Regulation of Yeast Ectoapyrase Ynd1p Activity by Activator Subunit Vma13p of Vacuolar H + -ATPase

Abstract

CD39-like ectoapyrases are involved in protein and lipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae, By using a two-hybrid screen, we found that air activator subunit (Vma13p) of yeast vacuolar HC ATPase:CV-ATPase) binds to the cytoplasmic domain of Ynd1p, a yeast ectoapyrase. Interaction of Ynd1p with Vma13p was demonstrated by direct binding and coimmunoprecipitation, Surprisingly, the membrane-bound ADPase activity of Ynd1p in a vma13 Delta mutant was drastically increased compared with that of Ynd1p in VMA13 cells. A similar increase in the apyrase activity of Ynd1p was found in a vma1 Delta mutant, in which the catalytic subunit A of V-ATPase is missing, and the membrane peripheral subunits including Vma13p are dissociated from the membranes. However, the E286Q mutant of VMA1, which assembles inactive V-ATPase complex including Vma13p in the membrane, retained wild type levels' of Ynd1p activity, demonstrating that the presence of Vmal13p rather than the function of V-ATPase in the membrane represses Ynd1p activity. These results suggest that association of Vma13p with the cytoplasmic domain of Ynd1p regulates its apyrase activity in the Golgi lumen.