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Query: EC:3.6.3.14 (ATP synthase)
 7,042 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Respiratory deficient mutants of Saccharomyces cerevisiae previously assigned to complementation group G59 are pleiotropically deficient in respiratory chain components and in mitochondrial ATPase. This phenotype has been shown to be a consequence of mutations in a nuclear gene coding for mitochondrial leucyl-tRNA synthetase. The structural gene (MSL1) coding for the mitochondrial enzyme has been cloned by transformation of two different G59 mutants with genomic libraries of wild type yeast nuclear DNA. The cloned gene has been sequenced and shown to code for a protein of 894 residues with a molecular weight of 101,936. The amino-terminal sequence (30-40 residues) has a large percentage of basic and hydroxylated residues suggestive of a mitochondrial import signal. The cloned MSL1 gene was used to construct a strain in which 1 kb of the coding sequence was deleted and substituted with the yeast LEU2 gene. Mitochondrial extracts obtained from the mutant carrying the disrupted MSL1::LEU2 allele did not catalyze acylation of mitochondrial leucyl-tRNA even though other tRNAs were normally charged. These results confirmed the correct identification of MSL1 as the structural gene for mitochondrial leucyl-tRNA synthetase. Mutations in MSL1 affect the ability of yeast to grow on nonfermentable substrates but are not lethal indicating that the cytoplasmic leucyl-tRNA synthetase is encoded by a different gene. The primary sequence of yeast mitochondrial leucyl-tRNA synthetase has been compared to other bacterial and eukaryotic synthetases. Significant homology has been found between the yeast enzyme and the methionyl- and isoleucyl-tRNA synthetases of Escherichia coli. The most striking primary sequence homology occurs in the amino-terminal regions of the three proteins encompassing some 150 residues. Several smaller domains in the more internal regions of the polypeptide chains, however, also exhibit homology. These observations have been interpreted to indicate that the three synthetases may represent a related subset of enzymes originating from a common ancestral gene.
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PMID:Homology of yeast mitochondrial leucyl-tRNA synthetase and isoleucyl- and methionyl-tRNA synthetases of Escherichia coli. 282 65

The role of H211 of the yeast F1-ATPase beta-subunit was investigated by site-specific mutagenesis and characterization of the resulting enzymes. Five amino acids (N, D, I, K, and A) were substituted for H211 of the ATP2 gene. The mutated genes were expressed in an atp2::LEU2 host, and only yeast expressing H211N respired aerobically. The respiratory phenotypes of the other four mutants were suppressed by a second site mutation (L203F). The ATPases from the single mutant strains were unstable when removed from the mitochondrial inner membrane, preventing purification. Submitochondrial particles were prepared from each strain and the activities were stable under a variety of conditions, allowing determination of Vmax and Km for ATP hydrolysis. Mutations of H211 caused increases in Km of 3.7- to 7.4-fold, while L203F had little effect. The suppressive effect of the L203F mutation was also expressed in the Km values of the double mutant strains. The ATPases from the H211 mutants had diminished sensitivity to oligomycin, and their pH optima were 1.5-2.0 units less than the wild-type optimum. Values of pKa for the groups involved in catalysis were estimated for the wild-type enzyme and three H211 mutants (N, D, and K). Each mutant enzyme showed a substantial decrease in the pKa of the group(s) which serves as a base in acid-base catalysis. The results of this study demonstrate that H211 is important in maintaining the structure of the wild-type enzyme complex and also contributes to the structure of the active site. L203 is also required for the stability of the enzyme complex and may have a structural or functional interaction with H211. Neither H211 nor L203 is required for catalysis by F1.
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PMID:Mutations at histidine 211 of the yeast F1-ATPase beta-subunit affect the stability and assembly of the ATPase and the structure of the active site. 857 60

Four sets of plasmid vectors for the budding yeast Kluyveromyces lactis (Kl) have been constructed. All plasmids are pUC19-based shuttle vectors having multiple unique sites in their multiple cloning site (MCS) within the bacterial lacZ gene. The first set of vectors contains Klori, the origin of replication for Kl isolated from Kluyveromyces plasmid pKD1, and one of the selectable nutritional markers, URA3, TRP1 or LEU2. These markers from the yeast, Saccharomyces cerevisiae (Sc), can complement the uraA1, trp1 and leu2 mutations of Kl. The second set of vectors, in addition to Klori, contains the ARS (autonomously replicating sequence) and centromeric sequences of Sc, and are able to replicate in both Sc and Kl. The third group of plasmids is centromeric vectors that are maintained in Kl at low copy number. The last family of vectors was designed for gene overexpression. As they contain the bacterial kanamycin-resistance-encoding gene (kan), plasmid copy number can be amplified to over 100 copies per cell in Kl by growing cells in the presence of the antibiotic G418 (Geneticin). This type of vector has been used to study the high-copy-lethality phenotype of a truncated version of the Kl MGI2 gene encoding the alpha-subunit of the mitochondrial F1F0-ATP synthase.
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PMID:Low- and high-copy-number shuttle vectors for replication in the budding yeast Kluyveromyces lactis. 865 73