The glycerophosphocholine acyltransferase Gpc1 is part of a phosphatidylcholine (PC)-remodeling pathway that alters PC species in yeast

Abstract

Phospholipase B-mediated hydrolysis of phosphatidylcholine (PC) results in the formation of free fatty acids and glycerophosphocholine (GPC) in the yeast Saccharomyces cerevisiae GPC can be reacylated by the glycerophosphocholine acyltransferase Gpc1, which produces lysophosphatidylcholine (LPC), and LPC can be converted to PC by the lysophospholipid acyltransferase Ale1. Here, we further characterized the regulation and function of this distinct PC deacylation/reacylation pathway in yeast. Through in vitro and in vivo experiments, we show that Gpc1 and Ale1 are the major cellular GPC and LPC acyltransferases, respectively. Importantly, we report that Gpc1 activity affects the PC species profile. Loss of Gpc1 decreased the levels of monounsaturated PC species and increased those of diunsaturated PC species, whereas Gpc1 overexpression had the opposite effects. Of note, Gpc1 loss did not significantly affect phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine profiles. Our results indicate that Gpc1 is involved in postsynthetic PC remodeling that produces more saturated PC species. qRT-PCR analyses revealed that GPC1 mRNA abundance is regulated coordinately with PC biosynthetic pathways. Inositol availability, which regulates several phospholipid biosynthetic genes, down-regulated GPC1 expression at the mRNA and protein levels and, as expected, decreased levels of monounsaturated PC species. Finally, loss of GPC1 decreased stationary phase viability in inositol-free medium. These results indicate that Gpc1 is part of a postsynthetic PC deacylation/reacylation remodeling pathway (PC-DRP) that alters the PC species profile, is regulated in coordination with other major lipid biosynthetic pathways, and affects yeast growth.

Keywords: Ale1; Gpc1; acyltransferase; glycerophosphocholine; lipid remodeling; membrane turnover; phosphatidylcholine; phospholipid metabolism; yeast.

Figure 1.

Schematic outline of PC metabolism in yeast. The PC-DRP is indicated by red arrows. The PC-DRP includes deacylation of PC to GPC by PLB1 or NTE1 followed by stepwise reacylation of GPC to LPC by GPC1 and LPC to PC by ALE1. The de novo PC synthesis routes are indicated by dotted arrows. Gene names are in italics. PLA, phospholipase A; PLB, phospholipase B.

Figure 2.

The role of Gpc1 in PC metabolism. A, the indicated strains were grown to log phase in the presence of 5 μm [¹⁴C]choline-GPC. The cells were harvested, phospholipids were extracted and separated, and PC was quantified as described under “Experimental procedures.” B and C, indicated strains contained plasmid in which GIT1 was constitutively expressed under the control of the ADH1 promoter. The cells were grown in the presence of [¹⁴C]choline-GPC for 4 h and harvested, and phospholipids were extracted and analyzed as described under “Experimental procedures.” The data are normalized to the WT containing ADH1-GIT1. Roughly 100-fold more PC was detected as compared with LPC in the WT strain. The data represent the averages of three independent cultures ± S.D. A t test was performed to determine significance as indicated. *, p ≤ 0.05; **, p ≤ 0.005; ***, p ≤ 0.0005.

Figure 3.

Gpc1 impacts PC molecular species profile. A and B, the indicated strains were grown to late log phase, the cultures were harvested, and the lipids were extracted. PC species were separated and analyzed using LC-MS/MS, as described under “Experimental procedures.” The data represent averages of three independent cultures ± S.D. A t test was performed to determine significance as indicated. *, p ≤ 0.05.

Figure 4.

Loss of PCT1 or ALE1 has minor effects on PC species profile. The indicated strains were grown to late log phase, the cultures were harvested, and the lipids were extracted. PC species were separated and analyzed as described under “Experimental procedures.” The data represent averages of three independent cultures ± S.D. A two-way analysis of variance was performed to determine significance for each strain compared with the WT. *, p ≤ 0.05; **, p ≤ 0.004; ***, p ≤ 0.0001.

Figure 5.

Loss of GPC1 has no significant effects on PE, PS, and PI species profiles and slightly decreases the total C16:0 acyl chain content. The indicated strains were grown to log phase and harvested, and the lipids were extracted. A–C, PE (A), PS (B), and PI (C) species were analyzed by MS as detailed under “Experimental procedures.” D, total acyl chain composition was determined using GC. The data represent averages of four independent cultures ± S.D. A t test was performed to determine significance as indicated. *, p = 0.0008.

Figure 6.

GPC1 transcript is increased by attenuation of PC and by inositol limitation. A and B, the indicated strains were grown to log phase, cells were harvested, and RNA was extracted. GPC1 transcripts were quantified by qRT-PCR. The data were normalized to the amount of the endogenous control mRNA, SNR17, and expressed relative to the WT strain. B, WT grown in the presence of 75 μm inositol (WT +Inositol) compared with WT grown in the absence of inositol (WT −Inositol). The experiments were performed in biological triplicate and assayed in experimental triplicate. A t test was performed to establish significance. **, p ≤ 0.005; ***, p ≤0.0005.

Figure 7.

Inositol supplementation affects Gpc1 protein abundance and PC species profile. A, WT strain was grown in medium containing 75 μm inositol (+Inositol) or lacking inositol (−Inositol). Western blotting analysis was performed using anti-HA mouse IRdye 680 and goat anti-mouse IRdye 800. Glucose-6-phosphate dehydrogenase was used as a loading control. Protein bands were quantified using Image Studio^TM software. The data represent two biological replicates. B, the indicated strains were grown to late log phase before harvesting. Phospholipids were extracted, and PC species were analyzed using LC-MS/MS. The data represent averages of three independent cultures ± S.D. A t test was performed to establish significance. ****, p ≤ 0.0005. C, strains were cultured in synthetic medium, harvested, and resuspended in sterile water. 10-fold serial dilutions (5 μl) were spotted onto plates containing (+Inositol) or lacking (−Inositol) inositol.

Figure 8.

Loss of GPC1 impacts growth and stationary phase viability in inositol-free medium. A, indicated strains were grown in synthetic liquid medium lacking inositol, and growth was monitored by measuring optical density at A_{600 nm} over 40 h. The cultures were allowed to continue shaking at 30 °C until day 4 (roughly 96 h). B and C, on days 3 and 4, the cells were taken from the original cultures and inoculated in fresh YNB I− medium to an A_{600 nm} of 0.005. A_{600 nm} was determined at 24, 29, and 32 h. Experiments were performed with three biological replicates (all data points not shown).

Figure 9.

Gpc1 is expressed in logarithmic phase. WT strain was grown in medium lacking inositol. The cultures were harvested at log (14 h), early stationary (24 h), and late stationary (48 h) phases. Western blotting analysis was performed using anti-HA mouse IRdye 680 and goat anti-mouse IRdye 800. Glucose-6-phosphate dehydrogenase was used as a loading control. The experiment was performed in biological triplicate with identical results (no detectable expression in stationary phase).