Regulation of yeast ectoapyrase ynd1p activity by activator subunit Vma13p of vacuolar H+-ATPase.
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 an activator subunit (Vma13p) of yeast vacuolar H(+)-ATPase (V-ATPase) binds to the cytoplasmic domain of Ynd1p, a yeast ectoapyrase. Interaction of Ynd1p with Vma13p was demonstrated by direct binding and co-immunoprecipitation. Surprisingly, the membrane-bound ADPase activity of Ynd1p in a vma13Delta 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 vma1Delta 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 Vma13p 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.
Zhong
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Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Malhotra
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Guidotti
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D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001081:Apyrase / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000378:metabolism; D006056:Golgi Apparatus / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008954:Models, Biological; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013347:Subcellular Fractions / Q000737:chemistry; D020798:Two-Hybrid System Techniques
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D001081:Apyrase; D006180:Proton-Translocating ATPases