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. 1996 Feb;16(2):677–684. doi: 10.1128/mcb.16.2.677

Identification of a positive regulator of the cell cycle ubiquitin-conjugating enzyme Cdc34 (Ubc3).

J A Prendergast 1, C Ptak 1, D Kornitzer 1, C N Steussy 1, R Hodgins 1, M Goebl 1, M J Ellison 1
PMCID: PMC231047  PMID: 8552096

Abstract

The Cdc34 (Ubc3) ubiquitin-conjugating enzyme from Saccharomyces cerevisiae plays an essential role in the progression of cells from the G1 to S phase of the cell division cycle. Using a high-copy suppression strategy, we have identified a yeast gene (UBS1) whose elevated expression suppresses the conditional cell cycle defects associated with cdc34 mutations. The UBS1 gene encodes a 32.2-kDa protein of previously unknown function and is identical in sequence to a genomic open reading frame on chromosome II (GenBank accession number Z36034). Several lines of evidence described here indicate that Ubs1 functions as a general positive regulator of Cdc34 activity. First, overexpression of UBS1 suppresses not only the cell proliferation and morphological defects associated with cdc34 mutants but also the inability of cdc34 mutant cells to degrade the general amino acid biosynthesis transcriptional regulator, Gcn4. Second, deletion of the UBS1 gene profoundly accentuates the cell cycle defect when placed in combination with a cdc34 temperature-sensitive allele. Finally, a comparison of the Ubs1 and Cdc34 polypeptide sequences reveals two noncontiguous regions of similarity, which, when projected onto the three-dimensional structure of a ubiquitin-conjugating enzyme, define a single region situated on its surface. While cdc34 mutations corresponding to substitutions outside this region are suppressed by UBS1 overexpression, Ubs1 fails to suppress amino acid substitutions made within this region. Taken together with other findings, the allele specificity exhibited by UBS1 expression suggests that Ubs1 regulates Cdc34 by interaction or modification.

