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Proteins that interact with ubiquitin-conjugating enzyme 2, UBC-2, in the nematode Caenorhabditis elegans Groombridge, Marieke Tina 2002

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PROTEINS T H A T INTERACT WITH UBIQUITrN-CONJUGATING E N Z Y M E 2, UBC-2, IN T H E N E M A T O D E CAENORHABDITIS ELEGANS by Marieke Tina Groombridge B. Sc., Trinity Western University, 1999  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF T H E REQUIREMENTS FOR T H E D E G R E E OF Master of Science  in T H E F A C U L T Y OF G R A D U A T E STUDIES Department of Biochemistry and Molecular Biology  We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A May 2002 © Marieke Tina Groombridge, 2002  In presenting this thesis in partial fulfillment of the requirements for an advanced degree at The University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  Department of Biochemistry and Molecular Biology The University of British Columbia 2146 Health Sciences Mall Vancouver, B.C. V6T 1Z3 Date: May 15,2002  ABSTRACT A major route for protein degradation occurs via the ubiquitin-proteasome pathway.  Proteins are targeted for destruction by the proteasome through covalent  ubiquitin attachment. This tagging system involves the concerted action of an ubiquitinactivating enzyme (El), an ubiquitin-conjugating enzyme (E2), and an ubiquitin ligase (E3), with the latter two being involved in achieving target specificity.  UBC-2, an  essential E2 in the nematode C. elegans, is a functional homolog of Saccharomyces cerevisiae UBC4, a member of a branch of ubiquitin-conjugating enzymes required for the degradation of short-lived and abnormal proteins. In order to identify proteins that interact with UBC-2, a yeast two-hybrid analysis was employed.  Putative TJBC-2  interacting proteins identified include: a ubiquitin-like budding yeast DSK2 ortholog referred to as DSK-2, two RING finger proteins, a protein containing a F Y V E domain, a calcium ATPase called M C A - 1 , and ubiquitin. Since DSK-2 represented 60% of the putative interactors isolated from the screen, this project focused on the interaction of UBC-2 and DSK-2, and demonstrated a novel interaction between an E2 and an ubiquitin-domain protein (UDP) in vitro. The ability of DSK-2 to bind polyubiquitin and itself was also demonstrated. R N A interference (RNAi) was employed in an attempt to determine the biological function of UBC-2 interacting proteins in vivo.  RNAi with  mca-1 produced a subtle body shortening phenotype, although genes encoding UBC-2 interacting proteins appear to be non-essential.  ii  TABLE OF CONTENTS  ABSTRACT  '.  ii  T A B L E OF CONTENTS LIST OF T A B L E S  iii .  vii  LIST OF FIGURES  viii  LIST OF ABBREVIATIONS  ix  ACKNOWLEDGEMENTS  xi  I. INTRODUCTION  1  1. UBC-2 and a summary of the ubiquitin pathway  1  2.  3  Components of the ubiquitin pathway 2.1. Ubiquitin  3  2.2. Ubiquitin-activating enzyme  4  2.3. Ubiquitin-conjugating enzyme  4  2.4. Ubiquitin ligase  6  2.5. Multiubiquitin assembly factor  6  2.6. The 26S proteasome  7  3. UBC4/5-like E2s: diverse examples and functions in ubiquitylation  8  4. Ubiquitylation mediated by UBC4 family E2s and associated E3s  11  4.1. Ubiquitylation mediated by UBC4 family E2s and 4.2. and 4.3. and 4.4. and 4.4. and  H E C T domain E3s Ubiquitylation mediated by UBC4 the A P C E3 Ubiquitylation mediated by UBC4 SCF complex E3s Ubiquitylation mediated by UBC4 the V H L E3 complex Ubiquitylation mediated by UBC4 RING finger domain E3s  iii  11 family E2s 12 family E2s 13 family E2s 14 family E2s 14  4.5. Ubiquitylation mediated by UBC4 family E2s and the U-box E3s 5.  15  Study obj ectives  16  II. MATERIALS A N D METHODS  17  1. D N A techniques  17  1.1.  Standard polymerase chain reaction (PCR)  17  1.2.  Restriction endonuclease digestion of D N A  17  1.3.  Agarose gel electrophoresis  17  1.4.  D N A extraction from agarose  1.5.  Ligation of restriction endonuclease digested D N A  1.6.  Transformation of D N A into Escherichia coli  18  1.7.  Transformation of plasmid D N A into S. cerevisiae  18  1.8.  Plasmid preparation from E. coli  19  1.9.  Plasmid preparation from S. cerevisiae  19  18 18  1.10. D N A concentration determination  20  1.11. D N A sequencing  20  1.12. Preparation of the yeast two-hybrid bait plasmid  20  1.13. Conversion of bacteriophage library X ACT-RB2 into plasmid library pACT-RB2 2. Culturing C. elegans  20 21  3. Maintenance and mating of S. cerevisiae  21  4. X-gal filter lift assay  22  5. Computer analysis  23  iv  6. Protein and immunological techniques 6.1. T N T ® Quick Coupled Transcription/Translation of radiolabeled DSK-2  23  6.2.  Overexpression and purification of 6XHIS-UBC-2  23  6.3.  Overexpression and purification of GST: :DSK-2  25  6.4.  Preparation of S. cerevisiae protein extract  6.5.  Protein concentration determination  26  6.6.  SDS-polyacrylamide gel electrophoresis  26  6.7.  Western blot analysis  26  6.8.  Coprecipitation of UBC-2 and DSK-2*  27  6.9.  Coprecipitation of UBC-2 and DSK-2 fusion proteins  27  25  6.10. Cross-linking UBC-2 and DSK-2 fusion proteins  28  6.11. Coprecipitation of multi-ubiquitin and GST::DSK-2  28  7. R N A interference  28  7.1. Overexpression of double stranded R N A  28  7.2. Scoring phenotypes of R N A i treated nematodes  29  III. RESULTS A N D DISCUSSION  ;  1. The yeast two-hybrid screen to identify interacting proteins  30 30  2. Yeast two-hybrid false positive elimination  33  3. Proteins that exhibit a yeast two-hybrid interaction with UBC-2  37  3.1. UBC-2 interacts with a plasma membrane calcium ATPase  37  3.2. UBC-2 interacts with a F Y V E finger domain protein  39  3.3. UBC-2 interacts with RING finger proteins  40  3.4. UBC-2 interacts with a ubiquitin-domain protein  41  v  4. Confirming the interaction of UBC-2 with DSK-2 in vitro  45  4.1. Attempt to coprecipitate radiolabeled DSK-2 with 6XHIS-UBC-2  45  4.2. Expression and Purification of GST::DSK-2  47  4.3. Confirming the identity of GST::DSK-2 fusion protein  ....47  4.4. Co-precipitation of GST::DSK-2 with C-terminal 6XHIS-UBC-2  47  4.5. Expression and Purification of UBC-2  50  4.6. N-terminal 6XHIS-UBC-2 and C-terminal 6XHIS-UBC-2 bind TALON® resin 4.7. Co-precipitation of GST::DSK-2 with N-terminal 6XHIS-UBC-2 and C-terminal 6XHIS-UBC-2  52 52  5. GST::DSK-2 self-associates  52  6.  55  GST::DSK-2 interacts with polyubiquitin  7. R N A interference of genes encoding UBC-2 interacting proteins 8. Conclusions and future directions IV. REFERENCES  57 66 67  APPENDIX 1  81  vi  LIST OF T A B L E S Table 1. Yeast Two-Hybrid Assay Control Tests  34  Table 2.A. UBC-2 interacting G A L 4 A D fusion proteins do not interact with GAL4DBD CDK2 fusion protein  36  Table 2.B. UBC-2 interacting G A L 4 A D fusion proteins do not interact with GAL4DBD SNF1 fusion protein  36  Table 3. Fecundity of R N A i treated C. elgeans  62  Table 4. Fecundity of RNAi treated C. elegans grown in the absence of an environmental supply of calcium chloride  63  Table 5. Average length of R N A i treated C. elegans  65  vii  LIST O F F I G U R E S Figure 1. The ubiquitin pathway of protein degradation Figure 2. The yeast two-hybrid assay for interacting proteins  2 31  Figure 3. GAL4DBD::UBC-2 fusion protein is expressed in yeast strain Y190  32  Figure 4. Predicted domains of C. elegans proteins identified in the yeast two-hybrid screen for proteins that interact with UBC-2  38  Figure 5. Attempt to coprecipitate radiolabeled DSK-2 with C-terminal 6XHIS-UBC-2  46  Figure 6. Expression and Purification of GST::DSK-2  48  Figure 7. Confirming the identity of GST::DSK-2 fusion protein  49  Figure 8. Co-precipitation of GST::DSK-2 with 6XHIS-UBC-2  51  Figure 9. UBC-2 binds TALON® resin  53  Figure 10. Co-precipitation of DSK-2 with UBC-2  54  Figure 11. GST::DSK-2 self-associates in the presence of cross-linker  56  Figure 12. Polyubiquitin does not bind to TALON® resin or glutathione agarose  58  Figure 13. Polyubiquitin does not interfere with protein interaction with affinity resin  59  Figure 14. Polyubiquitin co-precipitation experiments  60  Figure 15. R N A i of mca-1: Adult mca-1 R N A i treated nematodes exhibit a reduction in body length  65  Vlll  LIST OF A B B R E V I A T I O N S  A  angstrom  ABC  ATP-binding cassette  AD  Alzheimer's disease  APC  anaphase promoting complex  3-AT  3-aminotriazole  ATP  adenosine triphosphate  p-TrCP  P-transducing repeat-containing protein  BLAST  basic local alignment search tool  BSA  bovine serum albumin  bp  base pairs  CDC  cell division cycle  CDK  cyclin-dependent kinase  cDNA  complementary deoxyribonucleic acid  DMSO  dimethyl sulfoxide  DNA  deoxyribonucleic acid  dNTPs  deoxyribonucleoside triphosphates  dsRNA  double stranded R N A  DSK  dominant suppressor of karl  DTT  dithiothreitol  El  ubiquitin-activating enzyme  E2  ubiquitin-conjugating enzyme  E3  ubiquitin ligase  ECL  enhanced chemiluminescence  EDTA  ethylene diamine tetra-acetic acid  EGF  epidermal-growth factor  ERAD  endoplasmic reticulum-associated degradation  GST  glutathione s-tranferase  HD  Huntington disease  HECT  homologous to E6-AP carboxyl-terminus  JPTG  isopropyl-P-D-thiogalactopyranoside  ix  IKB  inhibitor of N F K B  LB  Luria broth  LiAc  lithium acetate  LSB  loading sample buffer  NFKB  nuclear factor K B  NG  nutrient growth  OD  optical density  PAGE  polyacrylamide gel electrophoresis  PBS  phosphate buffered saline  PCR  polymerase chain reaction  PEG  polyethylene glycol  pfu  plaque forming units  PLIC  protein linking integrin associated protein with cytoskeleton  Rad  radiation sensitivity abnormal  RNA  ribonucleic acid  RNAi  R N A interference  rpm  revolutions per minute  SCF  Skpl-Cdc53-F-box protein  SDS  sodium dodecyl sulphate  SPB  spindle pole body  TGF-p  transforming growth factor-fj  Tris  tris(hydroxymethyl)aminomethane  UBA  ubiquitin associated  UBC  ubiquitin-conjugating enzyme  U-box  UFD-2 homology domain  UBL  ubiquitin-like protein  Ubp  ubiquitin protease  UDP  ubiquitin domain protein  UFD  ubiquitin fusion degradation  X-gal  5-bromo-4-chloro-3-indolyl-P-galactopyranoside  x  ACKNOWLEDGEMENTS I would like to thank my supervisor Peter Candido for providing me with the opportunity to learn about an exciting field in molecular biology and for giving valuable guidance (and Birthday cake!) throughout the duration of this project. I am grateful for my committee members Ross MacGillivray and Ivan Sadowski for their guidance and critical reading of my thesis in such a short period of time. I would also like to thank Don  Jones for technical assistance, for providing helpful answers to my persistent  questions, and for maintaining a jovial atmosphere in the lab. Many thanks to Emily Crowe, friend and 'mentor extraordinaire', who provided me with an immense amount of help with this project and taught me many things including the fact that many, chemicals in the lab are 'good for you because they challenge the system' and that dancing and singing in the lab are very positive ways to approach life at the bench. I would also like to thank Tracy, Lily, B.J., David, and Jim for valuable scientific discussions.  I am  grateful to members of the Biochemistry department who freely shared protocols, reagents, equipment, scientific advice, and friendship. I would also like to thank those who have believed in me, loved me, and taught me so much about life.. .God.. .my Mom and  my Dad, Grandma and Grandpa, Robert, Alan and Andrea...and my friends  Jacqueline, Rachelle, Shannon, Kate, Susan, Kara, Annie, Antonio, Azien, Anthony, Karen, Mark, Glenna, Andrea, Christina, France, Gael, Jason, Tina, Ute, and Gord.  XI  I. INTRODUCTION  1. UBC-2 and a summary of the ubiquitin pathway The ubiquitin-proteasome  pathway is a major route for selective protein  degradation in the cell (Figure 1). Ubiquitin is activated through A T P hydrolysis and an ubiquitin activating enzyme (El). Ubiquitin is attached via the carboxy-terminal glycyl residue to an active site cysteinyl residue on the E l , resulting in the formation of a thiolester bond.  Activated ubiquitin is subsequently transferred to an ubiquitin-  conjugating enzyme (E2).  Ubiquitin is attached through the carboxy-terminal glycyl  residue to an active site cysteinyl residue on the E2 through a thiolester bond. The ubiquitin protein ligase (E3) provides substrate specificity for this protein degradation pathway. The E3 either transfers activated ubiquitin to substrates directly or provides a scaffold to facilitate substrate protein ubiquitylation by placing the E2 and the substrate in close proximity. The carboxy-terminal glycyl residue of activated ubiquitin is attached to an internal lysyl residue of the substrate protein.  Subsequently, the E2 transfers  additional activated ubiquitin moieties onto the initial ubiquitin residue to form a multiubiquitin chain on the substrate protein. This chain is recognized by the proteasome as a signal for degradation. The ubiquitin chains are hydrolyzed by isopeptidases to facilitate recycling of ubiquitin in the cell. UBC-2, an ubiquitin-conjugating enzyme in Caenorhabditis elegans, is a member of the highly conserved and functionally overlapping "UBC4 branch" of E2s. In budding yeast, Saccharomyces cerevisiae, simultaneous mutation of all UBC4 branch members, UBC1, UBC4, and UBC5, produces lethality (Seufert et al, 1990). Budding yeast UBC4 members are implicated in the degradation of short-lived proteins such as mitotic cyclins and misfolded proteins such as those produced during the stress response (Seufert and Jentsch, 1990). The amino acid sequence of UBC-2 is 85% identical to UBC4 and UBC5 in S. cerevisiae and 95% identical to UbcDl in Drosophila melanogaster (Zhen et al., 1993). The importance of members of the UBC4 branch in vivo is underscored by the high degree of conservation between UBC4 branch members, the fact that UBC-2 is essential for larval development in C. elegans and that UbcDl is required for proper telomere behaviour in Drosophila (Cenci et al, 1997; Zhen et al, 1996). Furthermore,  1  Figure 1. The ubiquitin pathway ofprotein degradation Ubiquitin (Ub) is activated by an ubiquitin activating enzyme (El) and A T P hydrolysis. Activated ubiquitin is subsequently transferred to the ubiquitin-conjugating enzyme (E2). The ubiquitin protein ligase (E3) is thought to provide a scaffold for the E2 and the substrate to bring them into close proximity and facilitate substrate protein ubiquitylation. The carboxy-terminal glycyl residue of activated ubiquitin is attached to an internal lysyl residue of the substrate protein. The E2 transfers subsequent activated ubiquitins onto the initial ubiquitin molecule on the substrate protein to form a multi-ubiquitin chain which is recognized by the proteasome as a signal for degradation to peptides ( —) in an A T P dependant manner. The ubiquitin chains are hydrolyzed by isopeptidases, allowing recycling of ubiquitin in the cell.  2  UBC-2 is constitutively expressed and ubc-2 mutants exhibit pleiotropic defects which affect embryonic, somatic and germline development in C. elegans (Stevens, Ph.D. thesis, 1999; Zhen et al, 1996).  2.Components of the ubiquitin pathway 2.1. Ubiquitin Ubiquitin is a small 76 amino acid protein which exhibits high sequence homology in a wide variety of organisms studied to date. A comparison of amino acid sequences of ubiquitin in budding yeast versus in mammals reveals differences at only three residues (Ozkaynak et al., 1984). This high degree of conservation in eukaryotes underscores the essential function of ubiquitin in the cell. In vivo, ubiquitin exists in an unbound state or in covalent linkage to a diversity of proteins (Ozkaynak et al., 1984). Conjugation of ubiquitin to proteins serves as a signal for degradation via the proteasome in an ATP-dependent manner (Ciechanover et al, 1978; Hershko et al., 1980). The crystal structure of ubiquitin, determined to 1.8 A, revealed that the protein is compact and globular with a carboxy-terminal extension from the hydrophobic core.  This extension facilitates conjugation of the carboxy-terminal  glycine carboxyl group to the e-amino group of a lysyl residue on target proteins via an isopeptide bond (Vijay-Kumar et al., 1987). Ubiquitin itself is the target of ubiquitylation during multiubiquitin chain formation. Interestingly, ubiquitin chains linked between the carboxyl-terminal glycine of one ubiquitin and lysyl 63 of the next ubiquitin moiety appears to be implicated in D N A repair and protection from stress conditions (Arnason and Ellison, 1994; Spence et al., 1995). In S. cerevisiae, ubiquitin-conjugating enzymes UBC4 and UBC5, present at elevated levels in the cell under stress, are involved in lysyl-63-linked polyubiquitin formation (Arnason and Ellison, 1994; Seufert and Jentsch, 1990). However, isopeptide t  i  bond formation, mediated by UBC4 and UBC5, between the carboxyl-terminal glycine of one ubiquitin moiety and lysyl 48 residue of the next ubiquitin in a growing chain appears to be the most common linkage used as a signal for proteasomal targeting under normal conditions (Finley et al., 1994).  Interestingly, the S5a/Rpnl0 subunit of the  proteasome 19S particle interacts with lysyl-48-linked polyubiquitin chains of at least  3  four ubiquitin moieties in length (Devereaux et al, 1994). This indicates that ubiquitin chains target substrates for degradation potentially through direct physical interaction with the proteasome, although other factors are plausibly involved.  