Biology and Biochemistry of Bacterial Proteasomes

  • Samuel H. Becker
  • Huilin Li
  • K. Heran DarwinEmail author
Part of the Subcellular Biochemistry book series (SCBI, volume 93)


Proteasomes are a class of protease that carry out the degradation of a specific set of cellular proteins. While essential for eukaryotic life, proteasomes are found only in a small subset of bacterial species. In this chapter, we present the current knowledge of bacterial proteasomes, detailing the structural features and catalytic activities required to achieve proteasomal proteolysis. We describe the known mechanisms by which substrates are doomed for degradation, and highlight potential non-degradative roles for components of bacterial proteasome systems. Additionally, we highlight several pathways of microbial physiology that rely on proteasome activity. Lastly, we explain the various gaps in our understanding of bacterial proteasome function and emphasize several opportunities for further study.


Proteasome Proteolysis Pupylation Mycobacterium 



Proteasome research in the Darwin lab is supported by NIH grants HL92774 and AI088075 awarded to K.H.D. NIH grant T32AI007180 supported S.H.B. HL is supported by AI070285.


  1. Bai L, Hu K, Wang T, Jastrab JM, Darwin KH, Li H (2016) Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A.
  2. Bai L et al (2017) Structural analysis of Mycobacterium tuberculosis homologues of the eukaryotic proteasome assembly chaperone 2 (PAC2). J Bacteriol 199.
  3. Barandun J, Delley CL, Ban N, Weber-Ban E (2013) Crystal structure of the complex between prokaryotic ubiquitin-like protein and its ligase PafA. J Am Chem Soc 135:6794–6797. Scholar
  4. Becker SH et al (2019) The Mycobacterium tuberculosis Pup-proteasome system regulates nitrate metabolism through an essential protein quality control pathway. Proc Natl Acad Sci U S A (In press)Google Scholar
  5. Bolten M, Delley CL, Leibundgut M, Boehringer D, Ban N, Weber-Ban E (2016) Structural analysis of the bacterial proteasome activator Bpa in complex with the 20S proteasome. Structure 24:2138–2151.
  6. Boubakri H et al. (2015) The absence of pupylation (prokaryotic ubiquitin-like Protein modification) affects morphological and physiological differentiation in Streptomyces coelicolor. J Bacteriol 197:3388–3399
  7. Brannigan JA, Dodson G, Duggleby HJ, Moody PC, Smith JL, Tomchick DR, Murzin AG (1995) A protein catalytic framework with an N-terminal nucleophile is capable of self-activation. Nature 378:416–419. Scholar
  8. Burns KE et al (2012) Mycobacterium tuberculosis prokaryotic ubiquitin-like protein-deconjugating enzyme is an unusual aspartate amidase. J Biol Chem 287:37522–37529. Scholar
  9. Burns KE, Liu WT, Boshoff HI, Dorrestein PC, Barry CE 3rd (2009) Proteasomal protein degradation in Mycobacteria is dependent upon a prokaryotic ubiquitin-like protein. J Biol Chem 284:3069–3075. Scholar
  10. Burns KE, Cerda-Maira FA, Wang T, Li H, Bishai WR, Darwin KH (2010a) “Depupylation” of prokaryotic ubiquitin-like protein from mycobacterial proteasome substrates. Mol Cell 39:821–827.
  11. Burns KE, Pearce MJ, Darwin KH (2010b) Prokaryotic ubiquitin-like protein provides a two-part degron to Mycobacterium proteasome substrates. J Bacteriol 192:2933–2935. Scholar
  12. Burroughs AM, Balaji S, Iyer LM, Aravind L (2007) Small but versatile: the extraordinary functional and structural diversity of the beta-grasp fold. Biol Direct 2:18. Scholar
  13. Cerda-Maira FA, McAllister F, Bode NJ, Burns KE, Gygi SP, Darwin KH (2011) Reconstitution of the Mycobacterium tuberculosis pupylation pathway in Escherichia coli. EMBO Rep 12:863–870. Scholar
  14. Cerda-Maira FA, Pearce MJ, Fuortes M, Bishai WR, Hubbard SR, Darwin KH (2010) Molecular analysis of the prokaryotic ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium tuberculosis. Mol Microbiol 77:1123–1135.
