Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1993 Jan;13(1):521–532. doi: 10.1128/mcb.13.1.521

Suppressors of clathrin deficiency: overexpression of ubiquitin rescues lethal strains of clathrin-deficient Saccharomyces cerevisiae.

K K Nelson 1, S K Lemmon 1
PMCID: PMC358931  PMID: 8380227

Abstract

Clathrin-mediated vesicular transport is important for normal growth of the yeast Saccharomyces cerevisiae. Previously, we identified a genetic locus (SCD1) that influences the ability of clathrin heavy-chain-deficient (Chc-) yeast cells to survive. With the scd1-v allele, Chc- yeast cells are viable but grow poorly; with the scd1-i allele, Chc- cells are inviable. To identify the SCD1 locus and other genes that can rescue chc1 delta scd1-i cells to viability, a multicopy suppressor selection strategy was developed. A strain of scd1-i genotype carrying the clathrin heavy-chain gene under GAL1 control (GAL1:CHC1) was transformed with a YEp24 yeast genomic library, and colonies that could grow on glucose were selected. Plasmids from six distinct genetic loci, none of which encoded CHC1, were recovered. One of the suppressor loci was shown to be UBI4, the polyubiquitin gene. UBI4 rescues only in high copy number and is not allelic to SCD1. The conjugation of ubiquitin to intracellular proteins can mediate their selective degradation. Since UBI4 is required for survival of yeast cells under stress and is induced during starvation, ubiquitin expression in GAL1:CHC1 cells was examined. After a shift to growth on glucose to repress synthesis of clathrin heavy chains, UBI4 mRNA levels were elevated > 10-fold, whereas the quantity of free ubiquitin declined severalfold relative to that of Chc+ cells. In addition, novel higher-molecular-weight ubiquitin conjugates appeared in clathrin-deficient cells. We suggest that higher levels of ubiquitin are required for turnover of mislocalized or improperly processed proteins that accumulate in the absence of clathrin and that ubiquitin may play a general role in turnover of proteins in the secretory or endocytic pathway.

Full text

PDF

Images in this article

523Image
on p.523
524Image
on p.524
526Image
on p.526
528Image
on p.528
528Image
on p.528
529Image
on p.529
529Image
on p.529

Selected References

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

  1. Becker D. M., Guarente L. High-efficiency transformation of yeast by electroporation. Methods Enzymol. 1991;194:182–187. doi: 10.1016/0076-6879(91)94015-5. [DOI] [PubMed] [Google Scholar]
  2. Bender A., Pringle J. R. Multicopy suppression of the cdc24 budding defect in yeast by CDC42 and three newly identified genes including the ras-related gene RSR1. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9976–9980. doi: 10.1073/pnas.86.24.9976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boeke J. D., LaCroute F., Fink G. R. 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. doi: 10.1007/BF00330984. [DOI] [PubMed] [Google Scholar]
  4. Booher R., Beach D. Interaction between cdc13+ and cdc2+ in the control of mitosis in fission yeast; dissociation of the G1 and G2 roles of the cdc2+ protein kinase. EMBO J. 1987 Nov;6(11):3441–3447. doi: 10.1002/j.1460-2075.1987.tb02667.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boorstein W. R., Craig E. A. Structure and regulation of the SSA4 HSP70 gene of Saccharomyces cerevisiae. J Biol Chem. 1990 Nov 5;265(31):18912–18921. [PubMed] [Google Scholar]
  6. Botstein D., Falco S. C., Stewart S. E., Brennan M., Scherer S., Stinchcomb D. T., Struhl K., Davis R. W. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene. 1979 Dec;8(1):17–24. doi: 10.1016/0378-1119(79)90004-0. [DOI] [PubMed] [Google Scholar]
  7. Brodsky F. M. Living with clathrin: its role in intracellular membrane traffic. Science. 1988 Dec 9;242(4884):1396–1402. doi: 10.1126/science.2904698. [DOI] [PubMed] [Google Scholar]
  8. Burgess T. L., Kelly R. B. Constitutive and regulated secretion of proteins. Annu Rev Cell Biol. 1987;3:243–293. doi: 10.1146/annurev.cb.03.110187.001331. [DOI] [PubMed] [Google Scholar]
  9. Carlson M., Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. doi: 10.1016/0092-8674(82)90384-1. [DOI] [PubMed] [Google Scholar]
  10. Cenciarelli C., Hou D., Hsu K. C., Rellahan B. L., Wiest D. L., Smith H. T., Fried V. A., Weissman A. M. Activation-induced ubiquitination of the T cell antigen receptor. Science. 1992 Aug 7;257(5071):795–797. doi: 10.1126/science.1323144. [DOI] [PubMed] [Google Scholar]
  11. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ciechanover A., Gropper R., Schwartz A. L. The ubiquitin-activating enzyme is required for lysosomal degradation of cellular proteins under stress. Biomed Biochim Acta. 1991;50(4-6):321–332. [PubMed] [Google Scholar]
  13. Cleves A. E., Novick P. J., Bankaitis V. A. Mutations in the SAC1 gene suppress defects in yeast Golgi and yeast actin function. J Cell Biol. 1989 Dec;109(6 Pt 1):2939–2950. doi: 10.1083/jcb.109.6.2939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dietzel C., Kurjan J. The yeast SCG1 gene: a G alpha-like protein implicated in the a- and alpha-factor response pathway. Cell. 1987 Sep 25;50(7):1001–1010. doi: 10.1016/0092-8674(87)90166-8. [DOI] [PubMed] [Google Scholar]
  15. Doherty F. J., Osborn N. U., Wassell J. A., Heggie P. E., Laszlo L., Mayer R. J. Ubiquitin-protein conjugates accumulate in the lysosomal system of fibroblasts treated with cysteine proteinase inhibitors. Biochem J. 1989 Oct 1;263(1):47–55. doi: 10.1042/bj2630047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dunn W. A., Jr Studies on the mechanisms of autophagy: formation of the autophagic vacuole. J Cell Biol. 1990 Jun;110(6):1923–1933. doi: 10.1083/jcb.110.6.1923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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. doi: 10.1038/338394a0. [DOI] [PubMed] [Google Scholar]
  18. Finley D., Chau V. Ubiquitination. Annu Rev Cell Biol. 1991;7:25–69. doi: 10.1146/annurev.cb.07.110191.000325. [DOI] [PubMed] [Google Scholar]
  19. Finley D., Ciechanover A., Varshavsky A. Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85. Cell. 1984 May;37(1):43–55. doi: 10.1016/0092-8674(84)90299-x. [DOI] [PubMed] [Google Scholar]
  20. 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. doi: 10.1016/0092-8674(87)90711-2. [DOI] [PubMed] [Google Scholar]
  21. Goebl M. G., Yochem J., Jentsch S., McGrath J. P., Varshavsky A., Byers B. The yeast cell cycle gene CDC34 encodes a ubiquitin-conjugating enzyme. Science. 1988 Sep 9;241(4871):1331–1335. doi: 10.1126/science.2842867. [DOI] [PubMed] [Google Scholar]
  22. Goldberg A. L., Rock K. L. Proteolysis, proteasomes and antigen presentation. Nature. 1992 Jun 4;357(6377):375–379. doi: 10.1038/357375a0. [DOI] [PubMed] [Google Scholar]
  23. Griffiths G., Simons K. The trans Golgi network: sorting at the exit site of the Golgi complex. Science. 1986 Oct 24;234(4775):438–443. doi: 10.1126/science.2945253. [DOI] [PubMed] [Google Scholar]
  24. Gropper R., Brandt R. A., Elias S., Bearer C. F., Mayer A., Schwartz A. L., Ciechanover A. The ubiquitin-activating enzyme, E1, is required for stress-induced lysosomal degradation of cellular proteins. J Biol Chem. 1991 Feb 25;266(6):3602–3610. [PubMed] [Google Scholar]
  25. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Hoffman C. S., Winston F. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene. 1987;57(2-3):267–272. doi: 10.1016/0378-1119(87)90131-4. [DOI] [PubMed] [Google Scholar]
  28. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Jentsch S., McGrath J. P., Varshavsky A. The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme. Nature. 1987 Sep 10;329(6135):131–134. doi: 10.1038/329131a0. [DOI] [PubMed] [Google Scholar]
  30. Jentsch S. Ubiquitin-dependent protein degradation: a cellular perspective. Trends Cell Biol. 1992 Apr;2(4):98–103. doi: 10.1016/0962-8924(92)90013-d. [DOI] [PubMed] [Google Scholar]
  31. Johnston M., Davis R. W. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Aug;4(8):1440–1448. doi: 10.1128/mcb.4.8.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  33. Laszlo L., Doherty F. J., Osborn N. U., Mayer R. J. Ubiquitinated protein conjugates are specifically enriched in the lysosomal system of fibroblasts. FEBS Lett. 1990 Feb 26;261(2):365–368. doi: 10.1016/0014-5793(90)80593-8. [DOI] [PubMed] [Google Scholar]
  34. Lemmon S. K., Freund C., Conley K., Jones E. W. Genetic instability of clathrin-deficient strains of Saccharomyces cerevisiae. Genetics. 1990 Jan;124(1):27–38. doi: 10.1093/genetics/124.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lemmon S. K., Jones E. W. Clathrin requirement for normal growth of yeast. Science. 1987 Oct 23;238(4826):504–509. doi: 10.1126/science.3116672. [DOI] [PubMed] [Google Scholar]
  36. Lemmon S. K., Pellicena-Palle A., Conley K., Freund C. L. Sequence of the clathrin heavy chain from Saccharomyces cerevisiae and requirement of the COOH terminus for clathrin function. J Cell Biol. 1991 Jan;112(1):65–80. doi: 10.1083/jcb.112.1.65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lemmon S., Lemmon V. P., Jones E. W. Characterization of yeast clathrin and anticlathrin heavy-chain monoclonal antibodies. J Cell Biochem. 1988 Apr;36(4):329–340. doi: 10.1002/jcb.240360403. [DOI] [PubMed] [Google Scholar]
  38. Lenk S. E., Dunn W. A., Jr, Trausch J. S., Ciechanover A., Schwartz A. L. Ubiquitin-activating enzyme, E1, is associated with maturation of autophagic vacuoles. J Cell Biol. 1992 Jul;118(2):301–308. doi: 10.1083/jcb.118.2.301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Leung D. W., Spencer S. A., Cachianes G., Hammonds R. G., Collins C., Henzel W. J., Barnard R., Waters M. J., Wood W. I. Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature. 1987 Dec 10;330(6148):537–543. doi: 10.1038/330537a0. [DOI] [PubMed] [Google Scholar]
  40. Mori S., Heldin C. H., Claesson-Welsh L. Ligand-induced polyubiquitination of the platelet-derived growth factor beta-receptor. J Biol Chem. 1992 Mar 25;267(9):6429–6434. [PubMed] [Google Scholar]
  41. Mortimer R. K., Johnston J. R. Genealogy of principal strains of the yeast genetic stock center. Genetics. 1986 May;113(1):35–43. doi: 10.1093/genetics/113.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Mortimore G. E., Pösö A. R., Lardeux B. R. Mechanism and regulation of protein degradation in liver. Diabetes Metab Rev. 1989 Feb;5(1):49–70. doi: 10.1002/dmr.5610050105. [DOI] [PubMed] [Google Scholar]
  43. Mosrin C., Riva M., Beltrame M., Cassar E., Sentenac A., Thuriaux P. The RPC31 gene of Saccharomyces cerevisiae encodes a subunit of RNA polymerase C (III) with an acidic tail. Mol Cell Biol. 1990 Sep;10(9):4737–4743. doi: 10.1128/mcb.10.9.4737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Mueller S. C., Branton D. Identification of coated vesicles in Saccharomyces cerevisiae. J Cell Biol. 1984 Jan;98(1):341–346. doi: 10.1083/jcb.98.1.341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Munn A. L., Silveira L., Elgort M., Payne G. S. Viability of clathrin heavy-chain-deficient Saccharomyces cerevisiae is compromised by mutations at numerous loci: implications for the suppression hypothesis. Mol Cell Biol. 1991 Aug;11(8):3868–3878. doi: 10.1128/mcb.11.8.3868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Nakańo A., Muramatsu M. A novel GTP-binding protein, Sar1p, is involved in transport from the endoplasmic reticulum to the Golgi apparatus. J Cell Biol. 1989 Dec;109(6 Pt 1):2677–2691. doi: 10.1083/jcb.109.6.2677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Novick P., Field C., Schekman R. Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell. 1980 Aug;21(1):205–215. doi: 10.1016/0092-8674(80)90128-2. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Ossig R., Dascher C., Trepte H. H., Schmitt H. D., Gallwitz D. The yeast SLY gene products, suppressors of defects in the essential GTP-binding Ypt1 protein, may act in endoplasmic reticulum-to-Golgi transport. Mol Cell Biol. 1991 Jun;11(6):2980–2993. doi: 10.1128/mcb.11.6.2980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Ozkaynak E., Finley D., Solomon M. J., Varshavsky A. The yeast ubiquitin genes: a family of natural gene fusions. EMBO J. 1987 May;6(5):1429–1439. doi: 10.1002/j.1460-2075.1987.tb02384.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Payne G. S., Hasson T. B., Hasson M. S., Schekman R. Genetic and biochemical characterization of clathrin-deficient Saccharomyces cerevisiae. Mol Cell Biol. 1987 Nov;7(11):3888–3898. doi: 10.1128/mcb.7.11.3888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Payne G. S., Schekman R. A test of clathrin function in protein secretion and cell growth. Science. 1985 Nov 29;230(4729):1009–1014. doi: 10.1126/science.2865811. [DOI] [PubMed] [Google Scholar]
  53. Payne G. S., Schekman R. Clathrin: a role in the intracellular retention of a Golgi membrane protein. Science. 1989 Sep 22;245(4924):1358–1365. doi: 10.1126/science.2675311. [DOI] [PubMed] [Google Scholar]
  54. Pearse B. M., Robinson M. S. Clathrin, adaptors, and sorting. Annu Rev Cell Biol. 1990;6:151–171. doi: 10.1146/annurev.cb.06.110190.001055. [DOI] [PubMed] [Google Scholar]
  55. Sachs A. B., Davis R. W. The poly(A) binding protein is required for poly(A) shortening and 60S ribosomal subunit-dependent translation initiation. Cell. 1989 Sep 8;58(5):857–867. doi: 10.1016/0092-8674(89)90938-0. [DOI] [PubMed] [Google Scholar]
  56. Salminen A., Novick P. J. A ras-like protein is required for a post-Golgi event in yeast secretion. Cell. 1987 May 22;49(4):527–538. doi: 10.1016/0092-8674(87)90455-7. [DOI] [PubMed] [Google Scholar]
  57. Sandvig K., van Deurs B. Endocytosis without clathrin (a minireview). Cell Biol Int Rep. 1991 Jan;15(1):3–8. doi: 10.1016/0309-1651(91)90077-v. [DOI] [PubMed] [Google Scholar]
  58. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Schwartz A. L., Brandt R. A., Geuze H., Ciechanover A. Stress-induced alterations in autophagic pathway: relationship to ubiquitin system. Am J Physiol. 1992 Apr;262(4 Pt 1):C1031–C1038. doi: 10.1152/ajpcell.1992.262.4.C1031. [DOI] [PubMed] [Google Scholar]
  60. Schwartz A. L., Ciechanover A., Brandt R. A., Geuze H. J. Immunoelectron microscopic localization of ubiquitin in hepatoma cells. EMBO J. 1988 Oct;7(10):2961–2966. doi: 10.1002/j.1460-2075.1988.tb03158.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Schwartz A. L., Trausch J. S., Ciechanover A., Slot J. W., Geuze H. Immunoelectron microscopic localization of the ubiquitin-activating enzyme E1 in HepG2 cells. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5542–5546. doi: 10.1073/pnas.89.12.5542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Seeger M., Payne G. S. A role for clathrin in the sorting of vacuolar proteins in the Golgi complex of yeast. EMBO J. 1992 Aug;11(8):2811–2818. doi: 10.1002/j.1460-2075.1992.tb05348.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Seeger M., Payne G. S. Selective and immediate effects of clathrin heavy chain mutations on Golgi membrane protein retention in Saccharomyces cerevisiae. J Cell Biol. 1992 Aug;118(3):531–540. doi: 10.1083/jcb.118.3.531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. 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]
  65. Siegelman M., Bond M. W., Gallatin W. M., St John T., Smith H. T., Fried V. A., Weissman I. L. Cell surface molecule associated with lymphocyte homing is a ubiquitinated branched-chain glycoprotein. Science. 1986 Feb 21;231(4740):823–829. doi: 10.1126/science.3003913. [DOI] [PubMed] [Google Scholar]
  66. Silveira L. A., Wong D. H., Masiarz F. R., Schekman R. Yeast clathrin has a distinctive light chain that is important for cell growth. J Cell Biol. 1990 Oct;111(4):1437–1449. doi: 10.1083/jcb.111.4.1437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Simeon A., van der Klei I. J., Veenhuis M., Wolf D. H. Ubiquitin, a central component of selective cytoplasmic proteolysis, is linked to proteins residing at the locus of non-selective proteolysis, the vacuole. FEBS Lett. 1992 Apr 20;301(2):231–235. doi: 10.1016/0014-5793(92)81254-j. [DOI] [PubMed] [Google Scholar]
  68. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  69. Struhl K., Stinchcomb D. T., Scherer S., Davis R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. doi: 10.1073/pnas.76.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Tooze J., Tooze S. A. Clathrin-coated vesicular transport of secretory proteins during the formation of ACTH-containing secretory granules in AtT20 cells. J Cell Biol. 1986 Sep;103(3):839–850. doi: 10.1083/jcb.103.3.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Tschopp J. F., Emr S. D., Field C., Schekman R. GAL2 codes for a membrane-bound subunit of the galactose permease in Saccharomyces cerevisiae. J Bacteriol. 1986 Apr;166(1):313–318. doi: 10.1128/jb.166.1.313-318.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. 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]

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

RESOURCES