Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 May 15;17(10):2759–2766. doi: 10.1093/emboj/17.10.2759

Degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae.

T Gilon 1, O Chomsky 1, R G Kulka 1
PMCID: PMC1170616  PMID: 9582269

Abstract

Combinations of different ubiquitin-conjugating (Ubc) enzymes and other factors constitute subsidiary pathways of the ubiquitin system, each of which ubiquitinates a specific subset of proteins. There is evidence that certain sequence elements or structural motifs of target proteins are degradation signals which mark them for ubiquitination by a particular branch of the ubiquitin system and for subsequent degradation. Our aim was to devise a way of searching systematically for degradation signals and to determine to which ubiquitin system subpathways they direct the proteins. We have constructed two reporter gene libraries based on the lacZ or URA3 genes which, in Saccharomyces cerevisiae, express fusion proteins with a wide variety of C-terminal extensions. From these, we have isolated clones producing unstable fusion proteins which are stabilized in various ubc mutants. Among these are 10 clones whose products are stabilized in ubc6, ubc7 or ubc6ubc7 double mutants. The C-terminal extensions of these clones, which vary in length from 16 to 50 amino acid residues, are presumed to contain degradation signals channeling proteins for degradation via the UBC6 and/or UBC7 subpathways of the ubiquitin system. Some of these C-terminal tails share similar sequence motifs, and a feature common to almost all of these sequences is a highly hydrophobic region such as is usually located inside globular proteins or inserted into membranes.

Full Text

The Full Text of this article is available as a PDF (324.1 KB).

