723  -  -  14990 56235  0.723 0.723  1.66 4.0  1.14 1.15  -  experimental  A1-44 o fo rm fm r 21  1.92 2.99  x x  10" 10'  9  9  theoretical  HSP126. H StheP12conformational 6.molecular massparameters The were calculated as described inx Materials and Methods, using and the partial specific volume (v) values determined from the amino 12612  0.737  -  -  12231  0.737  1.43  1.12  -  -  -  1  100  experimental  1.81  a  10"  9  acid composition. Experimental points representing the distribution of each molecular form were decomposed into Gaussian curves. The area of each Gaussian is proportional to the concentration of the oligomer. Numerical values for the n-mer of each species were obtained by dividing the experimental molecular mass of each species by the corresponding theoretical molecular mass. s°20,w is the sedimentation coefficient;/and/o are the frictional coefficients and R the Stokes radius. (Data kindly provided by R. Melki and G. Batelier). s  151  


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