Characterization of the Yeast Actin Patch Protein App1p Phosphatidate Phosphatase

Department of Food Science, Rutgers, The State University of New Jersey, Нью-Брансуик, New Jersey, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 01/2013; 288(9). DOI: 10.1074/jbc.M112.449629
Source: PubMed


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. 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), Mg2+ (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 (KsA = 11 mm), interfacial PA binding (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.

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    • "An independent enzymatic assay confirmed that TORC1 inhibition resulted in roughly a 3.5-fold increase of the cellular levels of DAG and TAG combined, and that this increase depended mainly on Pah1, but not on any of the three other known PAP enzymes in yeast (i.e. App1, Dpp1, and Lpp1; Fig. 1C) [9], [44], [45], [46], [47]. In line with these data, the relative PAP activity of Pah1 increased more than 2-fold in app1Δ dpp1Δ lpp1Δ cells after a 1-h rapamycin treatment, while the basal PAP activity in pah1Δ cells provided by App1, Dpp1, and Lpp1 combined remained unaffected by the same treatment (Fig. 1D). "
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