Considerable gaps remain in our understanding of basic functional properties of proteasome assembly chaperones, including Pba1–Pba2/PAC1–PAC2. Here we demonstrate that the yeast Pba1–Pba2 chaperone utilizes C-terminal HbYX motifs to bind to proteasome precursors and promote 20S proteasome assembly. In proteasome activators, these motifs function in opening the α-ring gate to the 20S interior. In contrast, Pba1–Pba2 only binds detectably to 20S precursors and is not an appreciable 20S activator. Surprisingly, Pba1–Pba2 is conserved even in archaea despite the compositional simplicity of archaeal proteasomes. The related M. maripaludis PbaA and PbaB proteins also form a complex, and PbaA (and possibly a PbaA–PbaB complex) also preferentially associates with proteasome assembly intermediates containing unprocessed catalytic β subunits. The conserved PbaA HbYX motif binds to the same surface pocket in the α-ring that is bound by proteasomal activators. Strikingly, mature archaeal proteasomes can be induced to bind PbaA and PbaB when the β subunits are bound to active-site inhibitors. This supports a model in which PbaA (and PbaB) binding is allosterically regulated by occupation of the active sites by β-subunit propeptides. When these are processed at the end of proteasome assembly, chaperones dissociate from the distal α-ring surface.
Our results identify for the first time the region on the proteasome to which Pba1–Pba2 binds, namely, the outer surface of the α-ring. This explains the failure to observe a physical interaction between PAC1–PAC2 and hUmp17
, inasmuch as the two assembly factors bind to opposite faces of α-ring-containing intermediates, and the observation that C-terminally peptide-tagged versions of Pba1 and Pba2 are not able to fully complement the respective yeast deletion strains8
. Neither Pba1–Pba2 nor archaeal Pba proteins function as proteasome activators, at least in vitro
, consistent with our previous work showing Pba1–Pba2 only associates detectably with proteasome assembly intermediates in vivo6
. Interestingly, a metazoan proteasome inhibitor protein (PI31) that can compete with activators for binding to the core particle also contains a C-terminal HbYX motif37,38
. Although it has not yet been shown that PI31 utilizes this motif for 20S binding, it would appear that HbYX motifs are not utilized exclusively by proteasome activators. The C-termini of activators share a number of functional similarities in the manner in which they bind 20S39
. It will be interesting to determine whether marked differences exist between how HbYX motifs of activators and non-activators bind to the α-ring pockets. PbaA-proteasome association also requires the N-terminal α-subunit gate segments (), which might also bind PbaA directly.
Pba1–Pba2 and PAC1–PAC2 function as heterodimers6,7
. Additional experiments are required to ascertain the nature of the functional Pba species in archaea. Our gel-shift experiments indicate a tight, HbYX-based association of PbaA with the PHP. This specificity of binding, and its persistence throughout our Ni-NTA-based fractionation steps in high salt buffers, strongly suggests physiological relevance. By contrast, PbaB, which lacks a HbYX motif, was unable to cause a similar gel-shift with PHP. Nevertheless, PbaB was able to cause a weak gel shift of inhibitor-treated WT proteasomes, leaving open the nature of the functional species (see Supplementary Notes
The precise role of Pba1–Pba2/PAC1–PAC2 and archaeal Pba function in proteasome assembly remains to be determined, but one can envision several possibilities based on our findings. By preferentially binding to the outer α-ring surface of intermediates such as the PHP, these factors could prevent activators from prematurely binding to nascent assembly structures or may help sequester incorrectly formed species. Consistent with these models, we find that PbaA can distinguish different states of maturing archaeal 20S proteasomes. While peptidase assays indicate that PbaA interacts with both the partially matured S1 and S2 intermediate species, it only causes a detectable gel-shift of the 20S with the less completely matured S1 intermediates ( and Suppl. Fig. 11
). The drop in apparent affinity for S2 relative to S1 suggests that PbaA can “sense” the ratio of processed and unprocessed β subunits.
The ability of archaeal Pba proteins to bind preferentially to the α rings of assembly intermediates indicates that these factors recognize a feature(s) present in these α rings but not in those of mature 20S. Crystal structures of the PHP-equivalent T1G mutant and mature 20S did not reveal any obvious differences between α ring surfaces29
. However, dense packing typical of protein crystals can alter the gated structure of the α ring34
. Strong support for the idea that the binding of PbaA (and PbaB) to the 20S is regulated allosterically by changes in the β-subunit active sites comes from binding analysis of inhibitor-treated mature 20S proteasomes (). Unlike untreated 20S, inhibitor-bound 20S particles form discrete complexes with both PbaA and PbaB. We propose that covalent modification of active sites with inhibitors mimics the presence of the propeptides found in immature proteasomes. During assembly, the presence of the intact propeptides of the β subunits sends an allosteric signal to the α-subunit N termini, resulting in an altered conformation that can be decoded by Pba proteins. Allosteric signaling from engaged active sites has been postulated previously to explain the stability of 19S RP-20S core particle complexes under conditions of active protein degradation35
. We have not been able to detect Pba1–Pba2 binding to yeast 20S proteasomes treated with inhibitors (not shown). The salient point from the current study is that propeptide-dependent signaling occurs between the β-subunit active sites and the α-subunit ring, and this causes the α rings of immature or aberrant β-subunit-containing intermediates to adopt a conformation(s) recognized by Pba assembly chaperones.
20S proteasomes are also found in actinomycete bacteria and, intriguingly, so are DUF75 proteins. Genome linkage has been used to argue that actinomycete DUF75 proteins may somehow function in proteasome assembly40
. However, structural features of actinomycete DUF75 proteins, and the mechanism of actinomycete 20S assembly41
, make it unlikely that they function in the same manner as eukaryotic and archaeal Pba proteins (see Supplementary Notes and Suppl. Fig. 12
Alternative functions for Pba proteins, consistent with their preferential binding to proteasome precursors, can also be proposed. They may promote β-subunit maturation, a role analogous to that proposed for Blm1012
; however, we found no evidence for this (not shown). Pba proteins could also function during degradation. Peptide hydrolysis proceeds through intermediates that include covalent modification of the active site Thr1 residue, and an alternative view of inhibitor-treated 20S is that these species mimic such hydrolysis intermediates34
. Consequently, Pba proteins could be recruited to actively hydrolyzing proteasomes, perhaps forming hybrid complexes with other activators, although experimental evidence for this is lacking. Finally, archaeal Pba and eukaryotic Pba1–Pba2 proteins may have roles in proteasome quality control. Proteasomes that have not properly matured or have their active sites blocked might be recognized by the Pba factors, thereby preventing activator association and ultimately eliminating them from the pool of available 20S proteasomes.