Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Oct;15(10):5635–5644. doi: 10.1128/mcb.15.10.5635

Intragenic suppression among CDC34 (UBC3) mutations defines a class of ubiquitin-conjugating catalytic domains.

Y Liu 1, N Mathias 1, C N Steussy 1, M G Goebl 1
PMCID: PMC230814  PMID: 7565715

Abstract

Ubiquitin-conjugating (E2) enzymes contain several regions within their catalytic domains that are highly conserved. However, within some of these conserved regions are several residues that may be used to define different classes of catalytic domains for the E2 enzymes. One class can be defined by the Ubc1 protein, which contains K-65, D-90, and D-120, while the corresponding positions within the Cdc34 (Ubc3) protein, which defines a second class of enzymes, contain S-73, S-97, and S-139, respectively. The presence of these differences within otherwise highly conserved regions of this family suggests that these residues may be critical for the specificity of Cdc34 function or regulation. Therefore, we have constructed a series of cdc34 alleles encoding mutant proteins in which these serine residues have been changed to other amino acid residues, including alanine and aspartic acid. In vivo complementation studies showed that S-97, which lies near the active site C-95, is essential for Cdc34 function. The addition of a second mutation in CDC34, which now encoded both the S97D and S73K changes, restored partial function to the Cdc34 enzyme. Moreover, the deletion of residues 103 to 114 within Cdc34, which are not present in the Ubc1-like E2s, allowed the S73K/S97D mutant to function as efficiently as wild-type Cdc34 protein. Finally, the cloning and sequencing of the temperature-sensitive alleles of CDC34 indicated that A-62 is also unique to the Cdc34 class of E2 enzymes and that mutations at this position can be detrimental to Cdc34 function. Our results suggest that several key residues within conserved regions of the E2 enzyme family genetically interact with each other and define a class of E2 catalytic domains.

