• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. Aug 1994; 14(8): 5501–5509.
PMCID: PMC359070

Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant.

Abstract

The degradation of many proteins requires their prior attachment to ubiquitin. Proteolytic substrates are characteristically multiubiquitinated through the formation of ubiquitin-ubiquitin linkages. Lys-48 of ubiquitin can serve as a linkage site in the formation of such chains and is required for the degradation of some substrates of this pathway in vitro. We have characterized the recessive and dominant effects of a Lys-48-to-Arg mutant of ubiquitin (UbK48R) in Saccharomyces cerevisiae. Although UbK48R is expected to terminate the growth of Lys-48 multiubiquitin chains and thus to exert a dominant negative effect on protein turnover, overproduction of UbK48R in wild-type cells results in only a weak inhibition of protein turnover, apparently because the mutant ubiquitin can be removed from multiubiquitin chains. Surprisingly, expression of UbK48R complements several phenotypes of polyubiquitin gene (UB14) deletion mutants. However, UbK48R cannot serve as a sole source of ubiquitin in S. cerevisiae, as evidenced by its inability to rescue the growth of ubi1 ubi2 ubi3 ubi4 quadruple mutants. When provided solely with UbK48R, cells undergo cell cycle arrest with a terminal phenotype characterized by replicated DNA, mitotic spindles, and two-lobed nuclei. Under these conditions, degradation of amino acid analog-containing proteins is severely inhibited. Thus, multiubiquitin chains containing Lys-48 linkages play a critical role in protein degradation in vivo.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.9M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • 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. [PubMed]
  • Bartel B, Wünning I, Varshavsky A. The recognition component of the N-end rule pathway. EMBO J. 1990 Oct;9(10):3179–3189. [PMC free article] [PubMed]
  • Boeke JD, LaCroute F, Fink GR. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197(2):345–346. [PubMed]
  • Butt TR, Khan MI, Marsh J, Ecker DJ, Crooke ST. Ubiquitin-metallothionein fusion protein expression in yeast. A genetic approach for analysis of ubiquitin functions. J Biol Chem. 1988 Nov 5;263(31):16364–16371. [PubMed]
  • Chattoo BB, Sherman F, Azubalis DA, Fjellstedt TA, Mehnert D, Ogur M. Selection of lys2 Mutants of the Yeast SACCHAROMYCES CEREVISIAE by the Utilization of alpha-AMINOADIPATE. Genetics. 1979 Sep;93(1):51–65. [PMC free article] [PubMed]
  • Chau V, Tobias JW, Bachmair A, Marriott D, Ecker DJ, Gonda DK, Varshavsky A. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science. 1989 Mar 24;243(4898):1576–1583. [PubMed]
  • Ciechanover A, DiGiuseppe JA, Bercovich B, Orian A, Richter JD, Schwartz AL, Brodeur GM. Degradation of nuclear oncoproteins by the ubiquitin system in vitro. Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):139–143. [PMC free article] [PubMed]
  • Ciechanover A, Finley D, Varshavsky A. Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts85. Cell. 1984 May;37(1):57–66. [PubMed]
  • Cook WJ, Jeffrey LC, Carson M, Chen Z, Pickart CM. Structure of a diubiquitin conjugate and a model for interaction with ubiquitin conjugating enzyme (E2). J Biol Chem. 1992 Aug 15;267(23):16467–16471. [PubMed]
  • Cook WJ, Jeffrey LC, Kasperek E, Pickart CM. Structure of tetraubiquitin shows how multiubiquitin chains can be formed. J Mol Biol. 1994 Feb 18;236(2):601–609. [PubMed]
