期刊
MOLECULAR BIOLOGY OF THE CELL
卷 17, 期 12, 页码 4962-4971出版社
AMER SOC CELL BIOLOGY
DOI: 10.1091/mbc.E06-04-0311
关键词
-
类别
资金
- NEI NIH HHS [EY10572, P30 EY010572] Funding Source: Medline
- NHLBI NIH HHS [T32 HL007781, T32HL07781] Funding Source: Medline
- NIDDK NIH HHS [DK51818, R01 DK051818] Funding Source: Medline
- NIGMS NIH HHS [GM53457, R01 GM053457] Funding Source: Medline
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 alpha-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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据