Full Text

The Full Text of this article is available as a PDF (378.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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]
  2. Boeke J. D., LaCroute F., Fink G. R. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197(2):345–346. doi: 10.1007/BF00330984. [DOI] [PubMed] [Google Scholar]
  3. Bram R. J., Kornberg R. D. Specific protein binding to far upstream activating sequences in polymerase II promoters. Proc Natl Acad Sci U S A. 1985 Jan;82(1):43–47. doi: 10.1073/pnas.82.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Cook W. J., Jeffrey L. C., Xu Y., Chau V. Tertiary structures of class I ubiquitin-conjugating enzymes are highly conserved: crystal structure of yeast Ubc4. Biochemistry. 1993 Dec 21;32(50):13809–13817. doi: 10.1021/bi00213a009. [DOI] [PubMed] [Google Scholar]
  7. Cunningham B. C., Wells J. A. High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science. 1989 Jun 2;244(4908):1081–1085. doi: 10.1126/science.2471267. [DOI] [PubMed] [Google Scholar]
  8. Dohmen R. J., Madura K., Bartel B., Varshavsky A. The N-end rule is mediated by the UBC2(RAD6) ubiquitin-conjugating enzyme. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7351–7355. doi: 10.1073/pnas.88.16.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dumas L. B., Lussky J. P., McFarland E. J., Shampay J. New temperature-sensitive mutants of Saccharomyces cerevisiae affecting DNA replication. Mol Gen Genet. 1982;187(1):42–46. doi: 10.1007/BF00384381. [DOI] [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. Finley D., Chau V. Ubiquitination. Annu Rev Cell Biol. 1991;7:25–69. doi: 10.1146/annurev.cb.07.110191.000325. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Hingamp P. M., Arnold J. E., Mayer R. J., Dixon L. K. A ubiquitin conjugating enzyme encoded by African swine fever virus. EMBO J. 1992 Jan;11(1):361–366. doi: 10.1002/j.1460-2075.1992.tb05058.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Jungmann J., Reins H. A., Schobert C., Jentsch S. Resistance to cadmium mediated by ubiquitin-dependent proteolysis. Nature. 1993 Jan 28;361(6410):369–371. doi: 10.1038/361369a0. [DOI] [PubMed] [Google Scholar]
  18. Koken M., Reynolds P., Bootsma D., Hoeijmakers J., Prakash S., Prakash L. Dhr6, a Drosophila homolog of the yeast DNA-repair gene RAD6. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3832–3836. doi: 10.1073/pnas.88.9.3832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Mathias N., Bayés M., Tyler-Smith C. Highly informative compound haplotypes for the human Y chromosome. Hum Mol Genet. 1994 Jan;3(1):115–123. doi: 10.1093/hmg/3.1.115. [DOI] [PubMed] [Google Scholar]
  21. Plon S. E., Leppig K. A., Do H. N., Groudine M. Cloning of the human homolog of the CDC34 cell cycle gene by complementation in yeast. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10484–10488. doi: 10.1073/pnas.90.22.10484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Reynolds P., Weber S., Prakash L. RAD6 gene of Saccharomyces cerevisiae encodes a protein containing a tract of 13 consecutive aspartates. Proc Natl Acad Sci U S A. 1985 Jan;82(1):168–172. doi: 10.1073/pnas.82.1.168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rose M. D., Broach J. R. Cloning genes by complementation in yeast. Methods Enzymol. 1991;194:195–230. doi: 10.1016/0076-6879(91)94017-7. [DOI] [PubMed] [Google Scholar]
  26. Rothstein R. Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol. 1991;194:281–301. doi: 10.1016/0076-6879(91)94022-5. [DOI] [PubMed] [Google Scholar]
  27. Schuler G. D., Altschul S. F., Lipman D. J. A workbench for multiple alignment construction and analysis. Proteins. 1991;9(3):180–190. doi: 10.1002/prot.340090304. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Seufert W., Jentsch S. Ubiquitin-conjugating enzymes UBC4 and UBC5 mediate selective degradation of short-lived and abnormal proteins. EMBO J. 1990 Feb;9(2):543–550. doi: 10.1002/j.1460-2075.1990.tb08141.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Sharon G., Raboy B., Parag H. A., Dimitrovsky D., Kulka R. G. RAD6 gene product of Saccharomyces cerevisiae requires a putative ubiquitin protein ligase (E3) for the ubiquitination of certain proteins. J Biol Chem. 1991 Aug 25;266(24):15890–15894. [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Sung P., Berleth E., Pickart C., Prakash S., Prakash L. Yeast RAD6 encoded ubiquitin conjugating enzyme mediates protein degradation dependent on the N-end-recognizing E3 enzyme. EMBO J. 1991 Aug;10(8):2187–2193. doi: 10.1002/j.1460-2075.1991.tb07754.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sung P., Prakash S., Prakash L. The RAD6 protein of Saccharomyces cerevisiae polyubiquitinates histones, and its acidic domain mediates this activity. Genes Dev. 1988 Nov;2(11):1476–1485. doi: 10.1101/gad.2.11.1476. [DOI] [PubMed] [Google Scholar]
  36. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Treier M., Seufert W., Jentsch S. Drosophila UbcD1 encodes a highly conserved ubiquitin-conjugating enzyme involved in selective protein degradation. EMBO J. 1992 Jan;11(1):367–372. doi: 10.1002/j.1460-2075.1992.tb05059.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Van Nocker S., Vierstra R. D. Cloning and characterization of a 20-kDa ubiquitin carrier protein from wheat that catalyzes multiubiquitin chain formation in vitro. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10297–10301. doi: 10.1073/pnas.88.22.10297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Vassal A., Boulet A., Decoster E., Faye G. QRI8, a novel ubiquitin-conjugating enzyme in Saccharomyces cerevisiae. Biochim Biophys Acta. 1992 Sep 24;1132(2):211–213. doi: 10.1016/0167-4781(92)90015-r. [DOI] [PubMed] [Google Scholar]
  40. Wiebel F. F., Kunau W. H. The Pas2 protein essential for peroxisome biogenesis is related to ubiquitin-conjugating enzymes. Nature. 1992 Sep 3;359(6390):73–76. doi: 10.1038/359073a0. [DOI] [PubMed] [Google Scholar]
  41. Zhen M., Heinlein R., Jones D., Jentsch S., Candido E. P. The ubc-2 gene of Caenorhabditis elegans encodes a ubiquitin-conjugating enzyme involved in selective protein degradation. Mol Cell Biol. 1993 Mar;13(3):1371–1377. doi: 10.1128/mcb.13.3.1371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES