Yeast Proteasome Research Articles

Purification of 20S proteasomes.

Proteasomes are large multicatalytic proteinases located in the nuclei and cytoplasm of all eukaryotic cells. Proteasomes are composed of four heptameric rings stacked to form a hollow cylinder (length 16–20 nm, diameter 11–12 nm). The outer two rings are composed of α-subunits, while β-subunits, which contain the active sites, comprise the inner two rings. Proteasomes from archaebacteria contain only one type each of αand β-subunits. Eukaryotic proteasomes are more divergent; yeast proteasomes have seven different αand seven different β-subunits, each occupying a unique position in the ring. Only three of the seven yeast β-subunits contain the N-terminal threonine necessary for activity (1–2). Mammalian 20S proteasomes have seven different αand ten different β-subunits, and have been classified into two groups. The so-called “constitutive” proteasomes contain three catalytic β-subunits: PSMB5 (X or MB1), PSMB6 (Y or δ), and PSMB7 (Z). These subunits can be replaced in “immunoproteasomes” by the IFN-γ-inducible catalytic β-subunits PSMB8 (LMP7), PSMB9 (LMP2), and PSMB10 (MECL-1), respectively (1–3). Although there are eight possible combinations of catalytic subunits in the β rings, proteasomes with mixtures of constitutive and immune subunits are not favored (4). Replacement of constitutive catalytic subunits with the IFN-γ-inducible subunits has been shown to change the proteasome activities against fluorogenic peptide and protein substrates (2,5). It has been shown that proteasomes are responsible for generation of cytosolic peptides 7–13 amino acids in length, which are presented on cell surfaces in association with major histocompatibility complex class I (MHC-I) molecules (1,3). The IFN-γ-inducible subunits are not essential for MHC-I antigen presentation, but it is thought that the additional

A gated channel into the proteasome core particle.

The core particle (CP) of the yeast proteasome is composed of four heptameric rings of subunits arranged in a hollow, barrel-like structure. We report that the CP is autoinhibited by the N-terminal tails of the outer (alpha) ring subunits. Crystallographic analysis showed that deletion of the tail of the alpha 3-subunit opens a channel into the proteolytically active interior chamber of the CP, thus derepressing peptide hydrolysis. In the latent state of the particle, the tails prevent substrate entry by imposing topological closure on the CP. Inhibition by the alpha-subunit tails is relieved upon binding of the regulatory particle to the CP to form the proteasome holoenzyme.

Identification and characterization of a mammalian protein interacting with 20S proteasome precursors.

The assembly of individual mammalian proteasome subunits into catalytically active 20S proteasome is not well understood. Herein, we report the identification and characterization of human and mouse homologues of the yeast proteasome maturating factor Ump1p. We delineate the region of hUMP1 implicated in the specific interaction with proteasome precursors and show that hUMP1 protein is absent from the mature form of the 20S proteasome. We also show that the transcript level of mammalian UMP1 is increased after IFN-gamma treatment and that mammalian UMP1 is functionally related to but not interchangeable with its yeast homologue.

Characterisation of the newly identified human Ump1 homologue POMP and analysis of LMP7(β5i) incorporation into 20 S proteasomes

Biogenesis of mammalian 20 S proteasomes occurs via precursor complexes containing α and unprocessed β 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 γ. To analyse the role of the β5 propeptide for proper maturation and incorporation of the β5 subunit into the complex, human T2 cells, which highly express derivatives of the β5i subunit (LMP7), were studied. In contrast to yeast, the presence of the β5 propeptide is not essential for incorporation of LMP7 into the proteasome complex. Mutated LMP7 subunits either carrying the prosequence of β2i (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.

Rapid Isolation and Characterization of the Yeast Proteasome Regulatory Complex

The 26S proteasome, which catalyzes degradation of ubiquitinated proteins, is composed of the 20S proteasome and the 19S complex. Recently, it has been reported that the 26S complex can be dissociated into the lid complex and the 20S-proteasome-base complex in a mutant yeast and that the lid complex is required for ubiquitin-dependent proteolysis. In the present study, we established methods for rapid isolation of the 19S complex, the lid complex, and the base complex from wild-type yeast. The isolated 19S complex was capable of binding to the 20S proteasome to reconstitute the 26S proteasome. In contrast with the previously reported result showing that Rpn10, a multiubiquitin chain binding subunit, is a component of the base complex, we present evidence that the lid complex isolated from wild-type yeast contains Rpn10.

New crystal forms and low resolution structure analysis of 20S proteasomes from bovine liver.

20S proteasomes from higher eukaryotes have immunological functions rather than those from archibacteria or yeast. To clarify the mechanism of the sorting and production of antigen-presenting peptides, it is important and worthwhile to determine the structure of mammalian proteasomes using a third generation synchrotron radiation source. Here we report new crystal forms of 20S proteasomes from bovine liver and preliminary structure analysis of them. The crystals belong to the same space group but have different cell dimensions. One crystal (form I) belongs to space group P2(1)2(1)2(1) with unit cell dimensions of a = 124.8, b =197.4, c =323.8 A, and diffracts to 3.0 A resolution. The other crystal (form II) belongs to the same space group with a =115.1, b =205.6, c =316. 0 A, and diffracts to 4.0 A resolution. The diffraction data for the form I crystal provided an interpretable electron density map for presenting the structural differences from yeast proteasomes.

Proteases

AbstractThe processes of growth and remodeling of cells and tissues in multicellular organisms require the breakdown of old protein molecules, in concert with the synthesis of new ones. For example, many newly‐synthesized molecules require proteolytic processing to convert them to biologically active forms. Proteolysis can terminate the activity of a protein—e.g., capsases mediate apoptosis, which is a vital step in the life cycle of the cell. Proteolysis contributes to defense systems too, as the recognition of peptide fragments of foreign proteins triggers the immune response. Proteases are the class of enzymes involved in these important reactions. This unit discusses the general categories of proteases, and sets the stage for addition of overview units on cysteine proteases, aspartic proteases, and metalloproteases, as well as protocol units featuring techniques for analyzing mammalian and yeast proteasomes and protease inhibitors, among other topics.

Analysis of a Gene Encoding Rpn10 of the Fission Yeast Proteasome Reveals That the Polyubiquitin-binding Site of This Subunit Is Essential When Rpn12/Mts3 Activity Is Compromised

Substrates are targeted for proteolysis by the ubiquitin pathway by the addition of a polyubiquitin chain before being degraded by the 26 S proteasome. Previously, a subunit of the proteasome, S5a, was identified that was able to bind to polyubiquitin in vitro and thus proposed to act as a substrate recognition component. Deletion of the corresponding Saccharomyces cerevisiae gene, MCB1/RPN10, rendered cells viable indicating that other proteasomal polyubiquitin receptors must exist. In this study, we describe pus1(+), the fission yeast homologue of RPN10. This gene is also not required for cell viability; however, the Deltapus1 mutant is synthetically lethal with mutations in other proteasomal component-encoding genes, namely mts3, pad1, and mts4 (RPN12, RPN11, and RPN1). Overexpression of pus1(+) is able to rescue mts3-1 at 32 degrees C but overexpression of a cDNA encoding a version of Pus1 that does not bind to polyubiquitin cannot and leads to greatly reduced viability when used to rescue the mts3-1Deltapus1 double mutant. The Mts3 protein was unable to bind to polyubiquitin in vitro, but the Pus1 and Mts3 proteins were found to bind to one another in vitro, which taken together with the genetic data suggests that they are also closely associated in vivo.

Subcellular localization of proteasomes and their regulatory complexes in mammalian cells

Proteasomes can exist in several different molecular forms in mammalian cells. The core 20S proteasome, containing the proteolytic sites, binds regulatory complexes at the ends of its cylindrical structure. Together with two 19S ATPase regulatory complexes it forms the 26S proteasome, which is involved in ubiquitin-dependent proteolysis. The 20S proteasome can also bind 11S regulatory complexes (REG, PA28) which play a role in antigen processing, as do the three variable c-interferoninducible catalytic b-subunits (e.g. LMP7). In the present study, we have investigated the subcellular distribution of the different forms of proteasomes using subunit speci®c antibodies. Both 20S proteasomes and their 19S regulatory complexes are found in nuclear, cytosolic and microsomal preparations isolated from rat liver. LMP7 was enriched approximately two-fold compared with core a-type proteasome subunits in the microsomal preparations. 20S proteasomes were more abundant than 26S proteasomes, both in liver and cultured cell lines. Interestingly, some signi®cant differences were observed in the distribution of different subunits of the 19S regulatory complexes. S12, and to a lesser extent p45, were found to be relatively enriched in nuclear fractions from rat liver, and immuno¯uorescent labelling of cultured cells with anti-p45 antibodies showed stronger labelling in the nucleus than in the cytoplasm. The REG was found to be localized predominantly in the cytoplasm. Three- to six-fold increases in the level of REG were observed following cinterferon treatment of cultured cells but c-interferon had no obvious effect on its subcellular distribution. These results demonstrate that different regulatory complexes and subpopulations of proteasomes have different distributions within mammalian cells and, therefore, that the distribution is more complex than has been reported for yeast proteasomes.

Subcellular localization of proteasomes and their regulatory complexes in mammalian cells

Proteasomes can exist in several different molecular forms in mammalian cells. The core 20S proteasome, containing the proteolytic sites, binds regulatory complexes at the ends of its cylindrical structure. Together with two 19S ATPase regulatory complexes it forms the 26S proteasome, which is involved in ubiquitin-dependent proteolysis. The 20S proteasome can also bind 11S regulatory complexes (REG, PA28) which play a role in antigen processing, as do the three variable gamma-interferon-inducible catalytic beta-subunits (e.g. LMP7). In the present study, we have investigated the subcellular distribution of the different forms of proteasomes using subunit specific antibodies. Both 20S proteasomes and their 19S regulatory complexes are found in nuclear, cytosolic and microsomal preparations isolated from rat liver. LMP7 was enriched approximately two-fold compared with core alpha-type proteasome subunits in the microsomal preparations. 20S proteasomes were more abundant than 26S proteasomes, both in liver and cultured cell lines. Interestingly, some significant differences were observed in the distribution of different subunits of the 19S regulatory complexes. S12, and to a lesser extent p45, were found to be relatively enriched in nuclear fractions from rat liver, and immunofluorescent labelling of cultured cells with anti-p45 antibodies showed stronger labelling in the nucleus than in the cytoplasm. The REG was found to be localized predominantly in the cytoplasm. Three- to six-fold increases in the level of REG were observed following gamma-interferon treatment of cultured cells but gamma-interferon had no obvious effect on its subcellular distribution. These results demonstrate that different regulatory complexes and subpopulations of proteasomes have different distributions within mammalian cells and, therefore, that the distribution is more complex than has been reported for yeast proteasomes.

Nob1p, a new essential protein, associates with the 26S proteasome of growing saccharomyces cerevisiae cells.

Nob1p, which interacts with Nin1p/Rpn12, a subunit of the 19S regulatory particle (RP) of the yeast 26S proteasome, has been identified by two-hybrid screening. NOB1 was found to be an essential gene, encoding a protein of 459 amino acid residues. Nob1p was detected in growing cells but not in cells in the stationary phase. During the transition to the stationary phase, Nob1p was degraded, at least in part, by the 26S proteasome. Nob1p was found only in proteasomal fractions in a glycerol gradient centrifugation profile and immuno-coprecipitated with Rpt1, which is an ATPase component of the yeast proteasomes. These results suggest that association of Nob1p with the proteasomes is essential for the function of the proteasomes in growing cells.

Synthesis of bivalent inhibitors of eucaryotic proteasomes

Based on the peculiar spatial array of the active sites in the internal chamber of the multicatalytic proteasome, as derived from the X-ray structure of yeast proteasome, homo- and heterobivalent inhibitors were designed and synthesized to exploit the principle of multivalency for enhancing inhibition potency. Peptidic bis-aldehyde compounds of the octapeptide size were synthesized to address adjacent active sites, whilst a PEG spacer with a statistical length distribution of 19–25 monomers was used to link two identical or different tripeptide aldehydes as binding heads. These bis-aldehyde compounds were synthesized applying both methods in solution and solid phase peptide synthesis. Bivalent binding was observed only for the PEG-spaced inhibitors suggesting that binding from the primed side prevents hemiacetal formation with the active site threonine residue. Copyright © 2000 European Peptide Society and John Wiley & Sons, Ltd.

Identification of the rice 20S proteasome subunits and analysis of their N-terminal modification.

N-Acetylation, catalyzed with N-acetyltransferase (NAT), is one of the most common co- and post-translational modifications of the α amino group of eukaryotic proteins. The eukaryotic 20S proteasome contains seven α-type and seven β-type subunits. We have found that the N-terminus of α1, α2, α3, α4, α5, α6, α7, β3 and β4 subunits of yeast proteasome is acetylated with NAT. Experiments using the yeast NAT deletion mutant suggested that N-acetylation be related to the proteolytic activity of the 20S proteasome (Kimura et al. J Biol Chem 2000; 275: 4635-9). In the present study, the rice 20S proteasome was purified by ion-exchange chromatography, gel filtration and glycerol density gradient centrifugation from the rice bran, and 14 subunits contained in the 20S proteasome were separated by two-dimensional electrophoresis and the partial amino acid sequence of them was analyzed by Edman degradation. The sequence analysis indicated that among 14 subunits, the α1, α2, α3, α4, α5, α6, α7, β3 and β4 subunits are N-terminally blocked in rice as well as yeast, and besides these subunits, the β6 subunit is blocked in rice, but not in yeast. This suggests that the β6 subunit in rice may have different functions from that in yeast.

Synthesis of bivalent inhibitors of eucaryotic proteasomes.

Based on the peculiar spatial array of the active sites in the internal chamber of the multicatalytic proteasome, as derived from the X-ray structure of yeast proteasome, homo- and heterobivalent inhibitors were designed and synthesized to exploit the principle of multivalency for enhancing inhibition potency. Peptidic bis-aldehyde compounds of the octapeptide size were synthesized to address adjacent active sites, whilst a PEG spacer with a statistical length distribution of 19-25 monomers was used to link two identical or different tripeptide aldehydes as binding heads. These bis-aldehyde compounds were synthesized applying both methods in solution and solid phase peptide synthesis. Bivalent binding was observed only for the PEG-spaced inhibitors suggesting that binding from the primed side prevents hemiacetal formation with the active site threonine residue.

α5 subunit in Trypanosoma brucei proteasome can self-assemble to form a cylinder of four stacked heptamer rings

The proteasomes have a central role in catalysing protein degradation among both prokaryotes and eukaryotes. The 20 S proteasome constitutes their catalytic core. In studying the structure of Trypanosoma brucei 20 S proteasomes, we isolated by two-dimensional (2D) gel electrophoresis a 27 kDa subunit protein with an estimated pI of 4.7 and subjected it to mass spectrometric analysis. A tryptic peptide sequence from the protein was found identical with that of the rat α5 subunit. With the use of antiserum against T. brucei 20 S proteasomes to screen a T. b. rhodesiense λ expression cDNA library, we obtained a cDNA clone encoding a full-length protein of 246 amino acid residues with a calculated molecular mass of 27174 Da and a pI of 4.71. It bears 50.0% and 46.3% sequence identity with rat and yeast proteasome subunit α5 respectively, and matches all the peptide sequences derived from MS of the 2D gel-purified protein. The protein is thus designated the α5 subunit of T. brucei 20 S proteasome (TbPSA5). The recombinant protein, expressed in plasmid-transformed Escherichia coli, was found in a 27 kDa monomer form as well as polymerized forms with estimated molecular masses ranging from 190 to 800 kDa. Under the electron microscope, the most highly polymerized forms bear the appearance of cylinders of four-stacked heptamer rings with an estimated outer diameter of 14.5 nm and a length of 18 nm, which were immunoprecipitable by anti-(T. brucei 20 S proteasome) antiserum. In view of the documented self-assembly of the archaeon proteasome α subunit into double heptamer rings and the spontaneous assembly of the two α subunits from the 20 S proteasome of Rhodococcus erythropolis, the self-assembly of the T. brucei α subunit might reflect a common feature of proteasome biogenesis shared by prokaryotes and primitive eukaryotes such as the trypanosomes but apparently lost among the higher forms of eukaryote such as the yeast and the mammals.

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.

The base of the proteasome regulatory particle exhibits chaperone-like activity.

Protein substrates of the proteasome must apparently be unfolded and translocated through a narrow channel to gain access to the proteolytic active sites of the enzyme. Protein folding in vivo is mediated by molecular chaperones. Here, to test for chaperone activity of the proteasome, we assay the reactivation of denatured citrate synthase. Both human and yeast proteasomes stimulate the recovery of the native structure of citrate synthase. We map this chaperone-like activity to the base of the regulatory particle of the proteasome, that is, to the ATPase-containing assembly located at the substrate-entry ports of the channel. Denatured but not native citrate synthase is bound by the base complex. Ubiquitination of citrate synthase is not required for its binding or refolding by the base complex of the proteasome. These data suggest a model in which ubiquitin-protein conjugates are initially tethered to the proteasome by specific recognition of their ubiquitin chains; this step is followed by a nonspecific interaction between the base and the target protein, which promotes substrate unfolding and translocation.

Mutational analysis of subunit iβ2 (MECL-1) demonstrates conservation of cleavage specificity between yeast and mammalian proteasomes

Proteasomes are the major protein-degrading complexes in the cytosol and regulate many cellular processes. To examine the functional importance of the MC14/MECL-1 proteasome active site subunits, cell lines expressing a catalytically inactive form of MECL-1 were established. Whereas mutant MECL-1 was readily incorporated into cytosolic proteasomes, replacing the constitutive MC14 subunit, removal of the prosequence was incomplete indicating that its processing required autocatalytic cleavage. Functional analyses showed that the absence of the MC14/MECL-1 active sites abrogated proteasomal trypsin-like activity, but did not affect other catalytic activities. Our data demonstrate a conservation of cleavage specificity between mammalian and yeast proteasomes.

Subcellular Localization, Stoichiometry, and Protein Levels of 26 S Proteasome Subunits in Yeast

The 26 S proteasome of eukaryotes is responsible for the degradation of proteins targeted for proteolysis by the ubiquitin system. Yeast has been an important model organism for understanding eukaryotic proteasome structure and function. Toward a quantitative characterization of the proteasome, we have determined the localization, cellular levels, and stoichiometry of proteasome subunits. The subcellular localization of two ATPase components of the regulatory complex of the proteasome, Sug2/Rpt4 and Sug1/Rpt6, and a subunit of the 20 S proteasome, Pre1, were determined by immunofluorescence. In contrast to findings in multicellular organisms, these proteins are localized almost exclusively to the nucleus throughout the cell cycle. We have also determined the cellular abundance and stoichiometry of these proteasome subunits. Sug1/Rpt6, Sug2/Rpt4, and Pre1 are present in roughly equal stoichiometry with an abundance of 15,000-30,000 molecules/cell, corresponding to a concentration of 13-26 microM in the nucleus. Also, in contrast to mammalian cells, we find no evidence of a p27-containing "modulator" of the proteasome in yeast. This information will be useful in comparing and contrasting the yeast and mammalian proteasomes and should contribute to a mechanistic understanding of how this complex functions.

Bifunctional inhibitors of the trypsin-like activity of eukaryotic proteasomes

The 20S proteasome is a multicatalytic protease complex that exhibits trypsin-like, chymotrypsin-like and post-glutamyl-peptide hydrolytic activities associated with the active sites of the beta2, beta5 and beta1 subunits, respectively. Modulation of these activities using inhibitors is essential for a better understanding of the proteasome's mechanism of action. Although there are highly selective inhibitors of the proteasome's chymotryptic activity, inhibitors of similar specificity have not yet been identified for the other activities. The X-ray structure of the yeast proteasome reveals that the sidechain of Cys118 of the beta3 subunit protrudes into the S3 subsite of the beta2 active site. The location of this residue was exploited for the rational design of bidentated inhibitors containing a maleinimide moiety at the P3 position for covalent linkage to the thiol group and a carboxy-terminal aldehyde group for hemiacetal formation with the Thr1 hydroxyl group of the active site. Structure-based modelling was used to determine the optimal spacing of the maleinimide group from the P2-P1 dipeptide aldehydes and the specificity of the S1 subsite was exploited to limit the inhibitory activity to the beta2 active site. X-ray crystallographic analysis of a yeast proteasome-inhibitor adduct confirmed the expected irreversible binding of the inhibitor to the P3 subsite. Maleoyl-beta-alanyl-valyl-arginal is a new type of inhibitor that is highly selective for the trypsin-like activity of eukaryotic proteasomes. Despite the reactivity of the maleinimide group towards thiols, and therefore the limited use of this inhibitor for in vitro studies, it might represent an interesting new biochemical tool.