  15. Chen X, Li C, Wang L, Liu Y, Li C, Zhang J (2016) The mechanism of mycobacterium smegmatis PafA self-pupylation. PLoS ONE 11:e0151021. Scholar
  16. Chen X, Solomon WC, Kang Y, Cerda-Maira F, Darwin KH, Walters KJ (2009) Prokaryotic ubiquitin-like protein pup is intrinsically disordered. J Mol Biol 392:208–217. Scholar
  17. Choi WH et al (2016) Open-gate mutants of the mammalian proteasome show enhanced ubiquitin-conjugate degradation. Nat Commun 7:10963. Scholar
  18. Compton CL, Fernandopulle MS, Nagari RT, Sello JK (2015) Genetic and proteomic analyses of pupylation in Streptomyces coelicolor. J Bacteriol 197:2747–2753. Scholar
  19. Darwin KH, Ehrt S, Gutierrez-Ramos JC, Weich N, Nathan CF (2003) The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide. Science 302:1963–1966. Scholar
  20. Darwin KH, Lin G, Chen Z, Li H, Nathan CF (2005) Characterization of a Mycobacterium tuberculosis proteasomal ATPase homologue. Mol Microbiol 55:561–571. Scholar
  21. Delley CL, Laederach J, Ziemski M, Bolten M, Boehringer D, Weber-Ban E (2014) Bacterial proteasome activator bpa (rv3780) is a novel ring-shaped interactor of the mycobacterial proteasome. PLoS One 9:e114348.
  22. Djuranovic S et al (2009) Structure and activity of the N-terminal substrate recognition domains in proteasomal ATPases. Mol Cell 34:580–590. Scholar
  23. Elharar Y et al (2014) Survival of mycobacteria depends on proteasome-mediated amino acid recycling under nutrient limitation. EMBO J 33:1802–1814. Scholar
  24. Erzberger JP, Berger JM (2006) Evolutionary relationships and structural mechanisms of AAA+ proteins. Annu Rev Biophys Biomol Struct 35:93–114.
  25. Fascellaro G et al (2016) Comprehensive proteomic analysis of nitrogen-starved Mycobacterium smegmatis deltapup reveals the impact of pupylation on nitrogen stress response. J Proteome Res 15:2812–2825. Scholar
  26. Festa RA et al (2011) A novel copper-responsive regulon in Mycobacterium tuberculosis. Mol Microbiol 79:133–148. Scholar
  27. Festa RA, McAllister F, Pearce MJ, Mintseris J, Burns KE, Gygi SP, Darwin KH (2010) Prokaryotic ubiquitin-like protein (Pup) proteome of Mycobacterium tuberculosis [corrected]. PLoS One 5:e8589
  28. Finley D, Chen X, Walters KJ (2016) Gates, channels, and switches: elements of the proteasome machine. Trends Biochem Sci 41:77–93. Scholar
  29. Gandotra S, Lebron MB, Ehrt S (2010) The Mycobacterium tuberculosis proteasome active site threonine is essential for persistence yet dispensable for replication and resistance to nitric oxide. PLoS Pathog 6:e1001040. Scholar
  30. Gottesman S, Zipser D (1978) Deg phenotype of Escherichia coli lon mutants. J Bacteriol 133:844–851CrossRefGoogle Scholar
  31. Gouzy A et al (2013) Mycobacterium tuberculosis nitrogen assimilation and host colonization require aspartate. Nat Chem Biol 9:674–676. Scholar
  32. Gouzy A et al (2014a) Mycobacterium tuberculosis exploits asparagine to assimilate nitrogen and resist acid stress during infection. PLoS Pathog 10:e1003928. Scholar
  33. Gouzy A, Poquet Y, Neyrolles O (2014b) Nitrogen metabolism in Mycobacterium tuberculosis physiology and virulence, vol 12.
  34. Groll M et al (2000) A gated channel into the proteasome core particle. Nat Struct Biol 7:1062–1067. Scholar
  35. Groll M, Brandstetter H, Bartunik H, Bourenkow G, Huber R (2003) Investigations on the maturation and regulation of archaebacterial proteasomes. J Mol Biol 327:75–83CrossRefGoogle Scholar
  36. Groll M, Ditzel L, Lowe J, Stock D, Bochtler M, Bartunik HD, Huber R (1997) Structure of 20S proteasome from yeast at 2.4. A resolution. Nature 386:463–471.
