Characterization of two acidic proteins of Saccharomyces cerevisiae ribosome

In $\text{Saccharomyces}\text{cerevisiae}$ ribosomes two proteins, L44 and L45, of strong acidic character are detected. These proteins, presumably equivalent to bacterial L7 and L12, have been purified and have given a total cross reaction when tested by double immunodiffusion. Reaction with fluorescamine has shown that the amino terminal group of the polypeptide is blocked in protein L44 and free in protein L45. Tryptic analysis of the two proteins shows that three out of nine peptides are in identical position in both patterns, three more are easily related and the last three are clearly different. The data indicate that proteins L44 and L45 are closely related but not totally identical.     

Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins.

To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly homologous to a peptide in calf thymus UP1 and UP2 and a human heterogeneous nuclear ribonucleoprotein. Residues 125 to 162 constitute a fivefold tandem repeat of the sequence RGGFRG, the composition of which suggests a nucleic acid-binding site. Near the C terminus, residues 233 to 245 are homologous to several RNA-binding proteins. Of 18 C-terminal residues, 10 are acidic, a characteristic of the procaryotic single-stranded DNA-binding proteins and eucaryotic DNA- and RNA-binding proteins. In addition, examination of the subcellular distribution of SSB1 by immunofluorescence microscopy indicated that SSB1 is a nuclear protein, predominantly located in the nucleolus. Sequence homologies and the nucleolar localization make it likely that SSB1 functions in RNA metabolism in vivo, although an additional role in DNA metabolism cannot be excluded.     

Regulation of nuclear genes encoding mitochondrial proteins in Saccharomyces cerevisiae.

Selection for mutants which release glucose repression of the CYB2 gene was used to identify genes which regulate repression of mitochondrial biogenesis. We have identified two of these as the previously described GRR1/CAT80 and ROX3 genes. Mutations in these genes not only release glucose repression of CYB2 but also generally release respiration of the mutants from glucose repression. In addition, both mutants are partially defective in CYB2 expression when grown on nonfermentable carbon sources, indicating a positive regulatory role as well. ROX3 was cloned by complementation of a glucose-inducible flocculating phenotype of an amber mutant and has been mapped as a new leftmost marker on chromosome 2. The ROX3 mutant has only a modest defect in glucose repression of GAL1 but is substantially compromised in galactose induction of GAL1 expression. This mutant also has increased SUC2 expression on nonrepressing carbon sources. We have also characterized the regulation of CYB2 in strains carrying null mutation in two other glucose repression genes, HXK2 and SSN6, and show that HXK2 is a negative regulator of CYB2, whereas SSN6 appears to be a positive effector of CYB2 expression.     

Nucleolar and nuclear envelope proteins of the yeast Saccharomyces cerevisiae

We have developed a fast and reliable purification protocol to obtain yeast nuclei in intact and pure form and in a reasonable yield. The purified nuclei appear homogeneous at the light and electron microscopic level, are highly enriched in the nuclear marker histone H2B and devoid of mitochondrial, vacuolar and cytosolic marker proteins. On sodium dodecyl sulfate (SDS)-polyacrylamide gels, the nuclear fraction contains unique proteins which distinguishes them from the major yeast subcellular fractions. Yeast nuclei were separated by detergent/salt extraction into soluble, insoluble and membrane fractions. Antibodies raised against subnuclear fractions lead to the identification of an integral nuclear membrane protein and a high-abundance 38-kDa protein which is located in the yeast nucleolus.     

Calmodulin-binding proteins of Saccharomyces cerevisiae

The subcellular distribution of calmodulin-binding proteins in the soluble, plasma membrane, and nuclear fractions of Saccharomyces cerevisiae was analyzed with a gel binding assay using 125I-labeled calmodulin. Over 20 binding proteins were detected. The calmodulin-binding protein profiles were markedly different among the fractions. Calmodulin-binding proteins were most abundant in the nuclear fraction, followed by the membrane fraction and the soluble fraction in decreasing order. The amounts of certain calmodulin-binding proteins increased after treatment with alpha-mating factor.     

Mitochondrial genome maintenance: roles for nuclear nonhomologous end-joining proteins in Saccharomyces cerevisiae

Mitochondrial DNA (mtDNA) deletions are associated with sporadic and inherited diseases and age-associated neurodegenerative disorders. Approximately 85% of mtDNA deletions identified in humans are flanked by short directly repeated sequences; however, mechanisms by which these deletions arise are unknown. A limitation in deciphering these mechanisms is the essential nature of the mitochondrial genome in most living cells. One exception is budding yeast, which are facultative anaerobes and one of the few organisms for which directed mtDNA manipulation is possible. Using this model system, we have developed a system to simultaneously monitor spontaneous direct-repeat-mediated deletions (DRMDs) in the nuclear and mitochondrial genomes. In addition, the mitochondrial DRMD reporter contains a unique KpnI restriction endonuclease recognition site that is not present in otherwise wild-type (WT) mtDNA. We have expressed KpnI fused to a mitochondrial localization signal to induce a specific mitochondrial double-strand break (mtDSB). Here we report that loss of the MRX (Mre11p, Rad50p, Xrs2p) and Ku70/80 (Ku70p, Ku80p) complexes significantly impacts the rate of spontaneous deletion events in mtDNA, and these proteins contribute to the repair of induced mtDSBs. Furthermore, our data support homologous recombination (HR) as the predominant pathway by which mtDNA deletions arise in yeast, and suggest that the MRX and Ku70/80 complexes are partially redundant in mitochondria.     

Benomyl prevents nuclear fusion in Saccharomyces cerevisiae

Summary Benomyl prevents nuclear fusion in mating mixtures of Saccharomyces cerevisiae. Cytoductants, heterokaryons and diploids may be recovered from these mixtures.     

Mitochondria-mediated nuclear mutator phenotype in Saccharomyces cerevisiae

Using Saccharomyces cerevisiae as a model organism, we analyzed the consequences of disrupting mitochondrial function on mutagenesis of the nuclear genome. We measured the frequency of canavanine-resistant colonies as a measure of nuclear mutator phenotype. Our data suggest that mitochondrial dysfunction leads to a nuclear mutator phenotype (i) when oxidative phosphorylation is blocked in wild-type yeast at mitochondrial complex III by antimycin A and (ii) in mutant strains lacking the entire mitochondrial genome (rho⁰) or those with deleted mitochondrial DNA (rho^–). The nuclear mutation frequencies obtained for antimycin A-treated cells as well as for rho^– and rho⁰ cells were ~2- to 3-fold higher compared to untreated control and wild-type cells, respectively. Blockage of oxidative phosphorylation by antimycin A treatment led to increased intracellular levels of reactive oxygen species (ROS). In contrast, inactivation of mitochondrial activity (rho^– and rho⁰) led to decreased intracellular levels of ROS. We also demonstrate that in rho⁰ cells the REV1, REV3 and REV7 gene products, all implicated in error-prone translesion DNA synthesis (TLS), mediate mutagenesis in the nuclear genome. However, TLS was not involved in nuclear DNA mutagenesis caused by inhibition of mitochondrial function by antimycin A. Together, our data suggest that mitochondrial dysfunction is mutagenic and multiple pathways are involved in this nuclear mutator phenotype.     

Characterization of gamma-glutamylamidase-glutaminase activity in Saccharomyces cerevisiae

In a foregoing paper we have shown the presence in the yeast Saccharomyces cerevisiae of an enzyme catalyzing the hydrolysis of L-gamma-glutamyl-p-nitroanilide, but apparently distinct from gamma-glutamyltranspeptidase. The cellular level of this enzyme was not regulated by the nature of the nitrogen source supplied to the yeast cell. Purification was attempted, using ion exchange chromatography on DEAE Sephadex A 50, salt precipitations and successive chromatographies on DEAE Sephadex 6B and Sephadex G 100. The apparent molecular weight of the purified enzyme was 14,800 as determined by gel filtration. As shown by kinetic studies and thin layer chromatography, the enzyme preparation exhibited only hydrolytic activity against gamma-glutamylarylamide and L-glutamine with an optimal pH of about seven. Various gamma-glutamylaminoacids, amides, dipeptides and glutathione were inactive as substrates and no transferase activity was detected. The yeast gamma-glutamylarylamidase was activated by SH protective agents, dithiothreitol and reduced glutathione. Oxidized glutathione, ophtalmic acid and various gamma-glutamylaminoacids inhibited competitively the enzyme. The activity was also inhibited by L-gamma-glutamyl-o-(carboxy)phenylhydrazide and the couple serine-borate, both transition-state analogs of gamma-glutamyltranspeptidase. Diazooxonorleucine, reactive analog of glutamine, inactivated the enzyme. The physiological role of yeast gamma-glutamylarylamidase-glutaminase is still undefined but is most probably unrelated to the bulk assimilation of glutamine by yeast cells.     

Characterization of methylglyoxal synthase in Saccharomyces cerevisiae

Methylglyoxal synthase in Saccharomyces cerevisiae was purified approximately 300 folds from cell extracts with 20% of activity yield. During purification procedures, polymorphic behaviours of the enzyme were observed. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis and consisted of a single polypeptide chain of Mr = 26,000. The enzyme was most active at pH 9.5-10.5 and strictly specific to dihydroxyacetone phosphate with Km = 3 mM. Phosphoenolpyruvate, glyceraldehyde-3-phosphate, orthophosphate and thiol compounds were potent inhibitors of the enzyme.     

