title: Phosphorylation regulates the ubiquitin-independent degradation of yeast Pah1 phosphatidate phosphatase by the 20S proteasome.
abstract: Saccharomyces cerevisiae Pah1 phosphatidate phosphatase, which catalyzes the conversion of phosphatidate to diacylglycerol for triacylglycerol synthesis and simultaneously controls phosphatidate levels for phospholipid synthesis, is subject to the proteasome-mediated degradation in the stationary phase of growth. In this study, we examined the mechanism for its degradation using purified Pah1 and isolated proteasomes. Pah1 expressed in S. cerevisiae or Escherichia coli was not degraded by the 26S proteasome, but by its catalytic 20S core particle, indicating that its degradation is ubiquitin-independent. The degradation of Pah1 by the 20S proteasome was dependent on time and proteasome concentration at the pH optimum of 7.0. The 20S proteasomal degradation was conserved for human lipin 1 phosphatidate phosphatase. The degradation analysis using Pah1 truncations and its fusion with GFP indicated that proteolysis initiates at the N- and C-terminal unfolded regions. The folded region of Pah1, in particular the haloacid dehalogenase-like domain containing the DIDGT catalytic sequence, was resistant to the proteasomal degradation. The structural change of Pah1, as reflected by electrophoretic mobility shift, occurs through its phosphorylation by Pho85-Pho80, and the phosphorylation sites are located within its N- and C-terminal unfolded regions. Phosphorylation of Pah1 by Pho85-Pho80 inhibited its degradation, extending its half-life by ∼2-fold. The dephosphorylation of endogenously phosphorylated Pah1 by the Nem1-Spo7 protein phosphatase, which is highly specific for the sites phosphorylated by Pho85-Pho80, stimulated the 20S proteasomal degradation and reduced its half-life by 2.6-fold. These results indicate that the proteolysis of Pah1 by the 20S proteasome is controlled by its phosphorylation state.
authors: From the Department of Food Science, Rutgers Center for Lipid Research, and New Jersey Institute for Food, Nutrition, and Health, Rutgers University, New Brunswick, New Jersey 08901.
authors: Lu-Sheng
authors: LS
authors: Hsieh
authors: From the Department of Food Science, Rutgers Center for Lipid Research, and New Jersey Institute for Food, Nutrition, and Health, Rutgers University, New Brunswick, New Jersey 08901.
authors: Wen-Min
authors: WM
authors: Su
authors: From the Department of Food Science, Rutgers Center for Lipid Research, and New Jersey Institute for Food, Nutrition, and Health, Rutgers University, New Brunswick, New Jersey 08901.
authors: Gil-Soo
authors: GS
authors: Han
authors: From the Department of Food Science, Rutgers Center for Lipid Research, and New Jersey Institute for Food, Nutrition, and Health, Rutgers University, New Brunswick, New Jersey 08901 carman@aesop.rutgers.edu.
authors: George M
authors: GM
authors: Carman
mesh_terms: D006801:Humans; D010711:Phosphatidate Phosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; C079831:PAH1 protein, S cerevisiae; D010711:Phosphatidate Phosphatase; D046988:Proteasome Endopeptidase Complex
keywords: Diacylglycerol; Lipid; Phosphatase; Phosphatidate; Phosphatidate Phosphatase; Proteasome; Triacylglycerol; Yeast