  37. Guth E, Thommen M, Weber-Ban E (2011) Mycobacterial ubiquitin-like protein ligase PafA follows a two-step reaction pathway with a phosphorylated pup intermediate. J Biol Chem 286:4412–4419. Scholar
  38. Hayer-Hartl M, Bracher A, Hartl FU (2016) The GroEL-GroES chaperonin machine: a nano-cage for protein folding. Trends Biochem Sci 41:62–76. Scholar
  39. Heinemeyer W, Fischer M, Krimmer T, Stachon U, Wolf DH (1997) The active sites of the eukaryotic 20 S proteasome and their involvement in subunit precursor processing. J Biol Chem 272:25200–25209Google Scholar
  40. Hu K, Jastrab JB, Zhang S, Kovach A, Zhao G, Darwin KH, Li H (2018) Proteasome substrate capture and gate opening by the accessory factor PafE from Mycobacterium tuberculosis. J Biol Chem 293:4713–4723. Scholar
  41. Hu G, Lin G, Wang M, Dick L, Xu RM, Nathan C, Li H (2006) Structure of the Mycobacterium tuberculosis proteasome and mechanism of inhibition by a peptidyl boronate. Mol Microbiol 59:1417–1428. Scholar
  42. Huber EM, Heinemeyer W, Li X, Arendt CS, Hochstrasser M, Groll M (2016) A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome. Nat Commun 7:10900.
  43. Imkamp F, Rosenberger T, Striebel F, Keller PM, Amstutz B, Sander P, Weber-Ban E (2010a) Deletion of dop in Mycobacterium smegmatis abolishes pupylation of protein substrates in vivo. Mol Microbiol 75:744–754. Scholar
  44. Imkamp F, Striebel F, Sutter M, Ozcelik D, Zimmermann N, Sander P, Weber-Ban E (2010b) Dop functions as a depupylase in the prokaryotic ubiquitin-like modification pathway. EMBO Rep 11:791–797. Scholar
  45. Jastrab JB et al (2015) An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 112:1763–1772. Scholar
  46. Jastrab JB, Darwin KH (2015) Bacterial proteasomes. Annu Rev Microbiol 69:109–127. Scholar
  47. Jastrab JB, Samanovic MI, Copin R, Shopsin B, Darwin KH (2017) Loss-of-function mutations in HspR rescue the growth defect of a Mycobacterium tuberculosis proteasome accessory factor E (pafE) Mutant. J Bacteriol 199.
  48. Kim YE, Hipp MS, Bracher A, Hayer-Hartl M, Hartl FU (2013) Molecular chaperone functions in protein folding and proteostasis. Annu Rev Biochem 82:323–355. Scholar
  49. Kim YC, Snoberger A, Schupp J, Smith DM (2015) ATP binding to neighbouring subunits and intersubunit allosteric coupling underlie proteasomal ATPase function. Nat Commun 6:8520. Scholar
  50. Kuberl A, Franzel B, Eggeling L, Polen T, Wolters DA, Bott M (2014) Pupylated proteins in Corynebacterium glutamicum revealed by MudPIT analysis. Proteomics 14:1531–1542. Scholar
  51. Kuberl A, Polen T, Bott M (2016) The pupylation machinery is involved in iron homeostasis by targeting the iron storage protein ferritin. Proc Natl Acad Sci U S A 113:4806–4811. Scholar
  52. Kurakawa T et al (2007) Direct control of shoot meristem activity by a cytokinin-activating enzyme. Nature 445:652–655. Scholar
  53. 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 27:660–674.
  54. Lehmann G, Udasin RG, Livneh I, Ciechanover A (2017) Identification of UBact, a ubiquitin-like protein, along with other homologous components of a conjugation system and the proteasome in different gram-negative bacteria. Biochem Biophys Res Commun 483:946–950.
  55. Li D, Li H, Wang T, Pan H, Lin G, Li H (2010) Structural basis for the assembly and gate closure mechanisms of the Mycobacterium tuberculosis 20S proteasome. EMBO J 29:2037–2047.
  56. Lin G et al (2006) Mycobacterium tuberculosis prcBA genes encode a gated proteasome with broad oligopeptide specificity. Mol Microbiol 59:1405–1416. Scholar
  57. Lowe J, Stock D, Jap B, Zwickl P, Baumeister W, Huber R (1995) Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution. Science 268:533–539Google Scholar
  58. Lupas A, Zwickl P, Baumeister W (1994) Proteasome sequences in eubacteria. Trends Biochem Sci 19:533–534CrossRefGoogle Scholar
  59. MacMicking JD, North RJ, LaCourse R, Mudgett JS, Shah SK, Nathan CF (1997) Identification of nitric oxide synthase as a protective locus against tuberculosis. Proc Natl Acad Sci U S A 94:5243–5248CrossRefGoogle Scholar
  60. Maldonado AY, Burz DS, Reverdatto S, Shekhtman A (2013) Fate of pup inside the Mycobacterium proteasome studied by in-cell NMR. PLoS ONE 8:e74576. Scholar
  61. Martin A, Baker TA, Sauer RT (2008) Pore loops of the AAA+ ClpX machine grip substrates to drive translocation and unfolding. Nat Struct Mol Biol 15:1147–1151.
