Abstract
The 100 kDa a-subunit of the yeast vacuolar (H(+))-ATPase (V-ATPase) is encoded by two genes, VPH1 and STV1. These genes encode unique isoforms of the a-subunit that have previously been shown to reside in different intracellular compartments in yeast. Vph1p localizes to the central vacuole, whereas Stv1p is present in some other compartment, possibly the Golgi or endosomes. To compare the properties of V-ATPases containing Vph1p or Stv1p, Stv1p was expressed at higher than normal levels in a strain disrupted in both genes, under which conditions V-ATPase complexes containing Stv1p appear in the vacuole. Complexes containing Stv1p showed lower assembly with the peripheral V(1) domain than did complexes containing Vph1p. When corrected for this lower degree of assembly, however, V-ATPase complexes containing Vph1p and Stv1p had similar kinetic properties. Both exhibited a K(m) for ATP of about 250 microm, and both showed resistance to sodium azide and vanadate and sensitivity to nanomolar concentrations of concanamycin A. Stv1p-containing complexes, however, showed a 4-5-fold lower ratio of proton transport to ATP hydrolysis than Vph1p-containing complexes. We also compared the ability of V-ATPase complexes containing Vph1p or Stv1p to undergo in vivo dissociation in response to glucose depletion. Vph1p-containing complexes present in the vacuole showed dissociation in response to glucose depletion, whereas Stv1p-containing complexes present in their normal intracellular location (Golgi/endosomes) did not. Upon overexpression of Stv1p, Stv1p-containing complexes present in the vacuole showed glucose-dependent dissociation. Blocking delivery of Vph1p-containing complexes to the vacuole in vps21Delta and vps27Delta strains caused partial inhibition of glucose-dependent dissociation. These results suggest that dissociation of the V-ATPase complex in vivo is controlled both by the cellular environment and by the 100-kDa a-subunit isoform present in the complex.
Highlights
ATP-dependent proton pumps found in a variety of intracellular compartments that function in both endocytic and secretory pathways [1,2,3,4,5,6,7,8]
Vph1p and Stv1p are homologous proteins displaying 54% identity and 71% similarity [23, 24]. These proteins show distinct intracellular localization, with Vph1p localized to the vacuole and Stv1p normally localized to some other intracellular compartment, possibly Golgi or endosomes [24]
It has previously been shown that overexpression of Stv1p in a strain disrupted in both VPH1 and STV1 causes a significant amount of Stv1p to appear in the vacuolar membrane [24]
Summary
Materials and Strains—Zymolyase 100T was obtained from Seikagaku America, Inc. Concanamycin A was purchased from Fluka Chemical Corp. Analysis of the 100-kDa Subunit Expression and V-ATPase Assembly—Yeast were grown to log phase at 30 °C in selective medium, whole cell lysates were prepared as described previously [26], and the proteins were separated by SDS-PAGE on 8% acrylamide gels. Vacuoles isolated from the vph1⌬stv1⌬ strain expressing Vph1p (3 g of protein) or Stv1p (10 g of protein) were incubated in ATPase assay buffer (50 mM NaCl, 30 mM KCl, 20 mM HEPES-NaOH, pH 7.0, 0.2 mM EGTA, 10% glycerol, 1 mM MgCl2, 1.5 mM phosphoenolpyruvate, 0.35 mM NADH, 20 units/ml pyruvate kinase, and 10 units/ml lactate dehydrogenase) with 0.1% Me2SO or 1 M concanamycin A at room temperature for 10 min. To examine the effect of inhibitors on ATPase activity, isolated vacuolar membranes were incubated in ATPase buffer containing 0.1% Me2SO, 1 M concanamycin A, 0.1 mM sodium vanadate, or 0.5 mM sodium azide at room temperature for 10 min followed by initiation of the assay by the addition of 0.5 mM ATP. Blots were developed using the chemiluminescent detection system from Kirkegaard and Perry Laboratories
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