Characterisation of the newly identified human Ump1 homologue POMP and analysis of LMP7(beta 5i) incorporation into 20 S proteasomes

Biogenesis of mammalian 20 S proteasomes occurs via precursor complexes containing alpha and unprocessed beta subunits. A human homologue of the yeast proteasome maturation factor Ump1 was identified in 2D gels of 16 S precursor preparations and designated as POMP (proteasome maturation protein). We show that POMP is detected only in precursor fractions and not in fractions containing mature 20 S proteasome. Northern blot experiments revealed that expression of POMP is induced after treatment with interferon gamma. To analyse the role of the beta 5 propeptide for proper maturation and incorporation of the beta 5 subunit into the complex, human T2 cells, which highly express derivatives of the beta 5i subunit (LMP7), were studied. In contrast to yeast, the presence of the beta 5 propeptide is not essential for incorporation of LMP7 into the proteasome complex. Mutated LMP7 subunits either carrying the prosequence of beta 2i (LMP2) or containing a mutation in the active threonine site are incorporated like wild-type LMP7, while a LMP7 derivative lacking the prosequence completely is incorporated to a lesser extent. Although the absence of the prosequence does not affect incorporation of LMP7, its deletion leads to delayed proteasome maturation and thereby to an accumulation of precursor complexes. As a result of the precursor accumulation, an increased amount of the POMP protein can be detected in these cells.     

20S proteasome biogenesis

26S proteasomes are multi-subunit protease complexes responsible for the turnover of short-lived proteins. Proteasomal degradation starts with the autocatalytic maturation of the 20S core particle. Here, we summarize different models of proteasome assembly. 20S proteasomes are assembled as precursor complexes containing alpha and unprocessed beta subunits. The propeptides of the beta subunits are thought to prevent premature conversion of the precursor complexes into matured particles and are needed for efficient beta subunit incorporation. The complex biogenesis is tightly regulated which requires additional components such as the maturation factor Ump1/POMP, an ubiquitous protein in eukaryotic cells. Ump1/POMP is associated with precursor intermediates and degraded upon final maturation. Mammalian proteasomes are localized all over the cell, while yeast proteasomes mainly localize to the nuclear envelope/endoplasmic reticulum (ER) membrane network. The major localization of yeast proteasomes may point to the subcellular place of proteasome biogenesis.     

Dissecting beta-ring assembly pathway of the mammalian 20S proteasome

The 20S proteasome is the catalytic core of the 26S proteasome. It comprises four stacked rings of seven subunits each, alpha(1-7)beta(1-7)beta(1-7)alpha(1-7). Recent studies indicated that proteasome-specific chaperones and beta-subunit appendages assist in the formation of alpha-rings and dimerization of half-proteasomes, but the process involved in the assembly of beta-rings is poorly understood. Here, we clarify the mechanism of beta-ring formation on alpha-rings by characterizing assembly intermediates accumulated in cells depleted of each beta-subunit. Starting from beta2, incorporation of beta-subunits occurs in an orderly manner dependent on the propeptides of beta2 and beta5, and the C-terminal tail of beta2. Unexpectedly, hUmp1, a chaperone functioning at the final assembly step, is incorporated as early as beta2 and is required for the structural integrity of early assembly intermediates. We propose a model in which beta-ring formation is assisted by both intramolecular and extrinsic chaperones, whose roles are partially different between yeast and mammals.     

Intermediates in the formation of mouse 20S proteasomes: implications for the assembly of precursor beta subunits.

The assembly of individual proteasome subunits into catalytically active mammalian 20S proteasomes is not well understood. Using subunit-specific antibodies, we characterized both precursor and mature proteasome complexes. Antibodies to PSMA4 (C9) immunoprecipitated complexes composed of alpha, precursor beta and processed beta subunits. However, antibodies to PSMA3 (C8) and PSMB9 (LMP2) immunoprecipitated complexes made up of alpha and precursor beta but no processed beta subunits. These complexes possess short half-lives, are enzymatically inactive and their molecular weight is approximately 300 kDa. Radioactivity chases from these complexes into mature, long-lived approximately 700 kDa proteasomes. Therefore, these structures represent precursor proteasomes and are probably made up of two rings: one containing alpha subunits and the other, precursor beta subunits. The assembly of precursor proteasomes occurs in at least two stages, with precursor beta subunits PSMB2 (C7-I), PSMB3 (C10-II), PSMB7 (Z), PSMB9 (LMP2) and PSMB10 (LMP10) being incorporated before others [PSMB1 (C5), PSMB6 (delta), and PSMB8 (LMP7)]. Proteasome maturation (processing of the beta subunits and juxtaposition of the two beta rings) is accompanied by conformational changes in the (outer) alpha rings, and may be inefficient. Finally, interferon-gamma had no significant effect on the half-lives or total amounts of precursor or mature proteasomes.     

