Molecular cloning of the yeast proteasome PRS2 gene identical to the suppressor gene scl1+

The YC7-alpha gene encoding a subunit of yeast proteasomes named PRS2 has been isolated and sequenced. Southern blot analysis after electrophoretic separation of yeast chromosomal DNAs showed that the PRS2 gene is located in chromosome VII, unlike the PRS1 gene for subunit YC1, is located on chromosome XV. Surprisingly, the overall structure of the PRS2 gene was found to be identical to that of the suppressor scl1+ gene restoring the SCL1-1 mutation that suppresses crl3 cycloheximide-resistant, temperature-sensitive lethality. The identity of these two genes indicates that the suppressive role of the scl1+ gene product on crl3 mutation could be attributed to proteasomal function.     

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.     

Separation of yeast proteasome subunits. Immunoreactivity with antibodies against ATP-dependent protease Ti from Escherichia coli

On two-dimensional gel electrophoresis, proteasomes (multicatalytic proteinase complexes) from the yeast Saccharomyces cerevisiae were separated into a characteristic set of approximately 20 components with molecular weights of 21,000 to 31,000 and isoelectric points of 3.5 to 7.5. The main components were isolated by reverse-phase high performance liquid chromatography on a TSK gel phenyl-5PW RP column and named YC1 to YC11, in order of their elution. Immuno-blot analysis showed that two components (YC1-alpha and YC1-beta) with molecular weights of 30,800 and 28,300 strongly cross-reacted with antibody against the P-component of ATP-dependent protease Ti from Escherichia coli, but no components were found to react with antibodies against the A-component of protease Ti or another ATP-dependent protease La (the Ion gene product) of Escherichia coli. These results indicate a structural relationship between eukaryotic proteasomes and bacterial ATP-dependent protease Ti.     

AAR1/TUP1 protein, with a structure similar to that of the beta subunit of G proteins, is required for a1-alpha 2 and alpha 2 repression in cell type control of Saccharomyces cerevisiae.

We have cloned a DNA fragment complementing the aar1 mutation defective in the a1-alpha 2 repression of the alpha 1 cistron and haploid-specific genes in Saccharomyces cerevisiae. Nucleotide sequence and mapping data indicated that the AAR1 gene is identical with TUP1, which is allelic to the SFL2, FLK1, CYC9, UMR7, AMM1, and AER2 genes, whose mutations are known to confer a variety of phenotypes, such as thymidine uptake, flocculation, insensitivity to glucose repression, a defect in UV-induced mutagenesis, and a defect in ARS plasmid maintenance. The TUP1/AER2 protein is known to have significant similarity with the beta subunits of G proteins in the C-terminal half, in two glutamine-rich domains in the N-terminal half, and in a central region rich in serine and threonine residues. Disruption of the chromosomal AAR1 gene in alpha and a/alpha cells conferred the nonmating phenotype, and the a/alpha diploids could not sporulate. The AAR1/TUP1 gene is transcribed into a 2.5-kb mRNA independently of the mating-type information of the cell. These observations and mRNA analysis of cell-type-specific genes indicated that the AAR1/TUP1 protein is also indispensable for a1-alpha 2 repression of RME1 and for alpha 2 repression of a-specific genes.     

Genetic and biochemical characterization of ISP6, a small mitochondrial outer membrane protein associated with the protein translocation complex.

To search genetically for additional components of the protein translocation apparatus of mitochondria, we have used low fidelity PCR mutagenesis to generate temperature-sensitive mutants in the outer membrane translocation pore component ISP42. A high copy number suppressor of temperature-sensitive isp42 has been isolated and sequenced. This novel gene, denoted ISP6, encodes a 61 amino acid integral membrane protein of the mitochondrial outer membrane, which is oriented with its amino-terminus facing the cytosol. Disruption of the ISP6 gene is without apparent effect in wild type yeast cells, but is lethal in temperature-sensitive isp42 mutants. Immunoprecipitation of the gene product, ISP42p, from mitochondria solubilized under mild conditions reveals a multi-protein complex containing ISP6p and ISP42p.     

The immunosuppressant SR 31747 blocks cell proliferation by inhibiting a steroid isomerase in Saccharomyces cerevisiae.

SR 31747 is a novel immunosuppressant agent that arrests cell proliferation in the yeast Saccharomyces cerevisiae, SR 31747-treated cells accumulate the same aberrant sterols as those found in a mutant impaired in delta 8- delta 7-sterol isomerase. Sterol isomerase activity is also inhibited by SR 31747 in in vitro assays. Overexpression of the sterol isomerase-encoding gene, ERG2, confers enhanced SR resistance. Cells growing anaerobically on ergosterol-containing medium are not sensitive to SR. Disruption of the sterol isomerase-encoding gene is lethal in cells growing in the absence of exogenous ergosterol, except in SR-resistant mutants lacking either the SUR4 or the FEN1 gene product. The results suggest that sterol isomerase is the target of SR 31747 and that both the SUR4 and FEN1 gene products are required to mediate the proliferation arrest induced by ergosterol depletion.     

Proteasomes: protein and gene structures.

Proteasomes are ring- or cylinder-shaped particles that have a sedimentation coefficient of 20S and are composed of a characteristic set of small polypeptides. These particles have a latent multicatalytic proteinase activity. Recently, proteasomes were found to combine reversibly with multiple protein components to form 26S proteolytic complexes that catalyze ATP-dependent, selective breakdown of proteins ligated with ubiquitin. This suggests that the 26S complexes are a new type of ATP-requiring protease in eukaryotic cells. We have studied the structures of various eukaryotic proteasomes at the molecular level by physicochemical and recombinant DNA techniques and have proposed that the gross structures of proteasomes, such as their size and shape, have been highly conserved during evolution. Proteasome subunits appear to be encoded by a family of homologous genes named the "proteasome gene family," which may have evolved from a common ancestral gene. Evidence obtained by genetic analyses in yeast and studies on the levels of proteasome expression in various eukaryotic cells indicates that proteasomes have essential roles in the cell. In this review, we summarize available information on the protein and gene structures of proteasomes and discuss the biological functions of proteasomes.     

Genes for directing vacuolar morphogenesis in Saccharomyces cerevisiae. II. VAM7, a gene for regulating morphogenic assembly of the vacuoles.

VAM7 gene function has shown to be required for proper morphogenesis of the vacuole in yeast. The DNA fragments that complemented the defective vacuolar morphology of the vam7-1 mutation were isolated from a yeast genomic library. An overlapping 2.5-kilobase BglII-HindIII region was found to be sufficient for complementation of the vam7-1 phenotype. This fragment was integrated at the chromosomal VAM7 locus, indicating that it contained an authentic VAM7 gene. On nucleotide sequencing of the VAM7 gene, an open reading frame of 948 base pairs, coding for a hydrophilic polypeptide of 316 amino acid residues, was identified. The deduced amino acid sequence of the carboxyl-terminal region of the VAM7 gene product has heptad repeats and shows potential ability to form a coiled-coil structure. Disruption of VAM7 was not lethal; cells with a disrupted VAM7 gene did not, however, have a prominent large vacuoles but rather numerous small compartments that accumulated the histochemical marker molecule of the vacuolar compartment. They contained mature forms of the vacuolar marker proteins carboxypeptidase Y and vacuolar glycoprotein vgp72. A mutant with both vam7 and vam5 null mutations was constructed and shown to have neither vacuolar structures stained with ade-related fluorochrome nor mature forms of vacuolar marker proteins. These findings suggested that the VAM7 gene product functions in the process of morphogenic assembly of the vacuolar compartment but is not involved in the protein sorting and delivery to the vacuole.     

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.     

PRS3 encoding an essential subunit of yeast proteasomes homologous to mammalian proteasome subunit C5.

We found by computer analysis that a putative yeast proteasome subunit gene named PRS3 that encodes a protein very similar to subunit C5 of rat and human proteasomes is located immediately 3' to the ERD2 gene of Saccharomyces cerevisiae. The similarity of the primary structures of the two suggests that this subunit may have a common function in proteasomes of all eukaryotes. The protein, deduced from the open reading frame of PRS3, consists of 242 amino acid residues with a calculated molecular weight of 27,077. Chromosomal disruption of the PRS3 gene created a recessive lethal mutation. Physical mapping by hybridization to intact S. cerevisiae chromosomal DNA showed that the PRS3 gene is located on chromosome II, unlike two other subunit genes, PRS1 and PRS2, which are located on chromosomes XV and VII, respectively. These findings indicate that the PRS3 protein is a subunit of yeast proteasomes that is essential for cell viability.     

cDNA cloning and functional analysis of p28 (Nas6p) and p40.5 (Nas7p), two novel regulatory subunits of the 26S proteasome

We employed cDNA cloning to deduce the complete primary structures of p28 and p40.5, two novel subunits of PA700 (also called 19S complex), a 700 kDa multisubunit regulatory complex of the human 26S proteasome. These polypeptides consisted of 226 and 376 amino acids with calculated molecular masses of 24 428 Da and 42 945 Da, and isoelectric points of 5.68 and 5.46, respectively. Intriguingly, p28 contained five conserved motifs known as `ankyrin repeats', implying that this subunit may contribute to interaction of the 26S proteasome with other protein(s). Computer-assisted homology analysis revealed high sequence similarities of p28 and p40.5 with yeast proteins, termed Nas6p and Nas7p (non-ATPase subunits 6 and 7), respectively, whose functions are as yet unknown. Disruption of these yeast genes, NAS6 and NAS7, had no effect on cell viability, indicating that neither of the two subunits is essential for proliferation of yeast cells. However, the NAS7, but not NAS6, disruptant cells caused high sensitivity to heat stress, being unable to proliferate at 37°C.     

cDNA cloning and sequencing of component C8 of proteasomes from rat hepatoma cells

The primary structure of component C8 of rat proteasomes (multicatalytic proteinase complexes) has been determined by sequencing on isolated cDNA clone. C8 consists of 255 amino acid residues with a calculated molecular weight of 28,417. These values are consistent with those obtained by protein chemical analyses. Computer-assisted homology comparison showed that C8 is a new protein, differing from all proteins reported so far. The overall amino acid sequence of C8 resembles those of most other components of proteasomes reported, such as components C2, C3 and C9 of rat proteasomes and certain components of other eukaryotic proteasomes, such as those of Drosophila and yeast, but shows little similarity with component C5 of rat proteasomes. C8 showed particularly close structural similarity to component YC1 of yeast proteasomes, suggesting that C8 has been highly conserved during evolution and functions ubiquitously in all eukaryotes.     

Cloning and characterization of LOS1, a Saccharomyces cerevisiae gene that affects tRNA splicing.

Saccharomyces cerevisiae strains carrying los1-1 mutations are defective in tRNA processing; at 37 degrees C, such strains accumulate tRNA precursors which have mature 5' and 3' ends but contain intervening sequences. Strains bearing los1-1 and an intron-containing ochre-suppressing tRNA gene, SUP4(0), also fail to suppress the ochre mutations ade2-1(0) and can1-100(0) at 34 degrees C. To understand the role of the LOS1 product in tRNA splicing, we initiated a molecular study of the LOS1 gene. Two plasmids, YEpLOS1 and YCpLOS1, that complement the los1-1 phenotype were isolated from the YEp24 and YCp50 libraries, respectively. YEpLOS1 and YCpLOS1 had overlapping restriction maps, indicating that the DNA in the overlapping segment could complement los1-1 when present in multiple or single copy. Integration of plasmid DNA at the LOS1 locus confirmed that these clones contained authentic LOS1 sequences. Southern analyses showed that LOS1 is a single copy gene. The locations of the LOS1 gene within YEpLOS1 and YCpLOS1 were determined by deletion and gamma-delta mapping. Two genomic disruptions of the LOS1 gene were constructed, i.e., an insertion of a 1.2-kilobase fragment carrying the yeast URA3 gene, los1::URA3, and a 2.4-kilobase deletion from the LOS1 gene, los1-delta V. Disruption or deletion of most of the LOS1 gene was not lethal; cells carrying the disrupted los1 alleles were viable and had phenotypes similar to those of cells carrying the los1-1 allele. Thus, it appears that the los1 gene product expedites tRNA splicing at elevated temperatures but is not essential for this process.     

Negative regulation of mitosis by two functionally overlapping PTPases in fission yeast.

We have identified a third protein tyrosine phosphatase (PTPase) gene in fission yeast, pyp2, encoding an 85 kDa protein. Disruption of pyp2 has no impact on cell viability, but pyp2 is essential in strains lacking the 60 kDa pyp1 PTPase. The two pyp PTPases are approximately 42% identical in their C-terminal catalytic domains and share weak homology in their N-terminal regions. Both genes play a role in inhibiting the onset of mitosis. Disruption of either gene rescues the G2 arrest caused by mutation of the cdc25 mitotic inducer, though the effect of pyp1-disruption is more pronounced. Disruption of pyp1 advances mitosis, suppresses overexpression of the tyrosine kinase encoded by the wee1 mitotic inhibitor, and causes lethal mitotic catastrophe in cdc25 overproducer cells. Cells bearing inactive wee1 are unresponsive to disruption of pyp1. Overexpression of pyp1 or pyp2 delays the onset of mitosis by a wee1-dependent mechanism. These data reveal an unexpected second role for protein tyrosine phosphorylation in the mitotic control that acts by promoting the inhibitory wee1 pathway.     

Two Saccharomyces cerevisiae genes which control sensitivity to G1 arrest induced by Kluyveromyces lactis toxin.

The Kluyveromyces lactis toxin causes an arrest of sensitive yeast cells in the G1 phase of the cell division cycle. Two complementary genetic approaches have been undertaken in the yeast Saccharomyces cerevisiae to understand the mode of action of this toxin. First, two sequences conferring toxin resistance specifically in high copy number have been isolated and shown to encode a tRNA(Glu3) and a novel polypeptide. Disruption of the latter sequence in the yeast genome conferred toxin resistance and revealed that it was nonessential, while the effect of the tRNA(Glu)3 was highly specific and mediated resistance by affecting the toxin's target. An alpha-specific, copy number-independent suppressor of toxin sensitivity was also isolated and identified as MATa, consistent with the observation that diploid cells are partially resistant to the toxin. Second, in a comprehensive screen for toxin-resistant mutants, representatives of 13 complementation groups have been obtained and characterized to determine whether they are altered in the toxin's intracellular target. Of 10 genes found to affect the target process, one (KTI12) was found to encode the novel polypeptide previously identified as a multicopy resistance determinant. Thus, both loss of KTI12 function and elevated KTI12 copy number can cause resistance to the K. lactis toxin.     

Cloning and characterization of ERG8, an essential gene of Saccharomyces cerevisiae that encodes phosphomevalonate kinase.

Saccharomyces cerevisiae strains that contain the ery8-1 mutation are temperature sensitive for growth due to a defect in phosphomevalonate kinase, an enzyme of isoprene and ergosterol biosynthesis. A plasmid bearing the yeast ERG8 gene was isolated from a YCp50 genomic library by functional complementation of the erg8-1 mutant strain. Genetic analysis demonstrated that integrated copies of an ERG8 plasmid mapped to the erg8 locus, confirming the identity of this clone. Southern analysis showed that ERG8 was a single-copy gene. Subcloning and DNA sequencing defined the functional ERG8 regulon as an 850-bp upstream region and an adjacent 1,272-bp open reading frame. The deduced 424-amino-acid ERG8 protein showed no homology to known proteins except within a putative ATP-binding domain present in many kinases. Disruption of the chromosomal ERG8 coding region by integration of URA3 or HIS3 marker fragments was lethal in haploid cells, indicating that this gene is essential. Expression of the ERG8 gene in S. cerevisiae from the galactose-inducible galactokinase (GAL1) promoter resulted in 1,000-fold-elevated levels of phosphomevalonate kinase enzyme activity. Overproduction of a soluble protein with the predicted 48-kDa size for phosphomevalonate kinase was also observed in the yeast cells.     

Subunit compositions and catalytic properties of proteasomes from developmental temperature- sensitive mutants of Drosophila melanogaster.

Two dominant temperature-sensitive (DTS) Drosophila mutants are missense mutations of proteasome genes encoding beta-type subunits beta6/C5 (DTS5) and beta2/Z (DTS7). At nonpermissive temperature (29 degrees C), heterozygotes (DTS5/+ and DTS7/+) develop normally until metamorphosis; pupae fail to mature and die before eclosion. Proteasomes were purified from wild-type (WT) and heterozygous adult flies raised at permissive temperature (25 degrees C). Two-dimensional gel electrophoresis separated at least 28 proteins, 13 of which were identified with monospecific antibodies to alpha6/C2 (five species), alpha2/C3 (three species), alpha7/C8 (three species), alpha5/zeta, and beta1/Y subunits. Both quantitative and qualitative differences were observed between WT and DTS/+ proteasomes, with DTS5/+ deviating more from WT than DTS7/+ proteasomes. In DTS5/+ there was a shift to more acidic species of C2 and C3 and a shift to less acidic species of 32-kDa subunits (#3-#7) recognized by an anti-alpha subunit monoclonal antibody (MCP222) and were losses of two 32-kDa subunits (#2 and #3), decreases in Y (25 kDa; 2-fold) and 31-kDa (#9; 2-fold) subunits, and increases in 52-kDa (#1; 1.9-fold) and 24-kDa (#13; 2.3-fold) subunits. In DTS7/+ there was a less pronounced shift to acidic species of C3 and no pI shift in C2 species and subunits #3-#7 and were decreases in #9 (2.5-fold) and #14 (3-fold) and a loss of #2. The three C8 species were similar between WT, DTS5/+, and DTS7/+ proteasomes. Qualitatively, the most dramatic difference was the appearance of a new 24-kDa subunit (#16) in DTS/+ preparations, with about a 14-fold greater amount of #16 in DTS7/+ than in DTS5/+ proteasomes. Catalytically, WT and DTS/+ proteasomes had similar peptidase activities, although the DTS/+ proteasomes were slightly more sensitive to SDS and elevated temperatures in vitro. The incorporation of DTS subunits apparently altered proteasome assembly and/or processing at permissive temperature with little effect on catalytic activities. These data suggest that at nonpermissive temperature, assembly/processing is more severely affected, producing DTS-containing complexes that lack functions essential for cellular proliferation and differentiation at metamorphosis.     

Molecular cloning and functional analysis of three subunits of yeast proteasome.

The genes encoding three subunits of Saccharomyces cerevisiae proteasome were cloned and sequenced. The deduced amino acid sequences were homologous not only to each other (30 to 40% identity) but also to those of rat and Drosophila proteasomes (25 to 65% identity). However, none of these sequences showed any similarity to any other known sequences, including various proteases, suggesting that these proteasome subunits may constitute a unique gene family. Gene disruption analyses revealed that two of the three subunits (subunits Y7 and Y8) are essential for growth, indicating that the proteasome and its individual subunits play an indispensable role in fundamental biological processes. On the other hand, subunit Y13 is not essential; haploid cells with a disrupted Y13 gene can proliferate, although the doubling time is longer than that of cells with nondisrupted genes. In addition, biochemical analysis revealed that proteasome prepared from the Y13 disrupted cells contains tryptic and chymotryptic activities equivalent to those of nondisrupted cells, indicating that the Y13 subunit is not essential for tryptic or chymotryptic activity. However, the chymotryptic activity of the Y13 disrupted cells is not dependent on sodium dodecyl sulfate (SDS), an activator of proteasome, since nearly full activity was observed in the absence of SDS. Thus, the activity in proteasome of the Y13 disrupted cells might result in unregulated intracellular proteolysis, thus leading to the prolonged cell cycle. These results indicate that cloned proteasome subunits having similar sequences to the yeast Y13 subunit are structural, but not catalytic, components of proteasome. It is also suggested that two subunits (Y7 and Y8) might occupy positions essential to proteasome structure or activity, whereas subunit Y13 is in a nonessential but important position.     

Normal yeast tRNA(CAGGln) can suppress amber codons and is encoded by an essential gene

We have isolated a gene that can encode yeast tRNA(CAGGln). When present on a multicopy plasmid, this gene suppresses the phenotype of a number of amber mutants, but has no effect on the ocher mutants tested. We therefore conclude that the anticodon CUG in tRNA(CAGGln) can decode the amber codon UAG by G-U mispairing, possibly by wobble base-pairing in the first codon position. This represents the second example we have observed in this laboratory of nonsense suppression in yeast by natural tRNA(Gln), involving G-U mispairing in the first codon position. Replacing the genomic copy of the cloned gene with a disrupted tRNA gene results in recessive lethality in heterozygous diploids and is lethal to haploid cells. This lethality can be rescued by transformation of cells with a single copy plasmid containing the tRNA(CAGGln) gene. Thus, the gene encoding tRNA(CAGGln) is apparently essential for viability in yeast, suggesting that it is normally present as a single copy gene.