Proteasome Subunit Genes Research Articles

Rapid Evolution Through Gene Duplication and Subfunctionalization of the Testes-Specific α4 Proteasome Subunits in DrosophilaSequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AY542377, AY542378, AY542379, AY542380, AY542381, AY542382, AY542383, AY542384, AY542385, AY542386, AY542387, AY542388, AY542389, AY542390, AY542391, AY542392, AY542393, AY542394, AY542395, AY542396, AY542397, AY542398, AY542399, AY542400, AY542401, AY542402, AY542403, AY542404, AY542405, AY542406,

Gene duplication is an important mechanism for acquiring new genes and creating genetic novelty in organisms. Evidence suggests that duplicated genes are retained at a much higher rate than originally thought and that functional divergence of gene copies is a major factor promoting their retention in the genome. We find that two Drosophila testes-specific alpha4 proteasome subunit genes (alpha4-t1 and alpha4-t2) have a higher polymorphism within species and are significantly more diverged between species than the somatic alpha4 gene. Our data suggest that following gene duplication, the alpha4-t1 gene experienced relaxed selective constraints, whereas the alpha4-t2 gene experienced positive selection acting on several codons. We report significant heterogeneity in evolutionary rates among all three paralogs at homologous codons, indicating that functional divergence has coincided with genic divergence. Reproductive subfunctionalization may allow for a more rapid evolution of reproductive traits and a greater specialization of testes function. Our data add to the increasing evidence that duplicated genes experience lower selective constraints and in some cases positive selection following duplication. Newly duplicated genes that are freer from selective constraints may provide a mechanism for developing new interactions and a pathway for the evolution of new genes.

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.

Ubiquitin depletion as a key mediator of toxicity by translational inhibitors.

Cycloheximide acts at the large subunit of the ribosome to inhibit translation. Here we report that ubiquitin levels are critical for the survival of Saccharomyces cerevisiae cells in the presence of cycloheximide: ubiquitin overexpression confers resistance to cycloheximide, while a reduced ubiquitin level confers sensitivity. Consistent with these findings, ubiquitin is unstable in yeast (t(1/2) = 2 h) and is rapidly depleted upon cycloheximide treatment. Cycloheximide does not noticeably enhance ubiquitin turnover, but serves principally to block ubiquitin synthesis. Cycloheximide also induces UBI4, the polyubiquitin gene. The cycloheximide-resistant phenotype of ubiquitin overexpressors is also characteristic of partial-loss-of-function proteasome mutants. Ubiquitin is stabilized in these mutants, which may account for their cycloheximide resistance. Previous studies have reported that ubiquitin is destabilized in the absence of Ubp6, a proteasome-associated deubiquitinating enzyme, and that ubp6 mutants are hypersensitive to cycloheximide. Consistent with the model that cycloheximide-treated cells are ubiquitin deficient, the cycloheximide sensitivity of ubp6 mutants can be rescued either by ubiquitin overexpression or by mutations in proteasome subunit genes. These results also show that ubiquitin wasting in ubp6 mutants is proteasome mediated. Ubiquitin overexpression rescued cells from additional translational inhibitors such as anisomycin and hygromycin B, suggesting that ubiquitin depletion may constitute a widespread mechanism for the toxicity of translational inhibitors.

Global analysis of gene expression patterns during disuse atrophy in rat skeletal muscle

Muscular inactivity leads to atrophy, weakness, and decreased fatigue resistance. In order to provide a window into the dynamic processes that underlie muscle atrophy, we performed global gene expression analysis of rat soleus muscles using Affymetrix GeneChips at 1, 4, 7 and 14 days of hindlimb unloading. Expression of 309 known genes was significantly changed by at least 2-fold (212 upregulated, 97 downregulated). K-means clustering was used to divide these genes into co-regulated clusters based on the similarity of temporal expression patterns. This allowed the development of a timeline of the atrophy process with respect to the behaviour of genes in a broad array of functional categories. Regulatory genes were often upregulated early, in either a transient or sustained manner, but they also populated clusters with later patterns of activation, suggesting different phases of molecular adaptations. Other early events were the activation of ubiquitination genes and downregulation of protein chaperones. In comparison, clusters representing slightly delayed activation patterns included genes involved in fast contraction, glycolysis, translational inhibition, oxidative stress, protein degradation, and amino acid catabolism. Downregulated genes exhibited fewer unique expression patterns and included structural and regulatory genes of the extracellular matrix and cytoskeleton, and genes that define a slow-oxidative phenotype. Other novel findings include the tight co-activation of proteasome subunit and ubiquitination genes, differential regulation of serine proteases and serine protease inhibitors, and the identification of transcriptional, signalling, growth and cell cycle genes that probably play a role in the atrophy process. The present work has uncovered temporal patterns of gene expression that highlight the molecular processes that underlie muscle atrophy and provide new avenues for study.