2.2. Ubiquitin-activating enzyme Ubiquitin activation is accomplished through an ubiquitin-activating enzyme (El).  The E l attaches A M P to the carboxy-terminal glycyl residue of ubiquitin  subsequent to catalyzing hydrolysis of ATP to A M P and PPi.  The AMP-ubiquitin  intermediate is subsequently attacked by the E l sulfhydryl group of the active site cysteinyl residue, resulting in the formation of a thiolester bond (Ciechanover et al., 1981).  In S. cerevisiae, the UBA1 essential gene encodes a 114 kDa ubiquitin-  conjugating enzyme which contains a nucleotide-binding site (McGrath et al., 1991). Genes encoding E l enzymes with active site cysteinyl residues have been cloned and functionally analyzed in wheat (UBA1, UBA2, UBA3) (Hatfield et al, 1990; Hatfield and Vierstra, 1992), rabbit (Sun et al, 1997), and human (Handley et al, 1991). The E l enzyme is approximately 100 kDa in size, exhibits a G X G X X G nucleotide-binding motif (X is any residue), and contains an active site cysteinyl residue in the center of the primary amino acid sequence (Hatfield and Vierstra, 1992). In vivo, the 105 kDa E l was found to dimerize in reticulocyte lysates (Ciechanover et al, 1982).  2.3. Ubiquitin-conjugating enzyme Activated ubiquitin is transferred from the E l to an ubiquitin-conjugating enzyme (E2).  The E2 forms a thiolester bond with the carboxy-terminal glycyl residue of an  activated ubiquityl moiety through the active site cysteinyl residue.  The E2 transfers  ubiquitin to either a substrate protein lysyl residue or to an ubiquitin ligase active site cysteinyl residue. Genes encoding 20 E2s in C. elegans, 13 E2s in S. cerevisiae, 17 E2s in human and E2s in other eukaryotic organisms have been identified (Jones et al, 2002; reviewed in Peters et al, 1998; reviewed in Pickart, 2001). A l l E2s exhibit a highly conserved (35-40% identity) 150 amino acid ubiquitin-conjugating (UBC) domain which includes the FHPNIXXXGXICLDL consensus sequence (Haas and Siepmann, 1997; reviewed in Peters et al, 1998). This sequence contains the active site cysteinyl residue  4  necessary for thioester bond formation with ubiquitin and an H P N tripeptide essential for appropriate folding of the protein. Mutation of the active site cysteinyl residue of E2s results in the elimination of ubiquitin-conjugating activity (Sommer and Jentsch, 1993; Sung et al, 1991). Deletion analysis has suggested that the amino-terminal region of the U B C domain might be involved in binding the E l (Sullivan and Vierstra, 1997). However, a direct interaction between the E l and an E2 has not been demonstrated. Further E2 classification is based on the presence or absence of the aminoterminal and/or carboxy-terminal extensions beyond the U B C domain. The extensions are often required for enzyme function and are believed to be important for subcellular localization and/or interaction with substrate or ubiquitin ligase proteins.  Class I E2s  consist of the U B C domain alone and are approximately 16 kDa in size. The lack of sequence beyond the U B C domains in Class I E2s is consistent with their dependence on an ubiquitin ligase to transfer ubiquitin to a substrate protein (Girod and Vierstra, 1993). Examples of Class I E2s include UBC-2 from C. elegans (Zhen et al, 1993), UBC4 and UBC5 from S. cerevisiae (Seufert and Jentsch, 1990), and UbcDl from Drosophila (Treier et al, 1992). UBC4 and UBC5 are involved in the degradation of short-lived and abnormal proteins (Seufert and Jentsch, 1990).  Interestingly, UBC-2 or UbcDl can  functionally substitute for yeast UBC4 activity in ubc4 ubc5 double mutant cells (Treier et al, 1992; Zhen et al, 1993). Class II E2s contain the U B C domain in addition to a carboxyl-terminal extension.  UBC-1, a homolog of UBC2(RAD6), is an example of a Class I E2 in C.  elegans (Leggett et al,  1995).  Examples of Class II E2s in S. cerevisiae include  UBC2(RAD6), UBC3(CDC34), and UBC6.  The acidic carboxy-terminal tail of  UBC2(RAD6) is necessary for polyubiquitylation of histones H2A and H2B and is essential for sporulation (Sung et al,  1988).  The carboxyl-terminal extension of  UBC3(CDC34) was determined to be essential for progression of the cell cycle.  It is  thought that the carboxyl-terminal tail may mediate interaction with UBC3 substrates or specific E3s which determine the substrate selection of UBC3(CDC34) (Kolman et al, 1992; Silver et al, 1992). A hydrophobic region of the carboxy-terminal tail of UBC6 appears to be responsible for localization of this E2 at the endoplasmic reticulum membrane (Sommer and Jentsch, 1993).  5  Class HI E2s consist of the U B C domain with an amino-terminal extension. Members of this class have been identified in Drosophila, mouse and human. A n ability of Class HI E2s to partially complement ubc4 mutants indicates the possibility that they have overlapping functions with UBC4-mediated proteolysis (Matuschewski et al., 1996). Class IV E2s contain both amino-terminal and carboxy-terminal extensions beyond the U B C domain. The amino-terminal extension of U b c M l in mouse exhibits transmembrane domains, indicating a possible subcellular targeting function for this portion of the protein (S. Jentsch, pers. comm. in Stevens, Ph.D. thesis, 1999). The carboxy-terminal extension of a 230 kDa E2 in rabbit exhibits a cysteinyl residue, in addition to the active site cysteinyl residue of the U B C domain, possibly indicating a dual ubiquitin ligase function and ubiquitin conjugating activity of this E2 (Berleth and Pickart, 1996).  2.4. Ubiquitin ligase The ubiquitin ligase (E3) is a component involved in the final transfer of ubiquitin to a target substrate.  The E3 is thought to promote ubiquitylation by direct transfer of  ubiquitin to the target substrate or by acting as a scaffold to enable facile transfer of ubiquitin from the E2 to the substrate. In the former case, the E3 forms a thiolester bond with ubiquitin at an active site cysteinyl residue after transfer from the E2. The variety of E3s that have been identified and characterized is thought to reflect the  specificity  of E3s for a wide  range of  substrates  that  are targeted  for  multiubiquitylation and subsequent degradation by the proteasome (reviewed in Jackson et al., 2000). E3s are classified on the basis of their structure, composition, and substrate specificity.  These E3 classes include: H E C T domain proteins, the SCF complex, the  A P C complex, RING finger proteins, and U-box containing proteins.  2.5. Multiubiquitin assembly factor The multiubiquitin assembly factor (E4), known as UFD2 in S. cerevisiae, is a recent addition to current knowledge of component proteins involved in the ubiquitin pathway of selective protein degradation (Koegl et al., 1999). The E4 is not involved in  6  the process of transferring ubiquitin to the substrate but appears to be necessary for efficient assembly of multiubiquitin chains of sufficient length for recognition by the proteasome. UFD2 enhances the formation of multiubiquitin chains catalyzed by TJBC4 in vitro. It has been proposed that the E4 might promote multiubiquitylation through a proven ability to bind ubiquitin conjugated to substrate proteins and a possible function in directing proteolysis by dictating a preference for specific lysyl residues of ubiquitin in multiubiquitin chain formation. Homologues of yeast UFD2 have been identified in human, C. elegans, Dictyostelium, and S. pombe (Koegl et al., 1999; Pukatzki et al., 1998).  These proteins share high sequence similarity in the carboxy-terminal region  referred to as the UFD2-homology domain or U-box (Koegl et al, 1999).  2.6. The 26S proteasome In eukaryotes, the 26S proteasome consists of two 19S regulatory particles which enclose either end of a hollow, cylindrical 20S proteolytic core particle. The regulatory complexes are thought to function in the recognition and unfolding of ubiquitylated substrates in an ATP dependent manner prior to their degradation in the core particle (reviewed in Voges et al, 1999). The crystal structure of the S. cerevisiae 20S particle revealed that it contains four stacked rings each consisting of 7 subunits. The two outer rings consist of 7 unique a-type subunits (a 1-7) while the two inner rings consist of 7 unique P-type subunits  (Pl-7).  The side chains of the amino-terminal ends of a-type  subunits prevent non-specific entry of proteins into the proteolytic core compartment. Three p-type subunits possess the chymotrypsin-like, trypsin-like, or peptidyl-glutamyl peptide proteolytic activities (Groll et al, 1997). Interestingly, in mammals, y-interferon induces expression of new p-type subunits which replace three of the existing P-type subunits, thus effecting a change in proteolytic cleavage to allow the production of peptides for M H C class I presentation (Monaco, 1995; Pamer et al, 1998). Furthermore, y-interferon induces expression of PA28, which associates with the 20S particle to increase the production of antigenic peptides (Dick et al., 1996; Gray et al, 1994). In S. cerevisiae, each 19S regulatory complex consists of 17 unique subunits which exhibit ATPase, polyubiquitin binding, or deubiquitylating activity. Six of these subunits are A A A family ATPases (Rptl to Rpt6) (Glickman et al,  7  1998). The  S5a/RpnlO subunit interacts with lysyl-48-linked polyubiquitin chains no less than four ubiquitin moieties in length (Devereaux et al, 1994). In mammalian cells, subunits of the 19S particle have been identified which participate in deubiquitylation (Uspl4 and a 37 kDa protein) (Borodovsky et al, 2001; Lam et al, 1997). Recent evidence has demonstrated that the 26S proteasome interacts with components of the ubiquitin pathway. Various E2s, including UBC4, have been shown to associate with the proteasome, although it is not clear whether this interaction is direct or requires other factors (Tongaonkar et al., 2000).  The ability of UBC4 to bind the  proteasome is not contigent on its ubiquitylation activity. Since non-functional UBC4 can bind the proteasome, it is thought that ubiquitylation activity of UBC4 occurs after it binds the proteasome. A model has been proposed whereby the association of E2s with the proteasome may promote efficient degradation of substrate proteins by decreasing the possibility of multiubiquitin chain disassembly through the action of ubiquitin proteases (Ubps) and constraining ubiquitin conjugation to the site of protein degradation. The idea that multiubiquitin assembly takes place in situ at the proteasome is supported by the fact that ubiquitylation activity of UBC4 is not required for interaction with the proteasome. Interestingly, heat shock induces UBC4 expression and also enhances its ability to bind to the proteasome (Seufert and Jentsch, 1990; Tongaonkar et al, 2000). Thus, heat shock or other events may change the type of E2 associated with the proteasome in order to alter the selectivity of protein degradation by the proteasome (Tongaonkar et al, 2000). Other ubiquitin pathway enzymes with a demonstrated interaction with components of the 19S particle of the 26S proteasome include the E3s: Ubrlp, a RING finger domain protein, and a H E C T domain protein called Ufd4p (Xie and Varshavsky, 2000).  Close  association of E2s and E3s with the proteasome may therefore prove to be a general theme in the degradation of select target proteins via the ubiquitin pathway.  3. UBC4/5-like E2s: diverse examples and functions in ubiquitylation E2s similar to UBC4/5 in S. cerevisiae have been isolated in other eukaryotes and have been shown to exhibit multiubiquitylation activity. UBC4/5-like E2s have been cloned from various other organisms including: wheat (E2ISK) (Girod and Vierstra, 1993); Arabidiopsis thaliana (AtUBC8-12) (Girod et al, 1993); cow (E2 K) (Chen et al, 25  8  1991; Chen and Pickart, 1990); rat (UBC4-1, UBC4-2 and UBC4-testis) (Rajapurohitam et al, 1999; Wing and Jain, 1995; Wing et al., 1996), Schizosaccharomyces pombe and Candida albicans (Damagnez et al., 1995); Drosophila (UbcDl) (Treier et al, 1992); C. elegans (Zhen et al., 1993); and human (UbcH5A-C) (Jensen et al., 1995; Scheffher et al, 1994). Interestingly, it has become apparent that proteins similar to yeast UBC4/5 have distinct in vivo functionality depending on the organism in which they are expressed. Mutations in UbcDl appear to promote the fusion of chromosomal telomeres (Cenci et al., 1997).  It has been proposed that the association of chromosomes through their  telomeres may enhance chromosome breakage and recombination, both of which may be linked to tumor development (Hastie and Allshire, 1989).  It has been proposed that  UbcDl may be involved in the degradation of proteins interacting with telomeres to prevent abnormal telomere behaviour (Cenci et al., 1997). The elimination of mutated tumor suppressors through the ubiquitin pathway is believed to be linked to the development of various human cancers. For instance, human UbcH5a-c are responsible for the degradation of tumor suppressor transcription factors, Smad2 and Smad4, when they are mutated (Xu and Attisano, 2000). In addition, Smad2 and Smad3 promote the ubiquitylation and subsequent degradation of SnoN mediated by UbcH5 and the anaphase-promoting complex (APC) ubiquitin ligase (Stroschein et al, 2001). SnoN is a protein which represses genes induced in response to transforming growth factor-p  (TGF-P) and is found in elevated  levels in a variety of human cancers.  In rat, the UBC4/5 homologues UBC4-2 and UBC4-testis exhibit specific patterns of expression in the testis germ cells (Rajapurohitam et al, 1999; Wing and Jain, 1995; Wing et al,  1996).  Since UBC4-2 and UBC4-testis are present in the rat testis and  exhibit increased expression and ubiquitylation activity in spermatids, it is thought that these E2s play a major role in the degradation of cellular proteins required for production of haploid spermatids during the process of spermatogenesis. Proteins similar to budding yeast UBC4 have demonstrated roles in nervous system development in higher eukaryotes and have been linked to diseases affecting the nervous system in humans. Analysis of UBC-2 in C. elegans revealed ubiquitous expression in early developmental stages which became specific to the nervous system in  9  L4 stage larvae and adults (Zhen et al,  1996).  In addition, expression of UBC-2  appeared to be more crucial for viability in early stages of development.  UbcDl has a  demonstrated role in the neuronal differentiation of the R7 photoreceptor precursor cell through elimination of the transcriptional repressor of photoreceptor differentiation called Tramtrack (TTK88) (Tang et al, 1997). A model has been proposed for photoreceptor cell fate decision whereby stimulation of Sevenless, a receptor tyrosine kinase, causes a RAS 1/MAPK signaling cascade resulting in: the transcription of PHYL, the association of P H Y L with TTK88 and SIN A, and the interaction of UbcDl with SHNA to promote the degradation of TTK88 and differentiation of the R7 photoreceptor cell (Tang et al., 1997). In addition, another yeast UBC4-like protein in human called E2-25K exhibits higher expression levels in the caudate nucleus and putamen regions of the brain which also demonstrate neural degeneration in Huntington disease (HD) affected individuals (Kalchman et al., 1996). Interestingly, these affected regions of the brain in H D also reveal concomitant expression of a protein linked to H D called Huntingtin which interacts with E2-25K and is ubiquitylated (Kalchman et al., 1996). Ubiquitin conjugation to membrane receptor proteins appears to signal receptor endocytosis.  For instance, in the presence of excess extracellular zinc, the zinc  transporter (ZRT1) in S. cerevisiae  appears to be targeted for endocytosis and  degradation in the vacuole by ubiquitylation, mediated by UBC4/5 and the E3 RSP5 (Gitan and Eide, 2000). In addition, the a-factor (Ste3) and a-factor pheromone receptors (Ste2), required for inter-cellular communication prior to mating of a and a haploid cells types, and the A B C transporter Ste6, required for a-factor pheromone secretion, are ubiquitylated in a UBC4/5 dependent manner prior to endocytosis (Hicke and Riezman, 1996; Roth and Davis, 1996).  In rabbit reticulocyte lysate, herbimycin A treated  epidermal-growth factor (EGF) receptors are polyubiquitylated in vitro in the presence of recombinant human UBC4 and other ubiquitylation factors (Mori et al., 1997).  It is  thought that herbimycin A , a tyrosine kinase inhibitor, binds and induces conformational changes in the tyrosine kinase E G F receptor which are recognized as signals for degradation by components of the ubiquitin pathway. In S. cerevisiae, UBC4 can mediate the monoubiquitylation of calmodulin in a calcium and E3 dependent manner in vitro (Parag et al., 1993). When calcium binds to  10  calmodulin, structural changes are induced that enhance its interaction with other proteins (O'Neil and De Grado, 1990).  Since it has not been proven that monoubiquitylation  leads to degradation of calmodulin in vivo, it has been proposed that the addition of ubiquitin may be necessary to modify the ability of calmodulin to interact with other proteins (Parag et al., 1993). It is also possible that multiubiquitylation and degradation of calmodulin may be promoted only at certain stages of the cell cycle when intracellular calcium levels increase.  4. Ubiquitylation mediated by UBC4 family E2s and associated E3s 4.1. Ubiquitylation mediated by UBC4 family E2s and H E C T domain E3s A variety of E3s containing homology to the E6-AP C-terminus have been identified and grouped into an E3 family termed the H E C T domain proteins (Huibregtse et al., 1995).  Human papillomavirus (HPV) 16, believed to be a causative agent in  cervical carcinoma, produces a protein called E6 that binds and targets the p53 tumor suppressor protein for destruction via the ubiquitin pathway, thus, paving the way for proliferative tumor growth (Scheffher et al., 1990).  Human UbcH5, a homologue of  budding yeast UBC4, acts as an E2 while the viral E6 protein and the host E6-associated protein (E6-AP), in combination, act as an E3 in the ubiquitylation and degradation of p53 (Rolfe etal, 1995; Scheffher et al, 1993; Scheffher et al, 1994). E6-AP is the final receptor of ubiquitin in the E1-E2-E3 ubiquitin thiolester cascade prior to transfer of ubiquitin to p53 (Rolfe et al, 1995). The ability to bind ubiquitin through a conserved cysteinyl residue, located in the approximately 350 amino acid H E C T domain, appears to be a common feature of proteins containing homology to the C-terminal portion of E6-AP (Huibregtse et al, 1995; Scheffher et al, 1995). An in vitro E2-E3 interaction between UbcH5 and the H E C T domain proteins E6-AP and & cerevisiae RSP5 is mediated by the UbcH5 Cterminal region surrounding the active site cysteinyl residue and a phenylalanine residue (Nuber and Scheffher, 1999). These conserved regions appear in many members of the UBC4 branch of E2s.  It is thought that the N-terminal portion of H E C T domain proteins  may mediate interaction with the proteasome and substrate proteins targeted for degradation via the ubiquitin pathway (You and Pickart, 2001; Wang et al, 1999). It  11  should be noted that while in vitro experiments do indicate that Rsp5 and E6-AP interact specifically with the UbcH5 E2, in vivo yeast two-hybrid experiments reveal that E6-AP interacts with UbcH7 and not UbcH5 (Kumar et al., 1997; Schwarz et al., 1998). Therefore, a yeast two-hybrid screen for UBC-2 interacting proteins may preclude the identification of H E C T domain proteins.  4.2. Ubiquitylation mediated by UBC4 family E2s and the A P C E3 Exit from mitosis and entry into anaphase is mediated by the degradation of cyclins through the activity of E2s and a multicomponent E3 called the anaphasepromoting complex (APC). For instance, in Xenopus egg extracts, the ubiquitylation of cyclin B requires the presence of the ubiquitin conjugating enzyme UBC4, a homolog of S. cerevisiae UBC4, and the APC in association with an activator of cyclin B degradation called Cdhl (King et al., 1995; Visintin et al., 1997).  Many substrates of the APC,  including cyclin B, exhibit an N-terminal nine amino acid destruction box (D-box) motif required for degradation mediated by the APC (Glotzer et al., 1991; reviewed in Page and Hieter, 1999). It is estimated that the A P C contains 11 subunits in both budding yeast and vertebrates (Gmachl et al., 2000; reviewed in Page and Hieter, 1999). Some subunits of the budding yeast A P C include: a RING-H2 finger protein called A P C 11 and a cullin protein called APC2 (Yu et al., 1998; Zachariae et al., 1998). The similarity of APC2 and APC 11 to proteins found in the Skpl-cullin-F box protein complex (SCF) and the von Hippel Lindau complex (VHL) might indicate the importance of both subunits in mediating the E3 activity of the APC; however, it has been demonstrated that APC 11 is the only portion of the APC required for promoting ubiquitylation of cyclin B (Gmachl et al., 2000). Nevertheless, it is thought that efficient activity of the A P C likely requires all subunits of the complex including APC2 (Leverson et al., 2000). For instance, E3 activity reconstituted by APC2 and APC 11 demonstrates non-specificity in that it catalyzes ubiquitylation of a cyclin B lacking a D-box (Tang et al., 2001). Furthermore, the RJNGH2 finger domain of A P C 11 was determined to be necessary for ubiquitin ligase activity and for interaction with the ubiquitin conjugating enzyme UBC4 (Leverson et al., 2000). Since an in vitro interaction between APC 11 and UBC4 has been demonstrated, it is  12  possible that UBC-2 may exhibit a yeast two-hybrid interaction with the RING finger domain of a C. elegans APC11  budding yeast E3 homologue.  4.3. Ubiquitylation mediated by UBC4 family E2s and SCF complex E3s The  SCF complex family is composed of a group of E3s which contain the  following components in S. cerevisiae: Skpl, Cdc53, and an F-box containing protein (reviewed in Patton et al., 1998). In metazoans, homologues of budding yeast Cdc53 are termed cullins (Kipreos et al., 1996). The F-box motif enables interaction with Skpl while the remaining portion of the F-box protein is thought to be involved in recognizing and recruiting target proteins to the SCF for ubiquitylation (Bai et al., 1996; reviewed in Patton et al., 1998). F-box proteins are classified according to the type of protein-protein interaction domain present in the non-F-box portion of the protein that promotes recognition of ubiquitin pathway substrates. The F-box protein classes include: Fbws which contain WD-40 domains, Fbls which contain leucine-rich domains, and Fbxs which contain leucine zippers, ring fingers, helix-loop-helix domains, proline-rich repeats or Src homology 2 (SH2) domains (Cenciarelli et al, 1999). The most recently identified component of the SCF is a RING-H2 finger protein, called Rbxl or Hrtl, which stimulates E3 activity of the complex (Kamura et al, 1999; Seol et al., 1999; Skowyra et al,  1999).  Providing further clues as to how the subunits of the SCF assemble, in  Drosophila, homologues of human Skpl and Rbxl have demonstrated an ability to interact with homologues of Cull and the F-box protein Slmb (Bocca et al, 2001). Individual SCF complexes are distinguished by the F-box that they contain. Some examples of substrates targeted by the S C F  p T r C P  complex include: phosphorylated  IKBCX, an inhibitor of a transcription activator of pro-inflammatory genes called N F K B , in response to pro-inflammatory signals (Winston et al,  1999) and phosphorylated P-  catenin, a transcription factor which stimulates the activity of the oncogene c - M Y C in colorectal cancers and is required for regulation of development via the Wnt/Wingless pathway (He et al, 1999; Winston et al, 1999). Interestingly, members of the UBC4 branch of E2s, such as budding yeast UBC4 and human UbcH4, UbcH5b and UbcH5c, function in conjunction with the S C F  p T r C P  complex to mediate ubiquitylation of IxBa  (Gonen et al, 1999; Kawakami et al, 2001; Strack et al, 2000). In addition, human  13  NEDD8, a ubiquitin-like protein, binds the Cull subunit to stimulate interaction of monoubiquitylated U b c H 4 with the S C F  p T r C P  complex (Kawakami et al, 2001). For this  reason, the possibility exists that UBC-2 may exhibit a yeast two-hybrid interaction, in a Nedd8 dependent fashion, with components of a putative homologous S C F  |3TrCP  complex  in C. elegans. This possibility seems plausible given the fact that homologues of human SCF  p T r C P  , including the cullin dCull and the F-box protein Slmb, have been identified in  Drosophila and have demonstrated an interaction with UbcDl (Bocca et al., 2001).  4.4. Ubiquitylation mediated by U B C 4 family E2s and the V H L E3 complex The von Hippel-Lindau (VHL) tumor suppressor complex associates with the Elongin B C complex in mammals (Kibel et al, 1995). This large complex interacts with another complex of Cull (a cullin) and Rbxl to promote Ubc5 ubiquitylation activity (Kamura et al., 2000). Specifically, the Elongin BC complex acts as an adaptor to bridge the V H L complex with the Cull-Rbxl complex to allow ubiquitylation of the a subunits of hypoxia inducible factors HJF1 and HIF2. The V H L complex interacts with the HIF substrates through an oxygen-dependent degradation domain when a specific prolyl residue is hydroxylated (Ivan et al., 2001).  Since the Elongin B C complex can bind  various B C box-containing proteins, such as V H L , and since Elongin B C complex binds Cull/Rbxl to promote ubiquitylation activity, it is thought that Elongin BC may function to link a particular substrate recognizing BC-box containing protein with Cull/Rbxl to promote assembly of a functional E3 (Kamura et al., 2001).  Since the VHL-Elongin B C  complex can activate ubiquitylation activity of Ubc5, it is possible that UBC-2 may interact  in  a  yeast  two-hybrid screen  with  a  component  of  this  complex.  4.5. Ubiquitylation mediated by U B C 4 family E2s and RING finger domain E3s RING (for Really Interesting New Gene) finger domains are defined by a sequence containing eight cysteinyl and histidyl residues that chelate two zinc atoms E2s (reviewed in Borden, 2000). Proteins containing RING domains exhibit the following consensus sequence: CX2CX(g-39)CX(io)HX(2-3)C/HX2CX(4-48)CX2C where the cysteinyl and histidinyl residues represent zinc binding residues and the X represents any amino acid (reviewed in Freemont, 2000). The RING proteins are classified further as RING-  14  H2 or PJNG-HC if a histidyl residue or a cysteinyl residue, respectively, is present at the 5 zinc coordination site. RING domains in E3s appear to be necessary for ubiquitylation th  of substrates and for mediating interaction with E2s (reviewed in Joazeiro and Weissman, 2000; Lorick et al., 1999).  For instance, in vitro experiments have demonstrated  interactions between UbcH5, a homologue of S. cerevisiae UBC4, and RING finger proteins including: A07, BRCA1, Siah-1, TRC8, NF-X1, kf-1 and Prajal (Lorick et al., 1999). In addition, human homologues of S. cerevisiae UBC4 appear to be required for the E3 activity of the following RING proteins: the membrane protein Rmal, the receptor protein-tyrosine kinase adaptor c-Cbl, and the apoptosis inhibitors c-IAPl and XIAP (Joazeiro et al., 1999; Matsuda et al., 2001; Yang et al.., 2000). RING domains are found in a diversity of E3 multicomponent complexes in addition to single protein E3s (reviewed in Jackson et al., 2000). Examples of RING finger proteins that exist in E3 complexes include the previously described Apcl 1 subunit of the A P C complex and Rbxl/Hrtl of the SCF complex (Kamura et al., 1999; Leverson et al., 2000; Seol et al., 1999; Skowyra et al., 1999). As already mentioned, the RJNGH2 finger domain of A P C 11 is required for ubiquitin ligase activity and for interaction with UBC4 (Leverson et al., 2000). Another RING-H2 finger protein, called Rbxl or Hrtl, stimulates E3 activity of the SCF complex (Kamura et al., 1999; Seol et al., 1999; Skowyra et al., 1999). Since UbcH4 interacts with an SCF complex (Kawakami et al., 2001), and since a variety of RING fingers exhibit an ability to interact with E2s, it is possible that Rbxl binds and recruits UbcH4 to the SCF complex.  Therefore, it is  plausible that a yeast two-hybrid screen for proteins that interact with C. elegans UBC-2 may identify multiple RING finger proteins.  4.6.  Ubiquitylation  mediated  by  UBC4  family  E2s  and  the  U-box  E3s  Proteins containing U-box domains are thought to represent a new class of E3s due to their ability to catalyze multiubiquitin chain formation in the presence of E l and E2 (Hatakeyama et al., 2001). CHIP (carboxyl terminus of Hsc70-mteracting protein) is a U-box protein which interacts with UbcH5c and catalyzes multiubiquitylation of heat denatured protein in a U-box dependent manner (Jiang et al., 2001; Murata et al., 2001). The E3 activity of CHIP and the recognition of misfolded substrate protein by molecular  15  chaperones Hsc70 or Hsp90 indicates that molecular chaperones exhibit dual roles in protein folding and in targeting misfolded proteins for degradation via the ubiquitin pathway (Murata et al., 2001). In vitro experiments demonstrated that U-box containing murine proteins including: UFD2a, UFD2b, KIAA0860, and CHIP all exhibit the greatest ability to catalyze multiubiquitylation in the presence of UbcH5c, a member of the UBC4 family (Hatakeyama et al, 2001).  Therefore, the possibility exists that a yeast two-  hybrid screen for proteins that interact with C. elegans UBC-2 may identify U-box proteins.  5. Study objectives Given that UBC4 branch proteins have been implicated in a diversity of cellular roles and are responsible for the selective degradation of many cellular proteins, and since UBC-2 plays an essential role in the development of C. elegans, a study was initiated to identify proteins that function in conjunction with UBC-2 in order to increase our knowledge about this vital component of the ubiquitin pathway. As discussed in the previous sections, a variety of potential UBC-2 interacting proteins have been identified, including H E C T domain proteins, components of the A P C and SCF multi-subunit complexes, RING finger domain proteins, and U-box containing proteins.  In order to  identify proteins that interact with UBC-2 in C. elegans, a yeast two-hybrid screen was employed.  16  JX MATERIALS A N D METHODS  1. D N A techniques 1.1 Standard polymerase chain reaction (PCR) Polymerase chain reactions were performed in a total reaction volume of 50 pi using 1 pi of D N A template, 50 pmoles of each oligonucleotide primer, 50 u M each deoxyribonucleotide triphosphate, and 1 unit of Vent D N A polymerase, all in IX Vent D N A polymerase buffer as defined by the manufacturer (New England Biolabs). PCR conditions followed the same general protocol: 3 minutes at 94°C; 30 cycles of 45 seconds at 94°C, 45 seconds at 60°C, 1 minute at 72°C; 10 minutes at 72°C.  The  annealing temperature or each primer was in the range of 58°C to 62°C depending on the G C content and length of the oligonucleotide.  The TwinBlock™ system thermal cycler  (ERICOMP Inc.) was used to carry out PCR reactions.  1.2 Restriction endonuclease digestion of D N A Various restriction enzymes acquired from GD3CO/BRL, Pharmacia and New England Biolabs were used to digest D N A samples. Digested samples were analyzed on horizontal agarose gels.  For cloning purposes, alkaline phosphatase (Amersham  Pharmacia) was used to dephosphorylate appropriately restriction digested vectors prior to ligation to restriction digested D N A fragments. Alkaline phosphatase treatment of D N A was performed according to standard protocols (Sambrook et al., 1989).  1.3 Agarose gel electrophoresis Agarose powder was added to T B E buffer (10.8 mg/ml Tris base, 5.5 mg/ml boric acid, 0.9 mg/ml EDTA) to make gels in the range of 0.7% and 2.0% agarose.  After  heating the solution to solubilize the agarose and cooling the solution to approximately 55°C, ethidium bromide was added to a final concentration of 0.5 pg/ml. Concentrated gel loading buffer was added to the D N A samples prior to gel loading in order to achieve a final concentration of 0.04% Bromophenol blue, 0.04% Xylene cyanol FF, 2.5% Ficoll Type 4000, and 20 m M E D T A .  17  1.4 D N A extraction from agarose The Qiaex II kit (Qiagen, Inc.) was used to purify D N A bands of interest from agarose gels.  1.5 Ligation of restriction endonuclease digested D N A To perform D N A ligations of cut insert and plasmid D N A , T4 D N A ligase was used (Amersham Pharmacia).  D N A was performed according to standard protocols  (Sambrook et al, 1989).  1.6 Transformation of D N A into Escherichia coli Ligation reactions and purified plasmids were transformed into competent E. coli DH5a (GIBCO/BRL) according to the manufacturer's instructions.  Purified plasmids  were transformed into CaCL. treated competent E. coli BL21(DE3) cells according to a standard protocol (Sambrook et al., 1989). A volume of 2 pi of plasmid D N A isolated from yeast was used to transform 50 pi of electrocompetent E. coli HB101 cells.  0.1 cm electroporation cuvettes (BioRad,  Hercules, California) were used in the BioRad electroporator to transform cells according to the manufacturer's instructions with instrument settings at 1.8 V , resistance at 200 Q and capacitance at 25 F. After electroporation, 1ml of SOC medium (2% Bacto tryptone, 0.5% Bacto yeast extract, lOmM NaCl, 2.5mM KC1, 20mM MgSCVMgC^, 20mM glucose, pH 7.0) was added to the cells followed by an incubation at 37°C for one hour and subsequently spreading on antibiotic selection plates for overnight incubation.  1.7 Transformation of plasmid D N A into S. cerevisiae For 10 transformations, yeast cells from a 10 ml overnight culture were harvested by centrifugation at 500xg for 5 minutes and the cell pellet was resuspended in 1 ml T L (lOmM  Tris-HCl pH 7.5, ImM E D T A ,  lOOmM lithium acetate, pH 7.5).  Each  transformation reaction consisted of a 100 pi aliquot of cells, 44 pi D M S O , 60 pg sheared salmon sperm D N A (Eppendorf), 4 pg plasmid D N A and 400 pi TLP (lOmM Tris-HCl pH 7.5, ImM E D T A , lOOmM lithium acetate (LiAc), 44% polyethylene glycol (PEG) 4000, pH 7.5).  The cell suspensions were mixed, incubated at 30°C for 1 hour, and  18  mixed again. Cells were then heat shocked at 42°C in a water bath for 10 minutes and plated on selection media. For high efficiency transformations (used in the transformation of Y190 with the C. elegans plasmid cDNA library), a 10 ml overnight culture was counted using a hematocytometer and used to inoculate 50 ml of synthetic complete medium to a cell density of 5xl0 cells/ml. After the culture had reached a cell density of 2xl0 cells/ml, 6  7  cells were harvested at 3000xg for 5 minutes and the cell pellet was resuspended in 1 ml of 100 m M Li Ac.  Cells were pelleted and resuspended again to a final cell density of  2xl0 cells/ml. Transformation reactions consisted of a 50 pi aliquot of cells, 240 pi 50% 9  w/v P E G 4000, 36 pi 1.0 M LiAc, 100 pg sheared salmon sperm D N A , x pi of plasmid D N A and 34-x pi of water. Transformation reactions were mixed for 1 minute (Fisher Scientific Vortex Genie 2), incubated at 30°C for 30 minutes, and heat shocked at 42°C for 25 minutes prior to plating on selection media.  1.8 Plasmid preparationfromE. coli Plasmids were prepared for D N A sequencing and other manipulations requiring high purity D N A using the Qiagen kits according to the manufacturer's instructions. The alakaline lysis method of D N A purification was also employed (Birnboim and Doly, 1979).  1.9 Plasmid preparation from S. cerevisiae Yeast cells from a 1.5 ml overnight culture harbouring the pACT::C. elegans cDNA plasmid of interest were harvested at 12,000 rpm in a microcentrifuge and resuspended in 200 pi of breaking buffer (2% Triton X-100, 1% SDS, 100 m M NaCl, 10 mM Tris-Cl, 1 m M E D T A , pH 8.0) in 1.5 ml microfuge tubes. After adding 0.3 g acid washed  0.45  to  0.55  mm glass beads (Sigma)  and 200  pi  25:24:1  (v/v/v)  phenol/chloroform/isoamyl alcohol, samples were mixed at high speed in a Vortex multitube head for 3 minutes.  Following a 5 minute centrifugation at 12,000 rpm, the  aqueous layer was saved for transformation of electrocompetent E. coli HB101.  19  1.10. D N A concentration determination A Pharmacia Ultraspec 3000 spectrophotometer was used to determine the concentration of D N A samples knowing that 1  1.11. D N A  O D 2 6 0  = 50 pg/ml dsDNA.  sequencing  DNA  samples suspended in E B buffer, prepared using the Qiagen Miniprep kit,  were sequenced and analyzed by the Nucleic Acid Protein Services (NAPS) Unit at the University of British Columbia, Vancouver, B.C., Canada. D N A samples were supplied at a concentration of 100 ng/pl and primers were supplied at a concentration of 3.2 pmoles/ul.  Samples were sequenced by technicians at NAPS using the enzyme  Sequenase" Version 2.0 T7 D N A polymerase and the PRISM Dye Terminator Cycle Sequencing kit. Extension products were isolated using a Centri-Sep column and were analyzed on either the Applied Biosystems (ABI) Model 373 Stretch sequencer or the ABI Prism 377 D N A  sequencer.  1.12. Preparation of the yeast two-hybrid bait plasmid ubc-2 was cloned from pRSET-c-w&c-2 using primer oligonucleotides MG1 and MG2  (Appendix I) and inserted into the Ndel and BamHl restriction endonuclease sites  of the pAS2 vector (Matchmaker).  1.13. Conversion of bacteriophage library AACT-RB2 into plasmid library pACT-RB2 A C. elegans cDNA library called XACT-RB2 was previously constructed by Robert Barstead in the lambda vector A C T (kindly provided by Steve Elledge) using cDNA primed with random hexamers (Durfee et al, 1993; Elledge et al, 1991). The titer of the library AACT-RB2 previously amplified and frozen at -80  C by E. Crowe was  determined to be 1.2 plaque forming units (pfu)/ml. For phage titer determination, T M G buffer (10 m M Tris-HCl, pH 7.5; 10 m M M g S 0 ; 0.01% gelatin) was used to dilute the 4  bacteriophage and LB top agar was prepared with 0.7% agarose. E. coli was cultured in LB liquid media with 50 ug/ml kanamycin and 0.2%> maltose. 1.2xl0 pfu were used to 9  infect a total of 7.2xl0 log phase cells (OD600 = 0.6 9  3xl0 cells/ml at log phase for E. 8  coli RB4E) in the presence of 10 mM MgSC<4. Logarithmic phase cells, in 2 ml aliquots,  20  were infected with bacteriophage and grown at 28 C for 30 minutes. 2 ml L B medium was added to the bacteriophage infected culture and the infected cells were then agitated for one hour at 30 C. Infected cells were concentrated at 2,000 rpm in a clinical centrifuge for 8 minutes, resuspended in 200 pi, and plated on 150 mm L B plates containing 50 pg/ml ampicillin. A total of 5.5x10 cells were infected, plated and grown 7  overnight at 37 C. Colonies were resuspended in 30 ml LB and used to inoculate 3 L Terrific Broth containing 50 pg/ml ampicillin. Cells were harvested and the pACT-RB2 plasmid library was isolated using the Qiagen Maxi D N A preparation kit for low copy plasmids (pACT is a low copy pBR322 based plasmid).  2. Culturing C. elegans As a source of wild type nematodes for the R N A i experiments, C. elegans Bristol (N2) was cultured on N G plates (0.3% NaCl, 0.25% tryptone, 5 mg/ml cholesterol, ImM CaCl , ImM M g S 0 , 25mM K H P 0 , 1.7% agarose, pH 6.0) spread with a lawn of 2  4  2  4  Escherichia coli OP50 (Brenner, 1974). L4 stage larvae were transferred to new OP50 seeded plates to support the growth of the next generation. 3. Maintenance and mating of S. cerevisiae Yeast strains Y187 (MATa gal4 gal80 his3 trpl-901 ade2-\0\ ura3-52 leu23,112 + URA3::GAL lacZ LYS2::GAL(UAS) gal80 his3 trpl-901  ade2-\Q\  HIS3 cyh ) and Y190 (MATa gal4 x  ura3-52 leu2-3,-\\2 met- URA3::GAL lacZ) were  maintained on Y E P D plates (10 mg/ml Bacto yeast extract, 20 mg/ml Bacto tryptone peptone, 20 mg/ml glucose and 2% agarose). Yeast strains were cultured on plates and in liquid media at 30 C. Selection media contained 20 mg/ml glucose, yeast nitrogen base without amino acids (Difco), 2% agarose, and a portion of a synthetic complete (SC) mixture of amino acids lacking leucine (-Leu) (0.7 mg/ml), lacking tryptophan (-Tip) (0.7 mg/ml), lacking leucine and tryptophan (-Leu-Trp) (0.6mg/ml), or lacking leucine, tryptophan and histidine (-Leu-Trp-His) (0.6 mg/ml) (BiolOl). SC-Leu media was used to select for yeast transformed with pACT::cDNA plasmids or p S E l l l l control plasmid. SC-Trp media was used to select for yeast transformed with pAS2::ubc-2 or pSE1112 control plasmid.  SC-Leu-Trp was used to select for the presence of yeast doubly  21  transformed with plasmids containing these nutrient selection markers. SC-Leu-Trp-His was used to select for yeast colonies containing two plasmids containing these nutrient selection markers and to select for yeast colonies expressing interacting proteins. This SC dropout medium was supplemented with 25 m M 3-aminotriazole to inhibit growth of colonies expressing non-interacting proteins due to potential weak activation of the reporter genes while still permitting growth of colonies expressing interacting proteins. Following an attempt to lose the bait plasmid by non-selection by streaking colonies transformed with the bait plasmid (pAS2::ubc-2) and a particular pACT::cDNA plasmid on SC-Leu media, colonies were plated on SC-Leu containing 2.5 pg/ml cycloheximide. This drug was used to eliminate colonies transformed with pAS2::ubc-2 (which contains a marker for cycloheximide sensitivity) and a particular pACTy.cDNA plasmid while permitting growth of colonies containing only a single pACT: :cDNA plasmid.  This  facilitated isolation of the cDNA plasmid away from the bait plasmid to permit isolation of a single cDNA plasmid species for sequencing and manipulation. Yeast matings were used as a means to eliminate non-specific interactors of UBC-2. Y190 (MATa) transformed with a particular pACTy.cDNA plasmid was streaked in patches on SC-Leu media grown at 30 C for 1 day. Y187 (MATa) transformed with p A S l wsnfl or pAS 1 ::cdk2 was streaked on SC-Trp plates and grown at 30 C for 3 days. One Y187 yeast colony was resuspended in 1 ml YEPD and 250 pi of this mixture was spread on a Y E P D plate and grown at 30 C for 3 hours. Sterilized velvet was used to replica plate the patched Y190 from the SC-Leu plate onto a lawn of Y187 on the YEPD plate and yeast was grown at 30 C overnight. Yeast colonies were replica plated from the YEPD plate onto a SC-Trp-Leu plate and were grown at 30  C until colonies appeared.  An X-gal filter lift assay (described below) was performed to detect the activation of the reporter gene lacZ, and hence, a non-specific interaction between a UBC-2 interacting protein and SNF1 or CDK2.  4. X-gal filter lift assay Schleicher and Schuell BA85 45 pm circular nitrocellulose filters were used to lift yeast colonies expressing interacting proteins (i.e. they grew on SC-His media) from the plate media. Liquid nitrogen was used to permeabilize the cells to Z buffer (60 m M  22  N a H P 0 , 40 m M N a H P 0 , 10 mM KC1, 1 mM M g S 0 , 40 m M 2-mercaptoethanol, pH 2  7.0)  4  2  containing  4  4  1 mg/ml 5-bromo-4-chloro-3-indolyl-P-D-galactoside  in dimethyl  formamide. Filters were incubated at 30°C until a blue colour developed in situ in the positive controls (i.e. Y190 yeast strain transformed with pSEl 111 and pSEl 112).  5. Computer analysis DNA  Strider was  used  to  determine  cloning  strategies and to  design  oligonucleotide primers. B L A S T (http://www.ncbi.nlm.nih.gov/BLAST/) was employed to positively identify cDNA sequences encoding putative UBC-2 interacting proteins from the C. elegans plasmid cDNA library. proteins  were  obtained  Protein sequences of UBC-2 interacting  from  the  (http://www.ncbi.nlm.nih.gov/Database/index.htmD.  GenBank  web  site  The Simple Modular Architecture  Research Tool (SMART) was used to search protein sequences for protein domains (http://smart.embl-heidelberg.de/)  (Schultz et al., 1998; Schultz et al., 2000).  The  WormPD was used to find homologues for UBC-2 interacting proteins (Bateman et al., 1999; Genome sequence of the nematode C. elegans: a platform for investigating biology, 1998)  6. Protein and immunological techniques 6.1 TNT® Quick Coupled Transcription/Translation of radiolabeled DSK-2 The  cDNA  fragment  encoding  DSK-2  was  excised  from  plasmid  pACT::F15C11.2 (dsk-2) and cloned into the Xhol site of the multiple cloning site of pcDNA3.1/Hygro(+) [ S]methionine 35  (Promega).  (Invitrogen).  and a  TNT®  35  Quick  S-labeled  DSK-2  was  produced  using  Coupled Transcription/Translation System  Translation was performed according to the manufacturer's instructions  using 40 pi T N T ® Quick Master Mix, 20 uCi [ S]methionine, and 1 pg plasmid D N A 35  template (pcDNA3.1: :dsk-2).  6.2 Overexpression and purification of 6XHIS-UBC-2 C-terminal 6XHIS-UBC-2 fusion protein was expressed from a pRSET-c-ubc-2 (Invitrogen) construct which had been previously made by M . Zhen (Zhen, Ph.D. thesis,  23  1995). A n N-terminal 6X HIS-UBC-2 fusion protein was also prepared from the pET28a (+) expression vector (Novagen).  ubc-2 was cloned from pAS2::ubc-2 using primer  oligonucleotides MG7 and MG10 and inserted into the Ncol and Xhol sites of the pET28a (+) vector. The pRSETC:\ubc-2 and pET28-a(+)::wZ>c-2 constructs were transformed into competent BL21 (DE3) STAR (Invitrogen) E. coli. Fresh pRSETC::«6c-2 and pET28a(+)::ubc-2 transformed colonies were used to inoculate 10 ml of L B containing 100 pg/ml ampicillin or 50 pg/ml kanamycin, respectively. These cultures, grown overnight at 37°C, were used to inoculate 1 L of the same medium. After the OD oo reading had 6  reached 0.7, cells were induced with 1 m M LPTG, grown for another 3 hours and cell pellets were obtained following centrifugation at 2,000xg for 15 minutes at 4°C. Each pellet was resuspended in 40 ml of extraction/wash buffer (300 m M NaCl, 50 m M NaPC»4, pH 7.0).  Cell suspensions were sonicated using a 10 second pulse on and 20  second pulse off for 5 min. The cell extract was centrifuged at 1 l,000xg for 20 minutes at 4°C to pellet the insoluble portion. The soluble portion was incubated for 20 minutes at room temperature with 4 ml of a 50% slurry of TALON® resin equilibrated in extraction/wash buffer. The resin was pelleted at 700xg for 5 minutes and washed three times.  500 pi fractions were collected after adding the resin to a column and applying  elution buffer (extraction/wash buffer with 150 m M imidazole). Fractions were pooled and dialyzed overnight at 4°C in UBC-2 buffer (300 mM NaCl, 50 m M NaP04, pH 7.4) using M W C O 3350 dialysis tubing to remove imidazole. During dialysis, the C-terminal 6XHIS-UBC-2 precipitated out of solution.  The soluble portion of the protein  preparation was removed as the supernatant following centrifugation for 10 minutes at 12,000 rpm in a microcentrifuge. The protein concentration was determined using a BioRad microassay. One litre of culture yielded 12 mg of soluble C-terminal 6XHIS-UBC-2 fusion protein and 2 mg of N-terminal 6XHIS-UBC-2.  To reduce the total reaction  volume for the cross-linking experiments, N-terminal 6XHIS-UBC-2 was concentrated 10 fold on a Millipore Ultrafree-4 centrifugal filter device (Biomax 5K N M W L Membrane 4 ml volume).  24  6.3 Overexpression and purification of GST::DSK-2 N-terminal GST::DSK-2 fusion protein was expressed using a pGEX-4T-2 (Pharmacia Biotech.) construct containing the dsk-2 gene which was subcloned from the pACT vector (Matchmaker) into the Xhol site of the pGEX-4T-2  vector in the correct  orientation and reading frame. The pGEX-4T-2 construct was transformed into BL21 (DE3) STAR E. coli.  Fresh pGEX-4T-2::<M-2 transformed colonies were used to  inoculate 10 ml of L B containing 100 pg/ml ampicillin. These cultures grown overnight at 37°C were used to inoculate 1 L of the same medium. After the culture had reached an ODeoo reading of 0.4, cells were induced with 1 m M IPTG and grown for an additional 3 hours. The preparation of bacterial sonicates and the batch purification of GST::DSK-2 fusion protein was performed using procedure 11 and 12 from the GST Gene Fusion System (Third Edition, Revision 1; Amersham Pharmacia Biotech).  The buffer used  during protein purification included 1 mM D T T and 1 protease inhibitor cocktail minitablet (Complete™ Mini, E D T A free; Roche) for every 10 ml of buffer. The GST tag of N-terminal GST::DSK-2 was removed using Thrombin Protease (Amersham Pharmacia Biotech). Thirty pg of GST::DSK-2 were incubated with one unit (50 ng) of Thrombin Protease in IX phosphate buffered saline [PBS; 0.58 M N a H P 0 , 2  4  0.17 M N a H P 0 , 0.68 M NaCl, pH 7.4] overnight at room temperature. 2  4  6.4. Preparation of S. cerevisiae protein extract To confirm that the GAL4DBD::UBC-2 fusion protein was expressed from the pAS2::ubc-2 construct in yeast strain Y190, protein extracts were prepared from two 50 ml, 48 hour Y190 cultures, of which one expressed the GAL4DBD::UBC-2 fusion protein. Cell pellets were obtained after centrifugation for 10 minutes at 1500 rpm in a clinical centrifuge. Cells were washed with water, pelleted and resuspended in 200 pi of 2X SDS sample buffer.  A n equal volume of glass beads (Sigma) was added before  vigorous mixing for 10 minutes using a Vortex mixer. Twenty pi of this extract was boiled and separated on an SDS-PAGE gel prior to Western blot analysis using an antiUBC-2 antibody to detect the GAL4DBD::UBC-2 fusion protein.  25  6.5. Protein concentration determination The concentration of protein samples was determined using a BioRad microassay according to the manufacturer's instructions.  A B S A standard curve was used to  determine the protein concentration of unknown protein samples.  6.6. SDS-polyacrylamide gel electrophoresis Discontinuous  SDS-polyacrylamide gel  electrophoresis  (SDS-PAGE)  was  employed to separate proteins according to size (Laemmli, 1970). Protein samples were suspended in loading sample buffer (LSB) (50 m M Tris pH 6.8, 100 m M D T T , 2% SDS, 10% glycerol, 0.1% bromophenol blue) and boiled for 5 minutes prior to gel loading. Gels (0.75 mm X 7.5 cm X 8.0 cm) were poured and run using the Mini-PROTEAN II apparatus (BioRad) according to the manufacturer's instructions.  6.7. Western blot analysis Methanol soaked Immobilon-P membranes (Millipore) and SDS-PAGE gels containing separated proteins were soaked in transfer buffer (25mM Tris-HCl pH 8.5, 192 m M glycine, 20% methanol) for 5 minutes prior to transfer.  A sandwich of filter  paper-membrane-gel-filter paper was prepared prior to assembly in the BioRad transfer apparatus. Transfer of proteins took place at 250 raA for one hour. Western blots were agitated at room temperature in TBS-Tween (140 m M NaCl, 20 m M Tris-HCl, 0.05% Tween-20, pH 7.5) containing 10% milk powder for one hour. One hour of primary antibody incubation was followed by one hour of secondary antibody incubation. Blots were washed three times for 5 minutes in TBS-Tween after blocking and antibody incubations. Polyclonal primary antibodies used in Western blot analysis in this study were used at the following dilutions in TBS-Tween: anti-GST (a kind gift from I. J. Sadowski, The University of British Columbia, Canada) at 1:3,000, anti-HIS at 1:5,000 (Santa Cruz), anti-UBC-2 at 1:5,000 (prepared by T.A. Stevens), antiDSK2 (a kind gift from M . Rose, Princeton University, U.S.A.), and anti-ubiquitin at 1:500 (Santa Cruz).  A 1:10,000 dilution of peroxidase-labelled donkey anti-rabbit  antibody (Amersham Pharmacia Biotech) was used for secondary antibody incubation.  26  The enhanced chemiluminescence kit (ECL  -Amersham Pharmacia Biotech) was used  to visualize the protein-antibody complexes.  