Characterization of Tgl2, a putative lipase in the IMS of yeast mitochondria

Abstract

Yeast cells store excess lipids as steryl esters (SEs) and triacylglycerols (TAGs) in lipid droplets, preventing lipotoxicity and serving as energy reservoirs. When required, TAG lipases hydrolyze these TAGs into diacylglycerols (DAGs) and free fatty acids which can be used in various cellular pathways as signaling molecules, precursors for phospholipid biosynthesis, and more. The yeast Tgl (triacylglycerol lipase) family of lipases includes Tgl1 through Tgl5, of which all members except Tgl2 are found in lipid droplets. While Tgl3, Tgl4 and Tgl5 have dual functions of a TAG lipase as well as an acyltransferase, Tgl1 is primarily an SE hydrolase with minimal lipase activity. Tgl2 is an unusual member of the Tgl family due to its smaller size and mitochondrial localization. Predominantly homologous to bacterial lipases, Tgl2 shows lipolytic activity toward DAGs and TAGs and can complement for the loss of a DAG kinase in E. coli. The protein contains the conserved A/GXSXG lipase motif with a catalytically active serine, loss of which abrogates protein activity. Within the mitochondria, the protein localizes to the intermembrane space (IMS) and its import is regulated by the Mia40-dependent disulfide relay pathway. While a single deletion of TGL2 has no growth defect, a combined Loss of TGL2 and MCP2, an ERMES suppressor and was recently shown to be involved in CoQ metabolism, results in increased cellular TAG levels and a growth phenotype that can be rescued by reintroducing Tgl2. In this study, we characterized the protein in greater detail to get a better understanding of its molecular function. Tgl2 was found to homodimerize in the absence of reducing agents, likely due to disulfide bridges. Additionally, Tgl2 also forms a thiol-dependent complex in native PAGE. The lipase motif (A/GXSXG) mutant, S144A, which was previously reported to be essential for the protein's lipolytic activity was further characterized where the mutated protein mimicked the native variant on SDS- and native PAGE but fails to rescue the growth phenotype of Δmcp2/Δtgl2. We also identified the catalytic triad of Tgl2, consisting of Ser-Asp-His where mutating the aspartate residue (D259) resulted in instability and loss of enzymatic activity. Steady-state level analyses of non-functional Tgl2 variants revealed that Yme1 is the protease responsible for Tgl2's quality control. Finally, mitochondria lacking both MCP2 and TGL2 have increased TAG levels.