Selected References

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

  1. Altuvia Y., Berzofsky J. A., Rosenfeld R., Margalit H. Sequence features that correlate with MHC restriction. Mol Immunol. 1994 Jan;31(1):1–19. doi: 10.1016/0161-5890(94)90133-3. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Biederer T., Volkwein C., Sommer T. Degradation of subunits of the Sec61p complex, an integral component of the ER membrane, by the ubiquitin-proteasome pathway. EMBO J. 1996 May 1;15(9):2069–2076. [PMC free article] [PubMed] [Google Scholar]
  5. Biederer T., Volkwein C., Sommer T. Role of Cue1p in ubiquitination and degradation at the ER surface. Science. 1997 Dec 5;278(5344):1806–1809. doi: 10.1126/science.278.5344.1806. [DOI] [PubMed] [Google Scholar]
  6. Bonifacino J. S., Cosson P., Klausner R. D. Colocalized transmembrane determinants for ER degradation and subunit assembly explain the intracellular fate of TCR chains. Cell. 1990 Nov 2;63(3):503–513. doi: 10.1016/0092-8674(90)90447-m. [DOI] [PubMed] [Google Scholar]
  7. Bonifacino J. S., Cosson P., Shah N., Klausner R. D. Role of potentially charged transmembrane residues in targeting proteins for retention and degradation within the endoplasmic reticulum. EMBO J. 1991 Oct;10(10):2783–2793. doi: 10.1002/j.1460-2075.1991.tb07827.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen P., Johnson P., Sommer T., Jentsch S., Hochstrasser M. Multiple ubiquitin-conjugating enzymes participate in the in vivo degradation of the yeast MAT alpha 2 repressor. Cell. 1993 Jul 30;74(2):357–369. doi: 10.1016/0092-8674(93)90426-q. [DOI] [PubMed] [Google Scholar]
  9. Ciechanover A. The ubiquitin-proteasome proteolytic pathway. Cell. 1994 Oct 7;79(1):13–21. doi: 10.1016/0092-8674(94)90396-4. [DOI] [PubMed] [Google Scholar]
  10. Cox J. S., Walter P. A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response. Cell. 1996 Nov 1;87(3):391–404. doi: 10.1016/s0092-8674(00)81360-4. [DOI] [PubMed] [Google Scholar]
  11. Dohmen R. J., Wu P., Varshavsky A. Heat-inducible degron: a method for constructing temperature-sensitive mutants. Science. 1994 Mar 4;263(5151):1273–1276. doi: 10.1126/science.8122109. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Esnault Y., Feldheim D., Blondel M. O., Schekman R., Képès F. SSS1 encodes a stabilizing component of the Sec61 subcomplex of the yeast protein translocation apparatus. J Biol Chem. 1994 Nov 4;269(44):27478–27485. [PubMed] [Google Scholar]
  14. 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]
  15. Goffeau A., Barrell B. G., Bussey H., Davis R. W., Dujon B., Feldmann H., Galibert F., Hoheisel J. D., Jacq C., Johnston M. Life with 6000 genes. Science. 1996 Oct 25;274(5287):546, 563-7. doi: 10.1126/science.274.5287.546. [DOI] [PubMed] [Google Scholar]
  16. Gottesman S., Maurizi M. R. Regulation by proteolysis: energy-dependent proteases and their targets. Microbiol Rev. 1992 Dec;56(4):592–621. doi: 10.1128/mr.56.4.592-621.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Guarente L. Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol. 1983;101:181–191. doi: 10.1016/0076-6879(83)01013-7. [DOI] [PubMed] [Google Scholar]
  18. Heinemeyer W., Gruhler A., Möhrle V., Mahé Y., Wolf D. H. PRE2, highly homologous to the human major histocompatibility complex-linked RING10 gene, codes for a yeast proteasome subunit necessary for chrymotryptic activity and degradation of ubiquitinated proteins. J Biol Chem. 1993 Mar 5;268(7):5115–5120. [PubMed] [Google Scholar]
  19. 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]
  20. Hicke L., Riezman H. Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis. Cell. 1996 Jan 26;84(2):277–287. doi: 10.1016/s0092-8674(00)80982-4. [DOI] [PubMed] [Google Scholar]
  21. Hiller M. M., Finger A., Schweiger M., Wolf D. H. ER degradation of a misfolded luminal protein by the cytosolic ubiquitin-proteasome pathway. Science. 1996 Sep 20;273(5282):1725–1728. doi: 10.1126/science.273.5282.1725. [DOI] [PubMed] [Google Scholar]
  22. Hilt W., Enenkel C., Gruhler A., Singer T., Wolf D. H. The PRE4 gene codes for a subunit of the yeast proteasome necessary for peptidylglutamyl-peptide-hydrolyzing activity. Mutations link the proteasome to stress- and ubiquitin-dependent proteolysis. J Biol Chem. 1993 Feb 15;268(5):3479–3486. [PubMed] [Google Scholar]
  23. Hilt W., Wolf D. H. Proteasomes: destruction as a programme. Trends Biochem Sci. 1996 Mar;21(3):96–102. [PubMed] [Google Scholar]
  24. Hochstrasser M. Protein degradation or regulation: Ub the judge. Cell. 1996 Mar 22;84(6):813–815. doi: 10.1016/s0092-8674(00)81058-2. [DOI] [PubMed] [Google Scholar]
