Inhibition of the yeast V-type ATPase by cytosolic ADP.
The activity of the vacuolar H(+)-ATPase has been characterized in isolated vacuoles of the yeast Saccharomyces cerevisiae by means of the patch-clamp technique. With cytosolic calcium at virtually zero (<10(-9) M), Mg-ATP induced a transient, bafilomycin A(1)-sensitive current corresponding to the flow of positive charges from the cytoplasmic surface to the vacuolar lumen. The Mg-ATP-dependent current reached its maximum amplitude (30+/-8 mA m(-2) with 5 mM Mg-ATP, n=34) within 15-20 s and declined slowly over a period of about 15-20 min even in the continuous presence of Mg-ATP. This decline of pumping activity was independent of the cytosolic KCl concentration, suggesting an inhibitory mechanism different from the high salt-induced dissociation of V(0) and V(1) reported for the V-ATPase of plants and fungi. Cytosolic ADP was found to modulate the pump activity since Mg-ATP-induced pump current was smaller if monitored in the presence of 5 mM ADP and addition of 5 mM ADP in the presence of 5 mM Mg-ATP reduced the pump current by more than 50%. Furthermore, reduction of the cytosolic ADP concentration by the ATP-regenerating system creatine phosphate/creatine kinase partially relieved the endogenous inhibition of the V-ATPase, confirming that interaction of cytosolic ADP with the V-ATPase is the reason for the transient nature of the pump current in yeast vacuoles.
Kettner
Carsten
C
Botanisches Institut I, Universität Karlsruhe (TH), Kaiserstrasse 12, 76128 Karlsruhe, Germany. ckettner@beilstein-institut.de
Obermeyer
Gerhard
G
Bertl
Adam
A
D000244:Adenosine Diphosphate / Q000378:metabolism* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000096:biosynthesis / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D003402:Creatine Kinase / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism*; D004594:Electrophysiology; D018942:Macrolides; D018408:Patch-Clamp Techniques; D011189:Potassium Chloride / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013347:Subcellular Fractions / Q000737:chemistry; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014617:Vacuoles / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D018942:Macrolides; D000244:Adenosine Diphosphate; D011189:Potassium Chloride; C040929:bafilomycin A1; D000255:Adenosine Triphosphate; D003402:Creatine Kinase; D025262:Vacuolar Proton-Translocating ATPases; D002118:Calcium