  • Dohmen RJ, 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. [PMC free article] [PubMed]
  • Ecker DJ, Butt TR, Marsh J, Sternberg E, Shatzman A, Dixon JS, Weber PL, Crooke ST. Ubiquitin function studied by disulfide engineering. J Biol Chem. 1989 Jan 25;264(3):1887–1893. [PubMed]
  • Ecker DJ, Butt TR, Marsh J, Sternberg EJ, Margolis N, Monia BP, Jonnalagadda S, Khan MI, Weber PL, Mueller L, et al. Gene synthesis, expression, structures, and functional activities of site-specific mutants of ubiquitin. J Biol Chem. 1987 Oct 15;262(29):14213–14221. [PubMed]
  • Ecker DJ, Khan MI, Marsh J, Butt TR, Crooke ST. Chemical synthesis and expression of a cassette adapted ubiquitin gene. J Biol Chem. 1987 Mar 15;262(8):3524–3527. [PubMed]
  • Elledge SJ, Davis RW. A family of versatile centromeric vectors designed for use in the sectoring-shuffle mutagenesis assay in Saccharomyces cerevisiae. Gene. 1988 Oct 30;70(2):303–312. [PubMed]
  • Ellison MJ, Hochstrasser M. Epitope-tagged ubiquitin. A new probe for analyzing ubiquitin function. J Biol Chem. 1991 Nov 5;266(31):21150–21157. [PubMed]
  • Eytan E, Armon T, Heller H, Beck S, Hershko A. Ubiquitin C-terminal hydrolase activity associated with the 26 S protease complex. J Biol Chem. 1993 Mar 5;268(7):4668–4674. [PubMed]
  • Finley D, Bartel B, Varshavsky A. The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis. Nature. 1989 Mar 30;338(6214):394–401. [PubMed]
  • Finley D, Chau V. Ubiquitination. Annu Rev Cell Biol. 1991;7:25–69. [PubMed]
  • Finley D, Ozkaynak E, Varshavsky A. The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses. Cell. 1987 Mar 27;48(6):1035–1046. [PubMed]
  • Ghiara JB, Richardson HE, Sugimoto K, Henze M, Lew DJ, Wittenberg C, Reed SI. A cyclin B homolog in S. cerevisiae: chronic activation of the Cdc28 protein kinase by cyclin prevents exit from mitosis. Cell. 1991 Apr 5;65(1):163–174. [PubMed]
  • 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. [PubMed]
  • Glotzer M, Murray AW, Kirschner MW. Cyclin is degraded by the ubiquitin pathway. Nature. 1991 Jan 10;349(6305):132–138. [PubMed]
  • Gregori L, Poosch MS, Cousins G, Chau V. A uniform isopeptide-linked multiubiquitin chain is sufficient to target substrate for degradation in ubiquitin-mediated proteolysis. J Biol Chem. 1990 May 25;265(15):8354–8357. [PubMed]
  • Haas AL, Reback PB, Chau V. Ubiquitin conjugation by the yeast RAD6 and CDC34 gene products. Comparison to their putative rabbit homologs, E2(20K) AND E2(32K). J Biol Chem. 1991 Mar 15;266(8):5104–5112. [PubMed]
  • Haas A, Reback PM, Pratt G, Rechsteiner M. Ubiquitin-mediated degradation of histone H3 does not require the substrate-binding ubiquitin protein ligase, E3, or attachment of polyubiquitin chains. J Biol Chem. 1990 Dec 15;265(35):21664–21669. [PubMed]
  • Hershko A, Ciechanover A. The ubiquitin system for protein degradation. Annu Rev Biochem. 1992;61:761–807. [PubMed]
  • Hershko A, Ganoth D, Sudakin V, Dahan A, Cohen LH, Luca FC, Ruderman JV, Eytan E. Components of a system that ligates cyclin to ubiquitin and their regulation by the protein kinase cdc2. J Biol Chem. 1994 Feb 18;269(7):4940–4946. [PubMed]
  • Hershko A, Heller H. Occurrence of a polyubiquitin structure in ubiquitin-protein conjugates. Biochem Biophys Res Commun. 1985 May 16;128(3):1079–1086. [PubMed]
  • Hochstrasser M, Ellison MJ, Chau V, Varshavsky A. The short-lived MAT alpha 2 transcriptional regulator is ubiquitinated in vivo. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4606–4610. [PMC free article] [PubMed]
