Interaction with the Assembly Chaperone Ump1 Promotes Incorporation of the ß7 Subunit into Half-Proteasome Precursor Complexes Driving Their Dimerization.

Biogenesis of the eukaryotic 20S proteasome core particle (PC) is a complex process assisted by specific chaperones absent from the active complex. The first identified chaperone, Ump1, was found in a precursor complex (PC) called 15S PC. Yeast cells lacking Ump1 display strong defects in the autocatalytic processing of ß subunits, and consequently have lower proteolytic activity. Here, we dissect an important interaction of Ump1 with the ß7 subunit that is critical for proteasome biogenesis. Functional domains of Ump1 and the interacting proteasome subunit ß7 were mapped, and the functional consequences of their deletion or mutation were analyzed. Cells in which the first sixteen Ump1 residues were deleted display growth phenotypes similar to ump1∆, but massively accumulate 15S PC and distinct proteasome intermediate complexes containing the truncated protein. The viability of these cells depends on the transcription factor Rpn4. Remarkably, ß7 subunit overexpression re-established viability in the absence of Rpn4. We show that an N-terminal domain of Ump1 and the propeptide of ß7 promote direct interaction of the two polypeptides in vitro. This interaction is of critical importance for the recruitment of ß7 precursor during proteasome assembly, a step that drives dimerization of 15S PCs and the formation of 20S CPs.

Hsp70 and Hsp110 Chaperones Promote Early Steps of Proteasome Assembly.

Whereas assembly of the 20S proteasome core particle (CP) in prokaryotes apparently occurs spontaneously, the efficiency of this process in eukaryotes relies on the dedicated assembly chaperones Ump1, Pba1-Pba2, and Pba3-Pba4. For mammals, it was reported that CP assembly initiates with formation of a complete α-ring that functions as a template for ß subunit incorporation. By contrast, we were not able to detect a ring composed only of a complete set of α subunits in S. cerevisiae. Instead, we found that the CP subunits α1, α2, and α4 each form independent small complexes. Purification of such complexes containing α4 revealed the presence of chaperones of the Hsp70/Ssa and Hsp110/Sse families. Consistently, certain small complexes containing α1, α2, and α4 were not formed in strains lacking these chaperones. Deletion of the SSE1 gene in combination with deletions of PRE9 (α3), PBA3, or UMP1 genes resulted in severe synthetic growth defects, high levels of ubiquitin-conjugates, and an accumulation of distinct small complexes with α subunits. Our study shows that Hsp70 and Hsp110 chaperones cooperate to promote the folding of individual α subunits and/or their assembly with other CP subunits, Ump1, and Pba1-Pba4 in subsequent steps.

Proteasome assembly from 15S precursors involves major conformational changes and recycling of the Pba1-Pba2 chaperone.

The chaperones Ump1 and Pba1-Pba2 promote efficient biogenesis of 20S proteasome core particles from its subunits via 15S intermediates containing alpha and beta subunits, except beta7. Here we elucidate the structural role of these chaperones in late steps of core particle biogenesis using biochemical, electron microscopy, cross-linking and mass spectrometry analyses. In 15S precursor complexes, Ump1 is largely unstructured, lining the inner cavity of the complex along the interface between alpha and beta subunits. The alpha and beta subunits form loosely packed rings with a wider alpha ring opening than in the 20S core particle, allowing for the Pba1-Pba2 heterodimer to be partially embedded in the central alpha ring cavity. During biogenesis, the heterodimer is expelled from the alpha ring by a restructuring event that organizes the beta ring and leads to tightening of the alpha ring opening. In this way, the Pba1-Pba2 chaperone is recycled for a new round of proteasome assembly.

Biochemical and biophysical characterization of recombinant yeast proteasome maturation factor ump1.

