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W2059694583 abstract "The CDC34 gene of the yeastSaccharomyces cerevisiae encodes a ubiquitin-conjugating protein that transfers ubiquitin onto substrates to signal rapid degradation via the proteasome. Cdc34p has been implicated in signaling the destruction of a variety of substrates including the cyclin-dependent kinase inhibitor, Sic1p, which must be degraded for cells to enter S-phase. Mutants lacking CDC34activity fail to degrade Sic1p and fail to enter S-phase, a phenotype that is also shared with cells lacking CDC4 and CDC53 activity. Here we demonstrate that Cdc4p, Cdc34p, and Cdc53p interact in vivo. We have mapped a Cdc4p/Cdc53p-binding region on Cdc34p; this region is essential for S-phase entry and thus the association of these three proteins is required for Sic1p degradation. All three proteins migrate in gel filtration to sizes that greatly exceed their actual size suggesting that they form stable associations with other proteins and we observe Cdc4p, Cdc34p, and Cdc53p fractionating into overlapping families of high molecular weight complexes. Finally, we demonstrate that Cdc4p, Cdc34p, and Cdc53p are stable throughout the cell cycle and that Cdc34p permanently resides as part of a complex throughout the cell cycle. This suggests that all Cdc34p substrates are ubiquitinated by a similar high molecular weight complex. The CDC34 gene of the yeastSaccharomyces cerevisiae encodes a ubiquitin-conjugating protein that transfers ubiquitin onto substrates to signal rapid degradation via the proteasome. Cdc34p has been implicated in signaling the destruction of a variety of substrates including the cyclin-dependent kinase inhibitor, Sic1p, which must be degraded for cells to enter S-phase. Mutants lacking CDC34activity fail to degrade Sic1p and fail to enter S-phase, a phenotype that is also shared with cells lacking CDC4 and CDC53 activity. Here we demonstrate that Cdc4p, Cdc34p, and Cdc53p interact in vivo. We have mapped a Cdc4p/Cdc53p-binding region on Cdc34p; this region is essential for S-phase entry and thus the association of these three proteins is required for Sic1p degradation. All three proteins migrate in gel filtration to sizes that greatly exceed their actual size suggesting that they form stable associations with other proteins and we observe Cdc4p, Cdc34p, and Cdc53p fractionating into overlapping families of high molecular weight complexes. Finally, we demonstrate that Cdc4p, Cdc34p, and Cdc53p are stable throughout the cell cycle and that Cdc34p permanently resides as part of a complex throughout the cell cycle. This suggests that all Cdc34p substrates are ubiquitinated by a similar high molecular weight complex. Ubiquitin (Ub) 1The abbreviations used are: Ub, ubiquitin; Ubc, ubiquitin conjugating family of proteins; E1, ubiquitin activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.1The abbreviations used are: Ub, ubiquitin; Ubc, ubiquitin conjugating family of proteins; E1, ubiquitin activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis. is a highly conserved 76-amino acid protein that is found as a post-translational modification of other proteins. The best characterized role of Ub modification is as a means of regulating protein abundance. When a Ub chain forms on a particular substrate protein, that protein is rapidly degraded by the proteasome (for reviews, see Refs. 1Finley D. Chau V. Annu. Rev. Cell Biol. 1991; 7: 25-69Crossref PubMed Scopus (421) Google Scholar, 2Hershko A. Ciechanover A. Annu. Rev. Biochem. 1992; 61: 761-807Crossref PubMed Scopus (1191) Google Scholar, 3Jentsch S. Annu. Rev. Genet. 1992; 26: 179-207Crossref PubMed Scopus (450) Google Scholar, 4Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1586) Google Scholar, 5Coux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2221) Google Scholar, 6Hochstrasser M. Curr. Opin. Cell Biol. 1995; 7: 215-223Crossref PubMed Scopus (779) Google Scholar). Poly-Ub chain formation on a substrate is catalyzed by a member of the ubiquitin conjugating (Ubc) family of proteins, also known as E2 enzymes. Prior to poly-Ub chain formation, Ubc (E2) enzymes are charged with ubiquitin by a ubiquitin activating, or E1, enzyme which has the ability to capture free Ub. In some cases, the transfer of ubiquitin from an Ubc (E2) enzyme to a substrate proceeds via a third protein known as a ubiquitin ligase, or E3 (7Scheffner M. Nuber U. Huibregtse J.M. Nature. 1995; 373: 81-83Crossref PubMed Scopus (727) Google Scholar, 8Huibregtse J.M. Scheffner M. Beaudenon S. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Crossref PubMed Scopus (688) Google Scholar). Based on sequence similarity, there are 13 members of the Ubc (E2) family in yeast and these proteins have diverse roles including cell cycle progression, heavy metal resistance, peroxisome biogenesis, and DNA repair. Members of this family have highly related catalytic domains which receive ubiquitin from an E1 and pass it directly, or via an E3, on to a substrate. The noncatalytic carboxyl termini of Ubc enzymes are more variable and are found on only some members of the family. The function of this region is unknown, however, it may be required for substrate recognition or regulation of Ubc activity.Cdc34p, a member of the Ubc family of proteins, is essential for cell cycle progression and primarily acts at the G1 to S-phase transition (9Goebl M.G. Yochem J. Jentsch S. McGrath J.P. Varshavsky A. Byers B. Science. 1988; 241: 1331-1335Crossref PubMed Scopus (321) Google Scholar). Cell cycle progression in yeast is coordinated by the protein kinase, Cdc28p, and the activity of this kinase requires its association with another protein termed cyclin (for review, see Refs.10Nasmyth K. Curr. Opin. Cell Biol. 1993; 5: 166-179Crossref PubMed Scopus (407) Google Scholar, 11Nasmyth K. Trends in Genetics. 1996; 12: 405-412Abstract Full Text PDF PubMed Scopus (294) Google Scholar, 12Lew D.J. Reed S. Trends Cell Biol. 1992; 2: 77-81Abstract Full Text PDF PubMed Scopus (57) Google Scholar). It is thought that the cyclin will determine substrate specificity for the kinase or direct the kinase to particular locations in the cell. In yeast nine cyclins have been characterized for Cdc28p and different cyclins associate with the Cdc28p kinase during different phases of the cell cycle. For example, G1 progression is coordinated by the cyclins Cln1p, Cln2p, and Cln3p that bind and activate Cdc28p. To initiate DNA replication, or S-phase, Cdc28p associates with cyclins Clb5p or Clb6p. Cyclin kinase activity can be negatively regulated by the physical association of an inhibitor (13Hunter T. Cell. 1993; 75: 839-841Abstract Full Text PDF PubMed Scopus (369) Google Scholar, 14Nasmyth K. Hunt T. Nature. 1993; 366: 634-635Crossref PubMed Scopus (99) Google Scholar, 15Pines J. Trends Biochem. Sci. 1994; 19: 143-145Abstract Full Text PDF PubMed Scopus (74) Google Scholar). One such inhibitor in yeast is Sic1p and this protein negatively regulates the activity of Cdc28p-Clb5p and Cdc28p-Clb6p complexes (16Mendenhall M.D. Science. 1993; 259: 216-219Crossref PubMed Scopus (163) Google Scholar, 17Nugroho T.T. Mendenhall M.D. Mol. Cell. Biol. 1994; 14: 3320-3328Crossref PubMed Scopus (130) Google Scholar, 18Donovan J.D. Toyn J.H. Johnson A.L. Johnston L.H. Genes Dev. 1994; 8: 1640-1653Crossref PubMed Scopus (115) Google Scholar, 19Schwob E. Bohm T. Mendenhall M.D. Nasmyth K. Cell. 1994; 79: 233-244Abstract Full Text PDF PubMed Scopus (769) Google Scholar). Only upon Sic1p degradation are these complexes active and S-phase initiated.Sic1p is one candidate substrate for Cdc34p-mediated ubiquitin degradation (19Schwob E. Bohm T. Mendenhall M.D. Nasmyth K. Cell. 1994; 79: 233-244Abstract Full Text PDF PubMed