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Inhibition of the yeast V‐type ATPase by cytosolic ADP
Author(s) -
Kettner Carsten,
Obermeyer Gerhard,
Bertl Adam
Publication year - 2003
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(02)03886-3
Subject(s) - cytosol , atpase , vacuole , bafilomycin , biochemistry , biophysics , v atpase , chemistry , enzyme , biology , cytoplasm , apoptosis , autophagy
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.