Characterization of the Yeast Actin Patch Protein App1p Phosphatidate Phosphatase*

Background: 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. Conclusion: App1p PAP exhibits similar, but also distinct properties from other PAP enzymes. 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. 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.

DAG, lipid intermediates that govern the synthesis of triacylglycerol and phospholipids, as well as other aspects of cell physiology that include transcription, lipid signaling, and vesicular trafficking (1, 8 -27).
Two types of PAP activity are found in eukaryotic organisms; one type of PAP is dependent on Mg 2ϩ for activity, and the other type of PAP has no divalent cation requirement for activity (2)(3)(4). A DXDX(T/V) catalytic motif directs the Mg 2ϩ -dependent activity (28,29), whereas a three-domain catalytic motif consisting of the sequences KX 6 RP, PSGH, and SRX 5 HX 3 D directs the Mg 2ϩ -independent activity (30,31). Mg 2ϩ -dependent PAP is a soluble enzyme that associates with the membrane to utilize its substrate PA (2)(3)(4)(5)32). In contrast, Mg 2ϩ -independent PAP is an intrinsic membrane enzyme that utilizes PA as well as other lipid phosphates (e.g. LPA and DGPP) as substrates (2)(3)(4)(5)32).
Insights into the roles of PAP enzymes in cell physiology have come from studies using the yeast Saccharomyces cerevisiae. In this model eukaryote, Mg 2ϩ -dependent Pah1p PAP activity is responsible for producing at the nuclear/ER membrane a pool of DAG used for the synthesis of triacylglycerol (2,29,33,34). This activity also controls a pool of PA used for the synthesis of phospholipids via the liponucleotide intermediate CDP-DAG (2,29,33,34). The PA content, as controlled by Pah1p PAP activity, also regulates the expression (e.g. transcriptional mechanism) of phospholipid synthesis enzymes (2,26,29,(33)(34)(35). Two membrane-associated Mg 2ϩ -independent PAP enzymes, Dpp1p and Lpp1p, are thought to control the signaling functions of PA, LPA, and DGPP in vacuole and Golgi membranes, respectively (2-4, 36 -42).
The focus of the current work was to biochemically characterize the novel Mg 2ϩ -dependent PAP encoded by the APP1 gene (43). App1p (named for actin patch protein) is unique among Mg 2ϩ -dependent PAP enzymes in yeast and higher eukaryotes in that its reaction is not involved with de novo lipid synthesis (43). Instead, this PAP may regulate the local concentrations of PA and DAG at the membrane to control endocytic events (43). This assertion stems from the fact that App1p associates with endocytic proteins at cortical actin patches (2, 44 -51), which are the sites of endocytosis in yeast (52). Indeed, the PAP substrate PA and product DAG are known to facilitate membrane fission/fusion events (53)(54)(55)(56)(57)(58). Moreover, these lipids regulate enzymes (e.g. phosphatidylinositol 4-phosphate kinase) that govern vesicular movement (59 -63). Detailed knowledge of the biochemical properties of App1p PAP is required to elucidate the mode of action and regulation of the enzyme at its site of action. Toward this end, we purified App1p PAP from S. cerevisiae and characterized its enzymological, kinetic, and regulatory properties. These studies revealed similarities but also distinct differences between App1p and other PAP enzymes with respect to substrate specificity and regulatory properties.

EXPERIMENTAL PROCEDURES
Materials-All chemicals were reagent grade. Growth medium supplies were purchased from Difco. Restriction endonucleases, modifying enzymes, and recombinant VentR DNA polymerase were purchased from New England Biolabs. Plasmid isolation and gel extraction kits, Ni 2ϩ -NTA-agarose resin, and spin columns were purchased from Qiagen. The Yeastmaker yeast transformation kit was purchased from Clontech. Invitrogen was the source of the DNA size standards and His 6tagged tobacco etch virus protease. Protease inhibitor mixture tablets were from Roche Applied Science. Sepharose Fast-Flow, IgG-Sepharose, protein A-Sepharose CL-4B, and the Superdex 200 column were purchased from GE Healthcare. Protein assay reagent, electrophoresis reagents, and Poly-Prep chromatography columns were purchased from Bio-Rad. Radiochemicals were purchased from PerkinElmer Life Sciences. Bovine serum albumin, nucleotides, oligonucleotides, Triton X-100, anti-protein A antibodies, and molecular mass markers for gel filtration chromatography were purchased from Sigma. Malachite green was from Fisher. Dioleoyl-PA, dioleoyl-DGPP, dioleoyl-DAG, and oleoyl-LPA, as well as the other lipids used in this study, were obtained from Avanti Polar Lipids. Scintillation counting supplies were purchased from National Diagnostics.
Purification of App1p PAP-Yeast cells (ϳ3 g wet weight) that expressed the protein A-tagged App1p PAP were disrupted with glass beads (0.5-mm diameter) using a Biospec Products Bead Beater in lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM KCl, 5 mM MgCl 2 , 1% Triton X-100, and a mixture of protease inhibitors. Unbroken cells and glass beads were removed by centrifugation at 1,500 ϫ g for 10 min. The cell extract (supernatant) was precleared of contaminating proteins by incubation with 1 ml of Sepharose Fast-Flow resin in a centrifuge tube. The enzyme sample was obtained by centrifugation at 1,500 ϫ g for 10 min, and then it was passed through 0.2 ml of IgG-Sepharose resin packed in a Bio-Rad Poly-Prep chromatography column. The column was washed with 10 ml of lysis buffer followed by 10 ml of wash buffer containing 50 mM Tris-HCl (pH 8.0), 0.5 mM EDTA, 0.5 M NaCl. The resin was then transferred to a 1-ml spin column and incubated with an equal volume of elution buffer that contained 50 mM Tris-HCl (pH 8.0), 0.5 mM EDTA, 0.5 M NaCl, 3 mM glutathione, 0.3 mM oxidized glutathione, and 100 units of tobacco etch virus protease. After incubation for 5 h, the column was subjected to centrifugation at 1,500 ϫ g for 1 min, and the eluted enzyme (supernatant) was transferred to another 1-ml spin column containing a mixture of protein A-Sepharose resin (0.1 ml) and Ni 2 -NTA-agarose resin (0.1 ml) for the removal of contaminating IgG and tobacco etch virus protease. After a 30-min incubation, the spin column was centrifuged at 1,500 ϫ g for 1 min to obtain the purified App1p PAP enzyme. The protein concentration of the purified enzyme preparation was estimated by the method of Bradford (70) using bovine serum albumin as the standard. ImageQuant analysis of Coomassie Blue-stained SDS-polyacrylamide gels containing purified App1p and known amounts of bovine serum albumin was also used to estimate the concentration of the App1p preparation. Both methods of protein estimation gave similar results. The protein concentration of the purified enzyme was 62.5 g/ml.
SDS-PAGE and Liquid Chromatography/Tandem Mass Spectrometry-SDS-PAGE (71) was performed with 10% slab gels. Proteins in gels were visualized by staining with Coomassie Brilliant Blue R-250. An SDS-polyacrylamide gel slice containing purified App1p was subjected to trypsin digestion (72), followed by liquid chromatography/tandem mass spectrometry using a Thermo Fisher Scientific LTQ Obitrap Velos instru-ment (73). This work was performed at the Center for Advanced Proteomics Research facility of the University of Medicine and Dentistry of New Jersey (Newark, NJ).
Superdex 200 Chromatography-The native molecular mass of App1p PAP was estimated by gel filtration chromatography (74). A Superdex 200 column (1 ϫ 30 cm) attached to a GE Pharmacia Ä KTA fast protein liquid chromatography system was equilibrated and eluted with 50 mM Tris-HCl (pH 7.5) and 0.15 M NaCl. The column was calibrated with blue dextran 2000 (for the void volume), ␤-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa). Purified App1p was applied and eluted from the column at a flow rate of 40 ml/h. Fractions (0.5 ml) were collected and analyzed for App1p by measuring PAP activity. The molecular mass of App1p was calculated from a plot of log molecular mass versus the elution volume/void volume (V/V 0 ) of the standards.
