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.

  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The evolutionarily conserved target of rapamycin complex 1 (TORC1) controls growth-related processes such as protein, nucleotide, and lipid metabolism in response to growth hormones, energy/ATP levels, and amino acids. Its deregulation is associated with cancer, type 2 diabetes, and obesity. Among other substrates, mammalian TORC1 directly phosphorylates and inhibits the phosphatidate phosphatase lipin-1, a central enzyme in lipid metabolism that provides diacylglycerol for the synthesis of membrane phospholipids and/or triacylglycerol as neutral lipid reserve. Here, we show that yeast TORC1 inhibits the function of the respective lipin, Pah1, to prevent the accumulation of triacylglycerol. Surprisingly, TORC1 regulates Pah1 in part indirectly by controlling the phosphorylation status of Nem1 within the Pah1-activating, heterodimeric Nem1-Spo7 protein phosphatase module. Our results delineate a hitherto unknown TORC1 effector branch that controls lipin function in yeast, which, given the recent discovery of Nem1-Spo7 orthologous proteins in humans, may be conserved.
    Full-text · Article · Aug 2014 · PLoS ONE
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In the yeast Saccharomyces cerevisiae, the synthesis of phospholipids in the exponential phase of growth occurs at the expense of the storage lipid triacylglycerol. As exponential phase cells progress into the stationary phase, the synthesis of triacylglycerol occurs at the expense of phospholipids. Early work indicates a role of the phosphatidate phosphatase (PAP) in this metabolism; the enzyme produces the diacylglycerol needed for the synthesis of triacylglycerol and simultaneously controls the level of phosphatidate for the synthesis of phospholipids. Four genes (APP1, DPP1, LPP1, and PAH1) encode PAP activity in yeast, and it has been unclear which gene is responsible for the synthesis of triacylglycerol throughout growth. An analysis of lipid synthesis and composition, as well as PAP activity in various PAP mutant strains, showed the essential role of PAH1 in triacylglycerol synthesis throughout growth. Pah1p is a phosphorylated enzyme whose in vivo function is dependent on its dephosphorylation by the Nem1p-Spo7p protein phosphatase complex. nem1Δ mutant cells exhibited defects in triacylglycerol synthesis and lipid metabolism that mirrored those imparted by the pah1Δ mutation, substantiating the importance of Pah1p dephosphorylation throughout growth. An analysis of cells bearing PPAH1-lacZ and PPAH1-DPP1 reporter genes showed that PAH1 expression was induced throughout growth and that the induction in the stationary phase was stimulated by inositol supplementation. A mutant analysis indicated that the Ino2p/Ino4p/Opi1p regulatory circuit and transcription factors Gis1p and Rph1p mediated this regulation.
    Full-text · Article · Nov 2013 · Journal of Biological Chemistry
  • [Show abstract] [Hide abstract]
    ABSTRACT: Clinically important links have been established between mitochondrial function and cardiac physiology and disease in the context of signaling mechanisms, energy production, and muscle cell development. The proteins and processes that drive mitochondrial fusion and fission are now known to have emergent functions in intracellular calcium homeostasis, apoptosis, vascular smooth muscle cell proliferation, myofibril organization, and Notch-driven cell differentiation, all key issues in cardiac disease. Moreover, decreasing fission may confer protection against ischemic heart disease, particularly in the setting of obesity, diabetes, and heart failure. The importance of lipids in controlling mitochondrial fission and fusion is increasingly becoming appreciated. Roles for the bulk and signaling lipids cardiolipin, phosphatidylethanolamine, phosphatidic acid, diacylglycerol, and lysophosphatidic acid and the enzymes that synthesize or metabolize them in the control of mitochondrial shape and function are reviewed here. A number of diseases have been linked to loss-of-function alleles for a subset of the enzymes, emphasizing the importance of the lipid environment in this context.
    No preview · Article · Dec 2014 · Journal of Molecular Medicine
Show more