  62. Mc Cormack T et al (1997) Active site-directed inhibitors of Rhodococcus 20 S proteasome. Kinetics and mechanism. J Biol Chem 272:26103–26109CrossRefGoogle Scholar
  63. De Mot R (2007) Actinomycete-like proteasomes in a Gram-negative bacterium. Trends Microbiol 15:335–338. Scholar
  64. Murzin AG (1993) OB(oligonucleotide/oligosaccharide binding)-fold: common structural and functional solution for non-homologous sequences. EMBO J 12:861–867Google Scholar
  65. Nagy I, Tamura T, Vanderleyden J, Baumeister W, De Mot R (1998) The 20S proteasome of Streptomyces coelicolor. J Bacteriol 180:5448–5453Google Scholar
  66. Narberhaus F (1999) Negative regulation of bacterial heat shock genes. Mol Microbiol 31:1–8CrossRefGoogle Scholar
  67. Ozcelik D et al (2012) Structures of Pup ligase PafA and depupylase Dop from the prokaryotic ubiquitin-like modification pathway. Nat Commun 3:1014. Scholar
  68. Pearce MJ, Arora P, Festa RA, Butler-Wu SM, Gokhale RS, Darwin KH (2006) Identification of substrates of the Mycobacterium tuberculosis proteasome. EMBO J 25:5423–5432. Scholar
  69. Pearce MJ, Mintseris J, Ferreyra J, Gygi SP, Darwin KH (2008) Ubiquitin-like protein involved in the proteasome pathway of Mycobacterium tuberculosis. Science 322:1104–1107. Scholar
  70. Pouch MN, Cournoyer B, Baumeister W (2000) Characterization of the 20S proteasome from the actinomycete. Frankia Mol Microbiol 35:368–377Google Scholar
  71. Poulsen C et al (2010) Proteome-wide identification of mycobacterial pupylation targets. Mol Syst Biol 6:386. Scholar
  72. Rabl J, Smith DM, Yu Y, Chang SC, Goldberg AL, Cheng Y (2008) Mechanism of gate opening in the 20S proteasome by the proteasomal ATPases. Mol Cell 30:360–368.
  73. 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 92:489–499Google Scholar
  74. Regev O, Korman M, Hecht N, Roth Z, Forer N, Zarivach R, Gur E (2016) An extended loop of the Pup ligase, PafA, mediates interaction with protein targets. J Mol Biol 428:4143–4153. Scholar
  75. Regev O, Roth Z, Korman M, Khalaila I, Gur E (2015) A kinetic model for the prevalence of mono- over poly-pupylation. FEBS J 282:4176–4186. Scholar
  76. Roncarati D, Danielli A, Spohn G, Delany I, Scarlato V (2007) Transcriptional regulation of stress response and motility functions in Helicobacter pylori is mediated by HspR and HrcA. J Bacteriol 189:7234–7243. Scholar
  77. Samanovic MI et al (2015) Proteasomal control of cytokinin synthesis protects Mycobacterium tuberculosis against nitric oxide. Mol Cell 57:984–994. Scholar
  78. Sauer RT, Baker TA (2011) AAA+ proteases: ATP-fueled machines of protein destruction. Annu Rev Biochem 80:587–612.
  79. Shi X, Festa RA, Ioerger TR, Butler-Wu S, Sacchettini JC, Darwin KH, Samanovic MI (2014) The copper-responsive RicR regulon contributes to Mycobacterium tuberculosis virulence. MBio 5
  80. Smith DM, Chang SC, Park S, Finley D, Cheng Y, Goldberg AL (2007) Docking of the proteasomal ATPases’ carboxyl termini in the 20S proteasome’s alpha ring opens the gate for substrate entry. Mol Cell 27:731–744.
