Cytosolic Lysophosphatidic Acid Acyltransferase : Implications in Lipid Biosynthesis in Yeast, Plants and Human

Abstract

Cytosolic LPA acyltransferase in yeast An isooctane tolerant strain of S. cerevisiae KK-12 was reported to have increased saturated fatty acid content (Miura et. al., 2000). Amongst the various genes upregulated on isooctane treatment, ICT1 (Increased Copper Tolerance 1) was found to have maximal expression (Miura et. al., 2000; Matsui et. al., 2006). This gene in S. cerevisiae is encoded by YLR099C annotated as Ict1p. However, the physiological significance of Ict1p was not understood. Here we showed that an increase in the synthesis of phosphatidic acid (PA) is responsible for enhanced phospholipid synthesis, which confers organic solvent tolerance to S. cerevisiae. This increase in the PA formation is due to the upregulation of Ict1p, a soluble oleoyl-CoA dependent lysophosphatidic acid (LPA) specific acyltransferase. Analysis of Δict1 strain by in vivo [32P]orthophosphate labeling showed a drastic reduction in PA, suggesting the role of Ict1p in phospholipid biosynthesis. Overexpression of Ict1p in S. cerevisiae showed an increase in PA and the overall phospholipid content on organic solvent exposure. The purified recombinant enzyme was found to specifically acylate LPA. Specific activity of Ict1p was found to be higher for oleoyl-CoA as compared to palmitoyl-CoA and stearoyl-CoA. The study therefore, provides a mechanistic basis of solvent tolerance in S. cerevisiae.It is well known that phosphatidic acid (PA) is formed by the acylation of LPA by LPA acyltransferase. However, all the LPA acyltransferases characterized till date have distinct transmembrane domains and form a member of membrane bound biosynthetic machinery of phospholipid biosynthesis. They have a conserved signature motif, H(X)4D. Phosphatidic acid is an important precursor for the synthesis of glycerophospholipids and triacylglycerols. PA enters the biosynthetic pathway of phospholipids through a CTP-dependent activation catalyzed by CDPdiacylglycerol synthase. This enzyme forms CDP-diacylglycerol, which serves as a direct precursor for phosphatidylinositol, phosphatidylglycerol and cardiolipin. PA can also be dephosphorylated by phosphatidic acid phosphatase yielding diacylglycerol, which serves as a precursor for the formation of PE and PC through the CDP-ethanolamine and CDP-choline pathway or for the triacylglycerol synthesis through a dephosphorylation step followed by an acylation establishing it as a supreme molecule for the acylglycerol biosynthesis. Since, PA is an important intermediate and that there are mechanisms to synthesize PA, other than the conventional membrane bound pathways, we wanted to understand whether such a mechanism of PA biosynthesis is conserved across the plant and animal kingdom. Therefore, we resorted to analyze Ict1p like proteins in Arabidopsis and human whose complete genome sequence is available. Cytosolic LPA acyltransferase in Arabidopsis Homology search with ICT1 in Arabidopsis thaliana genome, led to the identification of At4g24160 as a close relative. In order to gain an insight into the significance of such proteins in plants we performed a genome wide survey of At4g24160 like proteins in Arabidopsis. We identified that A. thaliana genome encodes twenty four At4g24160 like proteins, most of which belong to the α/β- hydrolase family of proteins and possess a distinct lipase motif (GXS/NXG). Interestingly, amongst these twenty four, only At4g24160 has a conserved HX4D motif. Domain analysis of these proteins suggests a wide functional diversification during evolution. Gene expression studies revealed their importance during various abiotic stress. Bacterial expression of At4g24160 followed by its purification using Ni2+-NTA column chromatography and characterization revealed it to be a LPA acyltransferase. Expression analysis showed that it is highly expressed in the pollen grains followed by the root cap. In addition, the gene was found to be upregulated under salt stress conditions. Direct correlation between salt stress and phospholipid biosynthesis is well known in the literature. We envisage that At4g24160 might be one of the gene products involved in membrane repair when exposed to such a stressCytosolic LPA acyltransferase in human Homology search with Ict1p revealed another interesting candidate protein in Homo sapiens known as Comparative Gene Identification–58 (cgi-58). Mutations in CGI- 58 are known to be the causative reason for a rare autosomal recessive genetic disorder known as Chanarin-Dorfman syndrome characterized by the excessive TG accumulation and defective membrane phospholipid regulation in several tissues. It is known to be a coactivator of adipose triglyceride lipase (ATGL), promoting lipolysis of TG (Lass et. al., 2006). However, the exact biochemical role remains unknown. To understand the biochemical function of cgi-58, the gene was overexpressed in E. coli and the purified, recombinant protein was found to specifically acylate lysophosphatidic acid in an acyl-CoA dependent manner. Overexpression of CGI-58 in Δict1 rescued the metabolic defect of the strain. Heterologous overexpression of CGI-58 in S. cerevisiae followed by metabolic labeling with [32P]orthophosphate showed an increased biosynthesis of membrane phospholipids. Analysis of neutral lipid biosynthesis by [14C]acetate labeling showed an increase in DG and free fatty acids. However, marked decrease in the TG biosynthesis was seen. Decrease in TG was confirmed by ESI-MS. In addition, physiological significance of cgi-58 in the mice white adipose tissue is reported in this thesis. We found soluble lysophosphatidic acid acyltransferase activity in the mice white adipose tissue. Immunoblot with anti-Ict1p antibodies followed by MALDI-TOF analysis of the cross reacting protein in lipid droplets revealed its identity as cgi-58. These observations suggest the existence of an alternate cytosolic phosphatidic acid biosynthetic pathway in the white adipose tissue. Collectively, our observations suggest a possible involvement of cgi-58 in the phospholipid biosynthesis of adipocytes and its probable role in maintaining the TG homeostasis. In conclusion, the study reveals the significance of cytosolic lipid metabolic enzymes having conserved biochemical function, in maintaining homeostasis in living organisms across phylogeny.