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.     

Identification of Proteassemblin,a Mammalian Homologue of the Yeast Protein,Ump1p,That Is Required for Normal Proteasome Assembly

We have identified a mammalian homologue of yeast Ump1p by searching for similar proteins in human and mouse expressed sequence tag (EST) databases. Ump1p is an accessory protein that is required for normal proteasome assembly in yeast (1). A mammalian homologue, which we refer to as “proteassemblin,” is a constituent of proteasome assembly intermediates (preproteasomes), but not fully assembled 20S proteasomes, as is Ump1p in yeast. We also provide evidence that proteassemblin is a constituent of pre-immunoproteasomes that contain the precursor of the interferon-γ-inducible subunit LMP2. By analogy with Ump1p, we hypothesize that proteassemblin is required for normal mammalian proteasome assembly.     

Protein-protein interactions among human 20S proteasome subunits and proteassemblin

Immunoproteasomes and standard proteasomes assemble by alternative pathways that bias against the formation of certain "mixed" proteasomes. Differences between beta subunit propeptides contribute to assembly specificity and an assembly chaperone, proteassemblin, may be involved via differential propeptide interactions. We investigated possible mechanisms of biased proteasome assembly and the role of proteassemblin by identifying protein-protein interactions among human 20S proteasome subunits and proteassemblin using a yeast two-hybrid interaction assay. Forty-one interactions were detected, including five involving proteassemblin and contiguous beta subunits, which suggests that proteassemblin binds to preproteasomes via a beta subunit surface. Interaction between proteassemblin and beta5, but not beta5i, suggests that proteassemblin may be involved in the propeptide-dependent differential incorporation of these subunits. Interactions between proteassemblin and beta1, beta1i, and beta7 suggest that proteassemblin may regulate preproteasome dimerization via interactions with the C-termini of these subunits, which in the mature 20S structure extend to contact opposing beta subunit rings.     

Proteolytic processing and assembly of the C5 subunit into the proteasome complex

Assembly of mammalian 20 S proteasomes from individual subunits is beginning to be investigated. Proteasomes are made of four heptameric rings in the configuration alpha7beta7beta7alpha7. By using anti-proteasome and anti-subunit-specific antibodies, we characterized the processing and assembly of the beta subunit C5. The C5 precursor (25 kDa) remains as a free non-assembled polypeptide in the cell. The conversion of the C5 precursor to mature C5 (23 kDa) occurs concomitantly with its incorporation into 15 S proteasome intermediate and 20 S mature proteasome complexes. This processing is dependent on proteasome activity and takes place in the cytosol. These results are not fully compatible with the hypothesis that postulates that assembly of proteasomes takes place via a "half-proteasome" intermediate that contains one full alpha-ring and one full beta-ring of unprocessed beta subunit precursors.     

Eukaryotic 20S proteasome catalytic subunit propeptides prevent active site inactivation by N-terminal acetylation and promote particle assembly.

Proteins targeted for degradation by the ubiquitin-proteasome system are degraded by the 26S proteasome. The core of this large protease is the 20S proteasome, a barrel-shaped structure made of a stack of four heptameric rings. Of the 14 different subunits that make up the yeast 20S proteasome, three have proteolytic active sites: Doa3/beta5, Pup1/beta2 and Pre3/beta1. Each of these subunits is synthesized with an N-terminal propeptide that is autocatalytically cleaved during particle assembly. We show here that the propeptides have both common and distinct functions in proteasome biogenesis. Unlike the Doa3 propeptide, which is crucial for proteasome assembly, the Pre3 and Pup1 propeptides are dispensable for cell viability and proteasome formation. However, mutants lacking these propeptide-encoding elements are defective for specific peptidase activities, are more sensitive to environmental stresses and have subtle defects in proteasome assembly. Unexpectedly, a critical function of the propeptide is the protection of the N-terminal catalytic threonine residue against Nalpha-acetylation. For all three propeptide-deleted subunits, activity of the affected catalytic center is fully restored when the Nat1-Ard1 Nalpha-acetyltransferase is mutated. In addition to delineating a novel function for proteasome propeptides, these data provide the first biochemical evidence for the postulated participation of the alpha-amino group in the proteasome catalytic mechanism.     

Mass spectrometry reveals the missing links in the assembly pathway of the bacterial 20 S proteasome

The 20 S proteasome is an essential proteolytic particle, responsible for degrading short-lived and abnormal intracellular proteins. The 700-kDa assembly is comprised of 14 alpha-type and 14 beta-type subunits, which form a cylindrical architecture composed of four stacked heptameric rings (alpha7beta7beta7alpha7). The formation of the 20 S proteasome is a complex process that involves a cascade of folding, assembly, and processing events. To date, the understanding of the assembly pathway is incomplete due to the experimental challenges of capturing short-lived intermediates. In this study, we have applied a real-time mass spectrometry approach to capture transient species along the assembly pathway of the 20 S proteasome from Rhodococcus erythropolis. In the course of assembly, we observed formation of an early alpha/beta-heterodimer as well as an unprocessed half-proteasome particle. Formation of mature holoproteasomes occurred in concert with the disappearance of half-proteasomes. We also analyzed the beta-subunits before and during assembly and reveal that those with longer propeptides are incorporated into half- and full proteasomes more rapidly than those that are heavily truncated. To characterize the preholoproteasome, formed by docking of two unprocessed half-proteasomes and not observed during assembly of wild type subunits, we trapped this intermediate using a beta-subunit mutational variant. In summary, this study provides evidence for transient intermediates in the assembly pathway and reveals detailed insight into the cleavage sites of the propeptide.     

Backbone 1H, 13C,and 15N assignments of yeast Ump1, an intrinsically disordered protein that functions as a proteasome assembly chaperone

Eukaryotic proteasome assembly is a highly organized process mediated by several proteasome-specific chaperones, which interact with proteasome assembly intermediates. In yeast, Ump1 and Pba1-4 have been identified as assembly chaperones that are dedicated to the formation of the proteasome 20S catalytic core complex. The crystal structures of Pba chaperones have been reported previously, but no detailed information has been provided for the structure of Ump1. Thus, to better understand the mechanisms underlying Ump1-mediated proteasome assembly, we characterized the conformation of Ump1 in solution using NMR. Backbone chemical shift data indicated that Ump1 is an intrinsically unstructured protein and largely devoid of secondary structural elements.     

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.     

Novel propeptide function in 20 S proteasome assembly influences beta subunit composition

The assembly of eukaryotic 20 S proteasomes involves the formation of half-proteasomes where precursor beta-type subunits gather in position on an alpha-subunit ring, followed by the association of two half-proteasomes and beta-subunit processing. In vertebrates three additional beta-subunits (beta1i/LMP2, beta2i/MECL1, and beta5i/LMP7) can be synthesized and substituted for constitutive homologues (beta1/delta, beta2/Z, and beta5/X) to yield immunoproteasomes, which are important for generating certain antigenic peptides. We have shown previously that when all six beta-subunits are present, cooperative assembly mechanisms limit the diversity of proteasome populations. Specifically, LMP7 is incorporated preferentially over X into preproteasomes containing LMP2 and MECL1. We show here that the LMP7 propeptide is responsible for this preferential incorporation, and it also enables LMP7 to incorporate into proteasomes containing delta and Z. In contrast, the X propeptide restricts incorporation to proteasomes with delta and Z. Furthermore, we demonstrate that the LMP7 propeptide can function in trans when expressed on LMP2, and that its NH(2)-terminal and mid-regions are particularly critical for function. In addition to identifying a novel propeptide function, our results raise the possibility that one consequence of LMP7 incorporation into both immunoproteasomes and delta/Z proteasomes may be to increase the diversity of antigenic peptides that can be generated.     