Scopus (769) Google Scholar). Cells that lack CDC34 activity are unable to remove Sic1p and thus arrest without initiating DNA replication. This phenotype is shared by cells that lack CDC4 and CDC53 activity. Although their activity is required for Sic1p ubiquitination (19Schwob E. Bohm T. Mendenhall M.D. Nasmyth K. Cell. 1994; 79: 233-244Abstract Full Text PDF PubMed Scopus (769) Google Scholar), the precise function of these proteins is unknown. CDC53 encodes a protein that defines a family of evolutionary conserved proteins which may serve to recognize substrates that have been targeted for ubiquitination (20Kipreous E.T. Lander L.E. Wing J.P. He W.W. Hedgecock E.M. Cell. 1996; 85: 829-839Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 21Mathias N. Johnson S.L. Winey M. Adams A.E.M. Goetsch L. Pringle J.R. Byers B. Goebl M.G. Mol. Cell. Biol. 1996; 16: 6634-6643Crossref PubMed Scopus (182) Google Scholar, 22Willems A.R. Lanker S. Patton E.E. Craig K.L. Nason T.F. Mathias N. Koybayashi R. Wittenberg C. Tyers M. Cell. 1996; 86: 453-463Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). CDC4encodes a protein containing 7 WD-40 repeats, a motif that has been implicated in protein-protein interactions (23Yochem J. Byers B. J. Mol. Biol. 1987; 195: 233-245Crossref PubMed Scopus (75) Google Scholar, 24Sondek J. Bohm A. Lambright D.G. Hamm H.E. Sigler P.B. Nature. 1996; 379: 369-374Crossref PubMed Scopus (707) Google Scholar). Furthermore, Cdc4p also contains an F-Box, a domain that is the binding site for Skp1p (25Bai C. Sen P. Hofmann K. Ma L. Goebl M.G. Harper J.W. Elledge S.J. Cell. 1996; 86: 263-274Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar). Skp1p is required for the degradation of Sic1p, Cln2p, and Clb5p (25Bai C. Sen P. Hofmann K. Ma L. Goebl M.G. Harper J.W. Elledge S.J. Cell. 1996; 86: 263-274Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar). It has been speculated that a complex containing these proteins is required for the degradation of Sic1p and initiation of S-phase (26King R.W. Deshaies R.J. Peters J-M. Kirschner M.W. Science. 1996; 274: 1652-1659Crossref PubMed Scopus (1117) Google Scholar). Here, we define a region on Cdc34p that is necessary and sufficient for its binding Cdc4p and Cdc53p. We report that Cdc4p, Cdc34p, and Cdc53p form a high molecular weight complex and that this association is required for Cdc34p function. We also show that these three proteins are not only present throughout the cell cycle but that Cdc34p is part of a complex during this process.DISCUSSIONOne essential function of the ubiquitin-conjugating enzyme Cdc34p is to attach Ub onto the cyclin-dependent kinase inhibitor Sic1p and thus signal Sic1p for proteolysis. The precise mechanism by which Cdc34p transfers Ub onto Sic1p is not known, however, this process is dependent on at least two other proteins, Cdc4p and Cdc53p. In this report we map a Cdc4p- and Cdc53p-binding domain on Cdc34p and show the importance of in vivo interactions between these three proteins, strengthening the hypothesis that Sic1p ubiquitination is mediated by a multi-protein complex.The Association of Cdc34p with Cdc4p and Cdc53p Is Essential for Cdc34p ActivityWe have defined a Cdc4p/Cdc53p-binding domain on Cdc34p (Fig. 7). This region is not found in other Ubc proteins which suggests that these interactions are unique to Cdc34p. However, the efficiency of Cdc4p and Cdc53p binding to a fusion containing Cdc34p residues 1–209 or residues 171–209 is less than that of a fusion containing full-length Cdc34p. Therefore the remainder of the Cdc34p protein may act to stabilize its association with Cdc4p and Cdc53p. Ptak et al. (34Ptak C. Prendergast J.A. Hodgins R. Kay C.M. Chau V. Ellison M.J. J. Biol. Chem. 1994; 269: 26539-26545Abstract Full Text PDF PubMed Google Scholar) have shown that this region is required for Cdc34p dimerization in vitro