Characterization of the yeast actin patch protein App1p phosphatidate phosphatase

Minjung Chae, George M. Carman

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

Abstract

Yeast App1p is a phosphatidate phosphatase (PAP) that associates with endocytic proteins at cortical actin patches. App1p, which catalyzes the conversion of phosphatidate (PA) to diacylglycerol, is unique among Mg 2+-dependent PAP enzymes in that its reaction is not involved with de novo lipid synthesis. Instead, App1p PAP is thought to play a role in endocytosis because its substrate and product facilitate membrane fission/ fusion events and regulate enzymes that govern vesicular movement. App1p PAP was purified from yeast and characterized with respect to its enzymological, kinetic, and regulatory properties. Maximum PAP activity was dependent on Triton X-100 (20mM), PA (2mM), Mg2+ (0.5mM), and 2-mercaptoethanol (10 mM) at pH 7.5 and 30 °C. Analysis of surface dilution kinetics with Triton X-100/PA-mixed micelles yielded constants for surface binding (K sA=11mM), interfacialPAbinding (KmB = 4.2 mol %), and catalytic efficiency (Vmax = 557 μmol/min/mg). The activation energy, turnover number, and equilibrium constant were 16.5 kcal/mol, 406 s-1, and 16.2, respectively. PAP activity was stimulated by anionic lipids (cardiolipin, phosphatidylglycerol, phosphatidylserine, and CDP-diacylglycerol) and inhibited by zwitterionic (phosphatidylcholine and phosphatidylethanolamine) and cationic (sphinganine) lipids, nucleotides (ATP and CTP),N-ethylmaleimide, propranolol, phenylglyoxal, and divalent cations (Ca2+, Mn2+, and Zn2+). App1p also utilized diacylglycerol pyrophosphate and lyso-PA as substrates with specificity constants 4- and 7-fold lower, respectively, when compared with PA.

Original languageEnglish (US)
Pages (from-to)6427-6437
Number of pages11
JournalJournal of Biological Chemistry
Volume288
Issue number9
DOIs
StatePublished - Mar 1 2013

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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