Identification of a novel GPCAT activity and a new pathway for phosphatidylcholine biosynthesis in S. cerevisiae

Abstract

Turnover of phospholipids in the yeast Saccharomyces cerevisiae generates intracellular glycerophosphocholine (GPC). Here we show that GPC can be reacylated in an acylCoA-dependent reaction by yeast microsomal membranes. The lysophosphatidylcholine that is formed in this reaction is efficiently further acylated to phosphatidylcholine (PC) by yeast microsomes, thus providing a new pathway for PC biosynthesis that can either recycle endogenously generated GPC or utilize externally provided GPC. Genetic and biochemical evidence suggests that this new enzymatic activity, which we call GPC acyltransferase (GPCAT), is not mediated by any of the previously known acyltransferases in yeast. The GPCAT activity has an apparent V max of 8.7 nmol/min/mg protein and an apparent K m of 2.5 mM. It has a neutral pH optimum, similar to yeast glycerol-3-phosphate acyltransferase, but differs from the latter in being more heat stable. The GPCAT activity is sensitive to N-ethylmaleimide, phenanthroline, and Zn 21 ions. In vivo experiments showed that PC is efficiently labeled when yeast cells are fed with [ 3 H]choline-GPC, and that this reaction occurs also in pct1 knockout strains, where de novo synthesis of PC by the CDP-choline pathway is blocked.This suggests that GPCAT can provide an alternative pathway for PC biosynthesis in vivo.-Stålberg, K., A. C. Neal, H. Ronne, and U. Ståhl. Identification of a novel GPCAT activity and a new pathway for phosphatidylcholine biosynthesis in S. cerevisiae.

Cloning of Glycerophosphocholine Acyltransferase (GPCAT) from Fungi and Plants

“…Stålberg et al (7) reported that yeast also possessed acyl-CoA:GPE acyltransferase activity. To test whether this activity was catalyzed by ScGPCAT, we performed assays with [ 14 C]GPE and 18:1 acyl-CoA in microsomal preparations of ale1 Δ gpc1 Δ strain transformed with GPC1 or empty vector (Fig.…”

“…Stålberg et al (7) demonstrated that the generally accepted assumption that GPC cannot be acylated and thus could not be a substrate for the synthesis of PC was incorrect. These authors (7) demonstrated acyl-CoA-dependent GPC acylation (GPCAT reaction) in microsomal fractions from yeast, and later Lager et al (8) showed the same enzyme reaction in a microsomal fraction from plants.…”

“…GPC is known to be catabolized to free choline and glycerol 3-phosphate (G3P) (3–5), and the resulting choline is used in the de novo synthesis of PC via the CDP-choline pathway (6). Importantly, direct acylation of GPC by an acyl-CoA-dependent activity was recently demonstrated in Saccharomyces cerevisiae cell-free extracts and microsomal preparations (7). The putative enzyme carrying out the acylation of GPC was named GPC acyltransferase (GPCAT) (see Fig.…”

Role of Aminoalcoholphosphotransferases 1 and 2 in Phospholipid Homeostasis in Arabidopsis

“…It is possible that some of the lysoPE is converted to its methylated form by a currently unknown mechanism and then reacylated to produce PC as the final product (Figure 12). In addition, the recently reported enzyme glycerophosphocholine acyltransferase (GPCAT), which acylates glycerol-3-phopshocholine to synthesize lysoPC (Stålberg et al, 2008;Lager et al, 2015), may contribute to the reproduction of lysoPC in AAPT-deficient plants. Glycerol-3-phopshocholine is believed to be produced from deacylation of PC, rather than through de novo PC biosynthesis.…”

Regulation of phospholipid synthesis in yeast

“…lysoPE and lysoPC are acylated to PE and PC, respectively, by the ALE1-encoded lysophospholipid acyltransferase (21,22,24,25). Short acyl chain PC, which is not incorporated into membranes, is remolded with 16-and 18-carbon acyl chains (26) through the activities of phospholipase B and lysophospholipid acyltransferase (27)(28)(29). Kennedy pathway mutants (e.g., cki1 eki1 and cpt1 ept1) defective in both the CDP-choline and CDPethanolamine branches synthesize PC only via the CDP-DAG pathway (19,30,31).…”