Beta 2 subunit propeptides influence cooperative proteasome assembly

Vertebrate proteasomes are structurally heterogeneous, consisting of both "constitutive" (or "standard") proteasomes and "immunoproteasomes." Constitutive proteasomes contain three ubiquitously expressed catalytic subunits, Delta (beta 1), Z (beta 2), and X (beta 5), whereas immunoproteasomes contain three interferon-gamma-inducible catalytic subunits, LMP2 (beta 1i), MECL (beta 2i), and LMP7 (beta 5i). We recently have demonstrated that proteasome assembly is biased to promote immunoproteasome homogeneity when both types of catalytic subunits are expressed in the same cell. This cooperative assembly is due in part to differences between the LMP7 (beta 5i) and X (beta 5) propeptides. In the current study we demonstrate that differences between the MECL (beta 2i) and Z (beta2) propeptides also influence cooperative assembly. Specifically, replacing the MECL propeptide with that of Z enables MECL incorporation into otherwise constitutive (Delta(+)/X(+)) proteasomes and facilitates X incorporation into otherwise immunoproteasomes (MECL(+)/LMP2(+)). We also show, using MECL(-/-) mice, that LMP2 incorporation does not require MECL, in contrast with previous suggestions that their incorporation is mutually codependent. These results enable us to refine our model for cooperative proteasome assembly by determining which combinations of inducible and constitutive subunits are favored over others, and we propose a mechanism for how propeptides mediate cooperative assembly.     

Blm3 is part of nascent proteasomes and is involved in a late stage of nuclear proteasome assembly

Proteasomes are multisubunit proteases that are responsible for regulated proteolysis. The degradation of the proteasomal maturation factor, named Ump1 in yeast, completes the autocatalytic processing of inactive precursor complexes into the proteolytically active core particle (CP) of the proteasome. We have identified Blm3, a conserved nuclear protein, as a new component of Ump1-associated precursor complexes. A lack of Blm3 resulted in an increased rate of precursor processing and an accelerated turnover of Ump1, which suggests that Blm3 prevents premature activation of proteasomal CPs. On the basis of biochemical fractionation experiments combined with in vivo localization studies, we propose that Blm3 joins nascent CPs inside the nucleus to coordinate late stages of proteasome assembly in yeast.     

Sequence information within proteasomal prosequences mediates efficient integration of beta-subunits into the 20 S proteasome complex.

The maturation of proteases is governed by prosequences. During the biogenesis of the highly oligomeric eukaryotic 20 S proteasome five different prosequence-containing subunits have to be integrated and processed either by autocatalysis or by neighbouring subunits. To analyse the functional impact of proteasomal prosequences during complex formation, the propeptide of the facultative subunit beta1i/LMP2 was truncated to nine amino acid residues or completely deleted. Additionally, the charged residues within the truncated beta1i/LMP2 version were replaced by neutral residues. While deletion did not affect subunit incorporation, the presence of charged residues within the truncated version of the LMP2 propeptide diminished incorporation efficiency, an effect that was restored upon replacement of the charged amino acids against neutral components. During immunoproteasome formation, incorporation and processing of inducible proteasome beta-subunits are cooperative processes. We demonstrate a linear correlation of the levels of beta2i/MECL1 and beta1i/LMP2 within 20 S proteasomes, suggesting a physical interaction to be the molecular basis for the biased incorporation of both subunits. In the absence of beta5i/LMP7, precursor complexes containing unprocessed beta1i/LMP2 accumulated. The contribution of beta5i/LMP7 on the cooperative formation of a homogeneous population of immunoproteasome is therefore most likely based on an acceleration of the beta1i/LMP2 and potentially of beta2i/MECL1 processing kinetics.     

Regulation of proteasome structure and function

Proteasomes are cylindrical particles made up of a stack of four heptameric rings. In animal cells the outer rings are made up of 7 different types of alpha subunits and the inner rings are composed of 7 out of 10 possible different beta subunits. Regulatory complexes can bind to the ends of the cylinder. We have investigated aspects of the assembly, activity and subunit composition of core proteasome particles and 26S proteasomes, the localization of proteasome subpopulations, and the possible role of phosphorylation in determining proteasome localization, activities and association with regulatory components.     

