New, compelling evidence is presented indicating that human mitochondria contain an acyl carrier protein (ACP)-dependent system for synthesis of fatty acids from malonyl-CoA. Sequence and functional analyses indicate that nuclear-encoded, mitochondrially-targeted fatty acid synthase (mitFAS) components are individual proteins resembling the FASs of prokaryotes and thus differ from the eukaryotic cytosolic FASs, in which all of the enzymes are present on a single polypeptide chain. The mitFAS ACP is associated with complex 1 of the respiratory chain. A similar system has recently been described in fungi; disruption of this mitFAS system results in respiratory deficient phenotypes, indicating that it is timely to determine whether this 'new' metabolic pathway may also play a critical role in human mitochondrial function. The proposal has three objectives: to identify, isolate and characterize individual components of the human mitFAS, to identify the products they generate and to assess the significance of the pathway to mitochondrial function. (1) Candidate mitFAS components, identified by BLAST searches of the human sequence database, will be cloned, expressed, purified and their substrate specificities determined. Confirmation that they are authentic mitochondrial proteins will be sought by demonstrating that mitFAS-green fluorescent-protein chimeras expressed in human cells are transported to the mitochondria only when putative N-terminal targeting sequences are present. (2) The products formed by the mitFAS pathway will be characterized by exposing permeabolized mitochondria to various radiolabeled substrates and identifying the products chromatographically. Particular attention will be paid to the possibility that the pathway generates octanoate, the precursor of lipoic acid, and/or long chain fatty acids that could be used in the biosynthesis of mitochondrial phospholipids. (3) The significance of the pathway to mitochondrial function will be assessed by silencing expression of mitFAS components, through RNA-mediated interference, and determining the effect on cellular morphology, mitochondrial respiratory capacity and mitochondrial phospholipid composition. Failure in mitochondrial function has been implicated in the pathogenesis of late developing neurodegenerative disorders such as Parkinson's, Alzheimer's, and Huntington's diseases, yet the role of many mitochondrial proteins is still unknown. Elucidation of the role of the mitFAS system in mitochondrial function may aid in understanding the etiology of these disorders.
