PA700, the 19 S regulatory complex of the 26 S proteasome, plays a central role in the recognition and efficient degradation of misfolded proteins. PA700 promotes degradation by recruiting proteasomal substrates utilizing polyubiquitin chains and chaperone-like binding activities and by opening the access to the core of the 20 S proteasome to promote degradation. Here we provide evidence that PA700 in addition to binding misfolded protein substrates also acts to remodel their conformation prior to proteolysis. Scrambled RNase A (scRNase A), a misfolded protein, only slowly refolds spontaneously into an active form because of the rate-limiting unfolding of misfolded disulfide isomers. Notably, PA700 accelerates the rate of reactivation of scRNase A, consistent with its ability to increase the exposure of these disulfide bonds to the solvent. In this regard, PA700 also exposes otherwise buried sites to digestion by exogenous chymotrypsin in a polyubiquitinated enzymatically active substrate, pentaubiquitinated dihydrofolate reductase, Ub(5)DHFR. The dihydrofolate reductase ligand methotrexate counters the ability of PA700 to promote digestion by chymotrypsin. Together, these results indicate that in addition to increasing substrate affinity and opening the access channel to the catalytic sites, PA700 activates proteasomal degradation by remodeling the conformation of protein substrates.
"Six of the ten base proteins (Rpt1–6) are AAA-ATPase subunits that are evolutionary homologs of the AAA-ATPases of the archaeal PAN  . The base AAA-ATPases interact directly with the 20S α-ring and their activity as a molecular chaperone independently of the proteasome appears to be responsible for protein unfolding by the base subcomplex   . The four other subunits within the base subcomplex are all non-ATPases, and are prefixed " Rpn " for " Regulatory Particle Non ATPase " . "
[Show abstract][Hide abstract] ABSTRACT: The 26S proteasome is a chambered protease in which the majority of selective cellular protein degradation takes place. Throughout evolution, access of protein substrates to chambered proteases is restricted and depends on AAA-ATPases. Mechanical force generated through cycles of ATP binding and hydrolysis is used to unfold substrates, open the gated proteolytic chamber and translocate the substrate into the active proteases within the cavity. Six distinct AAA-ATPases (Rpt1-6) at the ring base of the 19S regulatory particle of the proteasome are responsible for these three functions while interacting with the 20S catalytic chamber. Although high resolution structures of the eukaryotic 26S proteasome are not yet available, exciting recent studies shed light on the assembly of the hetero-hexameric Rpt ring and its consequent spatial arrangement, on the role of Rpt C-termini in opening the 20S 'gate', and on the contribution of each individual Rpt subunit to various cellular processes. These studies are illuminated by paradigms generated through studying PAN, the simpler homo-hexameric AAA-ATPase of the archaeal proteasome. The similarities between PAN and Rpts highlight the evolutionary conserved role of AAA-ATPase in protein degradation, whereas unique properties of divergent Rpts reflect the increased complexity and tighter regulation attributed to the eukaryotic proteasome.
"Instead, PA700 is sufficient for maximal Δgpαf retrotranslocation (Figures 3B and 3D). Since ATP is also required and NEM-treated PA700 was retrotranslocation defective, it seems likely that AAA ATPase components of PA700 are involved in powering substrate movement through the membrane via the chaperone-like properties of PA700 (Strickland et al., 2000; Liu et al., 2002). The roles of individual PA700 subunits in retrotranslocation will be clarified by future experiments. "
[Show abstract][Hide abstract] ABSTRACT: Secretory proteins unable to assemble into their native states in the endoplasmic reticulum (ER) are transported back or "retrotranslocated" into the cytosol for ER-associated degradation (ERAD). To examine the roles of different components in ERAD, one fluorescence-labeled ERAD substrate was encapsulated with selected lumenal factors inside mammalian microsomes. After mixing microsomes with fluorescence-quenching agents and selected cytosolic proteins, the rate of substrate efflux was monitored continuously in real time by the decrease in fluorescence intensity as cytosolic quenchers contacted dye-labeled substrates. The retrotranslocation kinetics of nonglycosylated pro-alpha factor were not significantly altered by replacing all lumenal proteins with only protein disulfide isomerase or all cytosolic proteins with only PA700, the 19S regulatory particle of the 26S proteasome. Retrotranslocation was blocked by antibodies against a putative retrotranslocation channel protein, derlin-1, but not Sec61alpha. In addition, pro-alpha factor photocrosslinked derlin-1, but not Sec61alpha. Thus, derlin-1 appears to be involved in pro-alpha factor retrotranslocation.
"Despite genetic, biochemical and functional evidence implicating the 19S RC and p97 in ER-associated degradation (ERAD), their precise roles remain unknown. AAA-ATPases (Rpt1–6) in the 19S RC collectively open the gate into the 20S core (Kohler et al, 2001), exhibit chaperone-like activity (Braun et al, 1999; Liu et al, 2002) and have been reported to facilitate 26S proteasome assembly and disassembly during the degradation cycle (Babbitt et al, 2005). Based on structural and functional homology to archaebacterial ATPdependent proteases PAN (Navon and Goldberg, 2001) and ClpX (Ortega et al, 2000), the eukaryotic 19S RC is thought to unfold and actively translocate substrates into the 20S catalytic chamber. "
[Show abstract][Hide abstract] ABSTRACT: The AAA-ATPase (ATPase associated with various cellular activities) p97 has been implicated in the degradation of misfolded and unassembled proteins in the endoplasmic reticulum (ERAD). To better understand its role in this process, we used a reconstituted cell-free system to define the precise contribution of p97 in degrading immature forms of the polytopic, multi-domain protein CFTR (cystic fibrosis transmembrane conductance regulator). Although p97 augmented both the rate and the extent of CFTR degradation, it was not obligatorily required for ERAD. Only a 50% decrease in degradation was observed in the complete absence of p97. Moreover, p97 specifically stimulated the degradation of CFTR transmembrane (TM) domains but had no effect on isolated cytosolic domains. Consistent with this, p97-mediated extraction of intact TM domains was independent of proteolytic cleavage and influenced by TM segment hydrophobicity, indicating that the relative contribution of p97 is partially determined by substrate stability. Thus, we propose that p97 functions in ERAD as a nonessential but important ancillary component to the proteasome where it facilitates substrate presentation and increases the degradation rate and efficiency of stable (TM) domains.
The EMBO Journal 11/2006; 25(19):4557-66. DOI:10.1038/sj.emboj.7601307 · 10.43 Impact Factor
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