  • Hodgins RR, Ellison KS, Ellison MJ. Expression of a ubiquitin derivative that conjugates to protein irreversibly produces phenotypes consistent with a ubiquitin deficiency. J Biol Chem. 1992 May 5;267(13):8807–8812. [PubMed]
  • Holloway SL, Glotzer M, King RW, Murray AW. Anaphase is initiated by proteolysis rather than by the inactivation of maturation-promoting factor. Cell. 1993 Jul 2;73(7):1393–1402. [PubMed]
  • Johnson ES, Bartel B, Seufert W, Varshavsky A. Ubiquitin as a degradation signal. EMBO J. 1992 Feb;11(2):497–505. [PMC free article] [PubMed]
  • Jungmann J, Reins HA, Schobert C, Jentsch S. Resistance to cadmium mediated by ubiquitin-dependent proteolysis. Nature. 1993 Jan 28;361(6410):369–371. [PubMed]
  • Murray AW, Solomon MJ, Kirschner MW. The role of cyclin synthesis and degradation in the control of maturation promoting factor activity. Nature. 1989 May 25;339(6222):280–286. [PubMed]
  • Nishizawa M, Okazaki K, Furuno N, Watanabe N, Sagata N. The 'second-codon rule' and autophosphorylation govern the stability and activity of Mos during the meiotic cell cycle in Xenopus oocytes. EMBO J. 1992 Jul;11(7):2433–2446. [PMC free article] [PubMed]
  • Ozkaynak E, Finley D, Solomon MJ, Varshavsky A. The yeast ubiquitin genes: a family of natural gene fusions. EMBO J. 1987 May;6(5):1429–1439. [PMC free article] [PubMed]
  • Parag HA, Raboy B, Kulka RG. Effect of heat shock on protein degradation in mammalian cells: involvement of the ubiquitin system. EMBO J. 1987 Jan;6(1):55–61. [PMC free article] [PubMed]
  • Pickart CM, Kasperek EM, Beal R, Kim A. Substrate properties of site-specific mutant ubiquitin protein (G76A) reveal unexpected mechanistic features of ubiquitin-activating enzyme (E1). J Biol Chem. 1994 Mar 11;269(10):7115–7123. [PubMed]
  • Rechsteiner M, Hoffman L, Dubiel W. The multicatalytic and 26 S proteases. J Biol Chem. 1993 Mar 25;268(9):6065–6068. [PubMed]
  • Reiss Y, Heller H, Hershko A. Binding sites of ubiquitin-protein ligase. Binding of ubiquitin-protein conjugates and of ubiquitin-carrier protein. J Biol Chem. 1989 Jun 25;264(18):10378–10383. [PubMed]
  • Rose IA, Warms JV. A specific endpoint assay for ubiquitin. Proc Natl Acad Sci U S A. 1987 Mar;84(6):1477–1481. [PMC free article] [PubMed]
  • Scheffner M, Huibregtse JM, Vierstra RD, Howley PM. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell. 1993 Nov 5;75(3):495–505. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Sung P, Berleth E, Pickart C, Prakash S, Prakash L. Yeast RAD6 encoded ubiquitin conjugating enzyme mediates protein degradation dependent on the N-end-recognizing E3 enzyme. EMBO J. 1991 Aug;10(8):2187–2193. [PMC free article] [PubMed]
  • Varshavsky A. The N-end rule. Cell. 1992 May 29;69(5):725–735. [PubMed]
  • Vijay-Kumar S, Bugg CE, Cook WJ. Structure of ubiquitin refined at 1.8 A resolution. J Mol Biol. 1987 Apr 5;194(3):531–544. [PubMed]
  • Vijay-Kumar S, Bugg CE, Wilkinson KD, Vierstra RD, Hatfield PM, Cook WJ. Comparison of the three-dimensional structures of human, yeast, and oat ubiquitin. J Biol Chem. 1987 May 5;262(13):6396–6399. [PubMed]
  • Wiebel FF, Kunau WH. The Pas2 protein essential for peroxisome biogenesis is related to ubiquitin-conjugating enzymes. Nature. 1992 Sep 3;359(6390):73–76. [PubMed]
  • Wilkinson KD, Cox MJ, O'Connor LB, Shapira R. Structure and activities of a variant ubiquitin sequence from bakers' yeast. Biochemistry. 1986 Sep 9;25(18):4999–5004. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...