Skip to main content
PMC home page
Genetics logoLink to Genetics
. 1999 Sep;153(1):69–79. doi: 10.1093/genetics/153.1.69

Pleiotropic defects caused by loss of the proteasome-interacting factors Rad23 and Rpn10 of Saccharomyces cerevisiae.

D Lambertson 1, L Chen 1, K Madura 1
PMCID: PMC1460738  PMID: 10471701

Abstract

Rad23 is a member of a novel class of proteins that contain unprocessed ubiquitin-like (UbL) domains. We showed recently that a small fraction of Rad23 can form an interaction with the 26S proteasome. Similarly, a small fraction of Rpn10 is a component of the proteasome. Rpn10 can bind multiubiquitin chains in vitro, but genetic studies have not clarified its role in vivo. We report here that the loss of both Rad23 and Rpn10 results in pleiotropic defects that are not observed in either single mutant. rad23Delta rpn10Delta displays slow growth, cold sensitivity, and a pronounced G2/M phase delay, implicating overlapping roles for Rad23 and Rpn10. Although rad23Delta rpn10Delta displays similar sensitivity to DNA damage as a rad23Delta single mutant, deletion of RAD23 in rpn10Delta significantly increased sensitivity to canavanine, a phenotype associated with an rpn10Delta single mutant. A mutant Rad23 that is unable to bind the proteasome ((DeltaUbL)rad23) does not suppress the canavanine or cold-sensitive defects of rad23Delta rpn10Delta, demonstrating that Rad23/proteasome interaction is related to these effects. Finally, the accumulation of multiubiquitinated proteins and the stabilization of a specific proteolytic substrate in rad23Delta rpn10Delta suggest that proteasome function is altered.

Full Text

The Full Text of this article is available as a PDF (391.3 KB).

