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. 1996 Jul;16(7):3679–3684. doi: 10.1128/mcb.16.7.3679

The molecular chaperone Ydj1 is required for the p34CDC28-dependent phosphorylation of the cyclin Cln3 that signals its degradation.

J A Yaglom 1, A L Goldberg 1, D Finley 1, M Y Sherman 1
PMCID: PMC231363  PMID: 8668184

Abstract

The G1 cyclin Cln3 of the yeast Saccharomyces cerevisiae is rapidly degraded by the ubiquitin-proteasome pathway. This process is triggered by p34CDC28-dependent phosphorylation of Cln3. Here we demonstrate that the molecular chaperone Ydj1, a DnaJ homolog, is required for this phosphorylation. In a ydj1 mutant at the nonpermissive temperature, both phosphorylation and degradation of Cln3 were deficient. No change was seen upon inactivation of Sis1, another DnaJ homolog. The phosphorylation defect in the ydj1 mutant was specific to Cln3, because no reduction in the phosphorylation of Cln2 or histone H1, which also requires p34CDC28, was observed. Ydj1 was required for Cln3 phosphorylation and degradation rather than for the proper folding of this cyclin, since Cln3 produced in the ydj1 mutant was fully active in the stimulation of p34CDC28 histone kinase activity. Moreover, Ydj1 directly associates with Cln3 in close proximity to the segment that is phosphorylated and signals degradation. Thus, binding of Ydj1 to this domain of Cln3 seems to be essential for the phosphorylation and breakdown of this cyclin. In a cell-free system, purified Ydj1 stimulated the p34CDC28-dependent phosphorylation of the C-terminal segment of Cln3 and did not affect phosphorylation of Cln2 (as was found in vivo). The reconstitution of this process with pure components provides evidence of a direct role for the chaperone in the phosphorylation of Cln3.

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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. Eukaryotic homologues of Escherichia coli dnaJ: a diverse protein family that functions with hsp70 stress proteins. Mol Biol Cell. 1993 Jun;4(6):555–563. doi: 10.1091/mbc.4.6.555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Caplan A. J., Langley E., Wilson E. M., Vidal J. Hormone-dependent transactivation by the human androgen receptor is regulated by a dnaJ protein. J Biol Chem. 1995 Mar 10;270(10):5251–5257. doi: 10.1074/jbc.270.10.5251. [DOI] [PubMed] [Google Scholar]
  5. Cegielska A., Georgopoulos C. Biochemical properties of the Escherichia coli dnaK heat shock protein and its mutant derivatives. Biochimie. 1989 Sep-Oct;71(9-10):1071–1077. doi: 10.1016/0300-9084(89)90113-2. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Cross F. R., Blake C. M. The yeast Cln3 protein is an unstable activator of Cdc28. Mol Cell Biol. 1993 Jun;13(6):3266–3271. doi: 10.1128/mcb.13.6.3266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cross F. R. DAF1, a mutant gene affecting size control, pheromone arrest, and cell cycle kinetics of Saccharomyces cerevisiae. Mol Cell Biol. 1988 Nov;8(11):4675–4684. doi: 10.1128/mcb.8.11.4675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cyr D. M., Douglas M. G. Differential regulation of Hsp70 subfamilies by the eukaryotic DnaJ homologue YDJ1. J Biol Chem. 1994 Apr 1;269(13):9798–9804. [PubMed] [Google Scholar]
  10. 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]
  11. Dirick L., Böhm T., Nasmyth K. Roles and regulation of Cln-Cdc28 kinases at the start of the cell cycle of Saccharomyces cerevisiae. EMBO J. 1995 Oct 2;14(19):4803–4813. doi: 10.1002/j.1460-2075.1995.tb00162.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gething M. J., Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33–45. doi: 10.1038/355033a0. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Itikawa H., Wada M., Sekine K., Fujita H. Phosphorylation of glutaminyl-tRNA synthetase and threonyl-tRNA synthetase by the gene products of dnaK and dnaJ in Escherichia coli K-12 cells. Biochimie. 1989 Sep-Oct;71(9-10):1079–1087. doi: 10.1016/0300-9084(89)90114-4. [DOI] [PubMed] [Google Scholar]
  15. Kimura Y., Yahara I., Lindquist S. Role of the protein chaperone YDJ1 in establishing Hsp90-mediated signal transduction pathways. Science. 1995 Jun 2;268(5215):1362–1365. doi: 10.1126/science.7761857. [DOI] [PubMed] [Google Scholar]
  16. Matts R. L., Hurst R. The relationship between protein synthesis and heat shock proteins levels in rabbit reticulocyte lysates. J Biol Chem. 1992 Sep 5;267(25):18168–18174. [PubMed] [Google Scholar]
  17. Matts R. L., Hurst R., Xu Z. Denatured proteins inhibit translation in hemin-supplemented rabbit reticulocyte lysate by inducing the activation of the heme-regulated eIF-2 alpha kinase. Biochemistry. 1993 Jul 27;32(29):7323–7328. doi: 10.1021/bi00080a001. [DOI] [PubMed] [Google Scholar]
  18. Matts R. L., Xu Z., Pal J. K., Chen J. J. Interactions of the heme-regulated eIF-2 alpha kinase with heat shock proteins in rabbit reticulocyte lysates. J Biol Chem. 1992 Sep 5;267(25):18160–18167. [PubMed] [Google Scholar]
  19. Nelson R. J., Heschl M. F., Craig E. A. Isolation and characterization of extragenic suppressors of mutations in the SSA hsp70 genes of Saccharomyces cerevisiae. Genetics. 1992 Jun;131(2):277–285. doi: 10.1093/genetics/131.2.277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Peter M., Herskowitz I. Direct inhibition of the yeast cyclin-dependent kinase Cdc28-Cln by Far1. Science. 1994 Aug 26;265(5176):1228–1231. doi: 10.1126/science.8066461. [DOI] [PubMed] [Google Scholar]
  21. Rowley A., Johnston G. C., Butler B., Werner-Washburne M., Singer R. A. Heat shock-mediated cell cycle blockage and G1 cyclin expression in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1993 Feb;13(2):1034–1041. doi: 10.1128/mcb.13.2.1034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rutherford S. L., Zuker C. S. Protein folding and the regulation of signaling pathways. Cell. 1994 Dec 30;79(7):1129–1132. doi: 10.1016/0092-8674(94)90003-5. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Stuart D., Wittenberg C. CLN3, not positive feedback, determines the timing of CLN2 transcription in cycling cells. Genes Dev. 1995 Nov 15;9(22):2780–2794. doi: 10.1101/gad.9.22.2780. [DOI] [PubMed] [Google Scholar]
  25. Tyers M., Tokiwa G., Futcher B. Comparison of the Saccharomyces cerevisiae G1 cyclins: Cln3 may be an upstream activator of Cln1, Cln2 and other cyclins. EMBO J. 1993 May;12(5):1955–1968. doi: 10.1002/j.1460-2075.1993.tb05845.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Wada M., Sekine K., Itikawa H. Participation of the dnaK and dnaJ gene products in phosphorylation of glutaminyl-tRNA synthetase and threonyl-tRNA synthetase of Escherichia coli K-12. J Bacteriol. 1986 Oct;168(1):213–220. doi: 10.1128/jb.168.1.213-220.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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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