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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)

Secretory vesicles that accumulate in the temperature-sensitive sec6-4 strain of yeast have been shown to contain a vanadate-sensitive ATPase, presumably en route to the plasma membrane (Walworth, N. C., and Novick, P. J. (1987) J. Cell Biol. 105, 163-174). We have now established this enzyme to be a fully functional form of the PMA1 [H+]ATPase, identical in its catalytic properties to that found in the plasma membrane. In addition, the secretory vesicles are sealed tightly enough to permit the measurement of ATP-dependent proton pumping with fluorescent probes. We have gone on to exploit the vesicles as an expression system for site-directed mutants of the ATPase. For this purpose, a sec6-4 strain has been constructed in which the chromosomal PMA1 gene is under control of the GAL1 promoter; the mutant pma1 allele to be studied is introduced on a centromeric plasmid under the control of a novel heat shock promoter. In galactose medium at 23 degrees C, the wild-type ATPase is produced and supports normal vegetative growth. When the cells are switched to glucose medium at 37 degrees C, however, the wild-type gene turns off, the mutant gene turns on, and secretory vesicles accumulate. The vesicles contain a substantial amount of newly synthesized, plasmid-encoded ATPase (5-10% of total vesicle protein), but only traces of residual wild-type PMA1 ATPase and no detectable mitochondrial ATPase, vacuolar ATPase, or acid or alkaline phosphatase. To test the expression strategy, we have made use of pma1-105 (Ser368----Phe), a vanadate-resistant mutant previously characterized by standard methods (Perlin, D. S., Harris, S. L., Seto-Young, D., and Haber, J. E. (1989) J. Biol. Chem. 264, 21857-21864). In secretory vesicles, as expected, the plasmid-borne pma1-105 allele gives rise to a mutant enzyme with a reduced rate of ATP hydrolysis and a 100-fold increase in Ki for vanadate. Proton pumping is similarly resistant to vanadate. Thus, the vesicles appear well suited for the production and characterization of mutant forms of the PMA1 [H+]ATPase. They should also aid the study of other yeast membrane proteins that are essential for growth as well as heterologous proteins whose appearance in the plasma membrane may be toxic to the cell.
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PMID:Expression of the yeast plasma membrane [H+]ATPase in secretory vesicles. A new strategy for directed mutagenesis. 182 8

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

The complete sequence of the apiculate wine yeast Hanseniaspora uvarum mtDNA has been determined and analysed. It is an extremely compact linear molecule containing the shortest functional region ever found in fungi (11 094 bp long), flanked by Type 2 telomeric inverted repeats. The latter contained a 2704-bp-long subterminal region and tandem repeats of 839-bp units. In consequence, a population of mtDNA molecules that differed at the number of their telomeric reiterations was detected. The functional region of the mitochondrial genome coded for 32 genes, which included seven subunits of respiratory complexes and ATP synthase (the genes encoding for NADH oxidoreductase subunits were absent), two rRNAs and 23 tRNA genes which recognized codons for all amino acids. A single intron interrupted the cytochrome oxidase subunit 1 gene. A number of reasons contributed towards its strikingly small size, namely: (1) the remarkable size reduction (by >40%) of the rns and rnl genes; (2) that most tRNA genes and five of the seven protein-coding genes were the shortest among known yeast homologs; and (3) that the noncoding regions were restricted to 5.1% of the genome. In addition, the genome showed multiple changes in the orientation of transcription and the gene order differed drastically from other yeasts. When all protein coding gene sequences were considered as one unit and were compared with the corresponding molecules from all other complete mtDNAs of yeasts, the phylogenetic trees constructed robustly supported its placement basal to the yeast species of the 'Saccharomyces complex', demonstrating the advantage of this approach over single-gene or multigene approaches of unlinked genes.
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PMID:The mitochondrial genome of the wine yeast Hanseniaspora uvarum: a unique genome organization among yeast/fungal counterparts. 1642 73