Proteomic and lipidomic analyses of lipid droplets in Aurantiochytrium limacinum ATCC MYA-1381

Abstract

Aurantiochytrium limacinum is a heterotrophic unicellular organism that develops cytosolic lipid droplets that store triacylglycerols (TGs) with a high docosahexaenoic acid (C22:6) content. Although the information on protein and lipid compositions in lipid droplets is useful for the understanding of storage lipid metabolism, it is not well known in this organism. In the present study, we purified lipid droplets by mild disruption of the cells using a hypo-osmotic buffer containing 0.05 % or 0.2 % NP-40 and analyzed the proteomics and lipidomics. In total, 29 putative lipid droplet-localizing proteins were identified. Approximately half of the lipid droplet protein content [54.0 % (w/w)] was accounted for by thraustochytrid-specific lipid droplet protein 1 (TLDP1), which might be a structural protein in the lipid droplet. The proteins included acyl-CoA synthetase (ACS), sterol 24-C methyltransferase (SMT1), diacylglycerol acyltransferase 2 (DGAT2), and heat-shock protein 70 kDa (HSP70), which are often found in lipid droplets isolated from other organisms. We also found putative orthologs of a lipid droplet-associated hydrolase which catalyzes the hydrolysis of cholesterol esters in macrophages and a monoglyceride lipase that has been identified in mammalian lipid droplets. The lipidomics showed that the major unsaturated TGs were TG60:12 [palmitic acid (C16:0)/C22:6/C22:6] and TG66:18 (C22:6/C22:6/C22:6). Phytosterol ester was more abundant in the lipid droplet fraction than the nonlipid droplet fraction. The accumulation of phytosterol ester in the lipid droplet might have a causal relationship to the localization of SMT1, which is considered to act in the phytosterol biosynthesis pathway. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were present, but the lipid droplet fraction was deficient in lysophospholipids such as lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). Through the proteome and lipidome analyses, the characteristics of proteins and lipids in A. limacinum lipid droplets were shown.