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.     

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.     

Mycobacterium tuberculosis prcBA genes encode a gated proteasome with broad oligopeptide specificity

Genes predicted to be associated with the putative proteasome of Mycobacterium tuberculosis (Mtb) play a critical role in defence of the bacillus against nitrosative stress. However, proteasomes are uncommon in eubacteria and it remains to be established whether Mtb's prcBA genes in fact encode a proteasome. We found that coexpression of recombinant PrcB and PrcA in Escherichia coli over a prolonged period at 37 degrees C allowed formation of an alpha(7)beta(7)beta(7)alpha(7), 750 kDa cylindrical stack of four rings in which all 14 beta-subunits were proteolytically processed to expose the active site threonine. In contrast to another Actinomycete, Rhodococcus erythropolis, Mtb's beta-chain propeptide was not required for particle assembly. Peptidolytic activity of the 750 kDa particle towards a hydrophobic oligopeptide was nearly two orders of magnitude less than that of the Rhodococcus 20S proteasome, and unlike eukaryotic and archaeal proteasomes, activity of the Mtb 750 kDa particle could not be stimulated by SDS, Mg(2+) or Ca(2+). Electron microscopy revealed what appeared to be obstructed alpha-rings in the Mtb 750 kDa particle. Deletion of the N-terminal octapeptide from Mtb's alpha-chain led to disappearance of the apparent obstruction and a marked increase of peptidolytic activity. Unlike proteasomes isolated from other Actinomycetes, the open-gate Mtb mutant 750 kDa particle cleaved oligopeptides not only after hydrophobic residues but also after basic, acidic and small, neutral amino acids. Thus, Mtb encodes a broadly active, gated proteasome that may work in concert with an endogenous activator.     

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.     

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.     

Proteasomes of the yeastS. cerevisiae: genes,structure and functions

Proteasomes are large multicatalytic protease complexes which fulfil central functions in major intracellular proteolytic pathways of the eukaryotic cell. 20S proteasomes are 700 kDa cylindrically shaped particles, found in the cytoplasm and the nucleus of all eukaryotes. They are composed of a pool of 14 different subunits (MW 22–25 kDa) arranged in a stack of 4 rings with 7-fold symmetry. In the yeastSaccharomyces cerevisiae a complete set of 14 genes coding for 20S proteasome subunits have been cloned and sequenced. 26S proteasomes are even larger proteinase complexes (about 1700 kDa) which degrade ubiquitinylated proteins in an ATP-dependent fashionin vitro. The 26S proteasome is build up from the 20S proteasome as core particle and two additional 19S complexes at both ends of the 20S cylinder. Recently existence of a 26S proteasome in yeast has been demonstrated. Several 26S proteasome specific genes have been cloned and sequenced. They share similarity with a novel defined family of ATPases. 20S and 26S proteasomes are essential for functioning of the eukaryotic cell. Chromosomal deletion of 20S and 26S proteasomal genes in the yeastS. cerevisiae caused lethality of the cell. Thein vivo functions of proteasomes in major proteolytic pathways have been demonstrated by the use of 20S and 26S proteasomal mutants. Proteasomes are needed for stress dependent and ubiquitin mediated proteolysis. They are involved in the degradation of short-lived and regulatory proteins. Proteasomes are important for cell differentiation and adaptation to environmental changes. Proteasomes have also been shown to function in the control of the cell cycle.     

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.     

Cooperation of multiple chaperones required for the assembly of mammalian 20S proteasomes

The 20S proteasome is a catalytic core of the 26S proteasome, a central enzyme in the degradation of ubiquitin-conjugated proteins. It is composed of 14 distinct gene products that form four stacked rings of seven subunits each, alpha(1-7)beta(1-7)beta(1-7)alpha(1-7). It is reported that the biogenesis of mammalian 20S proteasomes is assisted by proteasome-specific chaperones, named PAC1, PAC2, and hUmp1, but the details are still unknown. Here, we report the identification of a chaperone, designated PAC3, as a component of alpha rings. Although it can intrinsically bind directly to both alpha and beta subunits, PAC3 dissociates before the formation of half-proteasomes, a process coupled with the recruitment of beta subunits and hUmp1. Knockdown of PAC3 impaired alpha ring formation. Further, PAC1/2/3 triple knockdown resulted in the accumulation of disorganized half-proteasomes that are incompetent for dimerization. Our results describe a cooperative system of multiple chaperones involved in the correct assembly of mammalian 20S proteasomes.     

Posttranslational modification of the 20S proteasomal proteins of the archaeon Haloferax volcanii

20S proteasomes are large, multicatalytic proteases that play an important role in intracellular protein degradation. The barrel-like architecture of 20S proteasomes, formed by the stacking of four heptameric protein rings, is highly conserved from archaea to eukaryotes. The outer two rings are composed of alpha-type subunits, and the inner two rings are composed of beta-type subunits. The halophilic archaeon Haloferax volcanii synthesizes two different alpha-type proteins, alpha1 and alpha2, and one beta-type protein that assemble into at least two 20S proteasome subtypes. In this study, we demonstrate that all three of these 20S proteasomal proteins (alpha1, alpha2, and beta) are modified either post- or cotranslationally. Using electrospray ionization quadrupole time-of-flight mass spectrometry, a phosphorylation site of the beta subunit was identified at Ser129 of the deduced protein sequence. In addition, alpha1 and alpha2 contained N-terminal acetyl groups. These findings represent the first evidence of acetylation and phosphorylation of archaeal proteasomes and are one of the limited examples of post- and/or cotranslational modification of proteins in this unusual group of organisms.     

Catalytic components of proteasomes and the regulation of proteinase activity

The proteasome (multicatalytic proteinase complex) is a large multimeric complex which is found in the nucleus and cytoplasm of eukaryotic cells. It plays a major role in both ubiquitin-dependent and ubiquitin-independent nonlysosomal pathways of protein degradation. Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the and subunits of the simpler proteasome isolated fromThermoplasma acidophilum. Proteasomes have a cylindrical structure composed of four rings of seven subunits. The 26S form of the proteasome, which is responsible for ubiquitin-dependent proteolysis, contains additional regulatory complexes. Eukaryotic proteasomes have multiple catalytic activities which are catalysed at distinct sites. Since proteasomes are unrelated to other known proteases, there are no clues as to which are the catalytic components from sequence alignments. It has been assumed from studies with yeast mutants that -type subunits play a catalytic role. Using a radiolabelled peptidyl chloromethane inhibitor of rat liver proteasomes we have directly identified RC7 as a catalytic component. Interestingly, mutants in Prel, the yeast homologue of RC7, have already been reported to have defective chymotrypsin-like activity. These results taken together confirm a direct catalytic role for these -type subunits. Proteasome activities are sensitive to conformational changes and there are several ways in which proteasome function may be modulatedin vivo. Our recent studies have shown that in animal cells at least two proteasome subunits can undergo phosphorylation, the level of which is likely to be important for determining proteasome localization, activity or ability to form larger complexes. In addition, we have isolated two isoforms of the 26S proteinase.     

Biochemical properties of the proteasome from Thermoplasma acidophilum.

We have purified proteasomes to apparent homogeneity from the archaebacterium Thermoplasma acidophilum. This proteinase has a molecular mass of about 650 kDa and an isoelectric point of 5.6. The proteasome hydrolyses peptide substrates containing an aromatic residue adjacent to the reporter group, as well as [14C]methylated casein optimally at pH 8.5 and 90 degrees C. The enzyme activity is enhanced severalfold by Mg2+ and Ca2+ at 25-500 mM. This increase in activity results primarily from a change in Km. The serine-proteinase inhibitors diisopropylfluorophosphate and 3,4-dichloroisocoumarin irreversibly inhibit the enzyme, obviously by modification of both the alpha and beta subunits in the proteasome. The inhibition of proteasomal activity by the peptidylchloromethanes, Cbz-Leu-Leu-CH2Cl and Cbz-Ala-Ala-Phe-CH2Cl (Cbz, benzyloxycarbonyl), is reversible and predominantly of a competitive type. The enzyme is not activated by any of the compounds that typically stimulate the activities of the eukaryotic proteasome.     

