Involvement of the molecular chaperone Ydj1 in the ubiquitin-dependent degradation of short-lived and abnormal proteins in Saccharomyces cerevisiae (original) (raw)

Abstract

In Escherichia coli and mitochondria, the molecular chaperone DnaJ is required not only for protein folding but also for selective degradation of certain abnormal polypeptides. Here we demonstrate that in the yeast cytosol, the homologous chaperone Ydj1 is also required for ubiquitin-dependent degradation of certain abnormal proteins. The temperature-sensitive ydj1-151 mutant showed a large defect in the overall breakdown of short-lived cell proteins and abnormal polypeptides containing amino acid analogs, especially at 38 degrees C. By contrast, the degradation of long-lived cell proteins, which is independent of ubiquitin, was not altered nor was cell growth affected. The inactivation of Ydj1 markedly reduced the rapid, ubiquitin-dependent breakdown of certain beta-galactosidase (beta-gal) fusion polypeptides. Although degradation of N-end rule substrates (arginine-beta-gal and leucine-beta-gal) and the B-type cyclin Clb5-beta-gal occurred normally, degradation of the abnormal polypeptide ubiquitin-proline-beta-gal (Ub-P-beta-gal) and that of the short-lived normal protein Gcn4 were inhibited. As a consequence of reduced degradation of Ub-P-beta-gal, the beta-gal activity was four to five times higher in temperature-sensitive ydj1-151 mutant cells than in wild-type cells; thus, the folding and assembly of this enzyme do not require Ydj1 function. In wild-type cells, but not in ydj1-151 mutant cells, this chaperone is associated with the short-lived substrate Ub-P-beta-gal and not with stable beta-gal constructs. Furthermore, in the ydj1-151 mutant, the ubiquitination of Ub-P-beta-gal was blocked and the total level of ubiquitinated protein in the cell was reduced. Thus, Ydj1 is essential for the ubiquitin-dependent degradation of certain proteins. This chaperone may facilitate the recognition of unfolded proteins or serve as a cofactor for certain ubiquitin-ligating enzymes.

