A conserved 20S proteasome assembly factor requires a C-terminal HbYX motif for proteasomal precursor binding

Abstract

Dedicated chaperones facilitate eukaryotic proteasome assembly, yet how they function remains largely unknown. Here we demonstrate that a yeast 20S proteasome assembly factor, Pba1–Pba2, requires a previously overlooked C-terminal HbYX (hydrophobic-tyrosine-X) motif for function. HbYX motifs in proteasome activators open the 20S proteasome entry pore, but Pba1–Pba2 instead binds inactive proteasomal precursors. We discovered an archaeal ortholog of this factor, here named PbaA, that also binds preferentially to proteasomal precursors in a HbYX-dependent fashion using the same proteasomal α-ring surface pockets bound by activators. Remarkably, PbaA and the related PbaB protein can be induced to bind mature 20S proteasomes if the active sites in the central chamber are occupied by inhibitors. Our data suggest an allosteric mechanism in which proteasome active-site maturation determines assembly chaperone binding, potentially shielding assembly intermediates or misassembled complexes from non-productive associations until assembly is complete.

Inherent Asymmetry in the 26S Proteasome Is Defined by the Ubiquitin Receptor RPN13

“…The presence of a sensing mechanism in addition to the asymmetric distribution of Rpn13 may also enable cooperative sensing between the proteasome active sites and the 20S-19S interface as reported previously (36,37) and may also facilitate correct matching of RP to specific catalytic particle (data not shown). The molecular details of such regulatory mechanisms remain to be resolved.…”

The Ubiquitin–Proteasome System of Saccharomyces cerevisiae

“…Interestingly, Pba1 and Pba2 contain HbYX motifs, suggesting that they may suppress premature Rpt tail insertion into nascent CP species (Kusmierczyk et al 2011). The crystal structure of a Pba3-Pba4-a5 ternary complex indicates that these chaperones occlude interaction surfaces between the a and b rings (Yashiroda et al 2008).…”

Structural Analysis of Mycobacterium tuberculosis Homologues of the Eukaryotic Proteasome Assembly Chaperone 2 (PAC2)

“…The HbYX motif is conserved in archaeal and eukaryotic proteasome activators, whether they are ATP dependent (such as PAN and Rpt1 to Rpt6) or ATP independent (such as PA26 and PA28) (13). Both yeast Pba1 and Pba2 have the HbYX motif, and they form a heterodimer that binds mature 20S CP with those motifs (24,27). Known M. tuberculosis proteasome activators, such as the ATP-dependent Mpa and the ATPindependent PafE, have a C-terminal GQYL motif that is functionally equivalent to the archaeal and eukaryotic HbYX motif (14,31).…”

“…The heterodimeric Pba1-Pba2 and Pba3-Pba4 promote the assembly of the seven-membered ␣-ring (19)(20)(21), and UMP1 facilitates the incorporation of the ␤ subunits for assembly of the half-proteasomes and their subsequent dimerization to form mature 20S CPs (22,23). PbaA-PbaB, the archaeal homologue of the eukaryotic Pba1-Pba2 complex, associates with 20S CP assembly intermediates as well as with mature 20S CPs (24,25). A recent bioinformatic analysis discovered that the M. tuberculosis genome encodes two orthologs of the eukaryotic PAC2 protein, Rv2125 and Rv2714 (26).…”