Protein degradation is essential for maintaining cellular homeostasis. The proteasome is the central enzyme responsible for non-lysosomal protein degradation in eukaryotic cells. Although proteasome assembly is not yet completely understood, a number of cofactors required for proper assembly and maturation have been identified. Ump is a short-lived maturation factor required for the efficient biogenesis of the 20S proteasome. Upon the association of the two precursor complexes, Ump is encased and is rapidly degraded after the proteolytic sites in the interior of the nascent proteasome are activated. In order to further understand the mechanisms behind proteasomal maturation, we expressed and purified yeast Ump in E. coli for biophysical and structural analysis. We show that recombinant Ump is purified as a mixture of different oligomeric species and that oligomerization is mediated by intermolecular disulfide bond formation involving the only cysteine residue present in the protein. Furthermore, a combination of bioinformatic, biochemical and structural analysis revealed that Ump shows characteristics of an intrinsically disordered protein, which might become structured only upon interaction with the proteasome subunits.

Chaperone-assisted assembly of the proteasome core particle.

The 26S proteasome is a non-lysosomal protease in the cytosol and nucleus of eukaryotic cells. Its main function is to mediate ubiquitin-dependent proteolysis. The 26S proteasome is a multimeric complex composed by the 20S proteasome CP (core particle) and the 19S RPs (regulatory particles). Although the atomic structure of the 26S proteasome has not yet been determined, high-resolution structures are available for its CP. Studies on the complicated assembly pathway of the proteasome have revealed that it involves an unprecedented number of dedicated chaperones. Assembly of the CP alone involves three conserved proteasome-assembly chaperones [PAC1-PAC2, PAC3-PAC4 and UMP1 (ubiquitin-mediated proteolysis 1)]. Whereas the two heterodimeric PACs have been implicated in the formation of rings of the seven distinct alpha subunits, UMP1 is important for the formation and dimerization of proteasome precursor complexes containing beta subunits. Dimerization coincides with the incorporation of the last beta subunit (beta7). Additional modules important for the assembly of precursor complexes and their dimerization reside in the beta subunits themselves, either as transient or as permanent extensions. Particularly important domains are the propeptide of beta5 and the C-terminal extensions of beta2 and beta7. Upon maturation of the active sites by autocatalytic processing, UMP1 is degraded by the native proteasome.

PACemakers of proteasome core particle assembly.

The 26S proteasome mediates ubiquitin-dependent proteolysis in eukaryotic cells. A number of studies including very recent ones have revealed that assembly of its 20S catalytic core particle is an ordered process that involves several conserved proteasome assembly chaperones (PACs). Two heterodimeric chaperones, PAC1-PAC2 and PAC3-PAC4, promote the assembly of rings composed of seven alpha subunits. Subsequently, beta subunits join to form half-proteasome precursor complexes containing all but one of the 14 subunits. These complexes lack the beta7 subunit but contain UMP1, another assembly chaperone, and in yeast, at least to some degree, the activator protein Blm10. Dimerization of two such complexes is triggered by incorporation of beta7, whose C-terminal extension reaches out into the other half to stabilize the newly formed 20S particle. The process is completed by the maturation of active sites and subsequent degradation of UMP1 and PAC1-PAC2.

The C-terminal extension of the beta7 subunit and activator complexes stabilize nascent 20 S proteasomes and promote their maturation.

The eukaryotic 20 S proteasome is formed by dimerization of two precursor complexes containing the maturation factor Ump1. Beta7/Pre4 is the only one of the 14 subunits forming the 20 S proteasome that is absent from these precursor complexes in Saccharomyces cerevisiae. Increased expression of Pre4 leads to a reduction in the level of precursor complex, indicating that Pre4 incorporation into these complexes is rate-limiting for their dimerization. When we purified these precursor complexes, we observed co-purification of Blm10, a large protein known to attach to the alpha ring surface of proteasomes. In contrast to single mutants lacking either Blm10 or the C-terminal extension of Pre4, a mutant lacking both grew extremely poorly, accumulated very high levels of precursor complexes, and was impaired in beta subunit maturation. The effect of blm10Delta on proteasome biogenesis is modest, apparently because the 19 S regulatory particle is capable of substituting for Blm10, as long as precursor complex dimers are stabilized by the Pre4 C terminus. We found that a mutation (sen3/rpn2) affecting the Rpn2 subunit inhibits attachment of the 19 S activator to the 20 S particle or its precursors. Although the sen3 mutation alone had no apparent effect on precursor complex dimerization and active site maturation, the sen3 blm10 double mutant was impaired in these processes. Together these data demonstrate that Blm10 and the 19 S activator have a partially redundant function in stabilizing nascent 20 S proteasomes and in promoting their activation.