PAP Assay-PAP activity was measured for 20 min by following the release of water-soluble 32 P i from chloroform-soluble [ 32 P]PA (500 cpm/nmol) at 30°C (75). [ 32 P]PA was synthesized enzymatically from dioleoyl-DAG and [␥-32 P]ATP with E. coli DAG kinase (75). The radioactive PA was diluted with nonradioactive dioleoyl-PA to obtain the desired concentration of substrate used in the enzyme reactions. Alternatively, the formation of water-soluble P i from chloroform-soluble non-radioactive lipid phosphate substrates was measured with the malachite green-molybdate reagent (76) as described previously (77). The reaction mixture contained 50 mM Tris-HCl buffer (pH 7.5), 1 mM MgCl 2 , 2 mM PA, 20 mM Triton X-100, 10 mM 2-mercaptoethanol, and 1 ng of enzyme protein in a total volume of 0.1 ml. Enzyme assays were conducted in triplicate, and the average S.D. of the assays was Ϯ5%. All reactions were linear with time and protein concentration. A unit of enzyme activity was defined as the amount of enzyme that catalyzed the formation of 1 mol of product/min.
Preparation of Triton X-100/Lipid-mixed Micelles-Lipid in chloroform was transferred to a test tube, and the solvent was evaporated with nitrogen followed by drying under vacuum. Triton X-100 was added to the dried lipid to prepare Triton X-100/lipid-mixed micelles. The mol fraction of lipid in a Triton X-100/lipid-mixed micelle was calculated using the formula, mol fraction ϭ [lipid (molar)]/([lipid (molar)] ϩ [Triton X-100 (molar)]). The mol % was the mol fraction ϫ 100. The total lipid concentration in the Triton X-100/lipid-mixed micelles was kept below 15 mol % to ensure that the structure of the lipid-mixed micelles was similar to that of pure Triton X-100 micelles (78,79).
Data Analyses-Kinetic data were analyzed according to the Michaelis-Menten and Hill equations using the SigmaPlot enzyme kinetics module. Student's t test (SigmaPlot software) was used to determine statistical significance, and p values of Ͻ0.05 were taken as a significant difference.

Purification of App1p PAP and Determination of Its Native
Molecular Mass-A purified preparation of App1p was required to examine the enzymological and regulatory properties of its PAP activity under well defined conditions. Due to its very low abundance and lability, it has not been possible to obtain pure native enzyme from yeast by classical protein purification methods (43). The identity of the APP1 gene encoding PAP (43) permitted the molecular construction and overexpression of protein A-tagged App1p to facilitate its purification from yeast. The protein A-tagged enzyme was purified by IgG-Sepharose affinity chromatography, followed by tobacco etch virus protease cleavage to remove the protein A tag. The procedure yielded a highly purified preparation of App1p that migrated upon SDS-PAGE at the expected minimum subunit molecular mass of ϳ67 kDa (Fig. 1A). The identity of this protein as App1p was confirmed by trypsin digestion followed by analysis of peptides by liquid chromatography-tandem mass spectrometry. The purified enzyme was subjected to gel filtration chromatography with a Superdex 200 column to determine the native molecular mass (Fig. 1B). This analysis indicated a native size of ϳ67 kDa, and thus, the active form of the enzyme was monomeric. 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. 3 Effects of pH and Divalent Cations on App1p PAP Activity-The effect of pH on App1p PAP activity was examined using a Tris-maleate-glycine composite buffer ranging from pH 6 to 9. Maximum activity was found at pH 7.5 ( Fig. 2A). Mg 2ϩ (0.5-1 mM) was required for maximum PAP activity (Fig. 2B). Mn 2ϩ (40 M) could substitute for the Mg 2ϩ ion requirement, but the maximum activity obtained with Mn 2ϩ was nearly 2-fold lower than that obtained with Mg 2ϩ (Fig. 2B). Moreover, concentrations of Mn 2ϩ above 40 M were inhibitory to the PAP activity. Under standard assay conditions (i.e. with 1 mM MgCl 2 ), the addition of Ca 2ϩ , Mn 2ϩ , or Zn 2ϩ inhibited PAP activity by 81, 95, and 96%, respectively (Fig. 2C).