and it is possible that Cdc4p and Cdc53p bind to a Cdc34p dimer in vivo. Absence of this binding region results in loss of Cdc34p activity and cells are prevented from entering S-phase due to failure to degrade the CDK inhibitor Sic1p. Thus the association of Cdc34p with Cdc4p and Cdc53p is essential for Sic1p ubiquitination. What role does Cdc4p and Cdc53p play in Sic1p ubiquitination? Cdc34p residues 1–170 contain the catalytic domain of Cdc34p and its active site cysteine (Fig. 7). This region has been shown to be charged with ubiquitin by the ubiquitin-activating enzyme, E1 (9Goebl M.G. Yochem J. Jentsch S. McGrath J.P. Varshavsky A. Byers B. Science. 1988; 241: 1331-1335Crossref PubMed Scopus (321) Google Scholar), an event that does not require Cdc4p or Cdc53p association. Therefore Cdc4p and Cdc53p most likely play a role in transferring Ub from Cdc34p onto its substrates either by substrate recognition or by having a Ub-ligase (E3) activity. Ub-ligases (E3s) have been described (7Scheffner M. Nuber U. Huibregtse J.M. Nature. 1995; 373: 81-83Crossref PubMed Scopus (727) Google Scholar, 8Huibregtse J.M. Scheffner M. Beaudenon S. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Crossref PubMed Scopus (688) Google Scholar) yet the sequence comparison of these proteins do not resemble either Cdc4p or Cdc53p. Possibly Cdc53p is required to present the substrate to Cdc34p. Cdc53p has been found to associate with Cln2p, a G1 cyclin that is another candidate Cdc34p substrate (22Willems A.R. Lanker S. Patton E.E. Craig K.L. Nason T.F. Mathias N. Koybayashi R. Wittenberg C. Tyers M. Cell. 1996; 86: 453-463Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar).Cdc4p, Cdc34p, and Cdc53p Are Members of a Multiprotein ComplexGel filtration analysis shows that Cdc4p, Cdc34p, and Cdc53p are not found as monomers in yeast lysate and take part in associations resulting in the formation of high molecular mass complexes. Overlap of the three proteins' distribution suggests that the Cdc4p·Cdc34p·Cdc53p complex is approximately 400 kDa. This size is greater than that predicted for a complex containing one of each protein (218 kDa), that have a predicted molecular mass of 88, 34, and 96 kDa for Cdc4p, Cdc34p, and Cdc53p, respectively. Therefore either these proteins may act as multimers or additional proteins are part of this complex. Skp1p is required for the ubiquitination of Sic1p and has been shown to bind to Cdc4p through a Cdc4p domain called the F-box (25Bai C. Sen P. Hofmann K. Ma L. Goebl M.G. Harper J.W. Elledge S.J. Cell. 1996; 86: 263-274Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar). However, Skp1p is only 22 kDa and perhaps other such proteins are required for Cdc34p to interact with Cdc4p and Cdc53p. Significant amounts of Cdc4p and Cdc34p co-fractionate in the absence of Cdc53p and together with our data demonstrating the stabilization of Cdc4p when co-overproduced with Cdc34p suggest that these two proteins form a heterodimer. Again, this association, of approximately 200 kDa may occur in the presence of other proteins. Therefore it appears that components of the Cdc4p·Cdc34p·Cdc53p complex may be separated and be redistributed for distinct ubiquitinating activities.The Cdc34p Complex Is Present throughout the Cell CycleFinally we determined whether Cdc34p existed within a complex throughout the cell cycle. Many cell cycle regulators are themselves only present during specific times of the cell cycle. We have shown here that Cdc4p, Cdc34p, and Cdc53p are present throughout the cell cycle and that Cdc34p appears to reside as part of a complex throughout the cell cycle. Potentially, Cdc34p activity may be required throughout the cell cycle and not just at the G1/S transition in spite of the fact that some of its substrates are degraded in a cell cycle specific manner. Phosphorylation of Sic1p and Cln2p is a prerequisite for these substrates to be ubiquitinated by Cdc34p and Cdc34p activity may be solely regulated by recognizing phosphorylated substrates (38Lanker S. Valdivieso M.H. Wittenberg C. Science. 