Assays for proteasome assembly and maturation.

The 20S proteasome is a complex multisubunit protease that is present in all phylae of life. Eukaryotic 26S proteasomes, which are composed of 20S proteasomes and 19S activator complexes, mediate the degradation of ubiquitylated proteins. Biogenesis of proteasomes involves a coordinated expression of proteasome genes as well as numerous assembly and maturation steps. Activation of proteolytic sites occurs via autocatalytic processing of the N-terminal propeptides of beta subunits. This process is coupled to the dimerization of half-proteasome precursor complexes and, in eukaryotes, requires the presence of the Ump1 maturation factor to occur efficiently. After activation of proteolytic sites the encased Ump1 is degraded rapidly. Here we describe methods that track assembly and maturation of proteasomes in bacteria and eukaryotic cells. Assembly intermediates and mature forms of the proteasome present in cells at steady state are analyzed by gel filtration and immunoblotting after sodium dodecyl sulfate (SDS)- and native polyacrylamide gel electrophoresis (PAGE). The kinetics of proteasome assembly is followed by pulse chase detection of beta subunit maturation or of Ump1 degradation.

Regulatory mechanisms controlling biogenesis of ubiquitin and the proteasome.

Analysis of several Saccharomyces cerevisiae ump mutants with defects in ubiquitin (Ub)-mediated proteolysis yielded insights into the regulation of the polyubiquitin gene UBI4 and of proteasome genes. High-molecular weight Ub-protein conjugates accumulated in ump mutants with impaired proteasome function with a concomitant decrease in the amount of free Ub. In these mutants, transcriptional induction of UBI4 was depending in part on the transcription factor Rpn4. Deletion of UBI4 partially suppressed the growth defects of ump1 mutants, indicating that accumulation of polyubiquitylated proteins is deleterious to cell growth. Transcription of proteasome subunit genes was induced in ump mutants affecting the proteasome, as well as under conditions that mediate DNA damage or the formation of abnormal proteins. This induction required the transcriptional activator Rpn4. Elevated Rpn4 levels in proteasome-deficient mutants or as a response to abnormal proteins were due to increased metabolic stability. Up-regulation of proteasome genes in response to DNA damage, in contrast, is shown to operate via induction of RPN4 transcription.

Role of C-terminal extensions of subunits beta2 and beta7 in assembly and activity of eukaryotic proteasomes.

A close inspection of the crystal structure of the yeast 20 S proteasome revealed that a prominent connection between the two beta-rings is mediated by the subunit beta7/Pre4. Its C-terminal extension intercalates between the beta1/Pre3 and beta2/Pup1 subunits on the opposite ring. We show that the interactions promoted by the beta7/Pre4 tail are important to facilitate the formation of 20 S particles from two half-proteasome precursor complexes and/or to stabilize mature 20 S proteasomes. The deletion of 19 residues from the beta7/Pre4 C terminus leads to an accumulation of half-proteasome precursor complexes containing the maturation factor Ump1. The C-terminal extension of beta7/Pre4, which forms several hydrogen bonds with beta1/Pre3, is in addition required for the post-acidic activity mediated by the latter subunit. Deletion of the C-terminal tail of beta7/Pre4 results in an inhibition of beta1/Pre3 propeptide processing and abrogation of post-acidic activity. Our data obtained with yeast strains that expressed the mature form of Pre3 lacking its propeptide suggest that interactions between the Pre4 C terminus and Pre3 stabilize a conformation of its active site, which is essential for post-acidic activity. Deletion of the C-terminal extension of beta2/Pup1, which wraps around beta3/Pup3 within the same beta-ring, is lethal, indicating that this extension serves an essential function in proteasome assembly or stability.