  81. Song H, Niederweis M (2012) Uptake of sulfate but not phosphate by Mycobacterium tuberculosis is slower than that for Mycobacterium smegmatis. J Bacteriol 194:956–964. Scholar
  82. Stewart GR et al (2001) Overexpression of heat-shock proteins reduces survival of Mycobacterium tuberculosis in the chronic phase of infection. Nat Med 7:732–737. Scholar
  83. Stewart GR et al (2002) Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays. Microbiology 148:3129–3138. Scholar
  84. Streich FC Jr, Lima CD (2014) Structural and functional insights to ubiquitin-like protein conjugation. Annu Rev Biophys 43:357–379. Scholar
  85. Striebel F, Hunkeler M, Summer H, Weber-Ban E (2010) The mycobacterial Mpa-proteasome unfolds and degrades pupylated substrates by engaging Pup’s N-terminus. EMBO J 29:1262–1271. Scholar
  86. Striebel F, Imkamp F, Sutter M, Steiner M, Mamedov A, Weber-Ban E (2009a) Bacterial ubiquitin-like modifier Pup is deamidated and conjugated to substrates by distinct but homologous enzymes. Nat Struct Mol Biol 16:647–651. Scholar
  87. Striebel F, Kress W, Weber-Ban E (2009b) Controlled destruction: AAA+ ATPases in protein degradation from bacteria to eukaryotes. Curr Opin Struct Biol 19:209–217.
  88. Suraweera A, Munch C, Hanssum A, Bertolotti A (2012) Failure of amino acid homeostasis causes cell death following proteasome inhibition. Mol Cell 48:242–253. Scholar
  89. Sutter M, Striebel F, Damberger FF, Allain FH, Weber-Ban E (2009) A distinct structural region of the prokaryotic ubiquitin-like protein (Pup) is recognized by the N-terminal domain of the proteasomal ATPase Mpa. FEBS Lett 583:3151–3157.
  90. Tamura T et al (1995) The first characterization of a eubacterial proteasome: the 20S complex of Rhodococcus. Curr Biol 5:766–774Google Scholar
  91. Thrower JS, Hoffman L, Rechsteiner M, Pickart CM (2000) Recognition of the polyubiquitin proteolytic signal. EMBO J 19:94–102. Scholar
  92. Vabulas RM, Hartl FU (2005) Protein synthesis upon acute nutrient restriction relies on proteasome function. Science 310:1960–1963. Scholar
  93. Wang T et al (2009) Structural insights on the Mycobacterium tuberculosis proteasomal ATPase Mpa. Structure 17:1377–1385. Scholar
  94. Wang T, Darwin KH, Li H (2010) Binding-induced folding of prokaryotic ubiquitin-like protein on the Mycobacterium proteasomal ATPase targets substrates for degradation. Nat Struct Mol Biol 17:1352–1357. Scholar
  95. Watrous J et al (2010) Expansion of the mycobacterial “PUPylome”. Mol Biosyst 6:376–385.
  96. Wolf S et al (1998) Characterization of ARC, a divergent member of the AAA ATPase family from Rhodococcus erythropolis. J Mol Biol 277:13–25.
  97. Wu Y et al (2017) Mycobacterium tuberculosis proteasomal ATPase Mpa has a beta-grasp domain that hinders docking with the proteasome core protease. Mol Microbiol 105:227–241. Scholar
  98. Yu Y, Smith DM, Kim HM, Rodriguez V, Goldberg AL, Cheng Y (2010) Interactions of PAN’s C-termini with archaeal 20S proteasome and implications for the eukaryotic proteasome-ATPase interactions. EMBO J 29:692–702.
  99. Zhang S, Burns-Huang KE, Janssen GV, Li H, Ovaa H, Hedstrom L, Darwin KH (2017) Mycobacterium tuberculosis proteasome Accessory factor A (PafA) can transfer prokaryotic ubiquitin-like protein (Pup) between substrates. MBio 8.
  100. Ziemski M, Jomaa A, Mayer D, Rutz S, Giese C, Veprintsev D, Weber-Ban E (2018) Cdc48-like protein of actinobacteria (Cpa) is a novel proteasome interactor in mycobacteria and related organisms. Elife 7.
  101. Zuhl F, Seemuller E, Golbik R, Baumeister W (1997) Dissecting the assembly pathway of the 20S proteasome. FEBS Lett 418:189–194Google Scholar
  102. Zwickl P, Kleinz J, Baumeister W (1994) Critical elements in proteasome assembly. Nat Struct Biol 1:765–770CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Department of MicrobiologyNew York University School of MedicineNew YorkUSA
  2. 2.Van Andel Research InstituteCryo-EM Structural Biology LaboratoryGrand RapidsUSA

Personalised recommendations