title: Mutations at the putative junction sites of the yeast VMA1 protein, the catalytic subunit of the vacuolar membrane H(+)-ATPase, inhibit its processing by protein splicing.
abstract: A single gene, VMA1, encodes the 69-kDa subunit of the vacuolar membrane H(+)-ATPase in the yeast Saccharomyces cerevisiae. We have proposed that the subunit is synthesized as a precursor of 120 kDa (1,071 amino acids) and then converted to the 69-kDa form by an unusual processing reaction, which removes the internal domain of 454 amino acids (residues 284-737) and joins the N- and C-terminal domains. Cysteine to serine mutations at residues 284 and 738, the residues that bracket the internal domain, were introduced into the VMA1 gene by site-directed mutagenesis, and the mutant genes were expressed in a null vma1 mutant. Cells harboring either of the mutant vma1 genes accumulate nonfunctional fragments of the subunit. The mutation of Cys-284 inhibited the cleavage of the N-terminal junction site. Cys-738-->Ser mutation appeared to block the processing at both junction sites although the mutant gene yielded a small fraction of the functional 69-kDa subunit.
authors.affiliation: Department of Biology, Faculty of Science, University of Tokyo, Japan.
authors.forename: R
authors.initials: R
authors.lastname: Hirata
authors.forename: Y
authors.initials: Y
authors.lastname: Anraku
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D005800:Genes, Fungal; D007425:Intracellular Membranes / Q000201:enzymology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010641:Phenotype; D006180:Proton-Translocating ATPases / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D014617:Vacuoles / Q000201:enzymology*
chemical_list: D004271:DNA, Fungal; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D006180:Proton-Translocating ATPases