Global Organization and Function of Mammalian Cytosolic Proteasome Pools: Implications for PA28 and 19S Regulatory Complexes

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 ␣-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. INTRODUCTIONThe 20S proteasome is a cylindrical multicatalytic protease comprised of two stacked inner rings of proteolytically active ␤-subunits flanked by two outer rings of ␣-subunits (Lowe et al., 1995;Groll et al., 1997;Voges et al., 1999;Glickman and Ciechanover, 2002;Pickart and Cohen, 2004). In mammalian cells, proteasome activity is controlled by regulatory complexes (caps) that bind to the exposed ends of ␣-subunits and open the gate into and out of the catalytic chamber (Hoffman et al., 1992;DeMartino and Slaughter, 1999;Voges et al., 1999;Groll et al., 2000;Kloetzel and Ossendorp, 2004;Rechsteiner and Hill, 2005). One such cap, the 19S regulatory complex (PA700/RC), contains a hexameric ring of AAAATPases (base) and at least 12 additional subunits (lid) that recognize, unfold, and translocate polyubiquitinated proteins into the axial opening of the 20S core (Tanaka, 1998;Glickman et al., 1999;Strickland et al., 2000;Leggett et al., 2005;Liu et al., 2005). Two 19S RCs bind the 20S particle to form the doubly capped (30.3S) proteasomes (Yoshimura et al., 1993) that are generally believed to be the major species responsible for degrading polyubiquitinated proteins in the cell (Tanaka and Tsurumi, 1997;Voges et al., 1999;Wolf and Hilt, 2004). 19S RC binding to the 20S core particle requires ATP (Orino et al., 1991), and ATP hydrolysis transiently dissociates 20S and 19S particles, possibly to allow release of degradation products (Babbitt et al., 2005). Thus it has been proposed that the degradation cycle i...

Not4 E3 Ligase Contributes to Proteasome Assembly and Functional Integrity in Part through Ecm29

“…The plasticity of the proteasome is mediated by numerous factors, such as proteasome subunit modifications or subunit exchange (31). Alternative RPs have been isolated, and the assembly of various RPs with CP generates a dynamic repertoire of proteasome complexes exchanging RPs (51). Proteasome subunits can be organized in complexes, distinguishable from the 26S proteasome, that contribute to other cellular functions besides proteolysis, such as the regulation of gene transcription, DNA repair, chromatin remodeling, mRNA stability, and chaperone function (reviewed in references 9 and 31).…”

Inhibition of Proteasome Activity Induces Formation of Alternative Proteasome Complexes

“…In this latter case, transcriptional activation of PA200 is observed at late stages. To our knowledge such rapid recruitment of alternative proteasomal activators is a novel finding that has only been described for PA28␣␤ in rabbit reticulocyte lysates in vitro (29).…”

“…Recruitment of proteasomal activators to the 20S catalytic core gives rise to a variety of different alternative proteasome complexes consisting of singly or doubly capped 20S proteasomes as well as hybrid complexes of different activators attached to 26S proteasomes (23,(27)(28)(29). Recent evidence indicates that the 20S and its activators may function as building blocks that assemble rapidly into different proteasome complexes with diverse functions and substrate specificities, allowing fast and dynamic adaptation of proteasome activity to cellular needs (30 -32).…”

“…Recent publications suggested a protective effect of 19S reduction in response to catalytic proteasome inhibition, but involvement of the proteasome activators PA28␥ and PA200 in this response has not been investigated so far (35,36). Recruitment of PA28␣␤ in response to proteasome inhibition has been shown in reticulocytes lysates in vitro (29). Here, we show a so far unknown dynamic recruitment of PA28␥ and PA200 to 20S and 26S proteasomes in cells in response to either proteolytic inhibition or siRNA-mediated 26S/30S proteasome impairment.…”

Protein Aggregation and Proteasome Dysfunction After Brain Ischemia

“…To degrade polyubiproteins, the 20S proteasomes must cooperate with the 19S proteasomes that govern polyubiprotein movement into and out of the 20S catalytic chambers. 18 To enter the 20S proteasome catalytic chambers, polyubiproteins must be unfolded and then de-ubiquitinated by the 19S proteasomal subunit in an ATP-dependent manner. The peptidase activity, measured by the current and previous studies with the tetrapeptide succinyl-LLVY-AMC substrate, requires little unfolding and de-ubiquitination effort from the 19S proteasome subcomplex and thus, reflects only the peptidase portion of the 26S proteasome function.…”