Global organization and function of mammalian cytosolic proteasome pools: Implications for PA28 and 19S regulatory complexes

Toru Shibatani, Eric J. Carlson, Fredrick Larabee, Ashley L. McCormack, Klaus Frueh, William Skach

Research output: Contribution to journalArticle

58 Citations (Scopus)

Abstract

Proteolytic activity of the 20S proteasome is regulated by activators that govern substrate movement into and out of the catalytic chamber. However, the physiological relationship between activators, and hence the relative role of different proteasome species, remains poorly understood. To address this problem, we characterized the total pool of cytosolic proteasomes in intact and functional form using a single-step method that bypasses the need for antibodies, proteasome modification, or column purification. Two-dimensional Blue Native(BN)/SDS-PAGE and tandem mass spectrometry simultaneously identified six native proteasome populations in untreated cytosol: 20S, singly and doubly PA28-capped, singly 19S-capped, hybrid, and doubly 19S-capped proteasomes. All proteasome species were highly dynamic as evidenced by recruitment and exchange of regulatory caps. In particular, proteasome inhibition with MG132 markedly stimulated PA28 binding to exposed 20S α-subunits and generated doubly PA28-capped and hybrid proteasomes. PA28 recruitment virtually eliminated free 20S particles and was blocked by ATP depletion. Moreover, inhibited proteasomes remained stably associated with distinct cohorts of partially degraded fragments derived from cytosolic and ER substrates. These data establish a versatile platform for analyzing substrate-specific proteasome function and indicate that PA28 and 19S activators cooperatively regulate global protein turnover while functioning at different stages of the degradation cycle.

Original languageEnglish (US)
Pages (from-to)4962-4971
Number of pages10
JournalMolecular Biology of the Cell
Volume17
Issue number12
DOIs
StatePublished - Dec 2006

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Proteasome Endopeptidase Complex
Native Polyacrylamide Gel Electrophoresis
Tandem Mass Spectrometry
Cytosol
Polyacrylamide Gel Electrophoresis
Adenosine Triphosphate

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Global organization and function of mammalian cytosolic proteasome pools : Implications for PA28 and 19S regulatory complexes. / Shibatani, Toru; Carlson, Eric J.; Larabee, Fredrick; McCormack, Ashley L.; Frueh, Klaus; Skach, William.

In: Molecular Biology of the Cell, Vol. 17, No. 12, 12.2006, p. 4962-4971.

Research output: Contribution to journalArticle

Shibatani, Toru ; Carlson, Eric J. ; Larabee, Fredrick ; McCormack, Ashley L. ; Frueh, Klaus ; Skach, William. / Global organization and function of mammalian cytosolic proteasome pools : Implications for PA28 and 19S regulatory complexes. In: Molecular Biology of the Cell. 2006 ; Vol. 17, No. 12. pp. 4962-4971.
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