Abstract
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 is responsible for enhanced phospholipid synthesis that confers tolerance to the organic solvent in Saccharomyces cerevisiae. This increase in phosphatidic acid 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 (Miura, S., Zou, W., Ueda, M., and Tanaka, A. (2000) Appl. Environ. Microbiol. 66, 4883-4889); 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 ict1Delta strain showed a drastic reduction in phosphatidic acid suggesting the role of Ict1p in phosphatidic acid biosynthesis. Overexpression of Ict1p in S. cerevisiae showed an increase in phosphatidic acid 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. Specific activity of Ict1p was found to be higher for oleoyl-CoA as compared with palmitoyl- and stearoyl-CoAs. This study provides a mechanism for organic solvent tolerance from the point of membrane dynamics in S. cerevisiae.
Highlights
Ment in the ionic and the metabolic balances, pH gradient, electrical potential, etc., leading to cell lysis
Enhanced phospholipid biosynthesis was observed on treatment with 4% isooctane, suggesting a correlation between accumulation of membrane phospholipids and tolerance to organic solvent
These data are consistent with tolerance mechanisms exhibited by P. putida Idaho strain [3, 5], where the basal level of phospholipid biosynthesis was high on exposure to solvent
Summary
We initially determined the differences in the phospholipid composition in the presence and in the absence of isooctane. We studied the phospholipid profiles of ict1⌬ and ICT1 overexpressed strains on isooctane treatment. We elucidated the enzymatic function of the purified recombinant Ict1p. Our results clearly show that on treatment with 4% isooctane, the overall phospholipid biosynthesis in the cell was markedly enhanced. Ict1⌬ strain showed a reduced biosynthesis of major phospholipids both in the presence and in the absence of isooctane. The amount of phosphatidic acid (PA) was found to be drastically reduced in the ict1⌬ strain. Based on the above observations, we report here that Ict1p is a soluble lysophosphatidic acid acyltransferase that is highly expressed during organic solvent stress, to sustain enhanced requirement of membrane phospholipid for the repair of damaged membrane
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