Involvement of the Nonhomologous Region of Subunit A of the Yeast V-ATPase in Coupling and in Vivo Dissociation

• Published in: The Journal of biological chemistry 2004-11, Vol.279 (47), p.48663-48670
• Main Authors: Shao, Elim, Forgac, Michael
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - chemistry; Blotting, Western; Catalysis; Chloroquine - chemistry; Chloroquine - pharmacology; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Epitopes - chemistry; Escherichia coli - metabolism; Genetic Vectors; Glucose - chemistry; Glucose - metabolism; Hydrogen-Ion Concentration; Immunoprecipitation; Mutation; Plasmids - metabolism; Protein Binding; Protein Structure, Tertiary; Protons; Quinacrine - pharmacology; Saccharomyces cerevisiae - metabolism; Time Factors; Vacuolar Proton-Translocating ATPases - chemistry; Vacuoles - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M408278200

The catalytic nucleotide binding subunit (subunit A) of the vacuolar proton-translocating ATPase (or V-ATPase) is homologous to the β-subunit of the F-ATPase but contains a 90-amino acid insert not present in the β-subunit, termed the nonhomologous region. We previously demonstrated that mutations in this region lead to changes in coupling of proton transport and ATPase activity and to inhibition of in vivo dissociation of the V-ATPase complex, an important regulatory mechanism (Shao, E., Nishi T., Kawasaki-Nishi, S., and Forgac, M. (2003) J. Biol. Chem. 278, 12985–12991). Measurement of the ATP dependence of coupling for the wild type and mutant proteins demonstrates that the coupling differences are observed at ATP concentrations up to 1 m m . A decrease in coupling efficiency is observed at higher ATP concentrations for the wild type and mutant V-ATPases. Immunoprecipitation of an epitope-tagged nonhomologous region from cell lysates indicates that this region is able to bind to the integral V 0 domain in the absence of the remainder of the A subunit, an interaction confirmed by immunoprecipitation of V 0 . Interaction between the nonhomologous region and V 0 is reduced upon incubation of cells in the absence of glucose, suggesting that the nonhomologous region may act as a trigger to activate in vivo dissociation. Immunoprecipitation suggests that the epitope tag on the nonhomologous region becomes less accessible upon glucose withdrawal, possibly due to binding to another cellular target. In vivo dissociation of the V-ATPase in response to glucose removal is also blocked by chloroquine, a weak base that neutralizes the acidic pH of the vacuole. The results suggest that the dependence of in vivo dissociation of the V-ATPase on catalytic activity may be due to neutralization of the yeast vacuole, which in turn blocks glucose-dependent dissociation.