Global analysis of gene expression patterns during disuse atrophy in rat skeletal muscle.

Muscular inactivity leads to atrophy, weakness, and decreased fatigue resistance. In order to provide a window into the dynamic processes that underlie muscle atrophy, we performed global gene expression analysis of rat soleus muscles using Affymetrix GeneChips at 1, 4, 7 and 14 days of hindlimb unloading. Expression of 309 known genes was significantly changed by at least 2-fold (212 upregulated, 97 downregulated). K-means clustering was used to divide these genes into co-regulated clusters based on the similarity of temporal expression patterns. This allowed the development of a timeline of the atrophy process with respect to the behaviour of genes in a broad array of functional categories. Regulatory genes were often upregulated early, in either a transient or sustained manner, but they also populated clusters with later patterns of activation, suggesting different phases of molecular adaptations. Other early events were the activation of ubiquitination genes and downregulation of protein chaperones. In comparison, clusters representing slightly delayed activation patterns included genes involved in fast contraction, glycolysis, translational inhibition, oxidative stress, protein degradation, and amino acid catabolism. Downregulated genes exhibited fewer unique expression patterns and included structural and regulatory genes of the extracellular matrix and cytoskeleton, and genes that define a slow-oxidative phenotype. Other novel findings include the tight co-activation of proteasome subunit and ubiquitination genes, differential regulation of serine proteases and serine protease inhibitors, and the identification of transcriptional, signalling, growth and cell cycle genes that probably play a role in the atrophy process. The present work has uncovered temporal patterns of gene expression that highlight the molecular processes that underlie muscle atrophy and provide new avenues for study.

Targeted expression of dominant negative proteasome mutants in Drosophila melanogaster.

genesisVolume 34, Issue 1-2 p. 80-82 UAS LinesFree Access Targeted expression of dominant negative proteasome mutants in drosophila melanogaster John M. Belote, Corresponding Author John M. Belote [email protected] Department of Biology, Syracuse University, Syracuse, New YorkDepartment of Biology, Syracuse University, 130 College Place, Syracuse, NY 13244Search for more papers by this authorEric Fortier, Eric Fortier Department of Biology, Syracuse University, Syracuse, New YorkSearch for more papers by this author John M. Belote, Corresponding Author John M. Belote [email protected] Department of Biology, Syracuse University, Syracuse, New YorkDepartment of Biology, Syracuse University, 130 College Place, Syracuse, NY 13244Search for more papers by this authorEric Fortier, Eric Fortier Department of Biology, Syracuse University, Syracuse, New YorkSearch for more papers by this author First published: 12 September 2002 https://doi.org/10.1002/gene.10131Citations: 40AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. LITERATURE CITED Baumeister W, Walz J, Zühl F, Seemüller E. 1998. The proteasome: paradigm of a self-compartmentalizing protease. Cell 92: 367– 380. Brand AH, Perrimon N. 1993. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118: 401– 415. Chain DG, Schwartz JH, Hedge AN. 1999. Ubiquitin-mediated proteolysis in learning and memory. Mol Neurobiol 20: 125– 142. Covi JA, Belote JM, Mykles DL. 1999. Subunit compositions and catalytic properties of proteasomes from developmental temperature-sensitive mutants of Drosophila melanogaster. Arch Biochem Biophys 368: 85– 97. Freeman M. 1997. Personal communication to FlyBase available from http://flybase.bio.indiana.edu:82/.bin/fbidq.html?FBrf0091569. Hampton RY, Bhakta H. 1997. Ubiquitin-mediated regulation of 3-hydroxy-3-methylglutaryl-CoA reductase. Proc Natl Acad Sci USA 94: 12944– 12948 Hershko A, Ciechanover A. 1998. The ubiquitin system. Annu Rev Biochem 67: 425– 479. Holden JJ, Suzuki DT. 1973. Temperature-sensitive mutations in Drosophila melanogaster XII. The genetic and developmental effects of dominant lethals on chromosome 3. Genetics 73: 445– 458. Hoyt MA. 1997. Eliminating all obstacles: regulated proteolysis in the eukaryotic cell cycle. Cell 91: 149– 151. Huang Y, Baker RT, Fischer-Vize JA. 1995. Control of cell fate by a deubiquitinating enzyme encoded by the fat facets gene. Science 270: 1828– 1831. Jesenberger V, Jentsch S. 2002. Deadly encounter: ubiquitin meets apoptosis. Nat Rev Mol Cell Biol 3: 112– 121. Myung J, Kim KB, Crews CM. 2001. The ubiquitin-proteasome pathway and proteasome inhibitors. Med Res Rev 21: 245– 273. Naidoo N, Song W, Hunter-Ensor M, Sehgal A. 1999. A role for the proteasome in the light response of the timeless clock protein. Science 285: 1737– 1741. Saville KJ, Belote JM. 1993. Identification of an essential gene, l(3)73Ai, with a dominant temperature-sensitive lethal allele, encoding a Drosophila proteasome subunit. Proc Natl Acad Sci USA 90: 8842– 8846. Schweisguth F. 1999. Dominant-negative mutations in the β2 and β6 proteasome subunit genes affect alternative cell fate decisions in the Drosophila sense organ lineage. Proc Natl Acad Sci USA 96: 11382– 11386. Smyth KA, Belote JM. 1999. The dominant temperature-sensitive lethal DTS7 of Drosophila melanogaster encodes an altered 20S proteasome beta-type subunit. Genetics 151: 211– 220. Tsang, T, Copeland V, Bowden GT. 1991. A set of cloning vectors for rapid and versatile adaptation of restriction fragments. BioTechniques 10: 330. Zhou MY, Clark SE, Gomez-Sanchez CE. 1995. Universal cloning method by TA strategy. BioTechniques 19: 34– 35. Citing Literature Volume34, Issue1-2Special Issue: GAL4/UAS in Drosophila September ‐ October 2002Pages 80-82 ReferencesRelatedInformation

Reverse genetic analysis of the Caenorhabditis elegans 26S proteasome subunits by RNA interference.

Reverse genetic analysis was performed on the Caenorhabditis elegans 26S proteasome subunit genes by double-stranded RNA-mediated interference (RNAi). Embryonic and post-embryonic lethality was caused by interference of all of the eight tested 20S core subunits and all of the 19S regulatory particle subunits except for Ce-Rpn9, Ce-Rpn10, and Ce-Rpn12, where RNAi caused no abnormality. However, synthetic suppression of Ce-Rpn10 and Ce-Rpn12 was lethal, whereas neither the combination of Ce-Rpn9 with Ce-Rpn10 nor with Ce-Rpn12 resulted in abnormalities in RNAi. These results indicate that the 26S proteasome is indispensable for embryogenesis and post-embryonic development, although Ce-Rpn9, Ce-Rpn10, and Ce-Rpn12 are not essential, at least under the conditions used. Ce-Rpn10 and Ce-Rpn12 are considered to compensate for the suppression of each other.

A role for nuclear localised proteasomes in mediating auxin action.

A number of important cellular events in animals and yeast are regulated by protein degradation, and it is becoming apparent that such regulated proteolysis is involved in many facets of plant physiology and development. We have investigated the role of protein degradation by proteasomes in plants using NtPSA1, a tobacco gene that is predominantly expressed in young developing tobacco tissues and has extensive homology to yeast and human alpha-type proteasome subunit genes. The NtPSA1 cDNA was used to complement a lethal mutation of the yeast PRC1 alpha subunit gene indicating that NtPSA1 encodes a functional proteasome subunit, and transient expression of an NtPSA1::GUS protein fusion in onion cells confirmed that the nuclear localisation signal that is present in the NtPSA1 peptide sequence is active in plant cells. Plants transformed with an antisense NtPSA1 gene had reduced levels of NtPSA1 mRNA and exhibited reduced apical dominance. In addition, these low NtPSA1 plants displayed several morphological defects associated with auxin resistance such as reduced stamen length, and showed increased tolerance to high concentrations of auxin. These results support a role for nuclear localised proteasomes in floral development and auxin responses.

OsPAA2, a distinct α1 subunit gene for the 20S proteasome in rice (Oryza sativa L.)

The 20S proteasome is the proteolytic complex that is involved in removing abnormal proteins and other diverse biological functions. The 20S proteasome is constituted of 28 subunits arranged in four rings of seven subunits, and exists as a hollow cylinder. The two outer rings and the two inner rings are composed of seven different α and β type subunits, respectively, giving an α7β7β7α7 structure. We previously reported the primary structures of the 14 proteasomal subunit subfamilies in rice (Oryza sativa), representing the first set for all the subfamilies from monocot. In this study, a distinct cDNA sequence encoding the α1 subunit, OsPAA2, was identified. The amino acid sequence similarity between the two rice α1 subunits was as low as 59.6%, contrasting with those between paralogs of Arabidopsis proteasome subunit genes. The expression pattern of the OsPAA2 gene was different from that of another α1 gene, OsPAA1. These data suggest that OsPAA2 might play a distinct role from that of OsPAA1 in the 20S proteasome complex.

Essential Role of Human Leukocyte Antigen-encoded Proteasome Subunits in NF-κB Activation and Prevention of Tumor Necrosis Factor-α-induced Apoptosis

The multisubunit proteasome complex is the principal mediator of nonlysosomal protein degradation. The proteasome subunit varies minimally between cells with the exception of LMP2, LMP7, and LMP10 subunits in rodent and human cells. LMP2 and LMP7 subunits are encoded by the human lymphocyte antigen region, and they optimize proteolytic mediated antigen presentation. The proteasome is also important for the function of transcription factor nuclear factor-kappaB (NF-kappaB). It is required for NF-kappaB subunits p50 and p52 generation and catalyzes degradation of phosphorylated IkappaBalpha. These proteasome-mediated reactions have now been shown to be defective in T2 cells, a human lymphocyte cell line that lacks both LMP2 and LMP7. Although T2 cells contain normal expression of p100 and p105, the abundance of p50 and p52 was greatly reduced. Tumor necrosis factor-alpha (TNF-alpha) induced normal phosphorylation of IkappaBalpha but failed to induce degradation of phosphorylated IkappaBalpha. Both DNA binding assays and luciferase assays revealed that TNF-alpha-induced NF-kappaB activation is defective in T2 cells. Unlike parental cells, T2 cells were susceptible to TNF-alpha-induced apoptosis. These data indicate human leukocyte antigen-linked proteasome subunits are essential for NF-kappaB activation and protection of cells from TNF-alpha-induced apoptosis.

The Balance between Glucocorticoids and Insulin Regulates Muscle Proteolysis via the Ubiquitin-Proteasome Pathway

In uremia, accelerated muscle protein degradation results from activation of the ATP-ubiquitin proteasome proteolytic pathway. Like uremia, other conditions (e.g., acidosis and diabetes) activate this pathway in rat muscles and are associated with excess glucocorticoids (GC) and impaired insulin action. To define the stimuli responsible for muscle wasting in IDDM, the roles of glucocorticoids, insulinopenia and acidosis in streptozotocin (STZ) – induced diabetes were studied. Proteolysis in isolated epitrochlearis muscles from acutely (3d) diabetic rats was 52% higher than pair-fed, sham-injected rats; this increase was eliminated by an inhibitor of the proteasome or by blocking ATP synthesis. In muscles of STZ-diabetic rats, the levels of ubiquitin-conjugated proteins and mRNAs encoding ubiquitin, the ubiquitin-carrier protein, E2_14k and the C3, C5 and C9 proteasome subunits were increased. Transcription of ubiquitin and C3 proteasome subunit genes in muscle was also increased by IDDM. Oral NaHCO₃ eliminated acidemia but did not prevent accelerated muscle proteolysis. Corticosterone excretion was higher in IDDM rats and adrenalectomy (ADX) prevented these catabolic responses; physiologic doses of glucorcoticoids restored the excessive protein catabolism in ADX-STZ rats. Giving IDDM rats replacement insulin also normalized protein degradation in muscles. In conclusion, reduced insulin together with physiologic levels of glucocorticoids activate the ubiquitin-proteasome pathway by a mechanism that includes enhancing ubiquitin conjugation and proteolysis by the proteasome. The balance between these stimuli could regulate muscle proteolysis in uremia.

Invasion bySalmonella typhimuriumInduces Increased Expression of the LMP, MECL, and PA28 Proteasome Genes and Changes in the Peptide Repertoire of HLA-B27

We have analyzed proteasomal adaptation and associated changes in the B27-bound peptide repertoire in response to cellular invasion with Salmonella. The peptide repertoire of HLA-B27 complexes was analyzed by two different methods: (i) high-pressure liquid chromatography (HPLC) profiles of newly synthesized peptides eluted from B27 following metabolic labeling with arginine and (ii) reactivities with two B27 monoclonal antibodies, Ye-2 and B27.M2, sensitive to peptide-induced conformational changes. LMP, MECL, and PA28 expression was analyzed by reverse transcription-PCR (RT-PCR) of mRNA and by Western blot analysis for LMP2. Invasion of HLA-B27-transfected HeLa cells by Salmonella typhimurium induced significant changes in the reactivities of HLA-B27 with these two antibodies, which was accompanied by significant quantitative and qualitative changes in the HPLC profile of peptides eluted from HLA-B27. We also observed increases in the RT-PCR values for the LMP2, LMP7, and MECL proteasome subunit genes, as well as the proteasomal activator PA28alpha and -beta genes, and increased expression of the LMP2 protein by Western blotting. Upregulation of LMP2, but not LMP7, gene expression showed a close correlation with the changes in antibody reactivities observed upon bacterial invasion. We observed similar changes in reactivity with the Ye-2 or the B27.M2 antibody of lymphoblastoid cells upon gamma interferon treatment, which significantly correlated with the increased RT-PCR values for the LMP2 gene. This was accompanied by consistent HPLC profile changes for eluted peptides. Thus, Salmonella invasion leads to serologically recognizable changes in the B27-bound peptide repertoire, which may include peptides of host origin potentially through modulation of proteasome LMP2 subunit expression and, as a consequence, proteasomal activities.

Role of TNF receptor 1 in protein turnover during cancer cachexia using gene knockout mice

The implantation of the Lewis lung carcinoma (a fast-growing mouse tumour that induces cachexia) to both wild-type and gene-deficient mice for the TNF- α receptor type I protein (Tnfr1°/Tnfr1°), resulted in a considerable loss of carcass weight in both groups. However, while in the wild-type mice there was a loss of both fat and muscle, in the gene-knockout mice muscle wastage was not affected to the same extent. In both groups, tumour burden resulted in significant increases in circulating TNF- α, a cytokine which, as we have previously demonstrated, can induce protein breakdown in skeletal muscle. Muscle wastage in wild-type mice was accompanied by an increase in the fractional rate of protein degradation, while no changes were observed in protein synthesis. The result is a decreased rate of protein accumulation that accounts for the muscle weight loss observed as a result of tumour burden. In contrast, gene knockout mice did not have significantly lower rates of protein accumulation as a result of tumour implantation. The increase in protein degradation in the tumour-bearing wild mice was accompanied by an enhanced expression of both ubiquitin and proteasome subunit genes, all of them related to the activation of the ATP-dependent proteolytic system in skeletal muscle. Tumour-bearing gene-deficient mice did not show any increase in gene expression. It is concluded that TNF- α (alone or in combination with other cytokines) is responsible for the activation of protein breakdown in skeletal muscle of tumour-bearing mice.

Evolutionary conservation of a testes-specific proteasome subunit gene in Drosophila.

Proteasomes are large multisubunit particles that act as the proteolytic machinery for the ubiquitin-dependent proteolytic pathway. The core of this complex, the 20S proteasome, is made up of seven alpha-type and seven beta-type subunits, arranged in an (alpha1-alpha7)(beta1-beta7)(beta1-beta7)(alpha1-al pha7) configuration. Previous work had shown that there exist alternative isoforms of the Drosophila melanogaster alpha4-type subunit, encoded by two distinct genes, alpha4t1_dm and alpha4t2_dm, and that these are expressed exclusively in the germline of the testes. We sought to investigate the evolutionary conservation of this phenomenon by screening for orthologs of the alpha4-type gene family in the distantly related Drosophila species, D. virilis. We isolated the D. virilis orthologs of the somatically expressed gene, alpha4_dm, and the testes-specific gene, alpha4t2_dm. We failed to find an ortholog of the other testes-specific gene, alpha4t1_dm. The alpha4_dv gene maps to the X chromosome at 12A-C, its product shares 90% amino acid identity with alpha4_dm, and it is expressed at high levels in both males and females. The other gene, alpha4t_dv, encodes a protein most similar to the testes-specific alpha4t2_dm proteasome subunit (59% a.a. identity), and it maps to position 27 on chomosome 2. The expression of the alpha4t_dv gene is testes-specific, like that of alpha4t2_dm. The existence of testes-specific alpha4-type subunits in two widely diverged subgenera of Drosophila suggests that these subunit isoforms have important functional roles in spermatogenesis.

Molecular organization of the 20S proteasome gene family from Arabidopsis thaliana.

The 20S proteasome is the proteolytic complex in eukaryotes responsible for degrading short-lived and abnormal intracellular proteins, especially those targeted by ubiquitin conjugation. The 700-kD complex exists as a hollow cylinder comprising four stacked rings with the catalytic sites located in the lumen. The two outer rings and the two inner rings are composed of seven different alpha and beta polypeptides, respectively, giving an alpha7/beta7/beta7/alpha7 symmetric organization. Here we describe the molecular organization of the 20S proteasome from the plant Arabidopsis thaliana. From an analysis of a collection of cDNA and genomic clones, we identified a superfamily of 23 genes encoding all 14 of the Arabidopsis proteasome subunits, designated PAA-PAG and PBA-PBG for Proteasome Alpha and Beta subunits A-G, respectively. Four of the subunits likely are encoded by single genes, and the remaining subunits are encoded by families of at least 2 genes. Expression of the alpha and beta subunit genes appears to be coordinately regulated. Three of the nine Arabidopsis proteasome subunit genes tested, PAC1 (alpha3), PAE1 (alpha5) and PBC2 (beta3), could functionally replace their yeast orthologs, providing the first evidence for cross-species complementation of 20S subunit genes. Taken together, these results demonstrate that the 20S proteasome is structurally and functionally conserved among eukaryotes and suggest that the subunit arrangement of the Arabidopsis 20S proteasome is similar if not identical to that recently determined for the yeast complex.

Evolution of proteasome subunits delta and LMP2: complementary DNA cloning and linkage analysis with MHC in lower vertebrates.

The class II region of the mammalian MHC harbors two proteasome subunit genes, LMP2 and LMP7. These genes are induced by IFN-gamma, and their products are incorporated into proteasomes substituting for their closest relatives, the delta and X subunits, respectively. This substitution is believed to change the proteolytic specificity of proteasomes, making it more suitable for generation of peptides to be presented by class I molecules. To elucidate the phylogenetic origin of LMP2 and the linkage of its gene with the MHC, reverse transcriptase-PCR amplification of Xenopus laevis and lamprey liver mRNA was performed with primers designed to amplify both the mammalian LMP2 and delta sequences. Both LMP2 and delta were amplified from X. laevis, whereas only delta was amplified from lamprey, suggesting that delta/LMP2 gene duplication occurred after divergence of cyclostomes but before divergence of amphibians. The linkage between the LMP2 gene and the MHC was observed in a diploid Xenopus species, Xenopus tropicalis, but not in a tetraploid species, X. laevis, indicating that this linkage was established before the divergence of amphibian from higher vertebrates, but that this linkage was lost in X. laevis, probably by a gene reorganization accompanying the tetraploidization. The X. laevis LMP2 and LMP7 mRNA showed a similar tissue distribution, indicating that the genetic linkage is not required for apparently coordinated tissue-specific expression of these genes. Sequence and linkage analyses suggest that LMP2 may not play as vital a role as LMP7 in Ag presentation.

Identification of MECL-1 (LMP-10) as the third IFN-gamma-inducible proteasome subunit.

We show that six proteasome-associated proteins are induced by IFN-gamma, corresponding to three proteasome beta-type subunits and their precursors: the MHC-linked subunits (LMP-2 and LMP-7) and LMP-10. Concurrently, incorporation of LMP-9, LMP-17, and LMP-19 into proteasomes is reduced. LMP-10 appears to be the product of a previously cloned proteasome subunit gene, MECL-1. MECL-1 transcription is increased in the presence of IFN-gamma, whereas the transcription of two other proteasome genes, Lmp-15 and Lmp-3, is not affected. The three IFN-gamma-inducible subunits and their constitutively expressed counterparts contain most or all of the catalytic sites of the proteasome. Independent assortment of LMP-2, LMP-7, and LMP-10 into different proteasome complexes may thus generate up to 36 unique proteasome subsets. This may increase the repertoire of potentially antigenic peptides for presentation by MHC class I.

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The proteasome and protein degradation in yeast.

In 1984 a high molecular mass multisubunit protease complex was isolated from Saccharomyces cerevisiae [Achstetter et al. 1984] which proved to be the yeast homologue of the 20S proteasome complexes found in all eukaryotic cells [Kleinschmidt et al. 1988]. The yeast 20S proteasome is able to cleave chromo- and fluorogenic peptides at the carboxyterminus of hydrophobic, basic or acidic amino acids (chymotrypsin-like-, trypsin-like- and peptidyl-glutamyl-peptide hydrolyzing activity, respectively) [Heinemeyer et al. 1991]. The yeast 20S proteasome is composed of different subunits, showing a set of protein bands in the SDS-PAGE with molecular masses ranging from 20 to 35 kDa. They can be separated into 14 protein spots after two-dimensional gel electrophoresis [Heinemeyer et al. 1991]. Genes named Y7, Y13, PRS1 and PRS2 (independendly cloned as Y8 and SCL1) were cloned and sequenced on the basis of protein sequences of 20S proteasome subunits, genes named PRS3, PUP1, PUP2 and PUP3 were sequenced by chance [for summary see Hilt et al. 1993b]. We cloned the s-type genes PREI, PRE2, PRE3 and PRE4 by complementation of mutants defective in the chymotrypsin-like- (prel and pre2 mutants) or the PGPH-activity (pre3 and pre4 mutants) of the proteasome [Heinemeyer et al. 1991, Heinemeyer et al. 1993, Hilt et al. 1993a, Enenkel et al. 1994]. Additionally we cloned two α-type genes PRE5 and PRE6 using peptide sequences derived from purified proteasome subunits, extending the number of yeast 20S proteasome subunit genes to 14 [Heinemeyer et al. 1994].

Isolation and Characterization of the HC8 Subunit Gene of the Human Proteasome

For study of the molecular basis of regulation of proteasome gene expression, we isolated the gene encoding the α-type HC8 subunit of the human proteasome. About 2.3 kb of the 5′ flanking region of this gene was tested for promoter function by chloramphenicol acetyltransferase assay. This analysis revealed that CAAT and TATA boxes, but not a GC box, are essential for its promoter activity. These results differed from previous findings that the genes for the α-type HC3 and β-type HC5 subunits of the human proteasome have a TATA-less promoter and that two or three GC boxes function as the promoter sequences (Tamura, T. et al. (1994) J. Mel. Biol. 244, 1117-1124). We mapped the HC8 gene at q23 on human chromosome 14, which differs from the chromosomal locations of nine other proteasomal subunit genes mapped so far.