Abstract
PAH1-encoded phosphatidate phosphatase, which catalyzes the dephosphorylation of phosphatidate to produce diacylglycerol at the endoplasmic reticulum membrane, plays a major role in controlling the utilization of phosphatidate for the synthesis of triacylglycerol or membrane phospholipids. The conserved N-LIP and haloacid dehalogenase-like domains of Pah1 are required for phosphatidate phosphatase activity and the in vivo function of the enzyme. Its non-conserved regions, which are located between the conserved domains and at the C terminus, contain sites for phosphorylation by multiple protein kinases. Truncation analyses of the non-conserved regions showed that they are not essential for the catalytic activity of Pah1 and its physiological functions (e.g. triacylglycerol synthesis). This analysis also revealed that the C-terminal region contains a previously unrecognized WRDPLVDID domain (residues 637-645) that is conserved in yeast, mice, and humans. The deletion of this domain had no effect on the catalytic activity of Pah1 but caused the loss of its in vivo function. Site-specific mutational analyses of the conserved residues within WRDPLVDID indicated that Trp-637 plays a crucial role in Pah1 function. This work also demonstrated that the catalytic activity of Pah1 is required but is not sufficient for its in vivo functions.
Highlights
PAH1-encoded phosphatidate phosphatase, which catalyzes the dephosphorylation of phosphatidate to produce diacylglycerol at the endoplasmic reticulum membrane, plays a major role in controlling the utilization of phosphatidate for the synthesis of triacylglycerol or membrane phospholipids
For experiments to evaluate the effects of the mutations on PAP activity, the mutant enzymes were expressed in pah1⌬ app1⌬ dpp1⌬ lpp1⌬ quadruple mutant cells [43], which lack all of the endogenous PAP activities encoded by PAH1 [13], APP1 [43], DPP1 [44], and LPP1 [45] (Table 2)
The expression of Pah1 mutants was confirmed by immunoblot analysis with antibodies raised against N- and C-terminal peptides of the protein; the N-terminal antibody was used to detect the mutant enzymes with C-terminal truncations, and C-terminal antibody was used to detect those with the N-terminal truncations
Summary
Phosphatidylcholine and phosphatidylethanolamine, respectively, via the CDP-choline and CDP-ethanolamine branches of the Kennedy pathway when cells are supplemented with choline and ethanolamine [4, 5]. Yeast cells lacking Pah PAP activity accumulate PA due to a defect in its conversion to DAG used for TAG synthesis [13,14,15]. A conserved tryptophan is required for PA phosphatase function attenuate its cellular function by sequestering the enzyme to the cytosol apart from the location of its substrate PA as well as by inhibiting its PAP activity [30, 32,33,34]. Pah contains conserved N-LIP and HAD-like domains [13, 42] that are required for PAP catalytic activity and the in vivo function of the enzyme [14] (Fig. 1B). The tryptophan residue of the domain, which is conserved in orthologous PAP enzymes (i.e. lipins) from mice and humans, is essential for Pah function in TAG synthesis. The mutations of the tryptophan residue did not abolish the catalytic activity of Pah, demonstrating that PAP activity is required for, but not sufficient for, the physiological function of Pah
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