Molecular Genetics of the Ubiquitin-Proteasome System: Lessons from Yeast

  • M. Hochstrasser
  • M. Deng
  • A. R. Kusmierczyk
  • X. Li
  • S. G. Kreft
  • T. Ravid
  • M. Funakoshi
  • M. Kunjappu
  • Y. Xie
Conference paper
Part of the Ernst Schering Foundation Symposium Proceedings book series (SCHERING FOUND, volume 2008/1)


Our studies with the yeast Saccharomyces cerevisiae have uncovered a number of general principles governing substrate selectivity and proteolysis by the ubiquitin-proteasome system. The initial work focused on the degradation of a transcription factor, the MATα2 repressor, but the pathways uncovered have a much broader range of targets. At least two distinct ubiquitination mechanisms contribute to α2 turnover. One of them depends on a large integral membrane ubiquitin ligase (E3) and a pair of ubiquitin-conjugating enzymes (E2s). The transmembrane E3 and E2 proteins must travel from their site of synthesis in the ER to the inner nuclear membrane in order to reach nuclear substrates such as α2. The 26S proteasome is responsible for α2 degradation, and several important features of proteasome assembly and active site formation were uncovered. Most recently, we have delineated major steps in 20S proteasome assembly and have also identified several novel 20S proteasome assembly factors. Surprisingly, alterations in 20S proteasome assembly lead to defects in the assembly of the proteasome regulatory particle (RP). The RP associates with the 20S proteasome to form the 26S proteasome. Our data suggest that the 20S proteasome can function as an assembly factor for the RP, which would make it the first such factor for RP assembly identified to date.


Regulatory Particle Inner Nuclear Membrane Nuclear Substrate Proteasome Assembly Assembly Chaperone 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. Andrulis ED, Neiman AM, Zappulla DC, Sternglanz R (1998) Perinuclear localization of chromatin facilitates transcriptional silencing. [erratum appears in Nature 1998 Oct 1;395(6701):525]. Nature 394:592–595CrossRefPubMedGoogle Scholar
  2. Arendt CS, Hochstrasser M (1997) Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active-site formation. Proc Natl Acad Sci U S A 94:7156–7161CrossRefPubMedGoogle Scholar
  3. Arendt CS, Hochstrasser M (1999) Eukaryotic 20S proteasome catalytic subunit propeptides prevent active site inactivation by N-terminal acetylation and promote particle assembly. EMBO J 18:3575–3585CrossRefPubMedGoogle Scholar
  4. Bartee E, Mansouri M, Hovey Nerenberg BT, Gouveia K, Fruh K (2004) Downregulation of major histocompatibility complex class I by human ubiquitin ligases related to viral immune evasion proteins. J Virol 78:1109–1120CrossRefPubMedGoogle Scholar
  5. Biederer T, Volkwein C, Sommer T (1997) Role of Cue1p in ubiquitination and degradation at the ER surface. Science 278:1806–1809CrossRefPubMedGoogle Scholar
  6. Boname JM, Stevenson PG (2001) MHC class I ubiquitination by a viral PHD/LAP finger protein. Immunity 15:627–636CrossRefPubMedGoogle Scholar
  7. Brennan RJ, Schiestl RH (1996) Cadmium is an inducer of oxidative stress in yeast. Mutat Res 356:171–178PubMedGoogle Scholar
  8. Carvalho P, Goder V, Rapoport TA (2006) Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins. Cell 126:361–373CrossRefPubMedGoogle Scholar
  9. Chen P, Hochstrasser M (1995) Biogenesis, structure, and function of the yeast 20S proteasome. EMBO J 14:2620–2630PubMedGoogle Scholar
  10. Chen P, Hochstrasser M (1996) Autocatalytic subunit processing couples active site formation in the 20S proteasome to completion of assembly. Cell 86:961–972CrossRefPubMedGoogle Scholar
  11. Chen P, Johnson P, Sommer T, Jentsch S, Hochstrasser M (1993) Multiple ubiquitin-conjugating enzymes participate in the in vivo degradation of the yeast MATα2 repressor. Cell 74:357–369CrossRefPubMedGoogle Scholar
  12. Coscoy L, Sanchez DJ, Ganem D (2001) A novel class of herpesvirus-encoded membrane-bound E3 ubiquitin ligases regulates endocytosis of proteins involved in immune recognition. J Cell Biol 155:1265–1273CrossRefPubMedGoogle Scholar
  13. Deng M, Hochstrasser M (2006) Spatially regulated ubiquitin ligation by an ER/nuclear membrane ligase. Nature 443:827–831CrossRefPubMedGoogle Scholar
  14. Dodd RB, Allen MD, Brown SE, Sanderson CM, Duncan LM, Lehner PJ, Bycroft M, Read RJ (2004) Solution structure of the Kaposi's sarcoma-associated herpesvirus K3 N-terminal domain reveals a Novel E2-binding C4HC3-type RING domain. J Biol Chem 279:53840–53847CrossRefPubMedGoogle Scholar
  15. Emori Y, Tsukahara T, Kawasaki H, Ishiura S, Sugita H, Suzuki K (1991) Molecular cloning and functional analysis of three subunits of yeast proteasome. Mol Cell Biol 11:344–353PubMedGoogle Scholar
  16. Froment C, Uttenweiler-Joseph S, Bousquet-Dubouch MP, Matondo M, Borges JP, Esmenjaud C, Lacroix C, Monsarrat B, Burlet-Schiltz O (2005) A quantitative proteomic approach using two-dimensional gel electrophoresis and isotope-coded affinity tag labeling for studying human 20S proteasome heterogeneity. Proteomics 5:2351–2363CrossRefPubMedGoogle Scholar
  17. Fu H, Doelling JH, Arendt CS, Hochstrasser M, Vierstra RD (1998) Molecular organization of the 20S proteasome gene family from Arabidopsis thaliana. Genet 149:677–692Google Scholar
  18. Gilon T, Chomsky O, Kulka RG (2000) Degradation signals recognized by the Ubc6p-Ubc7p ubiquitin-conjugating enzyme pair. Mol Cell Biol 20:7214–7219CrossRefPubMedGoogle Scholar
  19. Goode BL, Wong JJ, Butty AC, Peter M, McCormack AL, Yates JR, Drubin DG, Barnes G (1999) Coronin promotes the rapid assembly and cross-linking of actin filaments and may link the actin and microtubule cytoskeletons in yeast. J Cell Biol 144:83–98CrossRefPubMedGoogle Scholar
  20. Gottesman S, Maurizi M (1992) Regulation by proteolysis: energy-dependent proteases and their targets. Microb Rev 56:592–621Google Scholar
  21. Groll M, Bajorek M, Kohler A, Moroder L, Rubin DM, Huber R, Glickman MH, Finley D (2000) A gated channel into the proteasome core particle. Nat Struct Biol 7:1062–1067CrossRefPubMedGoogle Scholar
  22. Herskowitz I, Rine J, Strathern J (1992) Mating-type determination and mating-type interconversion in Saccharomyces cerevisiae. The molecular and cellular biology of the yeast Saccharomyces: gene expression. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  23. Hiller MM, Finger A, Schweiger M, Wolf DH (1996) ER degradation of a misfolded luminal protein by the cytosolic ubiquitin-proteasome pathway. Science 273:1725–1728CrossRefPubMedGoogle Scholar
  24. Hirano Y, Hendil KB, Yashiroda H, Iemura S, Nagane R, Hioki Y, Natsume T, Tanaka K, Murata S (2005) A heterodimeric complex that promotes the assembly of mammalian 20S proteasomes. Nature 437:1381–1385CrossRefPubMedGoogle Scholar
  25. Hochstrasser M (1996) Ubiquitin-dependent protein degradation. Ann Rev Genet 30:405–439CrossRefPubMedGoogle Scholar
  26. Hochstrasser M, Varshavsky A (1990) In vivo degradation of a transcriptional regulator: the yeast α2 repressor. Cell 61:697–708CrossRefPubMedGoogle Scholar
  27. Hoyt MA, McDonough S, Pimpl SA, Scheel H, Hofmann K, Coffino P (2008) A genetic screen for Saccharomyces cerevisiae mutants affecting proteasome function, using a ubiquitin-independent substrate. Yeast 25:199–217CrossRefPubMedGoogle Scholar
  28. Inai Y, Nishikimi M (2002) Increased degradation of oxidized proteins in yeast defective in 26 S proteasome assembly. Arch Biochem Biophys 404:279–284CrossRefPubMedGoogle Scholar
  29. Johnson PR, Swanson R, Rakhilina L, Hochstrasser M (1998) Degradation signal masking by heterodimerization of MATα2 and MATa1 blocks their mutual destruction by the ubiquitin-proteasome pathway. Cell 94:217–227CrossRefPubMedGoogle Scholar
  30. King MC, Lusk CP, Blobel G (2006) Karyopherin-mediated import of integral inner nuclear membrane proteins. Nature 442:1003–1007CrossRefPubMedGoogle Scholar
  31. Kreft SG, Wang L, Hochstrasser M (2006) Membrane topology of the yeast endoplasmic reticulum-localized ubiquitin ligase Doa10 and comparison with its human ortholog TEB4 (MARCH-VI). J Biol Chem 281:4646–4653CrossRefPubMedGoogle Scholar
  32. Krogan NJ, Cagney G, Yu H, Zhong G, Guo X, Ignatchenko A, Li J, Pu S, Datta N, Tikuisis AP, Punna T, Peregrin-Alvarez JM, Shales M, Zhang X, Davey M, Robinson MD, Paccanaro A, Bray JE, Sheung A, Beattie B, Richards DP, Canadien V, Lalev A, Mena F, Wong P, Starostine A, Canete MM, Vlasblom J, Wu S, Orsi C, Collins SR, Chandran S, Haw R, Rilstone JJ, Gandi K, Thompson NJ, Musso G, St Onge P, Ghanny S, Lam MH, Butland G, Altaf-Ul AM, Kanaya S, Shilatifard A, O'Shea E, Weissman JS, Ingles CJ, Hughes TR, Parkinson J, Gerstein M, Wodak SJ, Emili A, Greenblatt JF (2006) Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature 440:637–643CrossRefPubMedGoogle Scholar
  33. Kusmierczyk AR, Kunjappu MJ, Funakoshi M, Hochstrasser M (2008) A multimeric assembly factor controls the formation of alternative 20S proteasomes. Nat Struct Mol Biol 15:237–244CrossRefPubMedGoogle Scholar
  34. Laney JD, Hochstrasser M (2003) Ubiquitin-dependent degradation of the yeast Matα2 repressor enables a switch in developmental state. Genes Dev 17:2259–2270CrossRefPubMedGoogle Scholar
  35. Laney JD, Hochstrasser M (2004) Ubiquitin-dependent control of development in Saccharomyces cerevisiae. Curr Opin Microbiol 7:647–654CrossRefPubMedGoogle Scholar
  36. Le Tallec B, Barrault MB, Courbeyrette R, Guerois R, Marsolier-Kergoat MC, Peyroche A (2007) 20S Proteasome assembly is orchestrated by two distinct pairs of chaperones in yeast and in mammals. Mol Cell 27:660–674CrossRefPubMedGoogle Scholar
  37. Lee SC, Shaw BD (2007) A novel interaction between N-myristoylation and the 26S proteasome during cell morphogenesis. Mol Microbiol 63:1039–1053CrossRefPubMedGoogle Scholar
  38. Li X, Kusmierczyk AR, Wong P, Emili A, Hochstrasser M (2007) Beta-subunit appendages promote 20S proteasome assembly by overcoming an Ump1-dependent checkpoint. EMBO J 26:2339–2349CrossRefPubMedGoogle Scholar
  39. Liu CW, Corboy MJ, DeMartino GN, Thomas PJ (2003) Endoproteolytic activity of the proteasome. Science 299:408–411CrossRefPubMedGoogle Scholar
  40. Mandart E, Dufour ME, Lacroute F (1994) Inactivation of SSM4, a new Saccharomyces cerevisiae gene, suppresses mRNA instability due to rna14 mutations. Mol Gen Genet 245:323–333CrossRefPubMedGoogle Scholar
  41. Marelli M, Lusk CP, Chan H, Aitchison JD, Wozniak RW (2001) A link between the synthesis of nucleoporins and the biogenesis of the nuclear envelope. J Cell Biol 153:709–724CrossRefPubMedGoogle Scholar
  42. Murata S, Sasaki K, Kishimoto T, Niwa S, Hayashi H, Takahama Y, Tanaka K (2007) Regulation of CD8+ T cell development by thymus-specific proteasomes. Science 316:1349–1353CrossRefPubMedGoogle Scholar
  43. Neuber O, Jarosch E, Volkwein C, Walter J, Sommer T (2005) Ubx2 links the Cdc48 complex to ER-associated protein degradation. Nat Cell Biol 7:993–998CrossRefPubMedGoogle Scholar
  44. Ohba T, Schirmer EC, Nishimoto T, Gerace L (2004) Energy- and temperature-dependent transport of integral proteins to the inner nuclear membrane via the nuclear pore. J Cell Biol 167:1051–1062CrossRefPubMedGoogle Scholar
  45. Palmer EA, Kruse KB, Fewell SW, Buchanan SM, Brodsky JL, McCracken AA (2003) Differential requirements of novel A1PiZ degradation deficient (ADD) genes in ER-associated protein degradation. J Cell Sci 116:2361–2373CrossRefPubMedGoogle Scholar
  46. Papa F, Hochstrasser M (1993) The yeast DOA4 gene encodes a deubiquitinating enzyme related to a product of the human tre-2 oncogene. Nature 366:313–319CrossRefPubMedGoogle Scholar
  47. Pickart CM (2001) Mechanisms underlying ubiquitination. Annu Rev Biochem 70:503–533CrossRefPubMedGoogle Scholar
  48. Ramos PC, Hockendorff J, Johnson ES, Varshavsky A, Dohmen RJ (1998) Ump1p is required for proper maturation of the 20S proteasome and becomes its substrate upon completion of the assembly. Cell 92:489–499CrossRefPubMedGoogle Scholar
  49. Ravid T, Kreft SG, Hochstrasser M (2006) Membrane and soluble substrates of the Doa10 ubiquitin ligase are degraded by distinct pathways. EMBO J 25:533–543CrossRefPubMedGoogle Scholar
  50. Rock KL, York IA, Saric T, Goldberg AL (2002) Protein degradation and the generation of MHC class I-presented peptides. Ad Immunol 80:1–70CrossRefGoogle Scholar
  51. Smalle J, Vierstra RD (2004) The ubiquitin 26S proteasome proteolytic pathway. Annu Rev Plant Biol 55:555–590CrossRefPubMedGoogle Scholar
  52. Sommer T, Jentsch S (1993) A protein translocation defect linked to ubiquitin conjugation at the endoplasmic reticulum. Nature 365:176–179CrossRefPubMedGoogle Scholar
  53. Swanson R, Hochstrasser M (2000) A viable ubiquitin-activating enzyme mutant for evaluating ubiquitin system function in Saccharomyces cerevisiae. FEBS Lett 477:193–198CrossRefPubMedGoogle Scholar
  54. Swanson R, Locher M, Hochstrasser M (2001) A conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both ER-associated and Matalpha2 repressor degradation. Genes Dev 15:2660–2674CrossRefPubMedGoogle Scholar
  55. Velichutina I, Connerly PL, Arendt CS, Li X, Hochstrasser M (2004) Plasticity in eucaryotic 20S proteasome ring assembly revealed by a subunit deletion in yeast. EMBO J 23:500–510CrossRefPubMedGoogle Scholar
  56. Weissman AM (2001) Themes and variations on ubiquitylation. Nat Rev Mol Cell Biol 2:169–178CrossRefPubMedGoogle Scholar
  57. Worman HJ, Courvalin JC (2000) The inner nuclear membrane. J Membr Biol 177:1–11CrossRefPubMedGoogle Scholar
  58. Yashiroda H, Mizushima T, Okamoto K, Kameyama T, Hayashi H, Kishimoto T, Niwa S, Kasahara M, Kurimoto E, Sakata E, Takagi K, Suzuki A, Hirano Y, Murata S, Kato K, Yamane T, Tanaka K (2008) Crystal structure of a chaperone complex that contributes to the assembly of yeast 20S proteasomes. Nat Struct Mol Biol 15:228–236CrossRefPubMedGoogle Scholar
  59. Yuan X, Miller M, Belote JM (1996) Duplicated proteasome subunit genes in Drosophila melanogaster encoding testes-specific isoforms. Genetics 144:147–157PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • M. Hochstrasser
    • 1
  • M. Deng
    • 1
  • A. R. Kusmierczyk
    • 1
  • X. Li
    • 1
  • S. G. Kreft
    • 1
  • T. Ravid
    • 1
  • M. Funakoshi
    • 1
  • M. Kunjappu
    • 1
  • Y. Xie
    • 1
  1. 1.Department of Molecular Biophysics and BiochemistryYale UniversityNew HavenUSA

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