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Selected References

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  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. Caplan A. J., Cyr D. M., Douglas M. G. YDJ1p facilitates polypeptide translocation across different intracellular membranes by a conserved mechanism. Cell. 1992 Dec 24;71(7):1143–1155. doi: 10.1016/s0092-8674(05)80063-7. [DOI] [PubMed] [Google Scholar]
  3. Chirico W. J., Waters M. G., Blobel G. 70K heat shock related proteins stimulate protein translocation into microsomes. Nature. 1988 Apr 28;332(6167):805–810. doi: 10.1038/332805a0. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Craig E. A., Gambill B. D., Nelson R. J. Heat shock proteins: molecular chaperones of protein biogenesis. Microbiol Rev. 1993 Jun;57(2):402–414. doi: 10.1128/mr.57.2.402-414.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cyr D. M., Langer T., Douglas M. G. DnaJ-like proteins: molecular chaperones and specific regulators of Hsp70. Trends Biochem Sci. 1994 Apr;19(4):176–181. doi: 10.1016/0968-0004(94)90281-x. [DOI] [PubMed] [Google Scholar]
  7. Cyr D. M., Lu X., Douglas M. G. Regulation of Hsp70 function by a eukaryotic DnaJ homolog. J Biol Chem. 1992 Oct 15;267(29):20927–20931. [PubMed] [Google Scholar]
  8. Deshaies R. J., Koch B. D., Werner-Washburne M., Craig E. A., Schekman R. A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature. 1988 Apr 28;332(6167):800–805. doi: 10.1038/332800a0. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Figueiredo-Pereira M. E., Berg K. A., Wilk S. A new inhibitor of the chymotrypsin-like activity of the multicatalytic proteinase complex (20S proteasome) induces accumulation of ubiquitin-protein conjugates in a neuronal cell. J Neurochem. 1994 Oct;63(4):1578–1581. doi: 10.1046/j.1471-4159.1994.63041578.x. [DOI] [PubMed] [Google Scholar]
  11. Georgopoulos C., Welch W. J. Role of the major heat shock proteins as molecular chaperones. Annu Rev Cell Biol. 1993;9:601–634. doi: 10.1146/annurev.cb.09.110193.003125. [DOI] [PubMed] [Google Scholar]
  12. Goldberg A. L. Functions of the proteasome: the lysis at the end of the tunnel. Science. 1995 Apr 28;268(5210):522–523. doi: 10.1126/science.7725095. [DOI] [PubMed] [Google Scholar]
  13. Goldberg A. L., St John A. C. Intracellular protein degradation in mammalian and bacterial cells: Part 2. Annu Rev Biochem. 1976;45:747–803. doi: 10.1146/annurev.bi.45.070176.003531. [DOI] [PubMed] [Google Scholar]
  14. Gragerov A., Nudler E., Komissarova N., Gaitanaris G. A., Gottesman M. E., Nikiforov V. Cooperation of GroEL/GroES and DnaK/DnaJ heat shock proteins in preventing protein misfolding in Escherichia coli. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10341–10344. doi: 10.1073/pnas.89.21.10341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Hendrick J. P., Hartl F. U. Molecular chaperone functions of heat-shock proteins. Annu Rev Biochem. 1993;62:349–384. doi: 10.1146/annurev.bi.62.070193.002025. [DOI] [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. Höhfeld J., Minami Y., Hartl F. U. Hip, a novel cochaperone involved in the eukaryotic Hsc70/Hsp40 reaction cycle. Cell. 1995 Nov 17;83(4):589–598. doi: 10.1016/0092-8674(95)90099-3. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Johnson E. S., Bartel B., Seufert W., Varshavsky A. Ubiquitin as a degradation signal. EMBO J. 1992 Feb;11(2):497–505. doi: 10.1002/j.1460-2075.1992.tb05080.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jones E. W. Three proteolytic systems in the yeast saccharomyces cerevisiae. J Biol Chem. 1991 May 5;266(13):7963–7966. [PubMed] [Google Scholar]
  22. Kandror O., Busconi L., Sherman M., Goldberg A. L. Rapid degradation of an abnormal protein in Escherichia coli involves the chaperones GroEL and GroES. J Biol Chem. 1994 Sep 23;269(38):23575–23582. [PubMed] [Google Scholar]
  23. Knittler M. R., Dirks S., Haas I. G. Molecular chaperones involved in protein degradation in the endoplasmic reticulum: quantitative interaction of the heat shock cognate protein BiP with partially folded immunoglobulin light chains that are degraded in the endoplasmic reticulum. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1764–1768. doi: 10.1073/pnas.92.5.1764. [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. Pratt G., Hough R., Rechsteiner M. Proteolysis in heat-stressed HeLa cells. Stabilization of ubiquitin correlates with the loss of proline endopeptidase. J Biol Chem. 1989 Jul 25;264(21):12526–12532. [PubMed] [Google Scholar]
  26. Rechsteiner M., Hoffman L., Dubiel W. The multicatalytic and 26 S proteases. J Biol Chem. 1993 Mar 25;268(9):6065–6068. [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Sherman MYu, Goldberg A. L. Involvement of the chaperonin dnaK in the rapid degradation of a mutant protein in Escherichia coli. EMBO J. 1992 Jan;11(1):71–77. doi: 10.1002/j.1460-2075.1992.tb05029.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Straus D. B., Walter W. A., Gross C. A. Escherichia coli heat shock gene mutants are defective in proteolysis. Genes Dev. 1988 Dec;2(12B):1851–1858. doi: 10.1101/gad.2.12b.1851. [DOI] [PubMed] [Google Scholar]
  31. Straus D., Walter W., Gross C. A. DnaK, DnaJ, and GrpE heat shock proteins negatively regulate heat shock gene expression by controlling the synthesis and stability of sigma 32. Genes Dev. 1990 Dec;4(12A):2202–2209. doi: 10.1101/gad.4.12a.2202. [DOI] [PubMed] [Google Scholar]
  32. Tilly K., Spence J., Georgopoulos C. Modulation of stability of the Escherichia coli heat shock regulatory factor sigma. J Bacteriol. 1989 Mar;171(3):1585–1589. doi: 10.1128/jb.171.3.1585-1589.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Wagner I., Arlt H., van Dyck L., Langer T., Neupert W. Molecular chaperones cooperate with PIM1 protease in the degradation of misfolded proteins in mitochondria. EMBO J. 1994 Nov 1;13(21):5135–5145. doi: 10.1002/j.1460-2075.1994.tb06843.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wild J., Altman E., Yura T., Gross C. A. DnaK and DnaJ heat shock proteins participate in protein export in Escherichia coli. Genes Dev. 1992 Jul;6(7):1165–1172. doi: 10.1101/gad.6.7.1165. [DOI] [PubMed] [Google Scholar]
  36. Yaglom J. A., Goldberg A. L., Finley D., Sherman M. Y. The molecular chaperone Ydj1 is required for the p34CDC28-dependent phosphorylation of the cyclin Cln3 that signals its degradation. Mol Cell Biol. 1996 Jul;16(7):3679–3684. doi: 10.1128/mcb.16.7.3679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zhong T., Arndt K. T. The yeast SIS1 protein, a DnaJ homolog, is required for the initiation of translation. Cell. 1993 Jun 18;73(6):1175–1186. doi: 10.1016/0092-8674(93)90646-8. [DOI] [PubMed] [Google Scholar]