The cellular functions of the yeast lipin homolog PAH1p are dependent on its phosphatidate phosphatase activity.
The Saccharomyces cerevisiae PAH1-encoded Mg2+-dependent phosphatidate phosphatase (PAP1, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol and Pi. This enzyme plays a major role in the synthesis of triacylglycerols and phospholipids in S. cerevisiae. PAP1 contains the DXDX(T/V) catalytic motif (DIDGT at residues 398-402) that is shared by the mammalian fat-regulating protein lipin 1 and the superfamily of haloacid dehalogenase-like proteins. The yeast enzyme also contains a conserved glycine residue (Gly80) that is essential for the fat-regulating function of lipin 1 in a mouse model. In this study, we examined the roles of the putative catalytic motif and the conserved glycine for PAP1 activity by a mutational analysis. The PAP1 activities of the D398E and D400E mutant enzymes were reduced by >99.9%, and the activity of the G80R mutant enzyme was reduced by 98%. The mutant PAH1 alleles whose products lacked PAP1 activity were nonfunctional in vivo and failed to complement the pah1Delta mutant phenotypes of temperature sensitivity, respiratory deficiency, nuclear/endoplasmic reticulum membrane expansion, derepression of INO1 expression, and alterations in lipid composition. These results demonstrated that the PAP1 activity of the PAH1 gene product is essential for its roles in lipid metabolism and cell physiology.
Han
Gil-Soo
GS
Department of Food Science and the Rutgers Center for Lipid Research, Rutgers University, New Brunswick, New Jersey 08901, USA.
Siniossoglou
Symeon
S
Carman
George M
GM
D000483:Alleles; D020816:Amino Acid Motifs / Q000502:physiology; D019943:Amino Acid Substitution; D026905:Amino Acid Transport Systems / Q000235:genetics / Q000378:metabolism; D000818:Animals; D004075:Diglycerides / Q000235:genetics / Q000378:metabolism; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism; D050356:Lipid Metabolism / Q000502:physiology*; D051379:Mice; D020125:Mutation, Missense; D007296:Myo-Inositol-1-Phosphate Synthase / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D000073718:Pancreatitis-Associated Proteins; D010710:Phosphates / Q000378:metabolism; D010711:Phosphatidate Phosphatase / Q000235:genetics / Q000378:metabolism*; D010743:Phospholipids / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014280:Triglycerides / Q000235:genetics / Q000378:metabolism
D026905:Amino Acid Transport Systems; C429560:BAP3 protein, S cerevisiae; D004075:Diglycerides; D009687:Nuclear Proteins; D000073718:Pancreatitis-Associated Proteins; D010710:Phosphates; D010743:Phospholipids; C000612141:REG3A protein, human; D029701:Saccharomyces cerevisiae Proteins; D014280:Triglycerides; D006867:Hydrolases; C418978:Lpin1 protein, mouse; C079831:PAH1 protein, S cerevisiae; D010711:Phosphatidate Phosphatase; C034507:2-haloacid dehalogenase; C519641:INO1 protein, S cerevisiae; D007296:Myo-Inositol-1-Phosphate Synthase