1996; 271: 1597-1601Crossref PubMed Scopus (195) Google Scholar, 39Schneider, B. L., Yang, Q. H., and Futcher, A. B.Science, 272, 560–562.Google Scholar). Other candidate Cdc34p substrates include Cdc6p (40Piatti S. Bohm T. Cocker J.H. Diffley J.F.X. Nasmyth K. Genes Dev. 1996; 10: 1516-1531Crossref PubMed Scopus (253) Google Scholar), Far1p (41Mckinney J.D. Chang F. Heinzt N. Cross F.R. Genes Dev. 1993; 7: 833-843Crossref PubMed Scopus (123) Google Scholar), and Gcn4p (42Kornitzer D. Raboy B. Kulka R.G. Fink G.R. EMBO J. 1994; 13: 6021-6030Crossref PubMed Scopus (219) Google Scholar). Both Cdc6p and Far1p are degraded in a cell cycle specific manner but outside the G1/S transition and therefore the Cdc34p complex must be active throughout the cell cycle. Ubiquitin (Ub) 1The abbreviations used are: Ub, ubiquitin; Ubc, ubiquitin conjugating family of proteins; E1, ubiquitin activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.1The abbreviations used are: Ub, ubiquitin; Ubc, ubiquitin conjugating family of proteins; E1, ubiquitin activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis. is a highly conserved 76-amino acid protein that is found as a post-translational modification of other proteins. The best characterized role of Ub modification is as a means of regulating protein abundance. When a Ub chain forms on a particular substrate protein, that protein is rapidly degraded by the proteasome (for reviews, see Refs. 1Finley D. Chau V. Annu. Rev. Cell Biol. 1991; 7: 25-69Crossref PubMed Scopus (421) Google Scholar, 2Hershko A. Ciechanover A. Annu. Rev. Biochem. 1992; 61: 761-807Crossref PubMed Scopus (1191) Google Scholar, 3Jentsch S. Annu. Rev. Genet. 1992; 26: 179-207Crossref PubMed Scopus (450) Google Scholar, 4Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1586) Google Scholar, 5Coux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2221) Google Scholar, 6Hochstrasser M. Curr. Opin. Cell Biol. 1995; 7: 215-223Crossref PubMed Scopus (779) Google Scholar). Poly-Ub chain formation on a substrate is catalyzed by a member of the ubiquitin conjugating (Ubc) family of proteins, also known as E2 enzymes. Prior to poly-Ub chain formation, Ubc (E2) enzymes are charged with ubiquitin by a ubiquitin activating, or E1, enzyme which has the ability to capture free Ub. In some cases, the transfer of ubiquitin from an Ubc (E2) enzyme to a substrate proceeds via a third protein known as a ubiquitin ligase, or E3 (7Scheffner M. Nuber U. Huibregtse J.M. Nature. 1995; 373: 81-83Crossref PubMed Scopus (727) Google Scholar, 8Huibregtse J.M. Scheffner M. Beaudenon S. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Crossref PubMed Scopus (688) Google Scholar). Based on sequence similarity, there are 13 members of the Ubc (E2) family in yeast and these proteins have diverse roles including cell cycle progression, heavy metal resistance, peroxisome biogenesis, and DNA repair. Members of this family have highly related catalytic domains which receive ubiquitin from an E1 and pass it directly, or via an E3, on to a substrate. The noncatalytic carboxyl termini of Ubc enzymes are more variable and are found on only some members of the family. The function of this region is unknown, however, it may be required for substrate recognition or regulation of Ubc activity. Cdc34p, a member of the Ubc family of proteins, is essential for cell cycle progression and primarily acts at the G1 to S-phase transition (9Goebl M.G. Yochem J. Jentsch S. McGrath J.P. Varshavsky A. Byers B. Science. 