Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1995 Jun 1;14(11):2620–2630. doi: 10.1002/j.1460-2075.1995.tb07260.x

Biogenesis, structure and function of the yeast 20S proteasome.

P Chen 1, M Hochstrasser 1
PMCID: PMC398376  PMID: 7781614

Abstract

Intracellular degradation of many eukaryotic proteins requires their covalent ligation to ubiquitin. We previously identified a ubiquitin-dependent degradation pathway in the yeast Saccharomyces cerevisiae, the DOA pathway. Independent work has suggested that a major mechanism of cellular proteolysis involves a large multisubunit protease(s) called the 20S proteasome. We demonstrate here that Doa3 and Doa5, two essential components of the DOA pathway, are subunits of the proteasome. Biochemical analyses of purified mutant proteasomes suggest functions for several conserved proteasome subunit residues. All detectable proteasome particles purified from doa3 or doa5 cells have altered physical properties; however, the mutant particles contain the same 14 different subunits as the wild-type enzyme, indicating that most or all yeast 20S proteasomes comprise a uniform population of hetero-oligomeric complexes rather than a mixture of particles of variable subunit composition. Unexpectedly, we found that the yeast Doa3 and Pre3 subunits are synthesized as precursors which are processed in a manner apparently identical to that of related mammalian proteasome subunits implicated in antigen presentation, suggesting that biogenesis of the proteasome particle is highly conserved between yeast and mammals.

Full text

PDF
2620

Images in this article

2621Image
on p.2621
2623Image
on p.2623
2624Image
on p.2624
2624Image
on p.2624
2625Image
on p.2625

