Functional analysis of proteins involved in mitochondrial lipid homeostasis
Biochemistry
Final Report Abstract
Mitochondria are organelles with multiple functions. During the endosymbiosis of an αproteobacterium, they became a compartment with two membranes, the mitochondrial outer and an inner membrane (MOM and MIM, respectively), both unique in their protein and lipid composition, separating two subcompartments, the matrix and the intermembrane space (IMS). Mitochondrial membranes are dynamic and undergo a continuous lipid exchange. Although cardiolipin and phosphatidylethanolamine are synthesised in mitochondria from phosphoglycerolipid (PGL) precursors, these precursors which are also membrane constituents and all other mitochondrial PGLs are imported, mainly from the ER. In the model organism S.cerevisiae this lipid exchange is manly mediated by the ER-mitochondria encounter structure (ERMES) complex. We have previously shown that the loss of this pathway in yeast can be partially rescued by overexpression of a MIM kinase facing the IMS, called Mdm10 complementing protein (Mcp) 2. Membrane lipids are then redistributed between the MOM and MIM across the IMS by lipid transfer proteins. The second family of cellular lipids are neutral lipids such as triacylglycerides (TAG). They play a crucial role in the storage of energy, fatty acids and PGL precursors. Although TAGs play a role in α-proteobacterial metabolism, little is known about whether they are important for mitochondrial processes. It was therefore a surprise when we identified in a systematic genetic array, the interaction of MCP2 with the gene for the predicted triacylglycerol lipase (Tgl) 2. Tgl2 is a protein of the IMS. It belongs to a family of lipases in yeast, whose other members are found in lipid droplets (LDs). Previous in vitro studies suggest a lipolytic activity of Tgl2 towards short-chain TAGs and DAGs and long-chain TAG substrates which play a relatively minor role in mitochondrial metabolism. Although the loss of Tgl2 does not affect cell growth, the loss of both Tgl2 and Mcp2 not only slows growth but also leads to an increase in total cellular TAG/PGL ratio of yeast cells, supporting an involvement of mitochondria in TAG metabolism. The main aim of our studies was to further characterise Tgl2 - its molecular structure and function - and to establish a comprehensive link between a putative TAG lipase in the IMS and cellular neutral lipid metabolism. We were able to show that Tgl2 forms a higher molecular weight complex and also forms intermolecular disulfide bridges in its native form. The protein is partially attached to the MIM and its predicted lipase/acyltransferase domain is required for function in vivo. Non-functional Tgl2 is degraded by the AAA protease Yme1 and we also observed a genetic functional relationship between MCP2, TGL2 and YME1. Our data suggest that the increased TAG/PGL ratio in cells lacking Tgl2 and Mcp2 is of mitochondrial origin.
Publications
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Characterization of Tgl2, a putative lipase in yeast mitochondria.
Tiku, Vitasta; Tatsuta, Takashi; Jung, Martin; Rapaport, Doron & Dimmer, Kai Stefan