Characterization of Magnaporthe oryzae chrysovirus 1 structural proteins and their expression in Saccharomyces cerevisiae

Magnaporthe oryzae chrysovirus 1 (MoCV1), which is associated with an impaired growth phenotype of its host fungus, harbors four major proteins: P130 (130 kDa), P70 (70 kDa), P65 (65 kDa), and P58 (58 kDa). N-terminal sequence analysis of each protein revealed that P130 was encoded by double-stranded RNA1 (dsRNA1) (open reading frame 1 [ORF1] 1,127 amino acids [aa]), P70 by dsRNA4 (ORF4; 812 aa), and P58 by dsRNA3 (ORF3; 799 aa), although the molecular masses of P58 and P70 were significantly smaller than those deduced for ORF3 and ORF4, respectively. P65 was a degraded form of P70. Full-size proteins of ORF3 (84 kDa) and ORF4 (85 kDa) were produced in Escherichia coli. Antisera against these recombinant proteins detected full-size proteins encoded by ORF3 and ORF4 in mycelia cultured for 9, 15, and 28 days, and the antisera also detected smaller degraded proteins, namely, P58, P70, and P65, in mycelia cultured for 28 days. These full-size proteins and P58 and P70 were also components of viral particles, indicating that MoCV1 particles might have at least two forms during vegetative growth of the host fungus. Expression of the ORF4 protein in Saccharomyces cerevisiae resulted in cytological changes, with a large central vacuole associated with these growth defects. MoCV1 has five dsRNA segments, as do two Fusarium graminearum viruses (FgV-ch9 and FgV2), and forms a separate clade with FgV-ch9, FgV2, Aspergillus mycovirus 1816 (AsV1816), and Agaricus bisporus virus 1 (AbV1) in the Chrysoviridae family on the basis of their RdRp protein sequences.     

DNA binding is not sufficient for nuclear localization of regulatory proteins in Saccharomyces cerevisiae.

We showed by immunofluorescence that the procaryotic DNA-binding protein LexA and a chimeric protein that contains the DNA-binding portion of LexA (amino acids 1 to 87) and a large portion (amino acids 74 to 881) of the Saccharomyces cerevisiae positive regulatory GAL4 protein (GAL4 gene product) are not preferentially localized in the nucleus in S. cerevisiae.     

Characterization of SIS1, a Saccharomyces cerevisiae homologue of bacterial dnaJ proteins

The Saccharomyces cerevisiae SIS1 gene was identified as a high copy number suppressor of the slow growth phenotype of strains containing mutations in the SIT4 gene, which encodes a predicted serine/threonine protein phosphatase. The SIS1 protein is similar to bacterial dnaJ proteins in the amino-terminal third and carboxyl-terminal third of the proteins. In contrast, the middle third of SIS1 is not similar to dnaJ proteins. This region of SIS1 contains a glycine/methionine-rich region which, along with more amino-terminal sequences, is required for SIS1 to associate with a protein of apparent molecular mass of 40 kD. The SIS1 gene is essential. Strains limited for the SIS1 protein accumulate cells that appear blocked for migration of the nucleus from the mother cell into the daughter cell. In addition, many of the cells become very large and contain a large vacuole. The SIS1 protein is localized throughout the cell but is more concentrated at the nucleus. About one-fourth of the SIS1 protein is released from a nuclear fraction upon treatment with RNase. We also show that overexpression of YDJ1, another yeast protein with similarity to bacterial dnaJ proteins, can not substitute for SIS1.     

Cloning and characterization of nuclear genes for two mitochondrial ribosomal proteins in Saccharomyces cerevisiae.

The genes for two large subunit proteins, YmL8 and YmL20, of the mitochondrial ribosome of Saccharomyces cerevisiae were cloned by hybridization with synthetic oligonucleotide mixtures corresponding to their N-terminal amino acid sequences. They were termed MRP-L8 and MRP-L20, respectively, and their nucleotide sequences were determined using a DNA sequencer. The MRP-L8 gene was found to encode a 26.8-kDa protein whose deduced amino acid sequence has a high degree of similarity to ribosomal protein L17 of Escherichia coli. The gene MRP-L20 was found to encode a 22.3-kDa protein with a presequence consisting of 18 amino acid residues. By Southern blot hybridization to the yeast chromosomes separated by field-inversion gel electrophoresis, the MRP-L8 and MRP-L20 genes were located on chromosomes X and XI, respectively. Gene disruption experiments indicate that their products, YmL8 and YmL20 proteins, are essential for the mitochondrial function and the absence of these proteins causes instability of the mitochondrial DNA.