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Selected References

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  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Bacon D. J., Anderson W. F. Multiple sequence alignment. J Mol Biol. 1986 Sep 20;191(2):153–161. doi: 10.1016/0022-2836(86)90252-4. [DOI] [PubMed] [Google Scholar]
  3. Banerjee A., Gregori L., Xu Y., Chau V. The bacterially expressed yeast CDC34 gene product can undergo autoubiquitination to form a multiubiquitin chain-linked protein. J Biol Chem. 1993 Mar 15;268(8):5668–5675. [PubMed] [Google Scholar]
  4. Bushman J. L., Foiani M., Cigan A. M., Paddon C. J., Hinnebusch A. G. Guanine nucleotide exchange factor for eukaryotic translation initiation factor 2 in Saccharomyces cerevisiae: interactions between the essential subunits GCD2, GCD6, and GCD7 and the regulatory subunit GCN3. Mol Cell Biol. 1993 Aug;13(8):4618–4631. doi: 10.1128/mcb.13.8.4618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Byers B., Goetsch L. Duplication of spindle plaques and integration of the yeast cell cycle. Cold Spring Harb Symp Quant Biol. 1974;38:123–131. doi: 10.1101/sqb.1974.038.01.016. [DOI] [PubMed] [Google Scholar]
  6. Cook W. J., Jeffrey L. C., Sullivan M. L., Vierstra R. D. Three-dimensional structure of a ubiquitin-conjugating enzyme (E2). J Biol Chem. 1992 Jul 25;267(21):15116–15121. doi: 10.2210/pdb1aak/pdb. [DOI] [PubMed] [Google Scholar]
  7. Deshaies R. J., Chau V., Kirschner M. Ubiquitination of the G1 cyclin Cln2p by a Cdc34p-dependent pathway. EMBO J. 1995 Jan 16;14(2):303–312. doi: 10.1002/j.1460-2075.1995.tb07004.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dobson M. J., Tuite M. F., Roberts N. A., Kingsman A. J., Kingsman S. M., Perkins R. E., Conroy S. C., Fothergill L. A. Conservation of high efficiency promoter sequences in Saccharomyces cerevisiae. Nucleic Acids Res. 1982 Apr 24;10(8):2625–2637. doi: 10.1093/nar/10.8.2625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ecker D. J., Khan M. I., Marsh J., Butt T. R., Crooke S. T. Chemical synthesis and expression of a cassette adapted ubiquitin gene. J Biol Chem. 1987 Mar 15;262(8):3524–3527. [PubMed] [Google Scholar]
  10. Ellison K. S., Gwozd T., Prendergast J. A., Paterson M. C., Ellison M. J. A site-directed approach for constructing temperature-sensitive ubiquitin-conjugating enzymes reveals a cell cycle function and growth function for RAD6. J Biol Chem. 1991 Dec 15;266(35):24116–24120. [PubMed] [Google Scholar]
  11. Ellison M. J., Hochstrasser M. Epitope-tagged ubiquitin. A new probe for analyzing ubiquitin function. J Biol Chem. 1991 Nov 5;266(31):21150–21157. [PubMed] [Google Scholar]
  12. Ghosh D. A relational database of transcription factors. Nucleic Acids Res. 1990 Apr 11;18(7):1749–1756. doi: 10.1093/nar/18.7.1749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Glotzer M., Murray A. W., Kirschner M. W. Cyclin is degraded by the ubiquitin pathway. Nature. 1991 Jan 10;349(6305):132–138. doi: 10.1038/349132a0. [DOI] [PubMed] [Google Scholar]
  14. Goebl M. G., Goetsch L., Byers B. The Ubc3 (Cdc34) ubiquitin-conjugating enzyme is ubiquitinated and phosphorylated in vivo. Mol Cell Biol. 1994 May;14(5):3022–3029. doi: 10.1128/mcb.14.5.3022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Goebl M. G., Yochem J., Jentsch S., McGrath J. P., Varshavsky A., Byers B. The yeast cell cycle gene CDC34 encodes a ubiquitin-conjugating enzyme. Science. 1988 Sep 9;241(4871):1331–1335. doi: 10.1126/science.2842867. [DOI] [PubMed] [Google Scholar]
  16. Hartwell L. H., Culotti J., Reid B. Genetic control of the cell-division cycle in yeast. I. Detection of mutants. Proc Natl Acad Sci U S A. 1970 Jun;66(2):352–359. doi: 10.1073/pnas.66.2.352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hershko A., Ciechanover A. The ubiquitin system for protein degradation. Annu Rev Biochem. 1992;61:761–807. doi: 10.1146/annurev.bi.61.070192.003553. [DOI] [PubMed] [Google Scholar]
  18. Hill J. E., Myers A. M., Koerner T. J., Tzagoloff A. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast. 1986 Sep;2(3):163–167. doi: 10.1002/yea.320020304. [DOI] [PubMed] [Google Scholar]
  19. Hochstrasser M. Ubiquitin and intracellular protein degradation. Curr Opin Cell Biol. 1992 Dec;4(6):1024–1031. doi: 10.1016/0955-0674(92)90135-y. [DOI] [PubMed] [Google Scholar]
  20. Hope I. A., Struhl K. Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast. Cell. 1986 Sep 12;46(6):885–894. doi: 10.1016/0092-8674(86)90070-x. [DOI] [PubMed] [Google Scholar]
  21. Jentsch S., McGrath J. P., Varshavsky A. The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme. Nature. 1987 Sep 10;329(6135):131–134. doi: 10.1038/329131a0. [DOI] [PubMed] [Google Scholar]
  22. Jentsch S. The ubiquitin-conjugation system. Annu Rev Genet. 1992;26:179–207. doi: 10.1146/annurev.ge.26.120192.001143. [DOI] [PubMed] [Google Scholar]