Identification of the Xenopus 20S proteasome alpha4 subunit which is modified in the meiotic cell cycle.

The proteasomes are large, multi-subunit particles that act as the proteolytic machinery for most of the regulated intracellular protein degradation in eukaryotic cells. To investigate the regulatory mechanism for the 26S proteasome in cell-cycle events, we purified this proteasome from immature and mature oocytes, and compared its subunits. Immunoblot analysis of 26S proteasomes showed a difference in the subunit of the 20S proteasome. A monoclonal antibody, GC3beta, cross-reacted with two bands in the 26S proteasome from immature oocytes (in G2-phase); however, the upper band was absent in the 26S proteasome from mature oocytes (in M-phase). These results suggest that changes in the subunits of 26S proteasomes are involved in the regulation of the meiotic cell cycle. Here we describe the molecular cloning of one of the alpha subunits of the 20S proteasome from a Xenopus ovarian cDNA library using an anti-GC3beta monoclonal antibody. From the screening, two types of cDNA are obtained, one 856bp, the other 984bp long. The deduced amino-acid sequences comprise 247 and 248 residues, respectively. These deduced amino-acid sequences are highly homologous to those of alpha4 subunits of other vertebrates. Phosphatase treatment of 26S proteasome revealed the upper band to be a phosphorylated form of the lower band. These results suggest that a part of the alpha4 subunit of the Xenopus 20S proteasome, alpha4_xl, is phosphorylated in G2-phase and dephosphorylated in M-phase.     

The proteasome 11S regulator subunit REG alpha (PA28 alpha) is a heptamer.

Activity of the 20S proteasome, which performs much of the cytosolic and nuclear proteolysis in eukaryotic cells, is controlled by regulatory complexes that bind to one or both ends of the cylindrical proteasome. One of these complexes, the 11S regulator (REG), is a complex of 28 kDa subunits that is thought to activate proteasomes toward the production of antigenic peptides. REG, purified from red blood cells, is a complex of REG alpha and REG beta subunits. We have crystallized recombinant REG alpha (rREG alpha) and collected diffraction data to 3.0 A resolution. The self-rotation function indicates that rREG alpha forms a heptameric ring in the crystal. Equilibrium sedimentation demonstrates that rREG alpha is a heptamer in solution also.     

Investigations on the maturation and regulation of archaebacterial proteasomes

The 20S proteasome (core particle, CP) is a multifunctional protease complex and composed of four heptameric subunit rings arranged in a hollow, barrel-shaped structure. Here, we report the crystal structure of the CP from Archaeoglobus fulgidus at 2.25A resolution. The analysis of the structure of early and late assembly intermediates of this CP gives new insights in the maturation of archaebacterial CPs and indicates similarities to assembly intermediates observed in eukaryotes. We also show a striking difference in mechanism and regulation of substrate access between eukaryotic and archaebacterial 20S proteasomes. While eukaryotic CPs are auto-inhibited by the N-terminal tails of the outer alpha-ring by imposing topological closure with a characteristic sequence motif (YDR-motif) and show regulatory gating this segment is disordered in the CP and differently structured in the alpha(7)-sub-complex of A.fulgidus leaving a pore leading into the particle with a diameter of 13A. Mutagenesis and functional studies indicate the absence of regulatory gating in the archaeal 20S proteasome.     

Intrinsic nucleoside diphosphate kinase-like activity is a novel function of the 20 S proteasome

The eukaryotic 20 S proteasome is the prototype of a new family of the N-terminal nucleophil hydrolases and is composed of numerous low molecular mass subunits arranged in a stack of four rings, each containing seven different alpha- or beta-subunits. Among the beta-type subunits in the yeast proteasome, three proteolytically active ones were identified, although the functions of the other beta- and alpha-type subunits remain to be clarified. We report here that the purified 20 S proteasome exhibits intrinsic nucleoside diphosphate (NDP) kinase-like activity. The proteasome exhibited a preference for ATP and dATP as phosphate donors, and a broad specificity for NDPs, other than GDP, as phosphate acceptors, unlike conventional NDP kinase, which catalyzes the transfer of gamma-phosphate between NDPs and nucleoside triphosphates. During the transfer of gamma-phosphate, the proteasome formed acid-labile phosphohistidine as autophosphorylated intermediates, and NDP-dependent dephosphorylation of the latter then occurred. These enzymatic properties are similar to those of the molecular chaperone, Hsp70, which also exhibits intrinsic NDP kinase-like activity, instead of ATPase activity. C5 among the beta-type subunits and C8 among the alpha-type subunits were autophosphorylated during the gamma-phosphate transfer reaction and were photoaffinity labeled with 8-azido-[alpha-(32)P]ATP, suggesting that the C5 and C8 subunits of the proteasome are responsible for the NDP kinase-like activity.     

Atomic force microscopy reveals two conformations of the 20 S proteasome from fission yeast

The proteasome is a major cytosolic proteolytic complex, indispensable in eukaryotic cells. The barrel-shaped core of this enzyme, the 20 S proteasome, is built from 28 subunits forming four stacked rings. The two inner beta-rings harbor active centers, whereas the two outer alpha-rings play a structural role. Crystal structure of the yeast 20 S particle showed that the entrance to the central channel was sealed. Because of this result, the path of substrates into the catalytic chamber has remained enigmatic. We have used tapping mode atomic force microscopy (AFM) in liquid to address the dynamic aspects of the 20 S proteasomes from fission yeast. We present here evidence that, when observed with AFM, the proteasome particles in top view position have either open or closed entrance to the central channel. The preferred conformation depends on the ligands present. Apparently, the addition of a substrate to the uninhibited proteasome shifts the equilibrium toward the open conformation. These results shed new light on the possible path of the substrate into the proteolytic chamber.     

Identification of the goldfish 20S proteasome beta6 subunit bound to nuclear matrix

Proteasomes are large, multisubunit particles that act as the proteolytic machinery for most of the regulated intracellular protein breakdown in eukaryotic cells. Proteasomes are present in both the nucleus and cytoplasm. When we analyzed the molecular composition of protein constituents of the nuclear matrix preparation of goldfish oocytes by two-dimensional polyacrylamide gel electrophoresis followed by sequence analysis, we found a 26 kDa spot identical in amino acid sequence to the beta6 subunits of the 20S proteasome. No spot of other subunits of 20S proteasome was detected. Here we describe the cloning, sequencing and expression analysis of Carassius auratus, beta6_ca, which encodes one of the proteasome beta subunits from goldfish ovary. From the screening of an ovarian cDNA library, two types of cDNA were obtained, one 941 bp and the other 884 bp long. The deduced amino acid sequences comprise 239 and 238 residues, respectively. These deduced amino acid sequences are highly homologous to those of beta6 subunits of other vertebrates. Immunoblot analysis of nuclear matrix using anti-proteasome antibodies showed only a spot of beta6_ca. These results suggest that the beta6 subunit of the goldfish 20S proteasome, beta6_ca, is responsible for anchoring proteasomes in the nucleus.     

Cloning and sequencing of the gene encoding the large (alpha-) subunit of the proteasome from Thermoplasma acidophilum

The gene encoding the alpha-subunit of the proteasome from the archaebacterium Thermoplasma acidophilum was cloned and sequenced. The gene encodes for a polypeptide with 233 amino acid residues and a calculated molecular weight of 25870. Sequence similarity of the alpha-subunit with the Saccharomyces cerevisiae wild-type suppressor gene scll+ encoded polypeptide, which is probably identical with the subunit YC7-alpha of the yeast proteasome, lends support to a putative role of proteasomes in the regulation of gene expression. The significant sequence similarity to the various subunits of eukaryotic proteasomes make it likely that proteasomal proteins are encoded by one gene family of ancient origin.