title: Pho85p-Pho80p phosphorylation of yeast Pah1p phosphatidate phosphatase regulates its activity, location, abundance, and function in lipid metabolism.
abstract: The yeast Pah1p phosphatidate phosphatase, which catalyzes the penultimate step in the synthesis of triacylglycerol and plays a role in the transcriptional regulation of phospholipid synthesis genes, is a cytosolic enzyme that associates with the nuclear/endoplasmic reticulum membrane to catalyze the dephosphorylation of phosphatidate to yield diacylglycerol. Pah1p is phosphorylated on seven (Ser-110, Ser-114, Ser-168, Ser-602, Thr-723, Ser-744, and Ser-748) sites that are targets for proline-directed protein kinases. In this work, we showed that the seven sites are phosphorylated by Pho85p-Pho80p, a protein kinase-cyclin complex known to regulate a variety of cellular processes. The phosphorylation of recombinant Pah1p was time- and dose-dependent and dependent on the concentrations of ATP (3.7 μm) and Pah1p (0.25 μm). Phosphorylation reduced (6-fold) the catalytic efficiency (V(max)/K(m)) of Pah1p and reduced (3-fold) its interaction (K(d)) with liposomes. Alanine mutations of the seven sites ablated the inhibitory effect that Pho85p-Pho80p had on Pah1p activity and on the interaction with liposomes. Analysis of pho85Δ mutant cells, phosphate-starved wild type cells, and cells expressing phosphorylation-deficient forms of Pah1p indicated that loss of Pho85p-Pho80p phosphorylation reduced Pah1p abundance. In contrast, lack of Nem1p-Spo7p, the phosphatase complex that dephosphorylates Pah1p at the nuclear/endoplasmic reticulum membrane, stabilized Pah1p abundance. Although loss of phosphorylation caused a decrease in abundance, a greater amount of Pah1p was associated with membranes when compared with phosphorylated enzyme, and the loss of phosphorylation allowed bypass of the Nem1p-Spo7p requirement for Pah1p function in the synthesis of triacylglycerol.
authors.affiliation: Department of Food Science and Rutgers Center for Lipid Research, Rutgers University, New Brunswick, New Jersey 08901, USA.
authors.forename: Hyeon-Son
authors.initials: HS
authors.lastname: Choi
authors.forename: Wen-Min
authors.initials: WM
authors.lastname: Su
authors.forename: Gil-Soo
authors.initials: GS
authors.lastname: Han
authors.forename: Devin
authors.initials: D
authors.lastname: Plote
authors.forename: Zhi
authors.initials: Z
authors.lastname: Xu
authors.forename: George M
authors.initials: GM
authors.lastname: Carman
mesh_terms: D001665:Binding Sites; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D050356:Lipid Metabolism; D008081:Liposomes / Q000378:metabolism; D009154:Mutation; D010711:Phosphatidate Phosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014280:Triglycerides / Q000096:biosynthesis / Q000378:metabolism
chemical_list: D016213:Cyclins; D008081:Liposomes; C066824:PHO80 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014280:Triglycerides; D018844:Cyclin-Dependent Kinases; C058605:PHO85 protein, S cerevisiae; C079831:PAH1 protein, S cerevisiae; D010711:Phosphatidate Phosphatase