1988; 241: 1331-1335Crossref PubMed Scopus (321) Google Scholar). Cell cycle progression in yeast is coordinated by the protein kinase, Cdc28p, and the activity of this kinase requires its association with another protein termed cyclin (for review, see Refs.10Nasmyth K. Curr. Opin. Cell Biol. 1993; 5: 166-179Crossref PubMed Scopus (407) Google Scholar, 11Nasmyth K. Trends in Genetics. 1996; 12: 405-412Abstract Full Text PDF PubMed Scopus (294) Google Scholar, 12Lew D.J. Reed S. Trends Cell Biol. 1992; 2: 77-81Abstract Full Text PDF PubMed Scopus (57) Google Scholar). It is thought that the cyclin will determine substrate specificity for the kinase or direct the kinase to particular locations in the cell. In yeast nine cyclins have been characterized for Cdc28p and different cyclins associate with the Cdc28p kinase during different phases of the cell cycle. For example, G1 progression is coordinated by the cyclins Cln1p, Cln2p, and Cln3p that bind and activate Cdc28p. To initiate DNA replication, or S-phase, Cdc28p associates with cyclins Clb5p or Clb6p. Cyclin kinase activity can be negatively regulated by the physical association of an inhibitor (13Hunter T. Cell. 1993; 75: 839-841Abstract Full Text PDF PubMed Scopus (369) Google Scholar, 14Nasmyth K. Hunt T. Nature. 1993; 366: 634-635Crossref PubMed Scopus (99) Google Scholar, 15Pines J. Trends Biochem. Sci. 1994; 19: 143-145Abstract Full Text PDF PubMed Scopus (74) Google Scholar). One such inhibitor in yeast is Sic1p and this protein negatively regulates the activity of Cdc28p-Clb5p and Cdc28p-Clb6p complexes (16Mendenhall M.D. Science. 1993; 259: 216-219Crossref PubMed Scopus (163) Google Scholar, 17Nugroho T.T. Mendenhall M.D. Mol. Cell. Biol. 1994; 14: 3320-3328Crossref PubMed Scopus (130) Google Scholar, 18Donovan J.D. Toyn J.H. Johnson A.L. Johnston L.H. Genes Dev. 1994; 8: 1640-1653Crossref PubMed Scopus (115) Google Scholar, 19Schwob E. Bohm T. Mendenhall M.D. Nasmyth K. Cell. 1994; 79: 233-244Abstract Full Text PDF PubMed Scopus (769) Google Scholar). Only upon Sic1p degradation are these complexes active and S-phase initiated. Sic1p is one candidate substrate for Cdc34p-mediated ubiquitin degradation (19Schwob E. Bohm T. Mendenhall M.D. Nasmyth K. Cell. 1994; 79: 233-244Abstract Full Text PDF PubMed Scopus (769) Google Scholar). Cells that lack CDC34 activity are unable to remove Sic1p and thus arrest without initiating DNA replication. This phenotype is shared by cells that lack CDC4 and CDC53 activity. Although their activity is required for Sic1p ubiquitination (19Schwob E. Bohm T. Mendenhall M.D. Nasmyth K. Cell. 1994; 79: 233-244Abstract Full Text PDF PubMed Scopus (769) Google Scholar), the precise function of these proteins is unknown. CDC53 encodes a protein that defines a family of evolutionary conserved proteins which may serve to recognize substrates that have been targeted for ubiquitination (20Kipreous E.T. Lander L.E. Wing J.P. He W.W. Hedgecock E.M. Cell. 1996; 85: 829-839Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 21Mathias N. Johnson S.L. Winey M. Adams A.E.M. Goetsch L. Pringle J.R. Byers B. Goebl M.G. Mol. Cell. Biol. 1996; 16: 6634-6643Crossref PubMed Scopus (182) Google Scholar, 22Willems A.R. Lanker S. Patton E.E. Craig K.L. Nason T.F. Mathias N. Koybayashi R. Wittenberg C. Tyers M. Cell. 1996; 86: 453-463Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). CDC4encodes a protein containing 7 WD-40 repeats, a motif that has been implicated in protein-protein interactions (23Yochem J. Byers B. J. Mol. Biol. 1987; 195: 233-245Crossref PubMed Scopus (75) Google Scholar, 24Sondek J. Bohm A. Lambright D.G. Hamm H.E. Sigler P.B. Nature. 1996; 379: 369-374Crossref PubMed Scopus (707) Google Scholar). Furthermore, Cdc4p also contains an F-Box, a domain that is the binding site for Skp1p (25Bai C. Sen P. Hofmann K. Ma L. Goebl M.G. Harper J.W. Elledge S.J. Cell. 1996; 86: 263-274Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar). Skp1p is required for the degradation of Sic1p, Cln2p, and Clb5p (25Bai C. Sen P. Hofmann K. Ma L. Goebl M.G. Harper J.W. Elledge S.J. Cell. 1996; 86: 263-274Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar). It has been speculated that a complex containing these proteins is required for the degradation of Sic1p and initiation of S-phase (26King R.W. Deshaies R.J. Peters J-M. Kirschner M.W. Science. 