Effects of N-Ethylmaleimide, 2-Mercaptoethanol, Propranolol, and Phenylglyoxal on App1p PAP Activity-The alkylating reagent N-ethylmaleimide (80) has been used to differentiate Mg 2ϩ -dependent and Mg 2ϩ -independent PAP enzymes (18,81). App1p PAP activity was insensitive to inhibition by N-ethylmaleimide at concentrations up to 5 mM, but the activity was inhibited (ϳ80%) at concentrations of 10 mM and above (Fig. 3A). In addition, the PAP activity was stimulated (87%) by the addition of 2-mercaptoethanol to the assay systems that did not contain N-ethylmaleimide (Fig. 3B). Thus, 2-mercaptoethanol was included in the standard reaction for the enzyme. Propranolol (a non-selective ␤-adrenergic receptor antagonist (80)) and phenylglyoxal (an arginine-reactive compound (80)) have been shown to inhibit Mg 2ϩ -dependent and Mg 2ϩ -independent forms of PAP (41,82,83). Both of these compounds inhibited App1p PAP activity; propranolol (IC 50 ϭ 0.3 mM) was a more potent inhibitor when compared with phenylglyoxal (IC 50 ϭ 12 mM) (Fig. 3, C and D).
Effects of Temperature on App1p PAP Activity and Stability-The effects of temperature on App1p PAP activity and stability were examined. Maximum PAP activity was observed at 30°C, but the activity was reduced at temperatures higher than 30°C (Fig. 4A). An Arrhenius plot analysis of the data from 0 to 30°C yielded an activation energy for the reaction of 16.5 kcal/mol. The thermal stability of App1p PAP was examined by incubating the enzyme at temperatures ranging from 0 to 70°C for 20 min. Following the incubation, the samples were incubated on ice to allow for renaturation and then assayed for PAP activity at 30°C. The enzyme was labile above 30°C, with total inactiva-   tion at 50°C (Fig. 4B). The purified enzyme was completely stable for at least 3 months of storage at Ϫ80°C and stable to three cycles of freeze-thawing.
Equilibrium Constant for App1p PAP-The PAP reaction was carried out to equilibrium using a 10-fold excess of App1p and an initial PA concentration of 2 mM (Fig. 5). At equilibrium, the concentrations of DAG and P i (as determined by the generation of 32 P i from [ 32 P]PA) were 1.8 mM each, and the equilibrium constant was calculated to be 16.2. This indicated that the forward reaction was favored in vitro.
Effects of PA and Triton X-100 on App1p PAP Activity-PAP activity was measured as a function of the molar concentration of PA (Fig. 6A). The water-insoluble PA substrate was solubilized with the nonionic detergent Triton X-100, which is known to form uniform Triton X-100/PA-mixed micelles (84). In this experiment, the molar ratio of Triton X-100 to PA was maintained at 10:1 (e.g. 9.1 mol % PA) because this ratio resulted in maximum PAP activity. With respect to PA, the maximum activity was obtained at a molar concentration of 2-3 mM. Analysis of the data according to the Michaelis-Menten equation yielded a V max of 463 mol/min/mg and a K m value for PA of 0.52 mM. When the concentration of PA was held constant at 2 mM and the concentration of Triton X-100 was varied from 20 to 170 mM (e.g. diluting the surface concentration of PA), there was a dose-dependent reduction in the PAP activity (Fig. 6B). These data indicated that App1p PAP activity was responsive to both the molar and surface concentrations of PA, kinetic properties that are characteristic of surface dilution kinetics (85,86).