Selected References

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

  1. Amon A., Irniger S., Nasmyth K. Closing the cell cycle circle in yeast: G2 cyclin proteolysis initiated at mitosis persists until the activation of G1 cyclins in the next cycle. Cell. 1994 Jul 1;77(7):1037–1050. doi: 10.1016/0092-8674(94)90443-x. [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. Biggins S., Ivanovska I., Rose M. D. Yeast ubiquitin-like genes are involved in duplication of the microtubule organizing center. J Cell Biol. 1996 Jun;133(6):1331–1346. doi: 10.1083/jcb.133.6.1331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chau V., Tobias J. W., Bachmair A., Marriott D., Ecker D. J., Gonda D. K., Varshavsky A. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science. 1989 Mar 24;243(4898):1576–1583. doi: 10.1126/science.2538923. [DOI] [PubMed] [Google Scholar]
  6. Deveraux Q., Ustrell V., Pickart C., Rechsteiner M. A 26 S protease subunit that binds ubiquitin conjugates. J Biol Chem. 1994 Mar 11;269(10):7059–7061. [PubMed] [Google Scholar]
  7. Deveraux Q., van Nocker S., Mahaffey D., Vierstra R., Rechsteiner M. Inhibition of ubiquitin-mediated proteolysis by the Arabidopsis 26 S protease subunit S5a. J Biol Chem. 1995 Dec 15;270(50):29660–29663. doi: 10.1074/jbc.270.50.29660. [DOI] [PubMed] [Google Scholar]
  8. Fu H., Sadis S., Rubin D. M., Glickman M., van Nocker S., Finley D., Vierstra R. D. Multiubiquitin chain binding and protein degradation are mediated by distinct domains within the 26 S proteasome subunit Mcb1. J Biol Chem. 1998 Jan 23;273(4):1970–1981. doi: 10.1074/jbc.273.4.1970. [DOI] [PubMed] [Google Scholar]
  9. Fujimuro M., Tanaka K., Yokosawa H., Toh-e A. Son1p is a component of the 26S proteasome of the yeast Saccharomyces cerevisiae. FEBS Lett. 1998 Feb 20;423(2):149–154. doi: 10.1016/s0014-5793(98)00084-2. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Gietz D., St Jean A., Woods R. A., Schiestl R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992 Mar 25;20(6):1425–1425. doi: 10.1093/nar/20.6.1425. [DOI] [PMC free article] [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. Glickman M. H., Rubin D. M., Coux O., Wefes I., Pfeifer G., Cjeka Z., Baumeister W., Fried V. A., Finley D. A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the COP9-signalosome and eIF3. Cell. 1998 Sep 4;94(5):615–623. doi: 10.1016/s0092-8674(00)81603-7. [DOI] [PubMed] [Google Scholar]
  14. Glickman M. H., Rubin D. M., Fried V. A., Finley D. The regulatory particle of the Saccharomyces cerevisiae proteasome. Mol Cell Biol. 1998 Jun;18(6):3149–3162. doi: 10.1128/mcb.18.6.3149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Groll M., Ditzel L., Löwe J., Stock D., Bochtler M., Bartunik H. D., Huber R. Structure of 20S proteasome from yeast at 2.4 A resolution. Nature. 1997 Apr 3;386(6624):463–471. doi: 10.1038/386463a0. [DOI] [PubMed] [Google Scholar]
  16. Guzder S. N., Bailly V., Sung P., Prakash L., Prakash S. Yeast DNA repair protein RAD23 promotes complex formation between transcription factor TFIIH and DNA damage recognition factor RAD14. J Biol Chem. 1995 Apr 14;270(15):8385–8388. doi: 10.1074/jbc.270.15.8385. [DOI] [PubMed] [Google Scholar]
  17. Guzder S. N., Habraken Y., Sung P., Prakash L., Prakash S. Reconstitution of yeast nucleotide excision repair with purified Rad proteins, replication protein A, and transcription factor TFIIH. J Biol Chem. 1995 Jun 2;270(22):12973–12976. doi: 10.1074/jbc.270.22.12973. [DOI] [PubMed] [Google Scholar]
  18. Guzder S. N., Sung P., Prakash L., Prakash S. Affinity of yeast nucleotide excision repair factor 2, consisting of the Rad4 and Rad23 proteins, for ultraviolet damaged DNA. J Biol Chem. 1998 Nov 20;273(47):31541–31546. doi: 10.1074/jbc.273.47.31541. [DOI] [PubMed] [Google Scholar]
  19. Hershko A. The ubiquitin pathway for protein degradation. Trends Biochem Sci. 1991 Jul;16(7):265–268. doi: 10.1016/0968-0004(91)90101-z. [DOI] [PubMed] [Google Scholar]
  20. Hochstrasser M. There's the rub: a novel ubiquitin-like modification linked to cell cycle regulation. Genes Dev. 1998 Apr 1;12(7):901–907. doi: 10.1101/gad.12.7.901. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Johnson E. S., Gonda D. K., Varshavsky A. cis-trans recognition and subunit-specific degradation of short-lived proteins. Nature. 1990 Jul 19;346(6281):287–291. doi: 10.1038/346287a0. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Johnson E. S., Schwienhorst I., Dohmen R. J., Blobel G. The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer. EMBO J. 1997 Sep 15;16(18):5509–5519. doi: 10.1093/emboj/16.18.5509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Koegl M., Hoppe T., Schlenker S., Ulrich H. D., Mayer T. U., Jentsch S. A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly. Cell. 1999 Mar 5;96(5):635–644. doi: 10.1016/s0092-8674(00)80574-7. [DOI] [PubMed] [Google Scholar]
