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The Saccharomyces cerevisiae Actin Patch Protein App1p Is a Phosphatidate Phosphatase Enzyme

Authors :
Gil-Soo Han
Minjung Chae
George M. Carman
Source :
Journal of Biological Chemistry. 287:40186-40196
Publication Year :
2012
Publisher :
Elsevier BV, 2012.

Abstract

Phosphatidate phosphatase (PAP) plays diverse roles in lipid metabolism and cell signaling.A novel yeast PAP is identified as the actin patch protein encoded by APP1.APP1 and other known genes (PAH1, DPP1, LPP1) are responsible for all detectable PAP activity in yeast.Identification of App1p as a PAP enzyme will facilitate the understanding of its cellular function. Phosphatidate phosphatase (PAP) catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol. In the yeast Saccharomyces cerevisiae, PAP is encoded by PAH1, DPP1, and LPP1. The presence of PAP activity in the pah1Δ dpp1Δ lpp1Δ triple mutant indicated another gene(s) encoding the enzyme. We purified PAP from the pah1Δ dpp1Δ lpp1Δ triple mutant by salt extraction of mitochondria followed by chromatography with DE52, Affi-Gel Blue, phenyl-Sepharose, MonoQ, and Superdex 200. Liquid chromatography/tandem mass spectrometry analysis of a PAP-enriched sample revealed multiple putative phosphatases. By analysis of PAP activity in mutants lacking each of the proteins, we found that APP1, a gene whose molecular function has been unknown, confers ~30% PAP activity of wild type cells. The overexpression of APP1 in the pah1Δ dpp1Δ lpp1Δ mutant exhibited a 10-fold increase in PAP activity. The PAP activity shown by App1p heterologously expressed in Escherichia coli confirmed that APP1 is the structural gene for the enzyme. Introduction of the app1Δ mutation into the pah1Δ dpp1Δ lpp1Δ triple mutant resulted in a complete loss of PAP activity, indicating that distinct PAP enzymes in S. cerevisiae are encoded by APP1, PAH1, DPP1, and LPP1. Lipid analysis of cells lacking the PAP genes, singly or in combination, showed that Pah1p is the only PAP involved in the synthesis of triacylglycerol as well as in the regulation of phospholipid synthesis. App1p, which shows interactions with endocytic proteins, may play a role in vesicular trafficking through its PAP activity.

Details

ISSN :
00219258
Volume :
287
Database :
OpenAIRE
Journal :
Journal of Biological Chemistry
Accession number :
edsair.doi.dedup.....f17c2e4864924299308748f4737d9d1c
Full Text :
https://doi.org/10.1074/jbc.m112.421776