App1p PAP Activity Follows Surface Dilution Kinetics with Triton X-100/PA-mixed Micelles-A kinetic analysis of App1p
PAP was performed according to the surface dilution kinetic model (85,86). In the bulk step (Reaction 1), the enzyme E (e.g. PAP) first associates with the mixed micelle A (e.g. Triton X-100 ϩ PA) to form an enzyme-mixed micelle complex EA. In the surface step, the enzyme associates with the mixed micelle and then interacts with the phospholipid substrate B (e.g. mol fraction of PA in the mixed micelle) to form the EAB complex. Catalysis occurs, the products Q (e.g. DAG and P i ) are formed, and EA is regenerated. The kinetic equation for the model is shown in Equation 1 (85), where K s A ϭ k Ϫ1 /k 1 and K m B ϭ (k Ϫ2 ϩ k 3 )/k 2 . The V max is the true V max at an infinite mol fraction and an infinite molar concentration of phospholipid substrate, K m B is the interfacial Michaelis constant (expressed in surface concentration), and K s A is the dissociation constant (expressed in molar concentration) for the mixed micelle binding site (85). The following assumptions are made with this model (85). The size and aggregation number of mixed micelles remain constant over the concentration range employed as the molar concentration or surface concentration of substrate is varied; the average surface area per Triton X-100 molecule is within the same range as the lipid substrate; both remain constant as the molar concentration or the surface concentration of substrate is varied; the micellar surface area per enzyme binding site remains constant; and the enzyme has access to the entire lipid pool.

Bulk step
Surface step App1p PAP activity was measured as a function of the sum of the molar concentrations of Triton X-100 ϩ PA (e.g. A of Equation 1) at a series of set mol fractions of PA (e.g. B of Equation 1). The PAP activity was dependent on the sum of molar concentrations of Triton X-100 ϩ PA at each surface concentration of PA (Fig. 7A). As the surface concentration of PA in the mixed micelle increased, there was an increase in the PAP activity (Fig. 7A). A double reciprocal plot of the data indicated that the enzyme exhibited saturation kinetics with respect to the molar concentrations of Triton X-100 ϩ PA at each surface concentration of PA (Fig. 7B). The family of lines shown in Fig.  7B was consistent with an equilibrium-ordered step, as predicted by Equation 1. A replot of the 1/activity intercepts versus 1/B (e.g. PA/Triton X-100 ϩ PA) (Fig. 7C) and a replot of the slopes versus 1/B (Fig. 7D) were linear, as predicted by Equation 1 (85,86). The intercept of the intercept axis and the intercept of the 1/B axis in Fig. 7C are the 1/V max and Ϫ1/K m B values, respectively, and the slope in Fig. 7D   86). The V max , K m B , and K s A values were calculated to be 557 mol/min/mg, 4.2 mol %, and 11 mM, respectively.
Substrate Specificity of App1p PAP-App1p was examined for its ability to utilize a variety of lipid phosphates as substrates. These included DGPP, LPA, ceramide 1-phosphate, sphinganine 1-phosphate, and sphingosine 1-phosphate. The PAP activity was also measured as a control. The lipids were delivered to the assay as uniform Triton X-100/lipid-mixed micelles, and the release of P i from the substrates was measured with the malachite green-molybdate reagent. Of these molecules, PA, DGPP, and LPA served as substrates. The kinetic analyses for these substrates were simplified by measuring the activities as a function of their surface concentrations (mol %) and maintaining their molar concentrations at a saturating level of 2 mM (Fig.  8). Under these conditions, the enzyme followed positive cooperative kinetics (Hill numbers of 2.3, 3.3, and 3.4, respectively) with respect to the surface concentrations of PA (V max ϭ 400 mol/min/mg, K m ϭ 2 mol %), DGPP (V max ϭ 143 mol/min/ mg, K m ϭ 2.9 mol %), and LPA (V max ϭ 139 mol/min/mg, K m ϭ 4.8 mol %). PA (200 mol/min/mg/mol %) was the preferred substrate of App1p with a specificity constant 4-fold and 7-fold greater, respectively, when compared with DGPP (49 mol/min/mg/mol %) and LPA (29 mol/min/mg/mol %).
Regulatory Effects of Lipids and Nucleotides on App1p PAP Activity-In addition to their roles as structural components of membranes, phospholipids and sphingolipids function as cofactors, inhibitors, and activators of several lipid metabolic enzymes (87)(88)(89)(90)(91)(92)(93)(94), including Mg 2ϩ -dependent Pah1p PAP (95)(96)(97). The nucleotides ATP and CTP are substrates in pathways leading to the synthesis of the lipid intermediates (e.g. CDP-DAG) (98,99). We questioned if these molecules had regulatory effects on App1p PAP activity. In these experiments, a surface concentration of PA (2.5 mol %) near its K m value was used to simultaneously observe stimulatory or inhibitory effects of lipids and nucleotides on PAP activity. Fig. 9 shows the effects of phospholipids (A) and sphingolipids (B) on PAP activity. At a surface concentration of 3.75 mol %, cardiolipin (90%), phosphatidylglycerol (62%), phosphatidylserine (50%), and CDPdiacylglycerol (25%) stimulated PAP activity. At 7.5 mol %, phosphatidylcholine (57%) and phosphatidylethanolamine (36%) inhibited PAP activity. The other phospholipids tested did not have a major effect on the enzyme. Of the sphingolipids

DISCUSSION
App1p is a novel Mg 2ϩ -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). Little is known about the mode of action of App1p PAP, and thus, the aim of this work was to gain information on its enzymological, kinetic, and regulatory properties. Our studies were performed under well defined conditions devoid of competing and/or modifying enzymes or molecules that might affect the interpretation of the results. IgG-Sepharose affinity chromatography of protein A-tagged App1p expressed in yeast afforded a nearly homogenous enzyme preparation. The purified enzyme was confirmed by mass spectrometry to be App1p. This further established that APP1 encodes a PAP enzyme in yeast (43). That the pure enzyme had such a high specific activity when compared with the activity in cell extracts indicated that App1p is a very low abundance PAP enzyme in yeast. In contrast, the other yeast Mg 2ϩ -dependent PAP (i.e. Pah1p) appears to be a much more abundant protein (29,100). Although App1p was less abundant than Pah1p, the turnover number (k cat ϭ 406 s Ϫ1 ) for its PAP activity was 34-fold greater than that (k cat ϭ 12 s Ϫ1 ) of Pah1p PAP activity (101). The importance of this relatively high turnover number might be related to App1p function at cortical actin patches.
The basic enzymological and regulatory properties for App1p were determined, and this information will be useful in future studies when the effects of PAP activity on membranes at cortical actin patches are examined. The pH optimum, Mg 2ϩ dependence, and sensitivities to divalent cations, propranolol, and phenylglyoxal of App1p PAP were generally similar to those shown for Pah1p PAP (29,100,102). However, there were some differences in enzymological properties that might be exploited to differentiate these enzymes in yeast extracts.
Although the Mg 2ϩ requirement for App1p PAP activity could be partially compensated by Mn 2ϩ , the Mg 2ϩ requirement for Pah1p PAP activity is absolute (29,100). In addition, App1p PAP activity was sensitive to inhibition by N-ethylmaleimide, but this reagent has no effect on Pah1p PAP activity (29). Differential effects of N-ethylmaleimide on Lpp1p (sensitive to inhibition) and Dpp1p (insensitive to inhibition) PAP activities might also be used to differentiate these Mg 2ϩ -independent enzymes in yeast extracts (36,41), yet the sensitivity to N-ethylmaleimide inhibition cannot be used to differentiate Mg 2ϩ -dependent and Mg 2ϩ -independent PAP activities in yeast. Likewise, inhibition by propranolol, a nonspecific ␤-blocker (80) that is commonly used to establish roles of PAP enzymes in various physiological processes (63,103,104), cannot be used to distinguish between App1p and Pah1p PAP enzymes or to distinguish between Mg 2ϩ -dependent and Mg 2ϩ -independent PAP enzymes because these PAP enzymes are sensitive to this reagent (41,77,82,105).
The kinetic analyses of enzymes using phospholipid substrates are conveniently carried out with uniform detergent/ phospholipid-mixed micelles (84 -86, 88, 92, 106 -109). This system provides an environment that resembles the physiological surface of the membrane (86) where the substrate PA resides. The kinetic analysis according to the surface dilution model of Deems et al. (85) was applicable to App1p PAP activity and yielded constants for micelle surface binding (K s A ), interfacial PA binding (K m B ), and catalytic efficiency (V max ). Notwithstanding the numerical values obtained from this analysis, the kinetic behavior can be extended to the in vivo condition, where soluble App1p PAP must first associate with the membrane surface at cortical actin patches followed by catalytically productive binding to PA at the membrane surface for DAG formation.
The kinetic analysis for App1p PAP was simplified by varying the PA surface concentration in Triton X-100 micelles at a set saturating molar concentration of PA. Although this approach has the limitation of not yielding a constant for micelle surface binding (K s A ), it has been useful for examining the kinetic properties of other PAP enzymes from yeast and mammalian cells and, in particular, has facilitated studies to examine the substrate specificities of these enzymes (29,36,41,77,110). Moreover, the K m (2 mol %) and V max (400 mol/min/mg) values determined for App1p PAP by the simplified kinetic approach are in the range of those values (K m ϭ 4.2 mol % and V max ϭ 557 mol/min/mg) obtained according to the more complicated surface dilution kinetic scheme. In addition, this kinetic analysis showed that App1p PAP activity followed positive cooperative kinetics with respect to the surface concentration of PA. We posit that this kinetic behavior is a reflection of cooperative substrate binding as opposed to a reflection of enzyme oligomerization. The gel filtration analysis of the purified enzyme indicates that native App1p is monomeric in nature.
Mutants defective in the synthesis/turnover of anionic (e.g. phosphatidylserine and phosphatidylinositol 4,5-bisphosphate) and cationic (e.g. sphinganine) lipids exhibit defects in endocytosis (74,(111)(112)(113). Lipids might contribute to the endocytic process by governing membrane structural properties (e.g. curvature) and/or recruiting endocytic proteins to the membrane. That anionic (stimulation) and cationic (inhibition) membrane lipids modulated App1p PAP activity, which by the nature of its reaction controls the balance of PA and DAG, indicated another level by which lipids may play a role in endocytosis. An explanation for the lipid-mediated regulation of PAP activity is that charge plays a role in enzyme interaction with the membrane surface and/or interaction with PA. Alternatively, the lipid effector molecules might allosterically interact with App1p to regulate its PAP activity. Regardless of the mechanism, the local concentrations of the lipid effector molecules might be expected to fluctuate during the endocytic process. A lipidomics analysis of the membrane at cortical actin patches would shed light on this notion. Nucleotides (e.g. ATP and CTP) were also shown to negatively affect App1p PAP activity. They might interact directly with App1p to allosterically regulate activity, or as in the case of Pah1p PAP (114), they might inhibit activity by simply chelating the enzyme cofactor Mg 2ϩ . The cellular concentrations of ATP (1-2 mM) and CTP (0.2-0.8 mM) (115)(116)(117) are within the range of the IC 50 values for PAP inhibition, and thus, this inhibition may be physiologically relevant. Whether or not these nucleotides fluctuate during the endocytic process to regulate PAP activity is also unknown.
An unexpected finding of this work was that App1p utilized DGPP and LPA as substrates. Substrate specificity is a major criterion for differentiating Mg 2ϩ -dependent and Mg 2ϩ -independent PAP enzymes (3,4,32,118). The Mg 2ϩ -dependent Pah1p PAP (29, 100) and its mammalian counterpart lipin 1 PAP (77) are specific for PA. Differences in substrate specificity between the Mg 2ϩ -dependent and Mg 2ϩ -independent PAP enzymes have been ascribed to their distinct catalytic motifs (3,4,32,118). Although App1p and Pah1p PAP activities are governed by the DXDX(T/V) catalytic motif (43,119), this sequence in Pah1p is contained within a conserved haloacid dehalogenase-like domain (29) that is not found in App1p (43). The DXDX(T/V) motif in App1p is contained in a conserved domain found only in fungi that has overlapping regions with only weak sequence similarity to the haloacid dehalogenaselike domain (120). That the haloacid dehalogenase-like domain found in Pah1p and its mammalian counterpart Mg 2ϩ -dependent PAP enzymes (e.g. lipins) is the basis of strict specificity for PA has not been established. Nonetheless, PA was clearly the preferred substrate for App1p with a specificity constant 4 -7fold greater than that of DGPP and LPA, respectively. In contrast, the yeast Mg 2ϩ -independent Dpp1p (36,121) and Lpp1p PAP (41) enzymes exhibit a much higher specificity for DGPP and LPA when compared with the App1p PAP enzyme. The lipidomics analysis of membranes at cortical actin patches might also shed light on whether App1p utilization of DGPP and LPA as substrates is physiologically relevant.