1996; 274: 1652-1659Crossref PubMed Scopus (1117) Google Scholar). Here, we define a region on Cdc34p that is necessary and sufficient for its binding Cdc4p and Cdc53p. We report that Cdc4p, Cdc34p, and Cdc53p form a high molecular weight complex and that this association is required for Cdc34p function. We also show that these three proteins are not only present throughout the cell cycle but that Cdc34p is part of a complex during this process. DISCUSSIONOne essential function of the ubiquitin-conjugating enzyme Cdc34p is to attach Ub onto the cyclin-dependent kinase inhibitor Sic1p and thus signal Sic1p for proteolysis. The precise mechanism by which Cdc34p transfers Ub onto Sic1p is not known, however, this process is dependent on at least two other proteins, Cdc4p and Cdc53p. In this report we map a Cdc4p- and Cdc53p-binding domain on Cdc34p and show the importance of in vivo interactions between these three proteins, strengthening the hypothesis that Sic1p ubiquitination is mediated by a multi-protein complex.The Association of Cdc34p with Cdc4p and Cdc53p Is Essential for Cdc34p ActivityWe have defined a Cdc4p/Cdc53p-binding domain on Cdc34p (Fig. 7). This region is not found in other Ubc proteins which suggests that these interactions are unique to Cdc34p. However, the efficiency of Cdc4p and Cdc53p binding to a fusion containing Cdc34p residues 1–209 or residues 171–209 is less than that of a fusion containing full-length Cdc34p. Therefore the remainder of the Cdc34p protein may act to stabilize its association with Cdc4p and Cdc53p. Ptak et al. (34Ptak C. Prendergast J.A. Hodgins R. Kay C.M. Chau V. Ellison M.J. J. Biol. Chem. 1994; 269: 26539-26545Abstract Full Text PDF PubMed Google Scholar) have shown that this region is required for Cdc34p dimerization in vitro and it is possible that Cdc4p and Cdc53p bind to a Cdc34p dimer in vivo. Absence of this binding region results in loss of Cdc34p activity and cells are prevented from entering S-phase due to failure to degrade the CDK inhibitor Sic1p. Thus the association of Cdc34p with Cdc4p and Cdc53p is essential for Sic1p ubiquitination. What role does Cdc4p and Cdc53p play in Sic1p ubiquitination? Cdc34p residues 1–170 contain the catalytic domain of Cdc34p and its active site cysteine (Fig. 7). This region has been shown to be charged with ubiquitin by the ubiquitin-activating enzyme, E1 (9Goebl M.G. Yochem J. Jentsch S. McGrath J.P. Varshavsky A. Byers B. Science. 1988; 241: 1331-1335Crossref PubMed Scopus (321) Google Scholar), an event that does not require Cdc4p or Cdc53p association. Therefore Cdc4p and Cdc53p most likely play a role in transferring Ub from Cdc34p onto its substrates either by substrate recognition or by having a Ub-ligase (E3) activity. Ub-ligases (E3s) have been described (7Scheffner M. Nuber U. Huibregtse J.M. Nature. 1995; 373: 81-83Crossref PubMed Scopus (727) Google Scholar, 8Huibregtse J.M. Scheffner M. Beaudenon S. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Crossref PubMed Scopus (688) Google Scholar) yet the sequence comparison of these proteins do not resemble either Cdc4p or Cdc53p. Possibly Cdc53p is required to present the substrate to Cdc34p. Cdc53p has been found to associate with Cln2p, a G1 cyclin that is another candidate Cdc34p substrate (22Willems A.R. Lanker S. Patton E.E. Craig K.L. Nason T.F. Mathias N. Koybayashi R. Wittenberg C. Tyers M. Cell. 1996; 86: 453-463Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar).Cdc4p, Cdc34p, and Cdc53p Are Members of a Multiprotein ComplexGel filtration analysis shows