  27. Kominami K., DeMartino G. N., Moomaw C. R., Slaughter C. A., Shimbara N., Fujimuro M., Yokosawa H., Hisamatsu H., Tanahashi N., Shimizu Y. Nin1p, a regulatory subunit of the 26S proteasome, is necessary for activation of Cdc28p kinase of Saccharomyces cerevisiae. EMBO J. 1995 Jul 3;14(13):3105–3115. doi: 10.1002/j.1460-2075.1995.tb07313.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kominami K., Okura N., Kawamura M., DeMartino G. N., Slaughter C. A., Shimbara N., Chung C. H., Fujimuro M., Yokosawa H., Shimizu Y. Yeast counterparts of subunits S5a and p58 (S3) of the human 26S proteasome are encoded by two multicopy suppressors of nin1-1. Mol Biol Cell. 1997 Jan;8(1):171–187. doi: 10.1091/mbc.8.1.171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lam Y. A., Xu W., DeMartino G. N., Cohen R. E. Editing of ubiquitin conjugates by an isopeptidase in the 26S proteasome. Nature. 1997 Feb 20;385(6618):737–740. doi: 10.1038/385737a0. [DOI] [PubMed] [Google Scholar]
  30. Madura K., Varshavsky A. Degradation of G alpha by the N-end rule pathway. Science. 1994 Sep 2;265(5177):1454–1458. doi: 10.1126/science.8073290. [DOI] [PubMed] [Google Scholar]
  31. Masutani C., Sugasawa K., Yanagisawa J., Sonoyama T., Ui M., Enomoto T., Takio K., Tanaka K., van der Spek P. J., Bootsma D. Purification and cloning of a nucleotide excision repair complex involving the xeroderma pigmentosum group C protein and a human homologue of yeast RAD23. EMBO J. 1994 Apr 15;13(8):1831–1843. doi: 10.1002/j.1460-2075.1994.tb06452.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Murakami Y., Matsufuji S., Kameji T., Hayashi S., Igarashi K., Tamura T., Tanaka K., Ichihara A. Ornithine decarboxylase is degraded by the 26S proteasome without ubiquitination. Nature. 1992 Dec 10;360(6404):597–599. doi: 10.1038/360597a0. [DOI] [PubMed] [Google Scholar]
  33. Murakami Y., Tanaka K., Matsufuji S., Miyazaki Y., Hayashi S. Antizyme, a protein induced by polyamines, accelerates the degradation of ornithine decarboxylase in Chinese-hamster ovary-cell extracts. Biochem J. 1992 May 1;283(Pt 3):661–664. doi: 10.1042/bj2830661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pickart C. M. Targeting of substrates to the 26S proteasome. FASEB J. 1997 Nov;11(13):1055–1066. doi: 10.1096/fasebj.11.13.9367341. [DOI] [PubMed] [Google Scholar]
  35. Piotrowski J., Beal R., Hoffman L., Wilkinson K. D., Cohen R. E., Pickart C. M. Inhibition of the 26 S proteasome by polyubiquitin chains synthesized to have defined lengths. J Biol Chem. 1997 Sep 19;272(38):23712–23721. doi: 10.1074/jbc.272.38.23712. [DOI] [PubMed] [Google Scholar]
  36. Ramos P. C., Höckendorff J., Johnson E. S., Varshavsky A., Dohmen R. J. Ump1p is required for proper maturation of the 20S proteasome and becomes its substrate upon completion of the assembly. Cell. 1998 Feb 20;92(4):489–499. doi: 10.1016/s0092-8674(00)80942-3. [DOI] [PubMed] [Google Scholar]
  37. Schauber C., Chen L., Tongaonkar P., Vega I., Lambertson D., Potts W., Madura K. Rad23 links DNA repair to the ubiquitin/proteasome pathway. Nature. 1998 Feb 12;391(6668):715–718. doi: 10.1038/35661. [DOI] [PubMed] [Google Scholar]
  38. Schauber C., Chen L., Tongaonkar P., Vega I., Madura K. Sequence elements that contribute to the degradation of yeast G alpha. Genes Cells. 1998 May;3(5):307–319. doi: 10.1046/j.1365-2443.1998.00192.x. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. Swerdlow P. S., Finley D., Varshavsky A. Enhancement of immunoblot sensitivity by heating of hydrated filters. Anal Biochem. 1986 Jul;156(1):147–153. doi: 10.1016/0003-2697(86)90166-1. [DOI] [PubMed] [Google Scholar]
  42. Varshavsky A. The ubiquitin system. Trends Biochem Sci. 1997 Oct;22(10):383–387. doi: 10.1016/s0968-0004(97)01122-5. [DOI] [PubMed] [Google Scholar]
  43. Watkins J. F., Sung P., Prakash L., Prakash S. The Saccharomyces cerevisiae DNA repair gene RAD23 encodes a nuclear protein containing a ubiquitin-like domain required for biological function. Mol Cell Biol. 1993 Dec;13(12):7757–7765. doi: 10.1128/mcb.13.12.7757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. van Nocker S., Deveraux Q., Rechsteiner M., Vierstra R. D. Arabidopsis MBP1 gene encodes a conserved ubiquitin recognition component of the 26S proteasome. Proc Natl Acad Sci U S A. 1996 Jan 23;93(2):856–860. doi: 10.1073/pnas.93.2.856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. van Nocker S., Sadis S., Rubin D. M., Glickman M., Fu H., Coux O., Wefes I., Finley D., Vierstra R. D. The multiubiquitin-chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover. Mol Cell Biol. 1996 Nov;16(11):6020–6028. doi: 10.1128/mcb.16.11.6020. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES