Cloning and mutational analysis of the gene encoding subunit C of yeast vacuolar H(+)-ATPase.
A DNA fragment containing the gene encoding subunit C of vaculor H(+)-ATPase (V-ATPase) was cloned from a yeast library. The predicted amino acid sequence indicated that the C subunit consists of 373 amino acids with a calculated molecular mass of 42,287 Da. The protein from yeast is 37% identical in its amino acid sequence to the C subunit of bovine V-ATPase. The DNA fragment that was cloned in this study contained two additional reading frames. At the 5' end an amino acid sequence that is homologous to Artemia elongation factor 1 was detected. At the 3' end the N-terminal part of a kinesin-like protein was observed. The gene encoding subunit C of the V-ATPase was interrupted, and the resulting mutant could not grow at high pH and was sensitive to low and high Ca2+ concentrations in the growth medium. Transformation of the mutant by a plasmid containing the gene encoding subunit C repaired the phenotype of the mutant. Substitution of more than half of the coding region by a corresponding DNA fragment encoding the bovine subunit C resulted in a phenotype indistinguishable from wild type. Immunological studies with the disruptant mutant revealed that subunit C is necessary for the assembly of the catalytic sector of the enzyme.
Beltrán
C
C
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
Kopecky
J
J
Pan
Y C
YC
Nelson
H
H
Nelson
N
N
D000595:Amino Acid Sequence; D015139:Blotting, Southern; D002384:Catalysis; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D010641:Phenotype; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D016415:Sequence Alignment; D014617:Vacuoles / Q000201:enzymology*
D004271:DNA, Fungal