Vacuole membrane topography of the DPP1-encoded diacylglycerol pyrophosphate phosphatase catalytic site from Saccharomyces cerevisiae.
The Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate phosphatase is a vacuole membrane-associated enzyme that catalyzes the removal of the beta-phosphate from diacylglycerol pyrophosphate to form phosphatidate, and it then removes the phosphate from phosphatidate to form diacylglycerol. The enzyme has six putative transmembrane domains and a hydrophilic region that contains a phosphatase motif required for its catalytic activity. In this work, we examined the topography of diacylglycerol-pyrophosphate phosphatase catalytic site within the transverse plane of the vacuole membrane. Results of protease protection analysis using endoproteinase Lys-C and labeling of cysteine residues using sulfhydryl reagents were consistent with a model where the catalytic site of diacylglycerol-pyrophosphate phosphatase was oriented to the cytosolic face of the vacuole membrane. In addition, diacylglycerol-pyrophosphate phosphatase activity was found with intact vacuoles. The phospholipids diacylglycerol pyrophosphate (0.6 mol %) and phosphatidate (1.4 mol %) were found in the vacuole membrane, and their levels decreased to an undetectable level and by 79%, respectively, when cells were depleted for zinc. The reduced levels of diacylglycerol pyrophosphate and phosphatidate correlated with the induced expression of diacylglycerol-pyrophosphate phosphatase. This work suggested that diacylglycerol pyrophosphate phosphatase functions to regulate the levels of diacylglycerol pyrophosphate and phosphatidate on the cytosolic face of the vacuole membrane.
Han
Gil-Soo
GS
Department of Food Science, Rutgers University, New Brunswick, New Jersey 08901, USA.
Johnston
Celeste N
CN
Carman
George M
GM
D020816:Amino Acid Motifs; D020134:Catalytic Domain; D002462:Cell Membrane / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D015151:Immunoblotting; D008666:Metalloendopeptidases / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013997:Time Factors; D014617:Vacuoles / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
D010455:Peptides; D010743:Phospholipids; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D011755:Pyrophosphatases; C097892:diacylglycerol pyrophosphate phosphatase; D015032:Zinc; D003545:Cysteine