Selected References

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

  1. Achstetter T., Ehmann C., Osaki A., Wolf D. H. Proteolysis in eukaryotic cells. Proteinase yscE, a new yeast peptidase. J Biol Chem. 1984 Nov 10;259(21):13344–13348. [PubMed] [Google Scholar]
  2. Akiyama K., Yokota K., Kagawa S., Shimbara N., Tamura T., Akioka H., Nothwang H. G., Noda C., Tanaka K., Ichihara A. cDNA cloning and interferon gamma down-regulation of proteasomal subunits X and Y. Science. 1994 Aug 26;265(5176):1231–1234. doi: 10.1126/science.8066462. [DOI] [PubMed] [Google Scholar]
  3. Baudin A., Ozier-Kalogeropoulos O., Denouel A., Lacroute F., Cullin C. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res. 1993 Jul 11;21(14):3329–3330. doi: 10.1093/nar/21.14.3329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brown M. G., Driscoll J., Monaco J. J. Structural and serological similarity of MHC-linked LMP and proteasome (multicatalytic proteinase) complexes. Nature. 1991 Sep 26;353(6342):355–357. doi: 10.1038/353355a0. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Chu-Ping M., Vu J. H., Proske R. J., Slaughter C. A., DeMartino G. N. Identification, purification, and characterization of a high molecular weight, ATP-dependent activator (PA700) of the 20 S proteasome. J Biol Chem. 1994 Feb 4;269(5):3539–3547. [PubMed] [Google Scholar]
  7. DeMartino G. N., Orth K., McCullough M. L., Lee L. W., Munn T. Z., Moomaw C. R., Dawson P. A., Slaughter C. A. The primary structures of four subunits of the human, high-molecular-weight proteinase, macropain (proteasome), are distinct but homologous. Biochim Biophys Acta. 1991 Aug 9;1079(1):29–38. doi: 10.1016/0167-4838(91)90020-z. [DOI] [PubMed] [Google Scholar]
  8. Enenkel C., Lehmann H., Kipper J., Gückel R., Hilt W., Wolf D. H. PRE3, highly homologous to the human major histocompatibility complex-linked LMP2 (RING12) gene, codes for a yeast proteasome subunit necessary for the peptidylglutamyl-peptide hydrolyzing activity. FEBS Lett. 1994 Mar 21;341(2-3):193–196. doi: 10.1016/0014-5793(94)80455-9. [DOI] [PubMed] [Google Scholar]
  9. Falkenburg P. E., Kloetzel P. M. Identification and characterization of three different subpopulations of the Drosophila multicatalytic proteinase (proteasome). J Biol Chem. 1989 Apr 25;264(12):6660–6666. [PubMed] [Google Scholar]
  10. Frentzel S., Pesold-Hurt B., Seelig A., Kloetzel P. M. 20 S proteasomes are assembled via distinct precursor complexes. Processing of LMP2 and LMP7 proproteins takes place in 13-16 S preproteasome complexes. J Mol Biol. 1994 Mar 4;236(4):975–981. doi: 10.1016/0022-2836(94)90003-5. [DOI] [PubMed] [Google Scholar]
  11. Friedman H., Snyder M. Mutations in PRG1, a yeast proteasome-related gene, cause defects in nuclear division and are suppressed by deletion of a mitotic cyclin gene. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2031–2035. doi: 10.1073/pnas.91.6.2031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Früh K., Gossen M., Wang K., Bujard H., Peterson P. A., Yang Y. Displacement of housekeeping proteasome subunits by MHC-encoded LMPs: a newly discovered mechanism for modulating the multicatalytic proteinase complex. EMBO J. 1994 Jul 15;13(14):3236–3244. doi: 10.1002/j.1460-2075.1994.tb06625.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Früh K., Yang Y., Arnold D., Chambers J., Wu L., Waters J. B., Spies T., Peterson P. A. Alternative exon usage and processing of the major histocompatibility complex-encoded proteasome subunits. J Biol Chem. 1992 Nov 5;267(31):22131–22140. [PubMed] [Google Scholar]
  14. Georgatsou E., Georgakopoulos T., Thireos G. Molecular cloning of an essential yeast gene encoding a proteasomal subunit. FEBS Lett. 1992 Mar 24;299(1):39–43. doi: 10.1016/0014-5793(92)80095-x. [DOI] [PubMed] [Google Scholar]
  15. Ghislain M., Udvardy A., Mann C. S. cerevisiae 26S protease mutants arrest cell division in G2/metaphase. Nature. 1993 Nov 25;366(6453):358–362. doi: 10.1038/366358a0. [DOI] [PubMed] [Google Scholar]
  16. Gietz R. D., Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. doi: 10.1016/0378-1119(88)90185-0. [DOI] [PubMed] [Google Scholar]
