Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1991 Aug;10(8):2187–2193. doi: 10.1002/j.1460-2075.1991.tb07754.x

Yeast RAD6 encoded ubiquitin conjugating enzyme mediates protein degradation dependent on the N-end-recognizing E3 enzyme.

P Sung 1, E Berleth 1, C Pickart 1, S Prakash 1, L Prakash 1
PMCID: PMC452907  PMID: 2065660

Abstract

The RAD6 gene of Saccharomyces cerevisiae encodes a 20 kd ubiquitin conjugating (E2) enzyme that is required for DNA repair, DNA damage-induced mutagenesis, and sporulation. Here, we demonstrate a novel activity of RAD6 protein--its ability to mediate protein degradation dependent on the N-end-recognizing ubiquitin protein ligase (E3). In reaction mixtures containing E1, E3 and the ubiquitin specific protease from rabbit reticulocytes, RAD6 is as effective as mammalian E214k in E3 dependent ubiquitin--protein conjugate formation and subsequent protein degradation. The ubiquitin conjugating activity of RAD6 is required for these reactions as indicated by the ineffectiveness of the rad6 Ala88 and rad6 Val88 mutant proteins, which lack the ability to form a thioester adduct with ubiquitin and therefore do not conjugate ubiquitin to substrates. We also show that the highly acidic carboxyl-terminus of RAD6 is dispensable for the interaction with E3, and that purified S. cerevisiae E2(30k), product of the UBC1 gene, does not function with E3. These findings demonstrate a specific interaction between RAD6 and E3, and highlight the strong conservation of the ubiquitin conjugating system in eukaryotes. We suggest a function for RAD6 mediated E3 dependent protein degradation in sporulation, and discuss the possible role of this activity during vegetative growth.

Full text

PDF
2187

Images in this article

Selected References

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

  1. Bachmair A., Finley D., Varshavsky A. In vivo half-life of a protein is a function of its amino-terminal residue. Science. 1986 Oct 10;234(4773):179–186. doi: 10.1126/science.3018930. [DOI] [PubMed] [Google Scholar]
  2. Bartel B., Wünning I., Varshavsky A. The recognition component of the N-end rule pathway. EMBO J. 1990 Oct;9(10):3179–3189. doi: 10.1002/j.1460-2075.1990.tb07516.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berleth E. S., Pickart C. M. Several mammalian ubiquitin carrier proteins, but not E2(20K), are related to the 20-kDa yeast E2, RAD6. Biochem Biophys Res Commun. 1990 Sep 14;171(2):705–710. doi: 10.1016/0006-291x(90)91203-5. [DOI] [PubMed] [Google Scholar]
  4. Chau V., Tobias J. W., Bachmair A., Marriott D., Ecker D. J., Gonda D. K., Varshavsky A. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science. 1989 Mar 24;243(4898):1576–1583. doi: 10.1126/science.2538923. [DOI] [PubMed] [Google Scholar]
  5. Ciechanover A., Elias S., Heller H., Hershko A. "Covalent affinity" purification of ubiquitin-activating enzyme. J Biol Chem. 1982 Mar 10;257(5):2537–2542. [PubMed] [Google Scholar]
  6. Evans A. C., Jr, Wilkinson K. D. Ubiquitin-dependent proteolysis of native and alkylated bovine serum albumin: effects of protein structure and ATP concentration on selectivity. Biochemistry. 1985 Jun 4;24(12):2915–2923. doi: 10.1021/bi00333a015. [DOI] [PubMed] [Google Scholar]
  7. Ferber S., Ciechanover A. Role of arginine-tRNA in protein degradation by the ubiquitin pathway. Nature. 1987 Apr 23;326(6115):808–811. doi: 10.1038/326808a0. [DOI] [PubMed] [Google Scholar]
  8. Finley D., Bartel B., Varshavsky A. The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis. Nature. 1989 Mar 30;338(6214):394–401. doi: 10.1038/338394a0. [DOI] [PubMed] [Google Scholar]
  9. Finley D., Ozkaynak E., Varshavsky A. The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses. Cell. 1987 Mar 27;48(6):1035–1046. doi: 10.1016/0092-8674(87)90711-2. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Gonda D. K., Bachmair A., Wünning I., Tobias J. W., Lane W. S., Varshavsky A. Universality and structure of the N-end rule. J Biol Chem. 1989 Oct 5;264(28):16700–16712. [PubMed] [Google Scholar]
  12. Haas A. L., Bright P. M. The resolution and characterization of putative ubiquitin carrier protein isozymes from rabbit reticulocytes. J Biol Chem. 1988 Sep 15;263(26):13258–13267. [PubMed] [Google Scholar]
  13. Haas A., Reback P. M., Pratt G., Rechsteiner M. Ubiquitin-mediated degradation of histone H3 does not require the substrate-binding ubiquitin protein ligase, E3, or attachment of polyubiquitin chains. J Biol Chem. 1990 Dec 15;265(35):21664–21669. [PubMed] [Google Scholar]
  14. Hershko A., Ciechanover A., Heller H., Haas A. L., Rose I. A. Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1783–1786. doi: 10.1073/pnas.77.4.1783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hershko A., Heller H., Elias S., Ciechanover A. Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown. J Biol Chem. 1983 Jul 10;258(13):8206–8214. [PubMed] [Google Scholar]
  16. Hershko A., Leshinsky E., Ganoth D., Heller H. ATP-dependent degradation of ubiquitin-protein conjugates. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1619–1623. doi: 10.1073/pnas.81.6.1619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hershko A. Ubiquitin-mediated protein degradation. J Biol Chem. 1988 Oct 25;263(30):15237–15240. [PubMed] [Google Scholar]
  18. 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]
  19. Jentsch S., Seufert W., Sommer T., Reins H. A. Ubiquitin-conjugating enzymes: novel regulators of eukaryotic cells. Trends Biochem Sci. 1990 May;15(5):195–198. doi: 10.1016/0968-0004(90)90161-4. [DOI] [PubMed] [Google Scholar]
  20. Klemperer N. S., Berleth E. S., Pickart C. M. A novel, arsenite-sensitive E2 of the ubiquitin pathway: purification and properties. Biochemistry. 1989 Jul 11;28(14):6035–6041. doi: 10.1021/bi00440a047. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Madura K., Prakash S., Prakash L. Expression of the Saccharomyces cerevisiae DNA repair gene RAD6 that encodes a ubiquitin conjugating enzyme, increases in response to DNA damage and in meiosis but remains constant during the mitotic cell cycle. Nucleic Acids Res. 1990 Feb 25;18(4):771–778. doi: 10.1093/nar/18.4.771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Morrison A., Miller E. J., Prakash L. Domain structure and functional analysis of the carboxyl-terminal polyacidic sequence of the RAD6 protein of Saccharomyces cerevisiae. Mol Cell Biol. 1988 Mar;8(3):1179–1185. doi: 10.1128/mcb.8.3.1179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ozkaynak E., Finley D., Solomon M. J., Varshavsky A. The yeast ubiquitin genes: a family of natural gene fusions. EMBO J. 1987 May;6(5):1429–1439. doi: 10.1002/j.1460-2075.1987.tb02384.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pickart C. M., Rose I. A. Functional heterogeneity of ubiquitin carrier proteins. J Biol Chem. 1985 Feb 10;260(3):1573–1581. [PubMed] [Google Scholar]
  27. Pickart C. M., Vella A. T. Levels of active ubiquitin carrier proteins decline during erythroid maturation. J Biol Chem. 1988 Aug 25;263(24):12028–12035. [PubMed] [Google Scholar]
  28. Reiss Y., Hershko A. Affinity purification of ubiquitin-protein ligase on immobilized protein substrates. Evidence for the existence of separate NH2-terminal binding sites on a single enzyme. J Biol Chem. 1990 Mar 5;265(7):3685–3690. [PubMed] [Google Scholar]
  29. Reiss Y., Kaim D., Hershko A. Specificity of binding of NH2-terminal residue of proteins to ubiquitin-protein ligase. Use of amino acid derivatives to characterize specific binding sites. J Biol Chem. 1988 Feb 25;263(6):2693–2698. [PubMed] [Google Scholar]
  30. Reynolds P., Koken M. H., Hoeijmakers J. H., Prakash S., Prakash L. The rhp6+ gene of Schizosaccharomyces pombe: a structural and functional homolog of the RAD6 gene from the distantly related yeast Saccharomyces cerevisiae. EMBO J. 1990 May;9(5):1423–1430. doi: 10.1002/j.1460-2075.1990.tb08258.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Schneider R., Eckerskorn C., Lottspeich F., Schweiger M. The human ubiquitin carrier protein E2(Mr = 17,000) is homologous to the yeast DNA repair gene RAD6. EMBO J. 1990 May;9(5):1431–1435. doi: 10.1002/j.1460-2075.1990.tb08259.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Sung P., Prakash S., Prakash L. Mutation of cysteine-88 in the Saccharomyces cerevisiae RAD6 protein abolishes its ubiquitin-conjugating activity and its various biological functions. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2695–2699. doi: 10.1073/pnas.87.7.2695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Treger J. M., Heichman K. A., McEntee K. Expression of the yeast UB14 gene increases in response to DNA-damaging agents and in meiosis. Mol Cell Biol. 1988 Mar;8(3):1132–1136. doi: 10.1128/mcb.8.3.1132. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES