Three Decades of Studies to Understand the Functions of the Ubiquitin Family

Part of the Methods in Molecular Biology book series (MIMB, volume 832)


Many intracellular proteins are metabolically unstable or can become unstable during their lifetime in a cell. The in vivo half-lives of specific proteins range from less than a minute to many days. Among the functions of intracellular proteolysis are the elimination of misfolded or otherwise abnormal proteins; maintenance of amino acid pools in cells affected by stresses such as starvation; and generation of protein fragments that act as hormones, antigens, or other effectors. One major function of proteolytic pathways is the selective destruction of proteins whose concentrations must vary with time and alterations in the state of a cell. Short in vivo half-lives of such proteins provide a way to generate their spatial gradients and to rapidly adjust their concentration or subunit composition through changes in the rate of their degradation. The regulated (and processive) degradation of intracellular proteins is carried out largely by the ubiquitin–proteasome system (Ub system), in conjunction with autophagy-lysosome pathways. Other contributors to intracellular proteolysis include cytosolic and nuclear proteases, such as caspases, calpains, and separases. They often function as “upstream” components of the Ub system, which destroys protein fragments that had been produced by these (nonprocessive) proteases. Ub, a 76-residue protein, mediates selective proteolysis through its enzymatic conjugation to proteins that contain primary degradation signals (degrons (1)), thereby marking such proteins for degradation by the 26S proteasome, an ATP-dependent multisubunit protease. Ub conjugation involves the formation of a poly-Ub chain that is linked (in most cases) to the ε-amino group of an internal Lys residue in a substrate protein. Ub is a “secondary” degron, in that Ub is conjugated to proteins that contain primary degradation signals.

Key words

Ubiquitin Proteolysis N-end rule N-recognin Arg/N-end rule pathway Ac/N-end rule pathway 



I thank R. Hoffman (University of California, San Diego, USA), C. Brower, A. Shemorry, and B. Wadas (California Institute of Technology, USA) for helpful comments on the manuscript. Studies in our laboratory are supported by grants from the National Institutes of Health and the March of Dimes Foundation.


  1. 1.
    Varshavsky A (1991) Naming a targeting signal. Cell 64:13–15.PubMedCrossRefGoogle Scholar
  2. 2.
    Hershko A, Ciechanover A, Varshavsky A (2000) The ubiquitin system. Nat Med 10:1073–1081.CrossRefGoogle Scholar
  3. 3.
    Varshavsky A (2006) The early history of the ubiquitin field. Pro Sci 15:647–654.CrossRefGoogle Scholar
  4. 4.
    Varshavsky A (2008) Discovery of cellular regulation by protein degradation. J Biol Chem 283: 34469–34489.PubMedCrossRefGoogle Scholar
  5. 5.
    Malynn B A, Ma A (2010) Ubiquitin makes its mark on immune regulation. Immunity 33:843–852.PubMedCrossRefGoogle Scholar
  6. 6.
    Liu F, Walters K J (2010) Multitasking with ubiquitin through multivalent interactions. Trends Biochem Sci 35:352–360.PubMedCrossRefGoogle Scholar
  7. 7.
    Gallastegui N, Groll M (2010) The 26S proteasome: assembly and function of a destructive machine. Trends Biochem Sci 35:634–642.PubMedCrossRefGoogle Scholar
  8. 8.
    Bohn S, Beck F, Sakata E et al. (2010) Structure of the 26S proteasome from Schizosaccharo­myces pombe at subnanometer resolution. Proc Natl Acad Sci USA 107:20992–20997.PubMedCrossRefGoogle Scholar
  9. 9.
    Ulrich H D, Walden H (2010) Ubiquitin signalling in DNA replication and repair. Nat Rev Mol Cell Biol 11:479–489.PubMedCrossRefGoogle Scholar
  10. 10.
    Stolz A, Wolf D H (2010) Endoplasmic reticulum-associated protein degradation: a chaperone-assisted journey to hell. Biochim Biophys Acta 1803:694–705.PubMedCrossRefGoogle Scholar
  11. 11.
    Lu Z, Hunter T (2009) Degradation of activated protein kinases by ubiquitination. Annu Rev Biochem 78:435–475.PubMedCrossRefGoogle Scholar
  12. 12.
    Hampton R Y, Garza R M (2009) Protein quality control as a strategy for cellular regulation: lessons from ubiquitin-mediated regulation of the sterol pathway. Chem Rev 109:1561–1574.PubMedCrossRefGoogle Scholar
  13. 13.
    Grabbe C, Dikic I (2009) Functional roles of ubiquitin-like domain (ULD) and ubiquitin-binding domain (UBD) containing proteins. Chem Rev 109:1481–1494.PubMedCrossRefGoogle Scholar
  14. 14.
    Daulni A, Tansey W P (2009) Damage control: DNA repair, transcription, and the ubiquitin-proteasome system. DNA Repair 8:444–448.CrossRefGoogle Scholar
  15. 15.
    Deshaies R J, Joazeiro C A P (2009) RING domain E3 ubiquitin ligases. Annu Rev Biochem 78:399–434.PubMedCrossRefGoogle Scholar
  16. 16.
    Finley D (2009) Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu Rev Biochem 78:477–513.PubMedCrossRefGoogle Scholar
  17. 17.
    Reyes-Turcu F E, Ventii K H, Wilkinson K D (2009) Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes. Annu Rev Biochem 78:363–397.PubMedCrossRefGoogle Scholar
  18. 18.
    Hirsch C, Gauss R, Horn S C et al. (2009) The ubiquitylation machinery of the endoplasmic reticulum. Nature 458:453–460.PubMedCrossRefGoogle Scholar
  19. 19.
    Marques A J, Palanimurugan R, Matias A C et al. (2009) Catalytic mechanism and assembly of the proteasome. Chem Rev 109:1509–1536.PubMedCrossRefGoogle Scholar
  20. 20.
    Ravid T, Hochstrasser M (2008) Diversity of degradation signals in the ubiquitin-proteasome system. Nat Rev Mol Cell Biol 9:679–689.PubMedCrossRefGoogle Scholar
  21. 21.
    Vembar S S, Brodsky J L (2008) One step at a time: endoplasmic reticulum-associated degradation. Nat Rev Mol Cell Biol 9:944–958.PubMedCrossRefGoogle Scholar
  22. 22.
    Dye B T, Schulman B A (2007) Structural mechanisms underlying posttranslational modification by ubiquitin-like proteins. Annu Rev Biophys Biomol Struct 36:131–150.PubMedCrossRefGoogle Scholar
  23. 23.
    Scheffner M, Staub O (2007) HECT E3s and human disease. BMC Biochemistry 8 (Suppl. I):S6.Google Scholar
  24. 24.
    Scott D C, Monda J K, Grace C R R et al. (2010) A dual mechanism for Rub1 ligation to Cdc53. Mol Cell 39:784–796.PubMedCrossRefGoogle Scholar
  25. 25.
    Loeb K R, Haas A L (1992) The interferon-inducible 15-kDa ubiquitin homolog conjugates to intracellular proteins. J Biol Chem 267:7806–7813.PubMedGoogle Scholar
  26. 26.
    Hochstrasser M (2009) Origin and function of ubiquitin-like proteins. Nature 458:422–429.PubMedCrossRefGoogle Scholar
  27. 27.
    Bawa-Khalfe T, Yeh E T (2010) SUMO losing balance: SUMO proteases disrupt SUMO homeostasis to facilitate cancer development and progression. Genes Cancer 1:748–752.PubMedCrossRefGoogle Scholar
  28. 28.
    Gareau J R, Lima C D (2010) The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition. Nat Rev Mol Cell Biol 11:861–871.PubMedCrossRefGoogle Scholar
  29. 29.
    Rubenstein E M, Hochstrasser M (2010) Redundancy and variation in the ubiquitin-mediated proteolytic targeting of a transcription factor. Cell Cycle 9:4282–4285.PubMedCrossRefGoogle Scholar
  30. 30.
    Merlet J, Burger J, Gomes J E et al. (2009) Regulation of cullin-RING E3 ubiquitin-ligases by neddylation and dimerization. Cell Mol Life Sci 66:1924–1938.PubMedCrossRefGoogle Scholar
  31. 31.
    Bergink S, Jentsch S (2009) Principles of ubiquitin and SUMO modifications in DNA repair. Nature 458:461–467.PubMedCrossRefGoogle Scholar
  32. 32.
    Burroughs A M, Balaji S, Iyer L M et al. (2007) Small but versatile: the extraordinary functional and structural diversity of the beta-grasp fold. Biol Direct 2:18.PubMedCrossRefGoogle Scholar
  33. 33.
    Iyer L M, Burroughs A M, Aravind L (2006) The prokaryotic antecedents of the ubiquitin-signaling system and the early evolution of ubiquitin-like beta-grasp domains. Genome Biol 7:R60.PubMedCrossRefGoogle Scholar
  34. 34.
    Uzunova K, Göttsche K, Miteva M et al. (2007) Ubiquitin-dependent proteolytic control of SUMO conjugates. J Biol Chem 282:34167–34175.PubMedCrossRefGoogle Scholar
  35. 35.
    Johnson E S (2004) Protein modification by SUMO. Annu Rev Biochem 73:355–382.PubMedCrossRefGoogle Scholar
  36. 36.
    Geoffroy M-C, Hay R T (2010) An additional role for SUMO in ubiquitin-mediated proteolysis. Nat Rev Mol Cell Biol 10:564–568.CrossRefGoogle Scholar
  37. 37.
    Zhao C, Hsiang T Y, Kuo R L et al. (2010) ISG15 conjugation system targets the viral NS1 protein in influenza A virus-infected cells. Proc Natl Acad Sci USA 107:2253–2258.PubMedCrossRefGoogle Scholar
  38. 38.
    Durfee L A, Lyon N, Seo K et al. (2010) The ISG15 conjugation system broadly targets newly synthesized proteins: implications for the antiviral function of ISG15. Mol Cell 38:722–732.PubMedCrossRefGoogle Scholar
  39. 39.
    Skaug B, Chen Z J (2010) Emerging role of ISG15 in antiviral immunity. Cell 143:187–190.PubMedCrossRefGoogle Scholar
  40. 40.
    Bachmair A, Finley D, Varshavsky A (1986) In vivo half-life of a protein is a function of its amino-terminal residue. Science 234:179–186.PubMedCrossRefGoogle Scholar
  41. 41.
    Hwang C-S, Shemorry A, Varshavsky A (2010) N-terminal acetylation of cellular proteins creates specific degradation signals. Science 327:973–977.PubMedCrossRefGoogle Scholar
  42. 42.
    Arnesen T, Van Damme P, Polevoda B et al. (2009) Proteomics analyses reveal the evolutionary conservation and divergence of N-terminal acetyltransferases from yeast to humans. Proc Natl Acad Sci USA 106:8157–8162.PubMedCrossRefGoogle Scholar
  43. 43.
    Helbig A O, Gauci S, Raijmakers R et al. (2010) Profiling of N-acetylated protein termini provides in-depth insights into the N-terminal nature of the proteome. Mol Cell Proteom 9:928–939.CrossRefGoogle Scholar
  44. 44.
    Polevoda B, Sherman F (2003) N-terminal acetyltransferases and sequence requirements for N-terminal acetylation of eukaryotic proteins. J Mol Biol 325:595–622.PubMedCrossRefGoogle Scholar
  45. 45.
    Goetze S, Qeli E, Mosimann C et al. (2009) Identification and functional characterization of N-terminally acetylated proteins in Drosophila melanogaster. PLoS Biol 7:e1000236.PubMedCrossRefGoogle Scholar
  46. 46.
    Moerschell R P, Hosokawa Y, Tsunasawa S et al. (1990) The specificities of yeast methionine aminopeptidase and acetylation of amino-terminal methionine in vivo. Processing of altered iso-1-cytochromes created by oligonucleotide transformation. J Biol Chem 265:19638–19643.PubMedGoogle Scholar
  47. 47.
    Frottin F, Martinez A, Peynot P et al. (2006) The proteomics of N-terminal methionine cleavage. Mol Cell Proteomics 5:2336–2349.PubMedCrossRefGoogle Scholar
  48. 48.
    Mullen J R, Kayne P S, Moerschell R P et al. (1989) Identification and characterization of genes and mutants for an N-terminal acetyltransferase from yeast. EMBO J 8:2067–2075.PubMedGoogle Scholar
  49. 49.
    Park E C, Szostak J W (1992) ARD1 and NAT1 proteins form a complex that has N-terminal acetyltransferase activity. EMBO J 11:2087–2093.PubMedGoogle Scholar
  50. 50.
    Gautschi M, Just S, Mun A et al. (2003) The yeast N-alpha-acetyltransferase NatA is quantitatively anchored to the ribosome and interacts with nascent polypeptides. Mol Cell Biol 23:7403–7414.PubMedCrossRefGoogle Scholar
  51. 51.
    Tasaki T, Kwon Y T (2007) The mammalian N-end rule pathway: new insights into its components and physiological roles. Trends Biochem Sci 32:520–528.PubMedCrossRefGoogle Scholar
  52. 52.
    Mogk A, Schmidt R, Bukau B (2007) The N-end rule pathway of regulated proteolysis: prokaryotic and eukaryotic strategies. Trends Cell Biol 17:165–172.PubMedCrossRefGoogle Scholar
  53. 53.
    Eisele F, Wolf D H (2008) Degradation of misfolded proteins in the cytoplasm by the ubiquitin ligase Ubr1. FEBS Lett 582:4143–4146.PubMedCrossRefGoogle Scholar
  54. 54.
    Heck J W, Cheung S K, Hampton R Y (2010) Cytoplasmic protein quality control degradation mediated by parallel actions of the E3 ubiquitin ligases Ubr1 and San1. Proc Natl Acad Sci USA 107:1106–1111.PubMedCrossRefGoogle Scholar
  55. 55.
    Hwang C-S, Varshavsky A (2008) Regulation of peptide import through phosphorylation of Ubr1, the ubiquitin ligase of the N-end rule pathway. Proc Natl Acad Sci USA 105:19188–19193.PubMedCrossRefGoogle Scholar
  56. 56.
    Hwang C-S, Shemorry A, Varshavsky A (2009) Two proteolytic pathways regulate DNA repair by co-targeting the Mgt1 alkyguanine transferase. Proc Natl Acad Sci USA 106:2142–2147.PubMedCrossRefGoogle Scholar
  57. 57.
    Hu R-G, Wang H, Xia Z et al. (2008) The N-end rule pathway is a sensor of heme. Proc Natl Acad Sci USA 105:76–81.PubMedCrossRefGoogle Scholar
  58. 58.
    Hu R-G, Sheng J, Xin Q et al. (2005) The N-end rule pathway as a nitric oxide sensor controlling the levels of multiple regulators. Nature 437:981–986.PubMedCrossRefGoogle Scholar
  59. 59.
    Wang H, Piatkov K I, Brower C S et al. (2009) Glutamine-specific N-terminal amidase, a component of the N-end rule pathway. Mol Cell 34:686–695.PubMedCrossRefGoogle Scholar
  60. 60.
    Graciet E, Wellmer F (2010) The plant N-end rule pathway: structure and functions. Trends Plant Sci 15:447–453.PubMedCrossRefGoogle Scholar
  61. 61.
    Brower C S, Varshavsky A (2009) Ablation of arginylation in the mouse N-end rule pathway: loss of fat, higher metabolic rate, damaged spermatogenesis, and neurological perturbations. PLoS One 4:e7757.PubMedCrossRefGoogle Scholar
  62. 62.
    Zenker M, Mayerle J, Lerch M M et al. (2005) Deficiency of UBR1, a ubiquitin ligase of the N-end rule pathway, causes pancreatic dysfunction, malformations and mental retardation (Johanson-Blizzard syndrome). Nat Genet 37:1345–1350.PubMedCrossRefGoogle Scholar
  63. 63.
    Hwang C-S S, M., Batygin O, Addor M C et al. (2011) Ubiquitin ligases of the N-end rule pathway: assessment of mutations in UBR1 that cause the Johanson-Blizzard syndrome. PLoS One 6:e24925.Google Scholar
  64. 64.
    Prasad R, Kawaguchi S, Ng D T W (2010) A nucleus-based quality control mechanism for cytosolic proteins. Mol Biol Cell 21:2117–2127.PubMedCrossRefGoogle Scholar
  65. 65.
    Kurosaka S, Leu N A, Zhang F et al. (2010) Arginylation-dependent neural crest cell migration is essential for mouse development. PLoS Genet 6:e1000878.PubMedCrossRefGoogle Scholar
  66. 66.
    Zhang F, Saha S, Shabalina S A et al. (2010) Differential arginylation of actin isoforms is regulated by coding sequence-dependent degradation. Science 329.Google Scholar
  67. 67.
    Baker R T, Varshavsky A (1991) Inhibition of the N-end rule pathway in living cells. Proc Natl Acad Sci USA 87:2374–2378.Google Scholar
  68. 68.
    Varshavsky A (1996) The N-end rule: functions, mysteries, uses. Proc Natl Acad Sci USA 93:12142–12149.PubMedCrossRefGoogle Scholar
  69. 69.
    Buchler N E, Gerland U, Hwa T (2005) Nonlinear protein degradation and the function of genetic circuits. Proc Natl Acad Sci USA 102:9559–9564.PubMedCrossRefGoogle Scholar
  70. 70.
    Lam Y W, Lamond A I, Mann M et al. (2007) Analysis of nucleolar protein dynamics reveals the nuclear degradation of ribosomal proteins. Curr Biol 17:749–760.PubMedCrossRefGoogle Scholar
  71. 71.
    Singh R K, Kabbaj M-H M, Paik J et al. (2009) Histone levels are regulated by phosphorylation and ubiquitylation-dependent proteolysis. Nat Cell Biol 11:925–933.PubMedCrossRefGoogle Scholar
  72. 72.
    Johnson E S, Gonda D K, Varshavsky A (1990) Cis-trans recognition and subunit-specific degradation of short-lived proteins. Nature 346:287–291.PubMedCrossRefGoogle Scholar
  73. 73.
    Hochstrasser M, Varshavsky A (1990) In vivo degradation of a transcriptional regulator: the yeast MATalpha2 repressor. Cell 61:697–708.PubMedCrossRefGoogle Scholar
  74. 74.
    Schrader E K, Harstad K G, Matouschek A (2009) Targeting proteins for degradation. Nat Chem Biol 5:815–822.PubMedCrossRefGoogle Scholar
  75. 75.
    Collins G A, Lipford J R, Deshaies R J et al. (2010) Gal4 turnover and transcription activation. Nature 461:E7-E8.CrossRefGoogle Scholar
  76. 76.
    Wang X, Muratani M, Tansey W P et al. (2010) Proteolytic instability and the action of nonclassical transcriptional activators. Curr Biol 20:868–871.PubMedCrossRefGoogle Scholar
  77. 77.
    Murray A W (2004) Recycling the cell cycle: cyclins revisited. Cell 116:221–234.PubMedCrossRefGoogle Scholar
  78. 78.
    Powers E T, Morimoto R I, Dillin A et al. (2009) Biological and chemical approaches to diseases of proteostasis deficiency. Annu Rev Biochem 78:959–991.PubMedCrossRefGoogle Scholar
  79. 79.
    Graciet E, Hu R G, Piatkov K et al. (2006) Aminoacyl-transferases and the N-end rule pathway of prokaryotic/eukaryotic specificity in a human pathogen. Proc Natl Acad Sci USA 103:3078–3083.PubMedCrossRefGoogle Scholar
  80. 80.
    Finley D, Bartel B, Varshavsky A (1989) The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis. Nature 338:394–401.PubMedCrossRefGoogle Scholar
  81. 81.
    Bedford L, Lowe J, Dick L R et al. (2011) Ubiquitin-like protein conjugation and the ubiquitin-proteasome system as drug targets. Nat Rev Drug Discov 10:29–46.PubMedCrossRefGoogle Scholar
  82. 82.
    Hwang C-S, Shemorry A, Varshavsky A (2010) The N-end rule pathway is mediated by a complex of the RING-type Ubr1 and HECT-type Ufd4 ubiquitin ligases. Nat Cell Biol 12:1177–1185.PubMedCrossRefGoogle Scholar
  83. 83.
    Xia Z, Webster A, Du F et al. (2008) Substrate-binding sites of UBR1, the ubiquitin ligase of the N-end rule pathway. J Biol Chem 283:24011–24028.PubMedCrossRefGoogle Scholar

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© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Division of BiologyCalifornia Institute of TechnologyPasadenaUSA

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