  23. Kolman C. J., Toth J., Gonda D. K. Identification of a portable determinant of cell cycle function within the carboxyl-terminal domain of the yeast CDC34 (UBC3) ubiquitin conjugating (E2) enzyme. EMBO J. 1992 Aug;11(8):3081–3090. doi: 10.1002/j.1460-2075.1992.tb05380.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kornitzer D., Raboy B., Kulka R. G., Fink G. R. Regulated degradation of the transcription factor Gcn4. EMBO J. 1994 Dec 15;13(24):6021–6030. doi: 10.1002/j.1460-2075.1994.tb06948.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McKinney J. D., Chang F., Heintz N., Cross F. R. Negative regulation of FAR1 at the Start of the yeast cell cycle. Genes Dev. 1993 May;7(5):833–843. doi: 10.1101/gad.7.5.833. [DOI] [PubMed] [Google Scholar]
  26. Molz L., Beach D. Characterization of the fission yeast mcs2 cyclin and its associated protein kinase activity. EMBO J. 1993 Apr;12(4):1723–1732. doi: 10.1002/j.1460-2075.1993.tb05817.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Myers A. M., Tzagoloff A., Kinney D. M., Lusty C. J. Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions. Gene. 1986;45(3):299–310. doi: 10.1016/0378-1119(86)90028-4. [DOI] [PubMed] [Google Scholar]
  28. Prendergast J. A., Helgason C. D., Bleackley R. C. Quantitative polymerase chain reaction analysis of cytotoxic cell proteinase gene transcripts in T cells. Pattern of expression is dependent on the nature of the stimulus. J Biol Chem. 1992 Mar 15;267(8):5090–5095. [PubMed] [Google Scholar]
  29. Prendergast J. A., Ptak C., Arnason T. G., Ellison M. J. Increased ubiquitin expression suppresses the cell cycle defect associated with the yeast ubiquitin conjugating enzyme, CDC34 (UBC3). Evidence for a noncovalent interaction between CDC34 and ubiquitin. J Biol Chem. 1995 Apr 21;270(16):9347–9352. doi: 10.1074/jbc.270.16.9347. [DOI] [PubMed] [Google Scholar]
  30. Ptak C., Prendergast J. A., Hodgins R., Kay C. M., Chau V., Ellison M. J. Functional and physical characterization of the cell cycle ubiquitin-conjugating enzyme CDC34 (UBC3). Identification of a functional determinant within the tail that facilitates CDC34 self-association. J Biol Chem. 1994 Oct 21;269(42):26539–26545. [PubMed] [Google Scholar]
  31. Scheffner M., Huibregtse J. M., Vierstra R. D., Howley P. M. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell. 1993 Nov 5;75(3):495–505. doi: 10.1016/0092-8674(93)90384-3. [DOI] [PubMed] [Google Scholar]
  32. Scheffner M., Werness B. A., Huibregtse J. M., Levine A. J., Howley P. M. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell. 1990 Dec 21;63(6):1129–1136. doi: 10.1016/0092-8674(90)90409-8. [DOI] [PubMed] [Google Scholar]
  33. Schwob E., Böhm T., Mendenhall M. D., Nasmyth K. The B-type cyclin kinase inhibitor p40SIC1 controls the G1 to S transition in S. cerevisiae. Cell. 1994 Oct 21;79(2):233–244. doi: 10.1016/0092-8674(94)90193-7. [DOI] [PubMed] [Google Scholar]
  34. Seufert W., Futcher B., Jentsch S. Role of a ubiquitin-conjugating enzyme in degradation of S- and M-phase cyclins. Nature. 1995 Jan 5;373(6509):78–81. doi: 10.1038/373078a0. [DOI] [PubMed] [Google Scholar]
  35. Seufert W., McGrath J. P., Jentsch S. UBC1 encodes a novel member of an essential subfamily of yeast ubiquitin-conjugating enzymes involved in protein degradation. EMBO J. 1990 Dec;9(13):4535–4541. doi: 10.1002/j.1460-2075.1990.tb07905.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Silver E. T., Gwozd T. J., Ptak C., Goebl M., Ellison M. J. A chimeric ubiquitin conjugating enzyme that combines the cell cycle properties of CDC34 (UBC3) and the DNA repair properties of RAD6 (UBC2): implications for the structure, function and evolution of the E2s. EMBO J. 1992 Aug;11(8):3091–3098. doi: 10.1002/j.1460-2075.1992.tb05381.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Struhl K., Stinchcomb D. T., Scherer S., Davis R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. doi: 10.1073/pnas.76.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sullivan M. L., Vierstra R. D. Cloning of a 16-kDa ubiquitin carrier protein from wheat and Arabidopsis thaliana. Identification of functional domains by in vitro mutagenesis. J Biol Chem. 1991 Dec 15;266(35):23878–23885. [PubMed] [Google Scholar]
  40. Treier M., Staszewski L. M., Bohmann D. Ubiquitin-dependent c-Jun degradation in vivo is mediated by the delta domain. Cell. 1994 Sep 9;78(5):787–798. doi: 10.1016/s0092-8674(94)90502-9. [DOI] [PubMed] [Google Scholar]
  41. Tyers M., Tokiwa G., Nash R., Futcher B. The Cln3-Cdc28 kinase complex of S. cerevisiae is regulated by proteolysis and phosphorylation. EMBO J. 1992 May;11(5):1773–1784. doi: 10.1002/j.1460-2075.1992.tb05229.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yaglom J., Linskens M. H., Sadis S., Rubin D. M., Futcher B., Finley D. p34Cdc28-mediated control of Cln3 cyclin degradation. Mol Cell Biol. 1995 Feb;15(2):731–741. doi: 10.1128/mcb.15.2.731. [DOI] [PMC free article] [PubMed] [Google Scholar]

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