  25. Hochstrasser M. Ubiquitin, proteasomes, and the regulation of intracellular protein degradation. Curr Opin Cell Biol. 1995 Apr;7(2):215–223. doi: 10.1016/0955-0674(95)80031-x. [DOI] [PubMed] [Google Scholar]
  26. Hochstrasser M. Ubiquitin-dependent protein degradation. Annu Rev Genet. 1996;30:405–439. doi: 10.1146/annurev.genet.30.1.405. [DOI] [PubMed] [Google Scholar]
  27. Hochstrasser M., Varshavsky A. In vivo degradation of a transcriptional regulator: the yeast alpha 2 repressor. Cell. 1990 May 18;61(4):697–708. doi: 10.1016/0092-8674(90)90481-s. [DOI] [PubMed] [Google Scholar]
  28. Jentsch S., Schlenker S. Selective protein degradation: a journey's end within the proteasome. Cell. 1995 Sep 22;82(6):881–884. doi: 10.1016/0092-8674(95)90021-7. [DOI] [PubMed] [Google Scholar]
  29. Johnson E. S., Ma P. C., Ota I. M., Varshavsky A. A proteolytic pathway that recognizes ubiquitin as a degradation signal. J Biol Chem. 1995 Jul 21;270(29):17442–17456. doi: 10.1074/jbc.270.29.17442. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Keiler K. C., Waller P. R., Sauer R. T. Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science. 1996 Feb 16;271(5251):990–993. doi: 10.1126/science.271.5251.990. [DOI] [PubMed] [Google Scholar]
  32. King R. W., Deshaies R. J., Peters J. M., Kirschner M. W. How proteolysis drives the cell cycle. Science. 1996 Dec 6;274(5293):1652–1659. doi: 10.1126/science.274.5293.1652. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  35. Lahav-Baratz S., Sudakin V., Ruderman J. V., Hershko A. Reversible phosphorylation controls the activity of cyclosome-associated cyclin-ubiquitin ligase. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9303–9307. doi: 10.1073/pnas.92.20.9303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lanker S., Valdivieso M. H., Wittenberg C. Rapid degradation of the G1 cyclin Cln2 induced by CDK-dependent phosphorylation. Science. 1996 Mar 15;271(5255):1597–1601. doi: 10.1126/science.271.5255.1597. [DOI] [PubMed] [Google Scholar]
  37. Lankford S. P., Cosson P., Bonifacino J. S., Klausner R. D. Transmembrane domain length affects charge-mediated retention and degradation of proteins within the endoplasmic reticulum. J Biol Chem. 1993 Mar 5;268(7):4814–4820. [PubMed] [Google Scholar]
  38. Papa F. R., Hochstrasser M. The yeast DOA4 gene encodes a deubiquitinating enzyme related to a product of the human tre-2 oncogene. Nature. 1993 Nov 25;366(6453):313–319. doi: 10.1038/366313a0. [DOI] [PubMed] [Google Scholar]
  39. Parag H. A., Raboy B., Kulka R. G. Effect of heat shock on protein degradation in mammalian cells: involvement of the ubiquitin system. EMBO J. 1987 Jan;6(1):55–61. doi: 10.1002/j.1460-2075.1987.tb04718.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Plemper R. K., Böhmler S., Bordallo J., Sommer T., Wolf D. H. Mutant analysis links the translocon and BiP to retrograde protein transport for ER degradation. Nature. 1997 Aug 28;388(6645):891–895. doi: 10.1038/42276. [DOI] [PubMed] [Google Scholar]
  41. Rechsteiner M., Rogers S. W. PEST sequences and regulation by proteolysis. Trends Biochem Sci. 1996 Jul;21(7):267–271. [PubMed] [Google Scholar]
  42. Rubin D. M., Finley D. Proteolysis. The proteasome: a protein-degrading organelle? Curr Biol. 1995 Aug 1;5(8):854–858. doi: 10.1016/s0960-9822(95)00172-2. [DOI] [PubMed] [Google Scholar]
  43. Rüther U., Müller-Hill B. Easy identification of cDNA clones. EMBO J. 1983;2(10):1791–1794. doi: 10.1002/j.1460-2075.1983.tb01659.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sadis S., Atienza C., Jr, Finley D. Synthetic signals for ubiquitin-dependent proteolysis. Mol Cell Biol. 1995 Aug;15(8):4086–4094. doi: 10.1128/mcb.15.8.4086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. 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]
  47. 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]
  48. Sikorski R. S., Boeke J. D. In vitro mutagenesis and plasmid shuffling: from cloned gene to mutant yeast. Methods Enzymol. 1991;194:302–318. doi: 10.1016/0076-6879(91)94023-6. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. Sommer T., Jentsch S. A protein translocation defect linked to ubiquitin conjugation at the endoplasmic reticulum. Nature. 1993 Sep 9;365(6442):176–179. doi: 10.1038/365176a0. [DOI] [PubMed] [Google Scholar]
  51. Varshavsky A. The N-end rule. Cell. 1992 May 29;69(5):725–735. doi: 10.1016/0092-8674(92)90285-k. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. Wiertz E. J., Tortorella D., Bogyo M., Yu J., Mothes W., Jones T. R., Rapoport T. A., Ploegh H. L. Sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction. Nature. 1996 Dec 5;384(6608):432–438. doi: 10.1038/384432a0. [DOI] [PubMed] [Google Scholar]
  54. 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]

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

RESOURCES