that Cdc4p, Cdc34p, and Cdc53p are not found as monomers in yeast lysate and take part in associations resulting in the formation of high molecular mass complexes. Overlap of the three proteins' distribution suggests that the Cdc4p·Cdc34p·Cdc53p complex is approximately 400 kDa. This size is greater than that predicted for a complex containing one of each protein (218 kDa), that have a predicted molecular mass of 88, 34, and 96 kDa for Cdc4p, Cdc34p, and Cdc53p, respectively. Therefore either these proteins may act as multimers or additional proteins are part of this complex. Skp1p is required for the ubiquitination of Sic1p and has been shown to bind to Cdc4p through a Cdc4p domain called the F-box (25Bai C. Sen P. Hofmann K. Ma L. Goebl M.G. Harper J.W. Elledge S.J. Cell. 1996; 86: 263-274Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar). However, Skp1p is only 22 kDa and perhaps other such proteins are required for Cdc34p to interact with Cdc4p and Cdc53p. Significant amounts of Cdc4p and Cdc34p co-fractionate in the absence of Cdc53p and together with our data demonstrating the stabilization of Cdc4p when co-overproduced with Cdc34p suggest that these two proteins form a heterodimer. Again, this association, of approximately 200 kDa may occur in the presence of other proteins. Therefore it appears that components of the Cdc4p·Cdc34p·Cdc53p complex may be separated and be redistributed for distinct ubiquitinating activities.The Cdc34p Complex Is Present throughout the Cell CycleFinally we determined whether Cdc34p existed within a complex throughout the cell cycle. Many cell cycle regulators are themselves only present during specific times of the cell cycle. We have shown here that Cdc4p, Cdc34p, and Cdc53p are present throughout the cell cycle and that Cdc34p appears to reside as part of a complex throughout the cell cycle. Potentially, Cdc34p activity may be required throughout the cell cycle and not just at the G1/S transition in spite of the fac" @default.
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W2059694583 date "1998-02-01" @default.
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W2059694583 title "An Essential Domain within Cdc34p Is Required for Binding to a Complex Containing Cdc4p and Cdc53p in Saccharomyces cerevisiae" @default.
W2059694583 cites W1426117618 @default.
W2059694583 cites W1546987546 @default.
W2059694583 cites W1566221754 @default.
W2059694583 cites W1897326544 @default.
W2059694583 cites W1964827998 @default.
W2059694583 cites W1967071341 @default.
W2059694583 cites W1978186903 @default.
W2059694583 cites W1994523623 @default.
W2059694583 cites W2008044453 @default.
W2059694583 cites W2015997780 @default.
W2059694583 cites W2028018309 @default.
W2059694583 cites W2031332408 @default.
W2059694583 cites W2039175174 @default.
W2059694583 cites W2039839254 @default.
W2059694583 cites W2040778486 @default.
W2059694583 cites W2051992774 @default.
W2059694583 cites W2052280808 @default.
W2059694583 cites W2057075902 @default.
W2059694583 cites W2057992913 @default.
W2059694583 cites W2059665827 @default.
W2059694583 cites W2064850779 @default.
W2059694583 cites W2071191421 @default.
W2059694583 cites W2074126694 @default.
W2059694583 cites W2075124527 @default.
W2059694583 cites W2088966640 @default.
W2059694583 cites W2091501337 @default.
W2059694583 cites W2092357174 @default.
W2059694583 cites W2093330988 @default.
W2059694583 cites W2098941196 @default.
W2059694583 cites W2108402398 @default.
W2059694583 cites W2117551036 @default.
W2059694583 cites W2132024812 @default.
W2059694583 cites W2175093343 @default.
W2059694583 cites W2178503716 @default.
W2059694583 cites W4251683570 @default.
W2059694583 cites W68591885 @default.
W2059694583 cites W971227723 @default.
W2059694583 doi "https://doi.org/10.1074/jbc.273.7.4040" @default.
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