Multiple chaperone-assisted formation of mammalian 20S proteasomes

Protein degradation is essential for maintenance of cellular homeostasis. The majority of proteins are selectively degraded in eukaryotic cells by the ubiquitin-proteasome system. The 26S proteasome selects target proteins that are covalently modified with polyubiquitin chains. The 26S proteasome is a multisubunit protease responsible for regulated proteolysis in eukaryotic cells. The catalytic activities are carried out by the core 20S proteasome. The eukaryotic 20S proteasome is composed of 28 subunits arranged in a cylindrical particle as four heteroheptameric rings, alpha1-7beta1-7beta1-7alpha1-7. Recent studies have revealed the mechanism responsible for the assembly of such a complex structure. This article recounts the observations that disclosed the biogenesis of 20S proteasomes and discusses the difference in the mechanism of assembly between archael, yeast, and mammalian 20S proteasomes.     

Understanding the separation of timescales in bacterial proteasome core particle assembly

The 20S proteasome core particle (CP) is a molecular machine that is a key component of cellular protein degradation pathways. Like other molecular machines, it is not synthesized in an active form but rather as a set of subunits that assemble into a functional complex. The CP is conserved across all domains of life and is composed of 28 subunits, 14 α and 14 β, arranged in four stacked seven-member rings (α₇β₇β₇α₇). While details of CP assembly vary across species, the final step in the assembly process is universally conserved: two half proteasomes (HPs; α₇β₇) dimerize to form the CP. In the bacterium Rhodococcus erythropolis, experiments have shown that the formation of the HP is completed within minutes, while the dimerization process takes hours. The N-terminal propeptide of the β subunit, which is autocatalytically cleaved off after CP formation, plays a key role in regulating this separation of timescales. However, the detailed molecular mechanism of how the propeptide achieves this regulation is unclear. In this work, we used molecular dynamics simulations to characterize HP conformations and found that the HP exists in two states: one where the propeptide interacts with key residues in the HP dimerization interface and likely blocks dimerization, and one where this interface is free. Furthermore, we found that a propeptide mutant that dimerizes extremely slowly is essentially always in the nondimerizable state, while the wild-type rapidly transitions between the two. Based on these simulations, we designed a propeptide mutant that favored the dimerizable state in molecular dynamics simulations. In vitro assembly experiments confirmed that this mutant dimerizes significantly faster than wild-type. Our work thus provides unprecedented insight into how this critical step in CP assembly is regulated, with implications both for efforts to inhibit proteasome assembly and for the evolution of hierarchical assembly pathways.     

The C terminus of Rpt3, an ATPase subunit of PA700 (19 S) regulatory complex, is essential for 26 S proteasome assembly but not for activation

PA700, the 19 S regulatory subcomplex of the 26 S proteasome, contains a heterohexameric ring of AAA subunits (Rpt1 to -6) that forms the binding interface with a heteroheptameric ring of α subunits (α1 to -7) of the 20 S proteasome. Binding of these subcomplexes is mediated by interactions of C termini of certain Rpt subunits with cognate binding sites on the 20 S proteasome. Binding of two Rpt subunits (Rpt2 and Rpt5) depends on their last three residues, which share an HbYX motif (where Hb is a hydrophobic amino acid) and open substrate access gates in the center of the α ring. The relative roles of other Rpt subunits for proteasome binding and activation remain poorly understood. Here we demonstrate that the C-terminal HbYX motif of Rpt3 binds to the 20 S proteasome but does not promote proteasome gating. Binding requires the last three residues and occurs at a dedicated site on the proteasome. A C-terminal peptide of Rpt3 blocked ATP-dependent in vitro assembly of 26 S proteasome from PA700 and 20 S proteasome. In HEK293 cells, wild-type Rpt3, but not Rpt3 lacking the HbYX motif was incorporated into 26 S proteasome. These results indicate that the C terminus of Rpt3 was required for cellular assembly of this subunit into 26 S proteasome. Mutant Rpt3 was assembled into intact PA700. This result indicates that intact PA700 can be assembled independently of association with 20 S proteasome and thus may be a direct precursor for 26 S proteasome assembly under normal conditions. These results provide new insights to the non-equivalent roles of Rpt subunits in 26 S proteasome function and identify specific roles for Rpt3.