6.8. Coprecipitation of UBC-2 and DSK-2* Ten pi or 30 pi of radiolabeled DSK-2* synthesized in a T N T kit was incubated with 0.5 pg C-terminal 6XHIS-UBC-2 in 200 pi UBC-2 buffer (50 m M N a H P 0 , 300 2  4  mM NaCl, 0.1% Triton X-100, pH 8.0) on a Nutator (Clay Adams® Brand) for 1.5 hours at 4°C. As a negative control to demonstrate the absence of non-specific interactions between DSK-2 and the resin, 10 pi DSK-2* was incubated with TALON® resin under the same reaction conditions.  As a positive control to demonstrate that N-terminal  6XHIS-UBC-2 was interacting with the TALON® resin, 0.5 pg C-terminal 6XHIS-UBC2 was incubated with TALON® resin under the same reaction conditions. Twenty pi of 50% TALON® resin in UBC-2 buffer was then added for 1 hour. Reactions were pelleted at 2,000 rpm in a microcentrifuge. Twenty pi of the supernatant was saved for analysis. TALON® resin pellets were washed twice in UBC-2 buffer before resuspension in 50 pi LSB. The supernatant and pellet fractions were divided and analyzed by autoradiography to detect DSK-2* and Western blotting to detect UBC-2. 6.9. Coprecipitation of UBC-2 and DSK-2 fusion proteins Seven pg N-terminal 6XHIS-UBC-2 or C-terminal 6XHIS-UBC-2 was incubated for two hours on a Nutator at 4 C with 40 pi 50% TALON® resin and 30 pg GST::DSK-2 in 200 pi of UBC-2 buffer supplemented with 1% Triton X-100 and 10 m M imidazole. Reactions were pelleted at 2,000 rpm in a microcentrifuge and pellets were washed three times in UBC-2 buffer. The same amounts of the UBC-2 fusion proteins and GST::DSK2 were incubated individually with the TALON® resin to demonstrate, respectively, positive and negative interaction of these proteins with the resin under co-precipitation reaction conditions.  Twenty pi of the supernatant, 20 pi of the wash, and the pellet  fractions of the co-precipitation experiments were separated on 10% and 12.5% SDSP A G E gels and respectively analyzed via Western blotting with anti-GST and anti-UBC2 primary antibodies.  27  6.10. Cross-linking UBC-2 and DSK-2 fusion proteins Three pg GST::DSK-2 (10 pi) and 0.2 pg C-terminal 6XHIS-UBC-2 (10 pi) were incubated together for 1 hour at room temperature followed by one hour on ice. BS (Pierce) cross-linker was added to a final concentration of 1 mM. Cross-linking reactions were quenched with 100 m M Tris-HCl pH 7.5 after 3, 5, 10, 15, 20, 25, and 30 minutes. As a control, three pg GST::DSK-2 were incubated in 10 pi UBC-2 buffer with crosslinker. Reactions were divided and subjected to Western blot analysis using anti-GST or anti-UBC-2 primary antibodies. 6.11. Coprecipitation of multi-ubiquitin and GST::DSK-2 Forty pi of a 50% slurry of TALON® resin was incubated with 2 pg polyubiquitin chains (Ub -7) (Affiniti) and 7 pg C-terminal 6XHIS-UBC-2 in 200 pi UBC-2 2  buffer supplemented with 0.5% Triton X-100 for 2 hours on a Nutator at 4 C. Forty pi of a 50% slurry of glutathione agarose was incubated with 2 pg polyubiquitin chains and 30 pg GST::DSK-2 in 200 pi UBC-2 buffer supplemented with 0.5% Triton X-100 for 2 hours on a Nutator at 4 C.  Negative control precipitation reactions involved the  incubation of polyubiquitin and GST::DSK-2 individually with TALON® resin. Positive control precipitation reactions involved the incubation of C-terminal 6XHIS-UBC-2 with TALON® resin and GST::DSK-2 with glutathione agarose.  Reactions were pelleted at  2,000 rpm in a microcentrifuge and washed three times in UBC-2 buffer. Twenty pi of the supernatant, 20 pi of the wash, and the pellet fractions of the co-precipitation experiments were analyzed via SDS-PAGE and Western blotting with anti-GST or antiUBC-2 primary antibodies.  7. R N A interference 7.1.Overexpression of double stranded R N A cDNAs with Xhol site cohesive ends, isolated from the Robert Barstead C. elegans cDNA plasmid library in the screen for UBC-2 interacting proteins, were cloned nondirectionally into the Timmons and Fire RNAi feeding vector (L4440) (Timmons and Fire, 1998). L4440 is an altered pBluescript vector with T7 promoters on either side of the polylinker region.  The following cDNAs were cloned into L4440: B0432.9,  28  C25D7.8, F15C11.2, R160.7, W02A11.3, mca-1, ubc-2, and ubiA. The rad-23 cDNA, a homologue of human Rad23, was cloned from yk81e3 (a kind gift from Y . Kohara, National Institute of Genetics, Japan) into t h e c a l and Kpnl sites of L4440. Constructs were transformed into HT115(DE3) E. coli, which lacks RNase HI activity and exhibits D?TG inducible T7 polymerase activity, and grown overnight on LB plates containing 100 pg/ml ampicillin.  Freshly transformed colonies were used to  inoculate 5 ml of LB containing 100 pg/ml ampicillin. Cultures grown overnight at 37°C for 8 to 16 hours were pelleted at 5,000 rpm in a microcentrifuge, resuspended in 100 pi and used to seed N G M plates supplemented with 25 pg/ml carbenicillin and ImM IPTG. Plates were left overnight at room temperature to induce T7 polymerase activity. L4 stage hermaphrodites were placed on these R N A i treatment plates for 72 hours at 15°C. Subsequently, treated nematodes were transferred to a second R N A i treatment plate for 24 hours at 15°C, after which adults were removed and the phenotypes of the progeny were recorded [Ahringer Lab R N A i Feeding Protocol (Version 10.8.00)] (Timmons and Fire, 1998).  7.2. Scoring phenotypes of RNAi treated nematodes The number of progeny per RNAi treated nematode (fecundity) and the average length of R N A i treated progeny was recorded. To measure the progeny, approximately 10 nematodes were picked from each RNAi treatment plate into 5 drops of M9 buffer (22mM K H P 0 , 22mM N a H P 0 , 85mM NaCl, ImM MgS0 ) on a slide. The slides 2  4  2  4  4  were heated for 30 seconds on a hot plate at 100  C. This treatment effectively killed the  worms and straightened their bodies enabling more accurate body length measurement. Nematodes were measured at 25X magnification using an ocular micrometer.  The  measurements were converted to millimeter units using a conversion factor 1 unit=0.4 mm. A Zeiss microscope was used to analyze and photograph R N A i treated worms.  29  LTJ. RESULTS A N D DISCUSSION  l.a. The yeast two-hybrid screen to identify interacting proteins To identify protein partners for UBC-2 in C. elegans, a yeast two-hybrid screen was employed. This method, performed in yeast strain Y190, involved expressing a bait protein consisting of the D N A binding domain (DBD) of the GAL4 transcription factor fused to UBC-2, and expressing a library of prey proteins consisting of C. elegans library proteins fused to the activation domain (AD) of the GAL4 transcription factor (Figure 2). The key idea of the yeast two-hybrid screen is that if a protein, such as UBC-2, interacts with a particular library protein, the GAL4 transcription factor will, be reconstituted and allow transcription of the yeast strain reporter genes under GAL4 transcriptional control, such as his3 and lacL in Y190 (Fields and Song, 1989).  Yeast colonies containing  interacting proteins grew on synthetic complete media lacking histidine and turned blue in the presence of X-gal. Prior to initiating the screen, it was determined that the bait protein was expressed in yeast strain Y190, and that it was the appropriate size (Figure 3). Various control tests were performed to confirm that neither the bait plasmid pAS2::ubc-2 nor the vectors pAS2 or pACT alone or in combination, caused autoactivation of the reporter genes (Table 1). It was determined that supplementation of the synthetic complete medium lacking histidine with 25 m M 3-aminotriazole (3-AT) was sufficient to prevent growth of colonies transformed with the pAS2: :ubc-2 construct. Colonies transformed with pAS2 and pACT together unexpectedly turned blue in the X gal filter lift assay probably due to leakiness of the lacZ reporter gene.  It has been  reported that once sequences are cloned into the pAS2 plasmid, this weak activation disappears, possibly due to sequences beyond the polylinker that are of no consequence once cDNAs are cloned in it (Elledge, 1993). In addition, positive control tests were performed to ensure that reporter gene activation could be detected using the known interacting GAL4DBD fusion proteins, SNF4 (encoded by plasmid pSEl 111) and SNF1 (encoded by plasmid pSEl 112).  Yeast  colonies containing both of these interacting proteins grew on synthetic complete media  30  Figure 2. The  yeast two-hybrid assay for interacting proteins  The yeast two-hybrid assay involved making a bait protein consisting of the D N A binding domain (DBD) of the G A L 4 transcription factor fused to UBC-2 and making a library of prey proteins consisting of C. elegans library proteins fused to the activation domain (AD) of the G A L 4 transcription factor. If UBC-2 interacts with a particular library protein, the G A L 4 transcription factor will be reconstituted and allow transcription of the yeast strain Y190 reporter genes, his3 and lacZ. Yeast colonies containing interacting proteins grow on synthetic complete medium lacking histidine and turn blue in the presence of X-gal.  Bait protein  UAS  HIS3  lacZ + Library of prey proteins  UAS  HIS3  lacZ  (Adapted from Fields and Bartel, 1997)  31  Figure 3. GAL4DBD:: UBC-2 fusion protein is expressed in yeast strain Y190 Yeast extract was prepared from cultures of yeast strains Y190 and Y190 transformed with the yeast two-hybrid bait encoding plasmid pAS2::ubc-2. Proteins were separated by electrophoresis on a 12.5% SDS-PAGE gel and subjected to Western blot analysis using anti-UBC-2 antibody (1:5,000). Note the higher molecular weight band in the Y190 extract + GAL4DBD::UBC-2 (lane 3) which likely corresponds to the 34 kDa bait fusion protein, GAL4DBD::UBC-2 The latter was used in the yeast two-hybrid screen for interacting proteins of the pACT-RB2 C. elegans c D N A library. To ensure the antibody could detect the bait protein, C-terminal 6XHISUBC-2 (23 kDa) was included as a positive control (lane 1). Endogenous UBC4/5 in yeast strain Y190 was detected by anti-UBC-2 antibody as seen in the lower molecular weight band in both Y190 extracts (lanes. 2 and 3)  kDa 43  GAL4DBD::UBC-2  29 18 -+-UBC4  14 6 3 1  2  3  32  lacking leucine and tryptophan to select for the vectors' nutrient marker and to select for activation of the protein interaction reporter genes his 3 and lacZ, by virture of their ability to grow in the absence of histidine and turn blue in the presence of X-gal (Table 1)-  To ensure that the C. elegans library was sufficiently screened for proteins that interact with UBC-2, it was necessary to screen at least 600,000 double transformants containing both the bait and prey proteins. In total, 980,000 double transformants were screened for activation of the his3 and lacL reporter genes. expressing interacting proteins were isolated.  Initially, 56 colonies  From the putative positive colonies,  plasmids encoding putative UBC-2 interacting proteins were separated from plasmids encoding the bait protein. This was accomplished through selection solely for the prey encoding plasmids, resulting in eventual loss of the bait encoding plasmid. Additionally, cycloheximide sensitivity selection against colonies hosting plasmids encoding the bait protein was employed.  Subsequently, the prey encoding plasmids were electroporated  into and amplified in Escherichia coli.  2. Yeast two-hybrid false positive elimination Four tests were employed to eliminate false positive interactors of UBC-2 (Table 1). The first test involved transforming yeast strain Y190 with pACT::cDNA plasmids encoding putative UBC-2 interacting G A L 4 A D fusion proteins.  Colonies expressing  putative interacting prey proteins grew on synthetic complete media lacking leucine to select for the prey encoding plasmid, pACT, but did not grow in the absence of histidine with 25 m M 3-AT present or turn blue in the X-gal filter lift assay. Therefore, it was concluded that putative UBC-2 interacting proteins do not independently activate the reporter genes lacL and his3 in the yeast two-hybrid system. The second test involved reconfirming the interaction between the putative U B C 2 interacting G A L 4 A D fusion proteins and the GAL4DBD::UBC-2 fusion protein. This test involved double transforming yeast strain Y190 with the pAS2::ubc-2 plasmid and one of the pACT::cDNA plasmids and selecting for activation of the reporter genes. Colonies expressing the bait protein and a putative UBC-2 interacting prey protein grew on synthetic complete medium lacking tryptophan and leucine to select for the bait  33  Table 1. Yeast Two-Hybrid Assay Control Tests Positive X-gal Assay  No  Growth on -TRP -LEU -HIS + 25 m M 3-AT n.d.  No  No  No  n.d.  No  Yes  No  No  n.d.  No  n.d.  n.d.  Yes  n.d.  No  Yes*  n.d.  n.d.  Yes  n.d.  No  No  No  Yes  No  No  n.d.  No  pSE1112  Yes  No  No  No  n.d.  No  pSEllll + pSE1112  n.d.  n.d.  Yes  n.d.  Yes  Yes  D N A used for Y190 transformation  Growth on -TRP  Growth on -LEU  Growth on -TRP -LEU  Growth on -HIS + 25 m M 3-AT  pAS2  Yes  No  No  pAS2 ::ubc-2  Yes  No  pACT  No  pAS2 + pACT pAS2: :ubc-2 + pACT pSEllll  * The colonies unexpectedly turned blue in the X-gal filter lift assay probably due to leakiness of the lacZ reporter gene. It has been reported that once sequences are cloned into the pAS2 plasmid, the weak activation appears to go away probably due to sequences beyond the polylinker that are of no consequence once cDNAs are cloned in it (Elledge, 1993).  p S E l l l l =pACT::s«/7 pSE1112=pASl::5«/4 n.d. = no data  34  encoding plasmid, pAS2, and the prey encoding plasmid, pACT. Twenty-nine of these double transformant colonies grew on synthetic complete medium lacking histidine supplemented with 25 m M 3-AT and turned blue in the X-gal filter lift assay. However, a yeast two-hybrid interaction could not be confirmed for the rest of the original pool of 56 putative UBC-2 interacting G A L 4 A D fusion proteins. The third test involved confirming that the putative UBC-2 interacting protein portion of the bait protein, not the GAL4 D N A binding domain portion, mediates the yeast two-hybrid interaction. Yeast strain Y190 was transformed with pAS2 and one of the pACT::cDNA plasmids.  Colonies expressing the GAL4 D N A binding domain  portion and a putative interacting prey protein grew on synthetic complete media lacking tryptophan and leucine to select for the bait encoding plasmid, pAS2, and the prey encoding plasmid, pACT. Doubly transformed colonies did not grow in the absence of histidine indicating that the his3 reporter gene was not activated; however, the colonies did unexpectedly turn blue in the X-gal filter lift assay probably due to leakiness of the lacZ reporter gene. It has been reported that once sequences are cloned into the pACT plasmid, the weak activation appears to be suppressed, probably due to sequences beyond the polylinker that are inactivated once cDNAs are cloned into it (Elledge, 1993). The fourth false positive test involved a yeast mating assay to eliminate putative UBC-2 interactors that bind the GAL4DBD::UBC-2 fusion protein nonspecifically. After mating yeast strain Y187(MATa)/pAS2:\cdk2 or Y187(MATa)/pAS2::s«/7 with yeast strain Y190(MATa)/pACT::cDNA and assaying for activation of the reporter gene lacZ, it was found that the UBC-2 interacting proteins did not bind to the CDK2 or SNF1 GAL4DBD fusion proteins (Tables 2.A. and B.). Therefore, the prey proteins identified in the yeast two-hybrid screen appear to be specific in their interaction with UBC-2. As a positive control for the mating assay, yeast strain Y187(MATa)/pAS2::u6c-2 was mated with yeast strain Y190(MATa)/pACT::cDNA.  The positive results of the X-gal assays  revealed that the mating assay was optimized to replicate most of the yeast two-hybrid interactions  between  the  GAL4DBD::UBC-2  fusion  protein  and  the  putative  GAL4AD::UBC-2 interacting proteins which were discovered in the initial screen.  35  Table 2.A. UBC-2 interacting GAL4AD fusion proteins do not interact with GAL4DBD::CDK2 fusion protein [Y187(MATa) + pAS2::cdk2] X [Y190 (MATa) + pACT::cDNA] cDNA in pACT W02A11.3 F15C11.2 B0432.9  mca-1 R160.7  Positive X-gal Assay  Growth on -TRP, -LEU Yes Yes Yes Yes Yes  No No No No No  Table 2.B. UBC-2 interacting GAL4AD fusion proteins do not interact with GAL4DBD SNF1 fusion protein [Y187(MATa) + pAS2::«i/l] X [Y190 (MATa) + pACTv.cDNA] cDNA in pACT W02A11.3 F15C11.2 B0432.9  mca-1 R160.7  Positive X-gal Assay  Growth on -TRP, -LEU Yes Yes Yes Yes Yes  No No No No No  36  3. Proteins that exhibit a yeast-two hybrid interaction with UBC-2 Plasmids (pACT::cDNA) encoding putative UBC-2 interactors were analyzed by restriction digests, sequencing, and BLAST.  In conclusion, six different proteins that  interact with UBC-2 were identified using the yeast-two hybrid assay. It was determined that UBC-2 interacts with the following proteins of interest: ubiquitin (encoded by UbiA); a ubiquitin-like protein encoded by F15C11.2 hereafter referred to as DSK-2 due to its similarity to S. cerevisiae DSK2; two RING finger proteins (encoded by B0432.9 and W02A11.3), a calcium ATPase (encoded by mca-\), and a F Y V E finger protein (encoded by R160.7) (Figure 4).  Four clones of the 29 clones encoding UBC-2  interacting proteins identified in the yeast two-hybrid screen, which passed false positive testing, did not exhibit homology to any known gene sequences. A protein encoded by the C25D7.8 gene and ubiquitin represented 1 and 3 clones, respectively, of the 29 clones encoding UBC-2 interacting proteins identified in the yeast two-hybrid screen which passed false positive testing.  A search for protein domains using the Simple Modular  Architecture Research Tool (SMART) revealed that the amino acid sequence of the protein encoded by C25D7.8 does not exhibit any previously characterized domains (Schultz et al, 1998; Schultz et al, 2000). The interaction between UBC-2 and ubiquitin was expected due to a previous experiment by M . Zhen which showed that UBC-2 can form a thiolester bond with ubiquitin (Zhen, Ph.D. thesis, 1995).  3.1. UBC-2 interacts with a plasma membrane calcium ATPase A plasma membrane calcium ATPase with 10 alpha-helical transmembrane domains, exhibits a yeast two-hybrid interaction with C. elegans UBC-2. MCA-1 represented 1 of 29 clones encoding UBC-2 interacting proteins identified in the yeast two-hybrid screen which passed false positive testing.  A search for protein domains  using the Simple Modular Architecture Research Tool (SMART) reveals that the 1228 amino acid sequence of MCA-1 contains ten transmembrane domains from residues 99116, 155-177, 357-379, 399-421, 856-878, 888-905, 935-957, 972-989, 1010-1032, and 1037-1059 (Schultz et al, 1998; Schultz et al, 2000).  A WormPD database search  reveals homologues of C. elegans MCA-1 in other organisms including: S. cerevisiae (the calcium ATPase Pmclp is 35% similar to MCA-1); Mus musculus (the calcium ATPase  37  Figure 4. Predicted domains of C. elegans proteins identified in the yeast two-hybrid screen proteins that interact with UBC-2 Gene/Size/  Predicted domains  Number of clones identified in the yeast two-hybrid screen  F15C11.2 (DSK-2); 60 kDa; 17  RING W02A11.3; 59 kDa; 1  RING  B0432.9; 51 kDa; 1  M M  Q  M  M  M M  \j  \j  mca-1; 147 kDa; 1  t  \j  \j  transmembrane domain  R160.7;  FYVE  79 kDa; 1  38  ATP2B2 is 55% similar to MCA-1), and H. sapiens (the calcium ATPases ATP2B1, ATP2B2, ATP2B3 are all 55% similar to MCA-1) (Bateman et al,  1999; Genome  sequence of the nematode C. elegans: a platform for investigating biology, 1998). Calcium-ATPases in eukaryotes pump calcium outside of the cell coupled with ATP hydrolysis. Previous analysis of this protein called MCA-1 in C. elegans revealed that it is a functional calcium ATPase, it has a serine-rich stretch of 11 residues between the 2  nd  and 3  rd  transmembrane domains and it interacts with human calmodulin through a  C-terminal calmodulin-binding domain (Kraev et al., 1999). Perhaps monoubiquitylation of calmodulin mediated by UBC4, albeit demonstrated only in vitro, (Parag et al., 1993) is enabled through interaction of UBC4 with M C A - 1 . In S. pombe, calmodulin is a calcium binding protein that localizes to the spindle pole body (SPB), stimulates plasma membrane calcium ATPases, and is required for chromosome segregation (Moser et al., 1997). Due to the fact that MCA-1 can bind calmodulin, it is possible that MCA-1 may be associated in some way with the centrosome.  Recently,  it has been determined that calcium, calmodulin, and  calcium/calmodulin-dependent protein kinase II (CaMKJI) are necessary for the first duplication of the centrosome in Xenopus (Matsumoto and Mailer, 2002). CaMKJI is activated by calcium to stimulate cyclin B degradation via the A P C to allow exit from mitosis (Lorca et al., 1993).  Since a sudden increase in calcium is required for  centrosome duplication at the Gi-S transition of the cell cycle (Matsumoto and Mailer, 2002), the maintenance of low levels of intracellular calcium, possibly through export by a calcium ATPase such as M C A - 1 , may provide a link between duplication of the centrosome and MCA-1 function. It is intriguing to note that two detected interactors of UBC-2, DSK-2 (discussed below), and M C A - 1 , have homologues in other organisms that are linked to duplication of microtubule organizing centres such as the SPB in yeast and the centrosome in metazoans.  3.2. UBC-2 interacts with a F Y Y E finger domain protein A F Y V E finger domain protein called R160.7 exhibits a yeast two-hybrid interaction with UBC-2. R160.7 represented 1 of 29 clones encoding UBC-2 interacting  39  proteins identified in the yeast two-hybrid screen which passed false positive testing. A search for protein domains using S M A R T revealed that the 658 amino acid sequence of R160.7 includes a F Y V E domain from residues 561 to 616 (Schultz et al, 1998; Schultz et al, 2000). A WormPD database search revealed homologues of C. elegans R160.7 in other organisms including: S. cerevisiae (Fablp is 50% similar to R160.7); M. musculus (PDCFYVE is 58% similar to R160.7), and H. sapiens (KIAA0993 is 58% similar to R160.7) (Bateman et al., 1999; Genome sequence of the nematode C. elegans: a platform for investigating biology, 1998). The F Y V E finger domain is a cysteine-rich conserved sequence that has a demonstrated ability to bind zinc and the membrane lipid phosphatidylinositol-3-phosphate (PtdIns(3)P) (Gaullier et al,  1998; Stenmark et al,  1996).  3.3. UBC-2 interacts with RING finger proteins The B0432.9 and W02A11.3 genes encode RING finger proteins that exhibit yeast two-hybrid interactions with UBC-2. B0432.9 and W02A11.3 each represented 1 of 29 clones encoding UBC-2 interacting proteins identified in the yeast two-hybrid screen which passed false positive testing. Interestingly, many E3s contain RING finger domains.  It seems plausible that UBC-2 can interact with RING proteins since, as  mentioned previously, RING finger domains in E3s appear to be necessary for mediating interaction with E2s and for ubiquitylation of substrates (reviewed in Joazeiro and Weissman, 2000; Lorick et al, 1999). A search for protein domains using S M A R T revealed that the 425 amino acid sequence of the B0432.9 protein includes a RING domain from residues 192 to 232 and that the 489 amino acid sequence of the W02A11.3 protein includes a RING domain from residues 433 to 473 (Schultz et al,  1998; Schultz et al, 2000).  A WormPD  database search revealed that S. cerevisiae Hrdlp is 27% similar to C. elegans B0432.9 and that S. cerevisiae A p c l l p and Sanlp are respectively 34% and 49% similar to C. elegans W02A11.3 (Bateman et al, 1999; Genome sequence of the nematode C. elegans: a platform for investigating biology, 1998). Interestingly, Hrdlp/Der3p in S. cerevisiae is an E3 which catalyzes ubiquitylation and endoplasmic reticulum-associated degradation (ERAD) of misfolded lumenal and membrane proteins in association with its interacting  40  E2s, UBC1 and UBC7 (Bays et al, 2001; Boradallo, 1998). Since UBC-2 interacts with a protein similar to Hrdlp, it is possible that UBC-2 may also play a previously unrecognized role in the E R A D of misfolded proteins. Furthermore, the RING-H2 finger domain of A P C 11 was determined to be necessary for E3 activity in the ubiquitylation of cyclin B to allow exit from mitosis and for interaction with the ubiquitin conjugating enzyme UBC4 in S. cerevisiae (Gmachl et al, 2000; Leverson et al, 2000). Since an in vitro interaction between A P C 11 and UBC4 has been demonstrated, it is plausible that UBC-2 exhibits a yeast two-hybrid interaction with an A P C 11-like protein to promote progression of the cell cycle.  Nevertheless, it should be noted that B0432.9 and  W02A11.3 belong to the RJNG-HC class while their respective counterparts Hrdlp and A P C 11 in S. cervisiae belong to the RING-H2 class. Whether these differences preclude the involvement of these UBC-2 interacting RING finger proteins in E R A D or cyclin B degradation remains to be determined.  3.4. UBC-2 interacts with an ubiquitin-domain protein An ubiquitin-domain protein was identified in the yeast two-hybrid screen for UBC-2 interacting proteins. This C. elegans protein encoded by F15C11.2 was given the name DSK-2 due to a 32% similarity to S. cerevisiae DSK2 (dominant suppressor of karl).  DSK-2 represented 17 of the 29 clones encoding UBC-2 interacting proteins  identified in the yeast two-hybrid screen which passed false positive testing. A search for protein domains using S M A R T revealed that the 502 amino acid sequence of DSK-2 includes an ubiquitin-like domain from residues 8 to 79 and an ubiquitin associated domain from residues 462 to 500 (Schultz et al, 1998; Schultz et al, 2000). A WormPD database search revealed homologues of C. elegans DSK-2 in other organisms including: S. cerevisiae (DSK2 is 32% similar to DSK-2); S. pombe (Dphlp is 23% similar to DSK2), M. Musculus (PLIC2 is 35% similar to DSK-2), and H. sapiens (UBQLN3 is 29% similar to DSK-2) (Bateman et al, 1999; Genome sequence of the nematode C. elegans: a platform for investigating biology, 1998). In & cerevisiae, the requirement of RAD23 and DSK2 for spindle pole body (SPB) duplication potentially links the ubiquitinproteasome pathway to processes involved in cell cycle progression (Biggins et 1996).  al,  It is interesting to note that in vivo expression of UBQLN3, the human  41  homologue of DSK-2, is restricted to the testis, as are UBC4-2 and UBC4-testis (Rajapurohitam et al, 1999; Wing and Jain, 1995; Wing et al, 1996), rat homologues of UBC-2.  The co-expression of DSK-2 and UBC-2 homologues in mammals provides  further support for the possibility of an in vivo interaction between UBC-2 and DSK-2. DSK-2 is a member of the ubiquitin-domain protein (UDP) class which is characterized by a ubiquitin-like domain and an inability to covalently bind other proteins through their C-termini, unlike the ubiquitin-like protein (UBL) class which exhibit this capability (reviewed in Jentsch and Pyrowolakis. 2000). Jentsch and Pyrowolakis have classified the following proteins as members of UDP class: S. cerevisiae RAD23, UBP6, and DSK2; Xenopus XDRP1 and Scythe; and mammalian HHR23A, HHR23B, PLIC1, PLIC2/Chapl, BAG-1L, BAG-IS, BAT3/Chap2, Parkin, UTP28/RBCK1, Elongin B, and Gdx. Ubiquitin-domain proteins demonstrate an ability to physically interact with components of the ubiquitin pathway.  For example, S. cerevisiae DSK2 and RAD23,  human BAG-IS, hHR23a, hHR23b, hPLIC-1 and hPLIC-2 can associate with the proteasome (Hiyami et al., 1999; Funakoshi et al., 2002; Liiders et al., 2000; Kleijnen et al., 2000; Schauber et al., 1998). In particular, hHR23a, hHR23b, and hPLIC-2 exhibit a specific interaction with the S5a subunit of the 19S regulatory complex of the proteasome (Hiyami et al,  1999; Walters et al,  2002).  Furthermore, hPLIC-2 interacts with  ubiquitin ligase E6-AP and associates with a large complex containing the proteasome and  E6-AP,  indicating the  possibility  that  ubiquitin-domain proteins  link  the  ubiquitylation machinery with the proteasome (Leijnen et al, 2000). Interestingly, the association of a molecular chaperone cofactor UDP called BAG-IS with the proteasome promotes the interaction of heat shock inducible Hsp70 and constitutively  expressed  Hsc70 molecular chaperones with the proteasome,  thus,  providing a relationship between protein degradation and folding processes (Liiders et al, 2000). It is intriguing to note that there is also a relationship between protein folding, protein degradation, and eukaryotic microtubule organizing centres in that the molecular chaperones Hsp70 and Hsp90 and active proteasomes associate with the centrosome, a homologue of the SPB in budding yeast, which is thought to serve a scaffolding function for protein degradation and folding (Fabunmi et al, 1999; Wigley et al, 1999).  42  Since budding yeast DSK2 is involved in SPB duplication and can bind the proteasome, perhaps DSK2 is responsible for recruiting the proteasome to the SPB, possibly through a direct or indirect interaction between DSK2 and the SPB that has not yet been demonstrated. Although an in vitro interaction between DSK2 and CDC31 has not been proven, DSK2 expression is required for proper localization of CDC31 to the SPB, perhaps indicating that DSK2 and CDC31 may interact transiently (Biggins et al, 1996) or require other factors for their interaction.  It is possible that proteasomes  localized at the SPB, perhaps through DSK2, may be involved in the degradation of specific proteins to allow SPB separation after SPB duplication. In disagreement with this idea, budding yeast lacking both DSK2 and RAD23 exhibit a defect in SPB duplication rather than separation, although, the SPBs were often enlarged in this double mutant strain (Biggins et al., 1996). Some UDPs exhibit an U B A (ubiquitin associated) domain in addition to an ubiquitin-like domain.  The U B A domain appears to function in promoting the  association of UDPs with multiubiquitin chains and possibly in stabilizing substrates of the ubiquitin pathway.  Proteins containing U B A domains which bind multiubiquitin  chains include: S. cerevisiae DSK2 and RAD23; S. pombe homologues of S. cerevisiae RAD23 and DSK2; and a protein encoded by the mudl gene in S. pombe (Chen et al., +  2001; Funakoshi et al., 2002; Wilkinson et al., 2001). In particular, the U B A domains of RAD23, DDI1, and DSK2 appear to selectively bind lysyl-48 and lysyl-29 linked mulitubiquitin chains and preferentially bind multiubiquitin chains at least four ubiquitin moieties in length over monoubiquitin (Rao and Sastry, 2002).  In vitro and in vivo  experimentation has revealed that RAD23 inhibits the assembly of multiubiquitin chains on substrates (Chen et al., 2001; Ortolan et al., 2000). Furthermore, a U B A containing UDP called ubiquilin, which localizes to neurofibrillary tangles and Lewy bodies in brains affected with Parkinson's disease and Alzheimer's disease (AD), interacts with and promotes the stability of proteins presenilin-1 and presenilin-2 which are implicated in the development of early-onset A D (Mah et al., 2000). Intriguingly, Xenopus XDRP1, a UDP homologue of S. cerevisiae DSK2, binds to and prevents the calcium induced degradation of cyclin A through its ubiquitin-like domain (Funakoshi et al., 1999). These  43  examples suggest the possibility that U B A domains UDPs inhibit degradation of certain substrates via the ubiquitin pathway. In contrast, the U B A domains of RAD23 and DSK2 are also required for promoting the degradation of substrates of the ubiquitin fusion degradation (UFD) pathway (Rao and Sastry, 2002). It is possible that UDP proteins which contain U B A domains bind multiubiquitin chains through their U B A domain to prevent chain degradation by deubiquitylating enzymes, and function as an adapter to link ubiquitylated substrate proteins to the proteasome (Rao and Sastry, 2002).  It is possible that the  preference of RAD23, DDI1, and DSK2 for lysyl-48 and lysyl-29 linked multiubiquitin chains provides a means for accomplishing selective protein degradation by the proteasome (Rao and Sastry, 2002).  Since UBA-containing UDP protein RAD23  interacts with D N A repair proteins and the proteasome, it has been suggested that the UBA-containing UDP protein DSK2 may link the proteasome to another cellular role such as cell cycle progression in the same manner that RAD23 links protein degradation to D N A repair by virtue of separate domains mediating separate protein-protein interactions in the same protein (Rao and Sastry, 2002). The fact that DSK2 is involved in SPB duplication (Biggins et al, 1996), binds the proteasome (Funakoshi et al, 2002), and binds multiubiquitin chains may indeed indicate that DSK2 functions in linking a progression of events leading to cell division with protein degradation. U B A domains have also demonstrated an ability to promote protein-protein interactions. For instance, the U B A domain mediates interaction of RAD23 with itself; interaction of RAD23 and DDI1; and interaction of RAD23 and DSK2 (Bertolaet et al, 2001).  Since dimerization of RAD23 abrogates the interaction of RAD23 with  multiubiquitin (Bertolaet et al, 2001), Bertolaet et al. have suggested that dimerization may be a means to eliminate the ability of RAD23 to prevent substrate degradation by interacting with multiubiquitin (Bertolaet et al, 2001; Ortolan et al, 2000). It is thought that U B A domains are prevented from binding multiubiquitin when they are involved in mediating protein-protein interactions (Bertolaet et al,  2001).  It remains to be  determined if dimerization of other U B A domain proteins eliminates their ability to bind multiubiquitin.  44  Interestingly, the ability of an ubiquitin-domain protein to bind an ubiquitin conjugating enzyme has not been previously demonstrated. It appears that the interaction of the ubiquitin-domain protein, DSK-2, with the ubiquitin conjugating enzyme, UBC-2, in C. elegans is a novel result which provides further evidence for the involvement of ubiquitin-domain proteins in the ubiquitin pathway. It remains to be determined if the U B A domain, U B L domain, or another region of DSK-2 mediates interaction with UBC-2. Further experimentation could also determine whether or not DSK-2 binds the proteasome and is involved in centrosome duplication in C. elegans.  Since DSK-2  represented 60% of the UBC-2 interacting proteins identified by the yeast-two hybrid screen, further effort was concentrated on studying this interaction in vitro.  4. Confirming the interaction of UBC-2 with DSK-2 in vitro 4.1. Attempt to coprecipitate radiolabeled DSK-2 with 6XHIS-UBC-2 Radiolabeled DSK-2 (DSK-2*) synthesized in a T N T kit was incubated with 6XHIS-UBC-2.  Control tests confirmed that 6XHIS-UBC-2 binds TALON® resin  (Figure 5.A. lane 4) and that DSK-2* alone does not bind TALON® resin (Figure 5.B. lane 3). Due to the fact that DSK-2* did not precipitate in the presence of 6XHIS-UBC-2 and TALON® resin, it appeared that DSK-2* could not bind 6XHIS-UBC-2.  The  possibility existed that conditions such as pH, salt concentration, temperature, time of incubation, and/or the presence of additional factors or modifications of the proteins normally present in vivo were not favorable to promote the interaction of DSK-2* and 6XHIS-UBC-2.  Interestingly, a reduction in binding of 6XHIS-UBC-2 to the TALON®  resin was observed in the presence of increasing amounts of DSK-2* (Figure 5.B. lanes 5 and 7). It was considered possible that the binding of DSK-2* to 6XHIS-UBC-2 might prevent TALON® resin from binding to the C-terminal 6XHIS-tag. For this reason, a N terminal 6XHIS-UBC-2 fusion protein was prepared and tested (Section 4.5, 4.6, and 4.7) in an attempt to prove an in vitro interaction between UBC-2 and DSK-2.  It was also  considered possible that a component of the TNT kit was preventing either the interaction of C-terminal 6XHIS-UBC-2 with the TALON® resin or the interaction of UBC-2 with DSK-2. To avoid this possibility, a different tagged version of DSK-2, a GST::DSK-2 fusion, was constructed and tested.  45  Figure 5. Attempt to coprecipitate radiolabeled DSK-2 with C-terminal 6XHIS-UBC-2 Radiolabeled DSK-2* synthesized in a TNT® Quick Coupled Transcription/Translation System Quick Master Mix was incubated with C-terminal 6XHIS-UBC-2 in UBC-2 buffer on a Nutator for 1.5 hours at 4°C. TALON® resin was added to this mixture for 1 hour. Resin pellets were isolated and washed in UBC-2 buffer. The pellet (P) and supernatant (S) fractions were separated on 12.5% SDS-PAGE gels, and analyzed by Western blotting to detect UBC-2 and autoradiography to detect DSK-2*. A. C-terminal 6XHIS-UBC-2 precipitates with TALON® resin less efficiently in the presence of increasing amounts of DSK-2* Western blot analysis [anti-HIS antibody (1:5,000)] showed that 23 kDa C-terminal 6XHIS-UBC2 binds TALON® resin efficiently. It was present in the P fraction (lane 3) but absent in the S fraction (lane 4). In the presence of radiolabeled DSK-2* (10 pi and 30 pi), less C-terminal 6XHIS-UBC-2 precipitated with TALON® resin as seen in the P fractions (lanes 5 and 7). C-6XHIS UBC-2  +  -  TNT DSK-2*  .  .  TNT*  +  .  +  +  +  +  +  kDa  P  S  P  S  P  S  <4—  C-6XHIS-UBC-2  1 2 3 4 5 6 7 8 B. DSK-2* does not precipitate in the presence of C-terminal 6XHIS-UBC-2 and TALON® resin An autoradiograph to detect S-labeled DSK-2* showed that 60 kDa DSK-2* was produced at the correct size in the TNT® Quick Master Mix (lane 1). DSK-2* was not found in the TALON® resin P fractions in the absence or presence of C-terminal 6XHIS-UBC-2 and TALON® resin (lanes 3,5, and 7). 35  C-6XHIS-UBC-2  TNT DSK-2* TNT*  .  .  .  + . - +  1  2  +  3  4  46  5  +  +  +  +  6  7  8  4.2. Expression and Purification of GST::DSK-2 A Xhol fragment which contained the entire open reading frame of dsk-2 was isolated from a pACT::cDNA plasmid and subcloned directly into pGEX-4T-2 (Pharmacia Biotech). This fragment, encoding a 60.3 kDa protein, was inserted in-frame with a sequence encoding a 26 kDa N-terminal glutathione S-transferase (GST) tag. After transforming this construct into E. coli BL21 (DE3) STAR (Invitrogen), a culture was grown to an OD oo of 0.4 and expression of the 86.3 kDa protein was induced with 6  the addition of 1 m M LPTG. GST::DSK-2 protein was batch purified using glutathione agarose  after determining that the fusion protein could be expressed in soluble form.  Purified fusion protein migrated according to the predicted size on a Coomassie stained SDS-PAGE gel (Figure 6 lane 8). A 2 L E. coli culture yielded 9.3 mg of fusion protein.  4.3. Confirming the identity of GST::DSK-2 fusion protein In addition to sequencing the pGEX-4T-2::dsk-2 construct to confirm the reading frame, I carried out thrombin protease cleavage and Western blot analysis to confirm the identity of the expressed and purified fusion protein.  Thrombin was successful in  removing the GST tag from the fusion protein (Figure 7.A.). The GST tag permitted facile purification of the protein and the ability to eliminate this tag could prove useful in the production and purification of antibody against DSK-2 in the future.  Since an  antibody against C. elegans DSK-2 was not available for studies of the interaction between DSK-2 and UBC-2, other available antibodies were tested for recognition of GST::DSK-2.  A polyclonal anti-GST antibody and a polyclonal antibody against &  cerevisiae DSK2 both recognized the 86.3 kDa GST::DSK-2 fusion protein following Western blot analysis (Figures 7.B. and C ) .  4.4. Co-precipitation of GST::DSK-2 with an C-terminal 6XHIS-UBC-2 preparation To confirm the yeast two-hybrid interaction of UBC-2 and DSK-2, an in vitro coprecipitation experiment was performed using equimolar amounts of GST::DSK-2 and Cterminal 6XHIS-UBC-2. The reaction buffer, UBC-2 buffer, was supplemented with 1% Triton X-100 to inhibit non-specific protein interactions with the TALON® resin. TALON® resin was pre-incubated with C-terminal 6XHIS-UBC-2 for 1 hour prior to  47  Figure 6. Expression and Purification of GST:: DSK-2 GST::DSK-2 fusion protein expression was induced by the addition of 1 m M IPTG to a culture of of 0.4. Glutathione agarose affinity chromatography was used to purify the fusion protein. Lane 1: total E. coli protein extract before IPTG induction; lane 2: total E. coli protein extract after IPTG induction; lane 3: total E. coli protein extract after sonication; lane 4: insoluble pellet of the post-sonication extract; lane 5: soluble portion of the post-sonication extract; lane 6: supernatant from the glutathione agarose batch incubation with soluble post-sonication extract; lane 7: wash of the glutathione agarose incubated with soluble extract; and lane 8: eluate of the glutathione agarose incubated with soluble extract. Purified GST::DSK-2 migrated in a heavy band that approximated the predicted size of 86 kDa on a Coomassie stained 10% SDS-PAGE gel (arrow, lane 8).  E. coli BL21 (DE3) S T A R grown to an O D  48  6 0 0  Figure 7. Confirming the identity of GST::DSK-2 fusion protein A. Thrombin protease cleavage of GST: DSK-2 (86 kDa) GST::DSK-2 (lane 1) incubation with Thrombin overnight at 22 C produced two bands on a Coomassie stained 10% SDS-PAGE gel (lane 2). These bands approximated the predicted sizes of fragments resulting from a GST::DSK-2 cleavage event: a 60 kDa DSK-2 portion and a 26 kDa GST portion of size similar to unpurified GST in E. coli lysate (lane 3).  1  2  3  B. GST::DSK-2 is recognized by anti-GST antibody 3 pg of GST::DSK-2 was separated on a 10% SDS-PAGE gel and subjected to Western blot analysis using anti-GST antibody (1:3,000 dilution in TBS-Tween). kDa  C. GST::DSK-2 is recognized by anti-DSK2 antibody 3 pg of GST::DSK-2 was separated on a 10% SDS-PAGE gel and subjected to Western blot analysis using anti-DSK2 antibody (1:3,000 dilution in TBS-Tween).  -GST:: DSK-2  49  incubation with GST::DSK-2 overnight on a Nutator at 4 C. The addition of 1% Triton X-100 to the co-precipitation buffer proved to be crucial in preventing non-specific interaction of GST::DSK-2 with the TALON® resin. The detergent concentration was low enough, however, to permit the interaction of GST::DSK-2 with C-terminal 6XHISUBC-2 in vitro.  Analysis of the supernatant, wash and pellet fractions of the co-  precipitation experiments via SDS-PAGE and Western blotting with anti-GST antibody revealed that GST::DSK-2 was found in the pellet fraction only in the presence of C terminal 6XHIS-UBC-2 and TALON® resin (Figure 8).  Thus, this co-precipitation  experiment confirmed the yeast two-hybrid interaction of UBC-2 and DSK-2. However, the in vitro interaction did not seem particularly strong as very little of the input GST::DSK-2 was found to precipitate with 6XHIS-UBC-2 in the presence of TALON® resin (lane 6). This preparation of C-terminal 6XHIS-UBC-2 had been previously frozen. Therefore, newly prepared UBC-2 fusion protein was prepared in the hope of improving the in vitro interaction of 6XHIS-UBC-2 and GST::DSK-2.  4.5. Expression and Purification of UBC-2 The potential negative effect of freezing on native folding of C-terminal 6XHISUBC-2 protein was considered as a possible cause for the weak interaction of UBC-2 with DSK-2 in the co-precipitation experiment.  Therefore, new 6XHIS-UBC-2 was  expressed and purified using the pRSETC-w&c-2 construct as previously described (Zhen, PhD. thesis, 1995). Previously, the possibility was considered that DSK-2* binding to 6XHIS-UBC-2 might occlude TALON® resin binding to the C-terminal 6XHIS tag. For this reason, a Cterminal 6XHIS-UBC-2 fusion protein was prepared in an attempt to prove an in vitro interaction between UBC-2 and DSK-2.  A fragment which contained the entire open  reading frame of ubc-2 was amplified from pRSETC::ubc-2. The restriction digested fragment was cloned into pET28-a (+) in frame with sequence encoding an N-terminal 6XHIS tag.  After transforming this construct into E. coli BL21 (DE3) STAR  (Invitrogen), a culture was induced to express the 86.3 kDa protein with the addition of 1 m M IPTG. 6XHIS-UBC-2 protein was batch purified using TALON® resin. Purified  50  Figure 8. Co-precipitation ofGST::DSK-2 with 6XHIS-UBC-2 Co-precipitation experiments were performed using equimolar amounts of GST::DSK-2 and 6XHIS-UBC-2 in UBC-2 buffer supplemented with 0.1% Triton X-100 in an overnight incubation on a Nutator at 4 C. Co-precipitation #1 included the GST::DSK-2 and TALON® resin while co-precipitation #2 included 6XHIS-UBC-2, GST::DSK-2, and TALON® resin. The supernatant (S), wash (W), and resin pellet (P) fractions of the co-precipitation reactions were separated on a 10% SDS-PAGE gel and analyzed by Western blotting using anti-GST antibody (1:3,000). Lanes 1, 2, and 3 contain the respective S, W, and P fractions of co-precipitation #1. Lanes 4, 5, and 6 contain the respective S, W, and P fractions of co-precipitation #2. GST::DSK2 (arrow) was localized to the TALON® resin P only in the presence of 6XHIS-UBC-2 indicating a positive interaction between GST::DSK-2 and 6XHIS-UBC-2 (lane 6, arrow).  C-6XHIS-UBC-2 + GST::DSK-2  GST::DSK-2  1  2  3  4  5  51  6  fusion protein migrated according to the predicted size, as detected on a Western blot probed with anti-UBC-2 antibody. 4.6. N-terminal 6XHIS-UBC-2 and C-terminal 6XHIS-UBC-2 bind TALON® resin The TALON® resin was incubated with a fresh preparation of either N-terminal 6XHIS tagged UBC-2 or C-terminal 6XHIS tagged UBC-2 in UBC-2 buffer containing 1% Triton X-100 for 2 hours on a Nutator at 4 C. Subsequently, the supernatant, wash and pellet fractions from the UBC-2 incubations with TALON® resin were analyzed by SDS-PAGE and Western blotting with anti-UBC-2 antibody.  Both N-terminal and C -  terminal 6XHIS tagged UBC-2 fusion proteins were precipitated by the TALON® resin (Figure 9.A. and B.).  4.7. Co-precipitation of GST::DSK-2 with fresh protein preparations of N-terminal 6XHIS-UBC-2 and C-terminal 6XHIS-UBC-2 In the place of frozen 6XHIS-UBC-2 prepared by Tracy Stevens, fresh protein preparations of 6XHIS tagged UBC-2 fusion proteins were utilized in a co-precipitation experiment in the hope of promoting a stronger in vitro interaction between UBC-2 with DSK-2. N-terminal 6XHIS tagged UBC-2 or C-terminal 6XHIS-UBC-2 was incubated for two hours on a Nutator at 4 C with TALON® resin and GST: :DSK-2 in UBC-2 buffer supplemented with 1% Triton X-100 and 10 m M imidazole to eliminate non-specific interactions of the proteins with the resin. Equimolar amounts of DSK-2 and UBC-2 fusion protein were used. Analysis of the supernatant, wash and pellet fractions of the co-precipitation experiments  via SDS-PAGE and Western blotting with anti-GST  antibody revealed that GST::DSK-2 is found in the pellet fraction only in the presence of TALON® resin and either N-terminal 6XHIS tagged UBC-2 or C-terminal 6XHIS tagged UBC-2 (Figure 10.A. and B.). Thus, these co-precipitation experiments confirmed the yeast two-hybrid interaction of UBC-2 and DSK-2. GST::DSK-2 degradation, visible in lanes 4 and 6 (versus in lane 1), is likely due to protease activity in the UBC-2 preparations (Figure 10.A. and B.).  52  Figure 9. UBC-2 binds TALON® resin N-terminal 6XHIS-UBC-2 or C-terminal 6XHIS-UBC-2 was incubated for two hours on a Nutator at 4 C with TALON® resin in UBC-2 buffer supplemented with 1% Triton X-100 and 10 m M imidazole to eliminate non-specific protein-resin interactions. Resin pellets were isolated and washed three times. The supernatant (S), wash (W), and pellet (P) fractions of the precipitation reactions were separated on 12.5% SDS-PAGE gels and were subsequently analyzed via Western blotting using anti-UBC-2 antibody (1:5,000).  A. N-terminal 6XHIS-UBC-2 binds TALON® resin 23 kDa N-terminal 6XHIS-UBC-2 is present in the P fraction (lane 3).  TALON  N-6XHIS-UBC-2  2  1  3  B. C-terminal 6XHIS-UBC-2 binds TALON® resin 23 kDa C-terminal 6XHIS-UBC-2 is present in the P fraction (lane 3). TALON  C-6XHIS-UBC-2 1  2  3  53  Figure 10. Co-precipitation of DSK-2 with UBC-2 Equimolar amounts of GST::DSK-2 and N-terminal 6XHIS-UBC-2 or C-terminal 6XHIS-UBC-2 were incubated together for two hours at 4 C on a Nutator. Co-precipitation reaction #1 (lanes 13) included GST::DSK-2 and TALON® resin while co-precipitation reaction #2 (lanes 4-6) included N-terminal or C-terminal 6XHIS-UBC-2, GST::DSK-2, and TALON® resin. The supernatant, wash and resin pellet fractions of the co-precipitation experiments were analyzed via SDS-PAGE and Western blot analysis with anti-GST antibody (1:3,000). Lanes 1, 2, and 3 contain the respective supernatant (S), wash (W), and pellet (P) fractions of co-precipitation #1. Lanes 4, 5, and 6 contain the respective S, W, and P fractions of co-precipitation #2 (N-terminal 6XHIS-UBC-2 in A . and C-terminal 6XHIS-UBC-2 in B.). Fractions of the precipitation reactions were separated on 10% SDS-PAGE gels and were subsequently analyzed via Western blotting using anti-GST antibody (1:3,000).  A. GST::DSK-2 interacts with N-terminal 6XHIS ::UBC-2 bound to TALON® resin GST::DSK-2 (86 kDa) localized to the TALON® resin pellet only in the presence of N-terminal 6XHIS-UBC-2 (P, lane 6).  GST:: DSK-2  N-6XHIS-UBC-2 + GST:: DSK-2  •GST::DSK-2  B. GST::DSK-2 interacts with C-terminal 6XHIS:: UBC-2 bound to TALON® resin GST::DSK-2 (86 kDa) localized to the TALON® resin pellet only in the presence of C-terminal 6XHIS-UBC-2 (P, lane 6).  54  5. GST::DSK-2 self-associates To demonstrate the interaction of UBC-2 and DSK-2 using another in vitro method, an attempt was made to cross-link GST::DSK-2 to C-terminal 6XHIS tagged UBC-2. Proteins were incubated for 1 hour at room temperature followed by one hour on ice before B S cross-linker (Pierce) was added to a final concentration of 1 mM. Cross3  linking reactions were quenched with 100 m M Tris-HCl pH 7.5 at various time points. Cross-linked proteins were analyzed via SDS-PAGE and Western blot analysis using anti-GST or anti-UBC-2 antibody. No difference was observed in the protein banding pattern of C-terminal 6XHIS-UBC-2 cross-linked to itself versus C-terminal 6XHISUBC-2 cross-linked in the presence of GST::DSK-2 on a Western blot probed with antiUBC-2 antibody (data not shown). In addition, no difference was observed in the protein banding pattern of GST::DSK-2 cross-linked to itself versus GST::DSK-2 cross-linked in the presence of C-terminal 6XHIS-UBC-2 on a Western blot probed with anti-GST antibody. Therefore, cross-linking failed to demonstrate an in vitro interaction between C-terminal 6XHIS tagged UBC-2 and GST::DSK-2. Interestingly, it was observed that GST::DSK-2 forms higher molecular weight conjugates with itself, perhaps in the form of homodimers or higher order multimers (Figure 1 l . A . and B.). U B A domains have been implicated in promoting protein-protein interactions. As mentioned previously, the U B A domain mediates the interaction of RAD23 with itself, with DDI1, and with DSK2 (Bertolaet et al., 2001). However, the interaction of DSK2 with itself has not previously been demonstrated.  Therefore, it appears that the self-  association of DSK-2, a C. elegans homologue of budding yeast DSK2, is a novel result. It remains to be determined if DSK-2 dimerization eliminates  interaction with  multiubiquitin in the same manner that dimerization of RAD23 eliminates the interaction of RAD23 with multiubiquitin (Bertolaet et al., 2001).  6. GST::DSK-2 interacts with polyubiquitin chains Recently, it was discovered that Schizosaccharomyces pombe proteins with U B A domains are capable of binding polyubiquitin chains (Wilkinson et al., 2001). particular, it was found that S. cerevisiae DSK2 can bind to polyubiquitin chains  55  In  Figure 11. GST..DSK-2 self-associates in the presence of cross-linker Equimolar amounts of GST::DSK-2 and C-terminal 6XHIS tagged UBC-2 were incubated together and then cross-linked with 1 m M B S for various time periods (cross-linking time in minutes is indicated above each lane). Cross-linking reactions were separated on 10% SDSP A G E gels and were subsequently analyzed via Western blotting using anti-GST antibody (1:3,000). There appears to be no difference in the protein band pattern between GST::DSK-2 cross-linked to itself (A.) versus GST::DSK-2 cross-linked in the presence of C-terminal 6XHISUBC-2 (B.). Interestingly, GST::DSK-2 forms higher molecular weight conjugates with itself only in the presence of B S cross-linker (lanes 2-8, A . and B.). 3  3  A. GST::DSK-2 cross-linked with BS  3  Time  0  3'  5'  10'  15'  20'  25'  1  2  3  4  5  6  7  30"  8  B. GST::DSK-2 cross-linked in the presence of 6XHIS-UBC-2 with BS  3  Time  0  3"  5'  10'  15"  20"  25'  30'  1  2  3  4  5  6  7  8  56  (Funakoshi et al., 2002).  Since C. elegans DSK-2 exhibits 32% similarity to S.  cerevisiae DSK2 and possesses a C-terminal U B A domain, a co-precipitation experiment was performed to determine if DSK-2 also binds polyubiquitin. Since UBC-2 was found to interact with DSK-2 in vivo and in vitro, the ability of UBC-2 to interact with polyubiquitin chains (Ub -7) was also tested. 2  Co-precipitation experiments were  performed in UBC-2 buffer supplemented with 0.5% Triton X-100 to eliminate nonspecific binding of proteins to TALON® resin or to glutathione agarose.  Negative  controls for the co-precipitation experiments revealed that polyubiquitin does not bind to TALON® resin or glutathione agarose in a non-specific fashion (Figure 12). Additional negative control tests showed that 6XHIS-UBC-2 binds TALON® resin and that GST::DSK-2 binds glutathione agarose in the presence of polyubiquitin (Figure 13.A. and B.). Co-precipitation experiments indicated that 6XHIS-UBC-2 bound to TALON® resin does not interact with polyubiquitin (Figure 14.A.). However, GST::DSK-2 bound to glutathione agarose did interact with polyubiquitin in a co-precipitation experiment (Figure 14.B.).  This result confirmed the hypothesis that U B A domain containing  proteins interact with polyubiquitin (Wilkinson et al., 2001).  7. R N A interference of genes encoding UBC-2 interacting proteins R N A interference experiments were performed to determine the biological function of genes encoding UBC-2 interacting proteins. Previously, it was demonstrated that injection of double stranded R N A (dsRNA) into C. elegans interferes with gene function (Fire et al., 1998). This method, termed R N A interference (RNAi), is used to determine the in vivo function of genes by generating a temporary null phenotype which is often identical to the null mutant phenotype. Evidence suggests that R N A i occurs by a post-transcriptional mechanism, although the exact mechanism has not yet been determined.  A gene called dicer-1 in Drosophila is believed to encode a protein  responsible for cleavage of the introduced dsRNA into 21 to 23 nucleotide long fragments to mediate an R N A i effect (Knight and Bass, 2001). Recently, it has been demonstrated that R N A i can be induced when C. elegans is fed with bacteria expressing target gene dsRNA (Timmons and Fire, 1998). Conveniently, this feeding method  57  Figure 12. Polyubiquitin does not bind to TALON resin or glutathione agarose 9  Polyubiquitin chains (Ub .7) were incubated with TALON® resin or glutathione agarose in UBC-2 buffer supplemented with 0.5% Triton X-100 for 2 hours on a Nutator at 4 C. The supernatant (S), wash (W), and pellet (P) fractions were analyzed via SDS-PAGE and Western blotting using a 1:500 dilution of anti-ubiquitin antibody (Santa Cruz). Lanes 1-3: S, W, and P fractions from an incubation of Ub .7 with TALON® resin. Lanes 4-6: S, W, and P fractions from an incubation of Ub .7 with glutathione agarose. Polyubiquitin was not visible in the pellet fractions indicating that it does not bind non-specifically to TALON® resin or glutathione agarose. 2  2  2  TALON kDa  S  W  1  2  glutathione ag. P  3  S  4  58  W  5  P  6  Figure 13. Polyubiquitin does not interfere with protein interaction with affinity resin Negative control precipitation experiments demonstrated that the presence of polyubiquitin (Ub _ ) did not prevent the interaction of C-terminal 6XHIS::UBC-2 with the TALON® resin or GST:: DSK-2 with glutathione agarose in UBC-2 buffer supplemented with 0.5% Triton X-100 for 2 hours on a Nutator at 4 C. The supernatant (S), wash (W), and pellet (P) fractions were analyzed via SDS-PAGE and Western blotting using a 1:5,000 dilution of anti-UBC-2 antibody. 2  7  A. C-terminal 6XHIS-UBC-2 binds TALON® resin in the presence ofpolyubiquitin Lanes 1-3: S, W, and P fractions from an incubation of C-terminal 6XHIS-UBC-2 with TALON® resin. Lanes 4-6: S, W, and P fractions from an incubation of Ub . , C-terminal 6XHIS-UBC-2, and TALON® resin. C-terminal 6XHIS-UBC-2 is found in the pellet fraction (lanes 3 and 6). 2  (Ub) . 2  C-6XHIS-UBC-2  7  7  C-6XHIS-UBC-2  C-6XHIS-UBC-2 1  2  3  4  5  6  B. GST:: DSK-2 binds glutathione agarose in the presence ofpolyubiquitin Lanes 1-3: S, W, and P fractions from an incubation of GST::DSK-2 with glutathione agarose. Lanes 4-6: S, W, and P fractions from an incubation of Ub . , GST:: DSK-2, and glutathione agarose. GST:: DSK-2 is found in the pellet fraction (lanes 3 and 6). 2  (Ub),. + GST::DSK-2 7  GST::DSK-2  1  2  3  4  5  6  59  7  Figure 14. Polyubiquitin co-precipitation experiments A. C-terminal 6XHIS-UBC-2 does not interact with polyubiquitin After attempting to co-precipitate Ub .7 with C-terminal 6XHIS-UBC-2 and TALON® resin in UBC-2 buffer supplemented with 0.5% Triton X-100 for 2 hours on a Nutator at 4 C, the supernatant (S), wash (W), and pellet (P) fractions were analyzed via SDS-PAGE and Western blotting using a 1:500 dilution of anti-ubiquitin antibody (Santa Cruz). Lane 1: 1 pg Ub _ , lanes 2-4: S, W, and P fractions from an incubation of Ub .7, C-terminal 6XHIS-UBC-2, and TALON® resin. The absence of Ub -7 in lane 4 indicated that 6XHIS-UBC-2 does not interact with polyubiquitin. 2  2  7  2  2  (Ub) . + 2  7  C-6XHIS-UBC-2 kDa  <ut>)  27  s  W  P  2  3  4  B. GST::DSK-2 interacts with polyubiquitin After attempting to co-precipitate U b . with GST::DSK-2 and glutathione agarose, the S, W, and P fractions were analyzed via SDS-PAGE and Western blotting using a 1:500 dilution of antiubiquitin antibody (Santa Cruz). Lane 1: 1 pg Ub . , lanes 2-4: S, W, and P fractions from an incubation of Ub . , GST::DSK-2, and glutathione agarose. The presence of U b . in lane 4 indicated that GST::DSK-2 interacts with polyubiquitin. 2  7  2  2  7  2  (Ub) . 2  7  GST::DSK-2  1  7  2  3  4  60  7  overcomes the difficulty and expense of single worm injections and enhances the efficiency of determining the function of many genes (Kamath et al., 2000). This method involved non-directional cloning of genes encoding UBC-2 interacting proteins, and genes for R N A i positive controls, into a double T7 promoter vector (pPD129.36).  Genes cloned into the pPD129.36 included: ubiA (encodes  ubiquitin); F15C11.2 (encodes a ubiquitin-like protein referred to as DSK-2); B0432.9 and W02A11.3 (encodes two RING finger proteins), mca-l (encodes a calcium ATPase), R160.7 (encodes a F Y V E finger protein) and C25D7.8 (encodes a C. elegans protein lacking any previously characterized protein domains). HT115 (DE3), a strain of E. coli with inducible T7 polymerase activity and an RNase HI deficiency, was transformed with one of these plasmids and induced with IPTG to express T7 polymerase which uses both T7 promoters to generate dsRNA against the target gene. Late larval L4 stage C. elegans hermaphrodite nematodes were transferred onto the induced plates for R N A i treatment. The rationale for using L4 stage nematodes was to induce R N A i in the developing eggs prior to egg shell formation (D. Jones, pers. comm.). removed and the progeny were scored for phenotypes. L4 stage worm was determined.  The treated nematodes were  The fecundity per R N A i treated  The number of progeny produced per R N A i treated  worm after the 72 hour treatment period varied between 70 and 119. Interestingly, the only R N A i treatment which reduced the fecundity below that of the negative control population was the one directed at mca-l (Table 3).  This gene  encodes a calcium ATPase which pumps calcium outside of the cell through A T P hydrolysis (Kraev et al., 1999).  Since dsk-2 function, in combination with rad23, is  required for SPB duplication, and calcium spikes appear to promote duplication of the metazoan microtubule organizing center (Biggins et al., 1996; Matsumoto and Mailer, 2002), I investigated the effect of calcium chloride depletion on R N A i treatments for mca-l, R160.7, and dsk-2.  It was reasoned that this might enhance subtle R N A i  phenotypes. R N A i treatments of other genes encoding UBC-2 interacting proteins were also performed in this calcium chloride-deficient background. In these experiments, only R N A i treatment of mca-l reduced the fecundity below that of the negative control population (Table 4).  61  Table 3. Fecundity of R N A i treated C. elegans Plasmid Used for HT115(DE3) £.co/z Transformation  Number of treated L4 stage nematodes  Number of viable progeny  Progeny per RNAi treated nematode  Number of R N A i progeny as percent difference from number of 'no plasmid' progeny [(A-74)/74]X100 0 +61% +34% +35% -5% +3%  222 74 3 no plasmid 594 119 pPD129.36::B0432.9 5 99 4 395 pPD129.36::C25D7.8 200 100 pPD129.36::<M-2 2 70* 4 281 pPD129.36::mca-7 4 304 76 pPD129.36::R160.7 — o ** 4 0 pPD129.36::«6c-2 +15% 255 85 3 pPD129.36 * R N A i treated nematodes vs. mca-\ resulted in progeny appearing to be at the L2 stage of development in comparison to the rest of the RNAi treated nematodes which were at the L4 stage. ** Positive control R N A i vs. ubc-2 is known to result in arrested development of fertilized eggs.  62  Table 4 Fecundity of RNAi treated C. elegans grown in the absence of an environmental supply of calcium chloride HT115(DE3) transformation for R N A i feeding method treatment  Number of treated L4 stage nematodes  Number of viable progeny  Progeny per RNAi treated nematode (A)  Number of RNAi progeny as percent difference from number of 'no plasmid' progeny [(A-86)/86]X100 0 +3% +42% +60% -14% +29%  432 86 5 no plasmid 4 350 88 pPD129.36::B0432.9 122 4 489 pPD129.36::C25D7.8 541 135 4 pPD129.36::<M-2 74* 371 5 pPD129.36::wca-7 111 4 444 pPD129.36::R160.7 ~ 4 0 o ** pPD129.36:\ubc-2 +1% 435 87 5 pPD129.36 * RNAi treated nematodes vs. mca-\ resulted in progeny appearing smaller than the other RNAi treated nematodes populations. ** RNAi treated nematodes vs. ubc-2 resulted in arrested development of fertilized eggs.  63  Results indicated that nematodes treated with dsRNA against dsk-2 appeared normal. In agreement with this result, dsk2 is a non-essential gene in S. cerevisiae (Biggins et al., 1996). Interestingly, rad22> in S. cerevisiae is also non-essential. However, it has been shown that a double dsk2rad23 mutation leads to cell arrest due to a block in spindle pole body duplication (Biggins et al., 1996). To determine if S. cerevisiae dsk2 and rad23 homologues in C. elegans have a similar essential cellular function, nematodes were treated with dsRNA against dsk2 and rad23 in combination. This double R N A i treatment did not produce a detectable phenotype. However, since experiments have demonstrated that RNAi treatments of multiple genes in combination result in a dramatic reduction in the phenotypes induced independently by each R N A i treatment (Kamath et al., 2000), this result remains inconclusive. Negative controls for the RNAi testing included feeding the nematodes with HT115 (DE3) E. coli alone or HT115 (DE3) E. coli transformed with the empty double T7 promoter vector. phenotype.  These negative RNAi controls did not produce a detectable  As a positive control, R N A i against ubc-2 produced an embryonic lethal  phenotype as previously demonstrated via the injection method (Jones et al., 2002; T.A. Stevens, Ph.D. thesis, 1999).  R N A i versus ubiA also produced an embryonic lethal  effect, and the treated adult worms exhibited a decrease in motility in comparison to negative control nematodes (data not shown). In addition, the small number of eggs laid by ubiA R N A i treated nematodes after the 72 hour treatment period did not hatch (data not shown). These two RNAi treatments provided good positive controls to demonstrate that the R N A i feeding method procedure was functioning as expected. mca-1 R N A i treated nematodes appeared to exhibit a reduction in body length in comparison to the negative control populations (Figure 15). To determine the existence or extent of body length reduction caused by RNAi of genes encoding UBC-2 interacting proteins, approximately ten progeny worms were picked from each R N A i treatment plate onto a slide containing five drops of M9 buffer. The slides were heated to effectively fix the nematodes in a straightened conformation and enable accurate body length measurement. R N A i versus the mca-l gene produced the most pronounced phenotype of all the genes encoding UBC-2 interacting proteins. Adult mca-l R N A i treated nematodes  64  Table 5. Average length of R N A i treated C. elegans Approximately 10 worms were picked from each RNAi treatment plate into five drops of M9 buffer on a slide. The slides were heated for 30 seconds on a hot plate at 100C. This treatment effectively killed the nematodes and straightened their bodies to enable accurate body length measurement. Nematodes were measured at 25X magnification using an ocular micrometer. The measurements were convert to millimeters using the conversion factor 1 unit=0.4 mm. Gene target of RNAi treatment pPD129.36 control  Average nematode length (mm) 1.42+.04  Percent of control length (%) 100±2.8  B0432.9  1.38±.05  97+3.5  C25D7.8  1.37+.07  99+4.9  dsk-2  1.29+04  91+2.8  mca-1  1.17+.05  82±3.5  R160.7  1.34+.06  94±4.2  W02A11.3  1.32±.08  93±5.6  Figure 15. R N A i vs. mca-1: Adult mca-1 RNAi treated nematodes exhibit a reduction in body length RNAi versus the mca-1 gene produced the most pronounced phenotype of all the genes encoding UBC-2 interacting proteins. Adult mca-1 RNAi treated nematodes are 82+3.5% of the control adult population (100+2.8%) which were fed HT115 (DE3) E. coli containing the empty feeding vector pPD 129.36.  65  were 82+3.5% of the control adult nematodes (100+2.8%) which were fed HT115 (DE3) E. coli containing the empty feeding vector pPD129.36 (Table 5).  9. Conclusions and future directions In summary, a yeast two-hybrid screen of a C. elegans cDNA library indicated that TJBC-2 interacts with ubiquitin, a calcium ATPase, a protein containing a F Y V E domain, two RING finger proteins, and a ubiquitin-like protein called DSK-2, which represented the majority of the UBC-2 interactions detected.  The interaction between  DSK-2 and UBC-2 was confirmed biochemically in vitro. The interaction of DSK-2 with multiubiquitin chains was demonstrated in vitro, in agreement with other studies using the budding yeast homologue of DSK-2. The self-association of DSK-2 in vitro was also demonstrated.  R N A i experimentation revealed that genes encoding UBC-2 interacting  proteins studied here are non-essential for viability. Future studies could determine the precise domains involved in mediating the interaction of UBC-2 with DSK-2, DSK-2 with itself, and DSK-2 with polyubiquitin chains. It would also be interesting to determine if DSK-2 exhibits a specific preference for ubiquitin chain linkages mediated by certain lysyl residues. The interaction of DSK-2 with the proteasome could also be investigated. It would be helpful to confirm the interaction of UBC-2 with other proteins identified in the screen using in vitro methods. Immunolocalization analysis of UBC-2 interacting proteins in the cell and in C. elegans might confirm their proposed roles in various cellular pathways as discussed previously.  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LIST OF OLIGONUCLEOTIDES A N D THEIR SEQUENCES MG1  5' G C G C T T G C A T A T G A T G G C T C T C A A A A G A A T C C A G 3'  Ndel MG2  5' C G G G A T C C T C A C A T A G C A T A C T T T T G C G T C 3'  BamHl MG3 (pAS2 sequencing primer)  5' T C G G A A G A G A G T A G T A A  3'  MG6 (pcDNA3.1 sequencing primer)  5' T G G G A G T G G C A C C T T C C 3'  MG 7  5' A T A T A C C A T G G C T C T C A A A A G A A T 3'  Ncol MG10  5' A T A T G C T C G A G C A T A G C G A C T T T T 3'  Xhol SEQEC1 (5' p A C T sequencing primer)  5' T T C G A T G A T G A A G A T A C C C C 3'  SEQEC2 (3' p A C T sequencing primer)  5' A G G C A A A A C G A T G T A T A A  pGex5' sequencing primer  5' G G G C T G G C A A G C C A C G T T T G G T G 3'  81  3'  B. S U M M A R Y O FB A C T E R I A A N D Y E A S T STRAINS A N D T H E I R G E N O T Y P E S  1. E. coli strains DH5o™  F - <D80d/acZAM15 A(/acZYA-argF)U169 deoR recAX endk\ hsdR\7(r -, m +)phoA supE44 X- thi-l gyrA96 relAX k  k  (GJJBCOBRL® LIFE T E C H N O L O G I E S T M )  BL21(DE3)  hsdSgal (Xc\ts857 indl Sam7 nin5 lacUV5-T7genel)  HT115(DE3)  F - mcrA mcrB JN(rrnD-rrnE)\ lambda-r«cl4::7nlO(DE3 lysogen: /avTJV5 promoter -T7polymerase) (IPTG-inducible T7 polymerase) (RNase in minus)  HB101  Z)(gpt-proA)(52 leuB6 thi-l lacYl hsdSB20 recA rpsL20 (Strr) ara-14 galK2 xyl-5 mtl-1 supE44 mcrBB  OP50  E. coli strain B uracil auxotroph  2. Yeast strains Y187  M A T a gal4 gal80 his3 trpl-90\ ade2-\0\ ura3-52 leu2-3, -112 + URA3::GAL lacZ LYS2::GAL(UAS) HIS3 cyh T  Y190  M A T a gal4 gal80 his3 trpl-901 ade2-\0\ ura3-52 leu2-3,-\\2 we?-URA3::GAL lacZ  82  

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