The Saccharomyces cerevisiae actin patch protein App1p is a phosphatidate phosphatase enzyme

Abstract

Background: Phosphatidate phosphatase (PAP) plays diverse roles in lipid metabolism and cell signaling.

Results: A novel yeast PAP is identified as the actin patch protein encoded by APP1.

Conclusion: APP1 and other known genes (PAH1, DPP1, LPP1) are responsible for all detectable PAP activity in yeast.

Significance: Identification of App1p as a PAP enzyme will facilitate the understanding of its cellular function. Phosphatidate phosphatase (PAP) catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol. In the yeast Saccharomyces cerevisiae, PAP is encoded by PAH1, DPP1, and LPP1. The presence of PAP activity in the pah1Δ dpp1Δ lpp1Δ triple mutant indicated another gene(s) encoding the enzyme. We purified PAP from the pah1Δ dpp1Δ lpp1Δ triple mutant by salt extraction of mitochondria followed by chromatography with DE52, Affi-Gel Blue, phenyl-Sepharose, MonoQ, and Superdex 200. Liquid chromatography/tandem mass spectrometry analysis of a PAP-enriched sample revealed multiple putative phosphatases. By analysis of PAP activity in mutants lacking each of the proteins, we found that APP1, a gene whose molecular function has been unknown, confers ~30% PAP activity of wild type cells. The overexpression of APP1 in the pah1Δ dpp1Δ lpp1Δ mutant exhibited a 10-fold increase in PAP activity. The PAP activity shown by App1p heterologously expressed in Escherichia coli confirmed that APP1 is the structural gene for the enzyme. Introduction of the app1Δ mutation into the pah1Δ dpp1Δ lpp1Δ triple mutant resulted in a complete loss of PAP activity, indicating that distinct PAP enzymes in S. cerevisiae are encoded by APP1, PAH1, DPP1, and LPP1. Lipid analysis of cells lacking the PAP genes, singly or in combination, showed that Pah1p is the only PAP involved in the synthesis of triacylglycerol as well as in the regulation of phospholipid synthesis. App1p, which shows interactions with endocytic proteins, may play a role in vesicular trafficking through its PAP activity.

FIGURE 1.

Elution profiles of PAP activity and protein after chromatography with Superdex 200 and SDS-PAGE of the purified enzyme. The MonoQ-purified PAP enzyme preparation was subjected to chromatography with Superdex 200. Fractions were collected and assayed for PAP activity (●) and protein (○). Fractions 24–30 were subjected to SDS-PAGE followed by staining with Coomassie Blue (above plot). The positions of electrophoresis molecular mass standards are indicated in the figure. The band derived from fraction 27 that was used for protein sequencing is indicated by the asterisk. mAU, milliabsorbance units.

FIGURE 2.

Motifs in App1p and its interacting endocytic cortical actin patch proteins. A, the diagram shows a linear representation of App1p with the approximate positions of the region that has similarity to a haloacid dehalogenase (HAD)-like domain (76), the DXDX(T/V) catalytic motif, and the PXXP motif for SH3 binding. B, the diagram shows that App1p interacts with the endocytic proteins Abp1p (–73), Bbc1p (70, 73), Bzz1p (70, 73, 74), Cof1p (69), Crn1p (67), Las17p (75), Rvs161p (67, 68, 70, 73, 74), Rvs167p (67, 68, 70, 73, 74), Sla1p (67, 73), Sla2p (67), and Ysc84p (70, 73). The proteins that possess SH3 binding domains are indicated by the parentheses after the name.

FIGURE 3.

SDS-PAGE and Western blot analysis of His₆-tagged App1p purified from E. coli and the PAP activity of the purified enzyme. A, molecular mass standards (Std) and His₆-tagged App1p (1 μg) purified from E. coli were subjected to SDS-PAGE and stained with Coomassie Blue (left). The purified His₆-tagged App1p (samples of 1 μg and 1 ng, respectively) was subjected to Western blot analysis using a 1:5000 dilution of anti-His₆ (αHis₆) and anti-App1p (αApp1p) antibodies, respectively (right). B, PAP activity of purified His₆-tagged App1p measured in the absence and presence of 20 mm N-ethylmaleimide (NEM). The data in A were representative of two experiments, whereas the data in B were the average of three experiments ± S.D. (error bars).

FIGURE 4.

Effects of the app1Δ mutation, APP1 overexpression, and APP1(D281E) mutation on PAP activity. A, PAP activity was measured in cell extracts prepared from the indicated mutants grown to the exponential and stationary phases of growth. B, cell extracts were prepared from the indicated cells at the exponential phase of growth and assayed for PAP activity. Inset, samples (50 μg protein) of the cell extracts were subjected to Western blot using a 1:500 dilution of anti-HA antibodies. Lanes a, b, and c correspond to the app1Δ pah1Δ dpp1Δ lpp1Δ quadruple mutant (QM), and the QM mutant overexpressing HA-APP1 and HA-APP1(D281E), respectively. The arrow indicates the position of HA-tagged App1p. The activity data shown in A and B were the average of three experiments ± S.D. (error bars), whereas the Western blot shown in B is representative of three experiments.

FIGURE 5.

Effects of the PAP gene mutations on DAG, TAG, and total phospholipids. The indicated cells were grown to the stationary phase of growth in the presence of [2-¹⁴C]acetate (1 μCi/ml). Lipids were extracted and separated by one-dimensional TLC, and the images were subjected to ImageQuant analysis. The percentages shown for the individual lipids were normalized to the total ¹⁴C-labeled chloroform-soluble fraction, which also contained sterols, fatty acids, and unidentified neutral lipids. Each data point represents the average of three experiments ± S.D. (error bars). PL, phospholipids.

FIGURE 6.

Effects of the PAP gene mutations on phospholipid composition. The indicated cells were grown to the exponential phase of growth in the presence of [³²P]P_i (10 μCi/ml). Phospholipids were extracted and separated by two-dimensional TLC, and the images were subjected to ImageQuant analysis. The percentages shown for the individual phospholipids were normalized to the total ³²P-labeled chloroform-soluble fraction that included sphingolipids and unidentified phospholipids. Each data point represents the average of three experiments ± S.D. (error bars). Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine.