Cloning and mutational analysis of the gene encoding subunit C of yeast vacuolar H+-ATPase

Carmen Beltrán, Jan Kopecky, Yu Ching E. Pan, Hannah Nelson, Nathan Nelson*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

69 Scopus citations

Abstract

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-ATP-ase 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.

Original languageEnglish
Pages (from-to)774-779
Number of pages6
JournalJournal of Biological Chemistry
Volume267
Issue number2
StatePublished - 15 Jan 1992
Externally publishedYes

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