10.4225/03/58d1d29b673b9 Ramakrishnan, Geetha Geetha Ramakrishnan Lysophosphatidic acid acyltransferases in yeast and human Monash University 2017 1959.1/476833 ICT1 Restricted access and full embargo monash:63601 thesis(doctorate) ATGL ethesis-20110225-031126 CGI-58 2011 Chanarin dorfman syndrome 2017-03-22 01:25:45 Thesis https://bridges.monash.edu/articles/thesis/Lysophosphatidic_acid_acyltransferases_in_yeast_and_human/4774876 One of the major determinants of organic solvent tolerance is the increase in membrane phospholipids. Here we report for the first time that an increase in the synthesis of phosphatidic acid (PA) is responsible for enhanced phospholipid synthesis that confers tolerance to the organic solvent in Saccharomyces cerevisiae. This increase in PA formation is because of the induction of Ict1p, a soluble oleoyl-CoA:lysophosphatidic acid acyltransferase. YLR099C (ICT1) was reported to be maximally expressed during solvent tolerance. However, its physiological significance was not understood. In silico analysis revealed the absence of any transmembrane domain in Ict1p. Domain analysis showed that it has a hydrolase/acyltransferase domain with a distinct lipid-binding motif and a lysophospholipase domain. Analysis of ict1Δ strain showed a drastic reduction in PA suggesting the role of ict1p in PA biosynthesis. Overexpression of Ict1p in S. cerevisiae showed an increase in PA and other phospholipids on organic solvent exposure. To understand the biochemical function of Ict1p, the gene was cloned and expressed in Escherichia coli. The purified recombinant enzyme was found to specifically acylate lysophosphatidic acid (LPA). This study provides a mechanism for organic solvent tolerance from the point of membrane dynamics in S. cerevisiae. Knowing the importance of Ict1p in phospholipid metabolism and combating stress, we wanted to understand the significance of Ict1p like proteins in Homo sapiens. BLAST analysis of human genome with Ict1p sequence, resulted in the identification of Comparative Gene Identification-58 (CGI-58) gene with unknown biochemical function. CGI-58 the gene responsible for Chanarin-Dorfman Syndrome (CDS) would be the first example of an association between a disorder and defects in the function of lipid droplets associated protein. CGI-58 coordinates with several proteins including Adipose TriGlyceride Lipase (ATGL), a putative rate-limiting enzyme for triacylglycerol (TG) degradation in adipocytes. CGI-58 and ATGL are the members of the α/β hydrolase family of proteins and they are the product of the causal gene of CDS. The mutations that affect this association of ATGL and CGI-58 hinder the equilibrium of biosynthesis and degradation of TG. Apart from the lipolytic defect, CDS patients also characterized by defective recycling of TG derived product to phospholipids. The biochemical role of these two genes still remains unknown. To understand the biochemical function of CGI- 58 and ATGL, the genes were overexpressed in E. coli and the purified, recombinant proteins were found to specifically acylate LPA in an acyl-CoA dependent manner. Overexpression of CGI-58 in (ict1Δ) and TriGlyceride Lipase defective yeast strain (tgl4Δ) rescued the metabolic defect of the strains. Heterologous overexpression of CGI-58 and ATGL in S. Cerevisiae showed an increased biosynthesis of membrane phospholipids and altered neutral lipid. The site directed mutants, generated for ATGL and CGI-58 have a significant effect on acyltransferase activities. Our observations suggest a possible involvement of CGI-58 and ATGL in phospholipid biosynthesis of adipocytes and its probable role in maintaining the TG homeostasis in lipid droplets.