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 (20 mm), PA (2 mm), Mg(2+) (0.5 mm), 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 (Ks(A) = 11 mm), interfacial PA binding (Km(B) = 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 (Ca(2+), Mn(2+), and Zn(2+)). App1p also utilized diacylglycerol pyrophosphate and lyso-PA as substrates with specificity constants 4- and 7-fold lower, respectively, when compared with PA.
Highlights
Yeast App1p is a phosphatidate phosphatase (PAP) that interacts with endocytic proteins at cortical actin patches
The specific activity of purified App1p PAP was typically 370 Ϯ 14 mol/min/mg. This was equivalent to a 185,000-fold purification of App1p PAP relative to the activity (2 nmol/min/mg) in the cell extract of the pah1⌬ dpp1⌬ lpp1⌬ triplet mutant devoid of other PAP enzyme activities
App1p is a novel Mg2ϩ-dependent PAP enzyme that associates with cortical actin patches and is postulated to play a role in endocytosis/vesicle movement in S. cerevisiae [43, 50]
Summary
Yeast App1p is a phosphatidate phosphatase (PAP) that interacts with endocytic proteins at cortical actin patches. Results: App1p PAP was purified from yeast and characterized with respect to its enzymological, kinetic, and regulatory properties. Significance: App1p PAP may regulate the balance of phosphatidate and diacylglycerol for endocytosis. 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 Mg2؉-dependent PAP enzymes in that its reaction is not involved with de novo lipid synthesis. 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. App1p utilized diacylglycerol pyrophosphate and lyso-PA as substrates with specificity constants 4- and 7-fold lower, respectively, when compared with PA
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