Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Nov;15(11):6311–6321. doi: 10.1128/mcb.15.11.6311

The yeast SEN3 gene encodes a regulatory subunit of the 26S proteasome complex required for ubiquitin-dependent protein degradation in vivo.

D J DeMarini 1, F R Papa 1, S Swaminathan 1, D Ursic 1, T P Rasmussen 1, M R Culbertson 1, M Hochstrasser 1
PMCID: PMC230883  PMID: 7565784

Abstract

The yeast Sen1 protein was discovered by virtue of its role in tRNA splicing in vitro. To help determine the role of Sen1 in vivo, we attempted to overexpress the protein in yeast cells. However, cells with a high-copy SEN1-bearing plasmid, although expressing elevated amounts of SEN1 mRNA, show little increase in the level of the encoded protein, indicating that a posttranscriptional mechanism limits SEN1 expression. This control depends on an amino-terminal element of Sen1. Using a genetic selection for mutants with increased expression of Sen1-derived fusion proteins, we identified mutations in a novel gene, designated SEN3. SEN3 is essential and encodes a 945-residue protein with sequence similarity to a subunit of an activator of the 20S proteasome from bovine erythrocytes, called PA700. Earlier work indicated that the 20S proteasome associates with a multisubunit regulatory factor, resulting in a 26S proteasome complex that degrades substrates of the ubiquitin system. Mutant sen3-1 cells have severe defects in the degradation of such substrates and accumulate ubiquitin-protein conjugates. Most importantly, we show biochemically that Sen3 is a subunit of the 26S proteasome. These data provide evidence for the involvement of the 26S proteasome in the degradation of ubiquitinated proteins in vivo and for a close relationship between PA700 and the regulatory complexes within the 26S proteasome, and they directly demonstrate that Sen3 is a component of the yeast 26S proteasome.

Full Text

The Full Text of this article is available as a PDF (732.7 KB).