  17. Glynne R., Kerr L. A., Mockridge I., Beck S., Kelly A., Trowsdale J. The major histocompatibility complex-encoded proteasome component LMP7: alternative first exons and post-translational processing. Eur J Immunol. 1993 Apr;23(4):860–866. doi: 10.1002/eji.1830230414. [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. Heinemeyer W., Kleinschmidt J. A., Saidowsky J., Escher C., Wolf D. H. Proteinase yscE, the yeast proteasome/multicatalytic-multifunctional proteinase: mutants unravel its function in stress induced proteolysis and uncover its necessity for cell survival. EMBO J. 1991 Mar;10(3):555–562. doi: 10.1002/j.1460-2075.1991.tb07982.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Heinemeyer W., Tröndle N., Albrecht G., Wolf D. H. PRE5 and PRE6, the last missing genes encoding 20S proteasome subunits from yeast? Indication for a set of 14 different subunits in the eukaryotic proteasome core. Biochemistry. 1994 Oct 11;33(40):12229–12237. doi: 10.1021/bi00206a028. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Lilley K. S., Davison M. D., Rivett A. J. N-terminal sequence similarities between components of the multicatalytic proteinase complex. FEBS Lett. 1990 Mar 26;262(2):327–329. doi: 10.1016/0014-5793(90)80220-d. [DOI] [PubMed] [Google Scholar]
  24. Lupas A., Koster A. J., Walz J., Baumeister W. Predicted secondary structure of the 20 S proteasome and model structure of the putative peptide channel. FEBS Lett. 1994 Oct 31;354(1):45–49. doi: 10.1016/0014-5793(94)01082-x. [DOI] [PubMed] [Google Scholar]
  25. Müller M., Mesyanzhinov V. V., Aebi U. In vitro maturation of prehead-like bacteriophage T4 polyheads: structural changes accompanying proteolytic cleavage and lattice expansion. J Struct Biol. 1994 May-Jun;112(3):199–215. doi: 10.1006/jsbi.1994.1021. [DOI] [PubMed] [Google Scholar]
  26. Nothwang H. G., Tamura T., Tanaka K., Ichihara A. Sequence analyses and inter-species comparisons of three novel human proteasomal subunits, HsN3, HsC7-I and HsC10-II, confine potential proteolytic active-site residues. Biochim Biophys Acta. 1994 Oct 18;1219(2):361–368. doi: 10.1016/0167-4781(94)90060-4. [DOI] [PubMed] [Google Scholar]
  27. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  28. Orlowski M. The multicatalytic proteinase complex, a major extralysosomal proteolytic system. Biochemistry. 1990 Nov 13;29(45):10289–10297. doi: 10.1021/bi00497a001. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Pühler G., Weinkauf S., Bachmann L., Müller S., Engel A., Hegerl R., Baumeister W. Subunit stoichiometry and three-dimensional arrangement in proteasomes from Thermoplasma acidophilum. EMBO J. 1992 Apr;11(4):1607–1616. doi: 10.1002/j.1460-2075.1992.tb05206.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Richter-Ruoff B., Heinemeyer W., Wolf D. H. The proteasome/multicatalytic-multifunctional proteinase. In vivo function in the ubiquitin-dependent N-end rule pathway of protein degradation in eukaryotes. FEBS Lett. 1992 May 11;302(2):192–196. doi: 10.1016/0014-5793(92)80438-m. [DOI] [PubMed] [Google Scholar]
  33. Richter-Ruoff B., Wolf D. H., Hochstrasser M. Degradation of the yeast MAT alpha 2 transcriptional regulator is mediated by the proteasome. FEBS Lett. 1994 Oct 31;354(1):50–52. doi: 10.1016/0014-5793(94)01085-4. [DOI] [PubMed] [Google Scholar]
  34. Rose M. D., Novick P., Thomas J. H., Botstein D., Fink G. R. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. doi: 10.1016/0378-1119(87)90232-0. [DOI] [PubMed] [Google Scholar]
  35. Schauer T. M., Nesper M., Kehl M., Lottspeich F., Müller-Taubenberger A., Gerisch G., Baumeister W. Proteasomes from Dictyostelium discoideum: characterization of structure and function. J Struct Biol. 1993 Sep-Oct;111(2):135–147. doi: 10.1006/jsbi.1993.1044. [DOI] [PubMed] [Google Scholar]
  36. Seufert W., Jentsch S. In vivo function of the proteasome in the ubiquitin pathway. EMBO J. 1992 Aug;11(8):3077–3080. doi: 10.1002/j.1460-2075.1992.tb05379.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tanaka K., Tamura T., Kumatori A., Kwak T. H., Chung C. H., Ichihara A. Separation of yeast proteasome subunits. Immunoreactivity with antibodies against ATP-dependent protease Ti from Escherichia coli. Biochem Biophys Res Commun. 1989 Nov 15;164(3):1253–1261. doi: 10.1016/0006-291x(89)91804-4. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Yoshimura T., Kameyama K., Takagi T., Ikai A., Tokunaga F., Koide T., Tanahashi N., Tamura T., Cejka Z., Baumeister W. Molecular characterization of the "26S" proteasome complex from rat liver. J Struct Biol. 1993 Nov-Dec;111(3):200–211. doi: 10.1006/jsbi.1993.1050. [DOI] [PubMed] [Google Scholar]
  40. Zwickl P., Lottspeich F., Baumeister W. Expression of functional Thermoplasma acidophilum proteasomes in Escherichia coli. FEBS Lett. 1992 Nov 9;312(2-3):157–160. doi: 10.1016/0014-5793(92)80925-7. [DOI] [PubMed] [Google Scholar]

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

RESOURCES