Selected References

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

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Amsterdam A., Pitzer F., Baumeister W. Changes in intracellular localization of proteasomes in immortalized ovarian granulosa cells during mitosis associated with a role in cell cycle control. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):99–103. doi: 10.1073/pnas.90.1.99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen P., Hochstrasser M. Biogenesis, structure and function of the yeast 20S proteasome. EMBO J. 1995 Jun 1;14(11):2620–2630. doi: 10.1002/j.1460-2075.1995.tb07260.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Culbertson M. R., Winey M. Split tRNA genes and their products: a paradigm for the study of cell function and evolution. Yeast. 1989 Nov-Dec;5(6):405–427. doi: 10.1002/yea.320050602. [DOI] [PubMed] [Google Scholar]
  7. DeMarini D. J., Winey M., Ursic D., Webb F., Culbertson M. R. SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae. Mol Cell Biol. 1992 May;12(5):2154–2164. doi: 10.1128/mcb.12.5.2154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. DeMartino G. N., Moomaw C. R., Zagnitko O. P., Proske R. J., Chu-Ping M., Afendis S. J., Swaffield J. C., Slaughter C. A. PA700, an ATP-dependent activator of the 20 S proteasome, is an ATPase containing multiple members of a nucleotide-binding protein family. J Biol Chem. 1994 Aug 19;269(33):20878–20884. [PubMed] [Google Scholar]
  9. Dingwall C., Laskey R. A. Nuclear targeting sequences--a consensus? Trends Biochem Sci. 1991 Dec;16(12):478–481. doi: 10.1016/0968-0004(91)90184-w. [DOI] [PubMed] [Google Scholar]
  10. Field J., Nikawa J., Broek D., MacDonald B., Rodgers L., Wilson I. A., Lerner R. A., Wigler M. Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol. 1988 May;8(5):2159–2165. doi: 10.1128/mcb.8.5.2159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Green N., Alexander H., Olson A., Alexander S., Shinnick T. M., Sutcliffe J. G., Lerner R. A. Immunogenic structure of the influenza virus hemagglutinin. Cell. 1982 Mar;28(3):477–487. doi: 10.1016/0092-8674(82)90202-1. [DOI] [PubMed] [Google Scholar]
  14. Hill J. E., Myers A. M., Koerner T. J., Tzagoloff A. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast. 1986 Sep;2(3):163–167. doi: 10.1002/yea.320020304. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Jones J. S., Prakash L. Yeast Saccharomyces cerevisiae selectable markers in pUC18 polylinkers. Yeast. 1990 Sep-Oct;6(5):363–366. doi: 10.1002/yea.320060502. [DOI] [PubMed] [Google Scholar]
  18. Kim Y. J., Björklund S., Li Y., Sayre M. H., Kornberg R. D. A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell. 1994 May 20;77(4):599–608. doi: 10.1016/0092-8674(94)90221-6. [DOI] [PubMed] [Google Scholar]
  19. Louvion J. F., Havaux-Copf B., Picard D. Fusion of GAL4-VP16 to a steroid-binding domain provides a tool for gratuitous induction of galactose-responsive genes in yeast. Gene. 1993 Sep 6;131(1):129–134. doi: 10.1016/0378-1119(93)90681-r. [DOI] [PubMed] [Google Scholar]
  20. Maicas E., Pluthero F. G., Friesen J. D. The accumulation of three yeast ribosomal proteins under conditions of excess mRNA is determined primarily by fast protein decay. Mol Cell Biol. 1988 Jan;8(1):169–175. doi: 10.1128/mcb.8.1.169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McCraith S. M., Phizicky E. M. An enzyme from Saccharomyces cerevisiae uses NAD+ to transfer the splice junction 2'-phosphate from ligated tRNA to an acceptor molecule. J Biol Chem. 1991 Jun 25;266(18):11986–11992. [PubMed] [Google Scholar]
  22. 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]
  23. Peters J. M., Cejka Z., Harris J. R., Kleinschmidt J. A., Baumeister W. Structural features of the 26 S proteasome complex. J Mol Biol. 1993 Dec 20;234(4):932–937. doi: 10.1006/jmbi.1993.1646. [DOI] [PubMed] [Google Scholar]
  24. Rauhut R., Green P. R., Abelson J. Yeast tRNA-splicing endonuclease is a heterotrimeric enzyme. J Biol Chem. 1990 Oct 25;265(30):18180–18184. [PubMed] [Google Scholar]
  25. 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]
  26. Riles L., Dutchik J. E., Baktha A., McCauley B. K., Thayer E. C., Leckie M. P., Braden V. V., Depke J. E., Olson M. V. Physical maps of the six smallest chromosomes of Saccharomyces cerevisiae at a resolution of 2.6 kilobase pairs. Genetics. 1993 May;134(1):81–150. doi: 10.1093/genetics/134.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Sandbaken M. G., Culbertson M. R. Mutations in elongation factor EF-1 alpha affect the frequency of frameshifting and amino acid misincorporation in Saccharomyces cerevisiae. Genetics. 1988 Dec;120(4):923–934. doi: 10.1093/genetics/120.4.923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Shapira S. K., Chou J., Richaud F. V., Casadaban M. J. New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacZ gene sequences encoding an enzymatically active carboxy-terminal portion of beta-galactosidase. Gene. 1983 Nov;25(1):71–82. doi: 10.1016/0378-1119(83)90169-5. [DOI] [PubMed] [Google Scholar]
  30. Swaffield J. C., Melcher K., Johnston S. A. A highly conserved ATPase protein as a mediator between acidic activation domains and the TATA-binding protein. Nature. 1995 Mar 2;374(6517):88–91. doi: 10.1038/374088a0. [DOI] [PubMed] [Google Scholar]
  31. Tsay Y. F., Thompson J. R., Rotenberg M. O., Larkin J. C., Woolford J. L., Jr Ribosomal protein synthesis is not regulated at the translational level in Saccharomyces cerevisiae: balanced accumulation of ribosomal proteins L16 and rp59 is mediated by turnover of excess protein. Genes Dev. 1988 Jun;2(6):664–676. doi: 10.1101/gad.2.6.664. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Udvardy A. Purification and characterization of a multiprotein component of the Drosophila 26 S (1500 kDa) proteolytic complex. J Biol Chem. 1993 Apr 25;268(12):9055–9062. [PubMed] [Google Scholar]
  34. 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]
  35. Winey M., Culbertson M. R. Mutations affecting the tRNA-splicing endonuclease activity of Saccharomyces cerevisiae. Genetics. 1988 Apr;118(4):609–617. doi: 10.1093/genetics/118.4.609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES