Molecular control of CPT-I and CD36 in the regulation of mitochondrial fatty acid transport

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Mitochondria influence several processes, including energy provision, apoptosis, calcium handling and redox balance, and as result are now recognized as a key organelle that influences homeostasis in almost every cell. A fundamental event influencing mitochondrial biology is the movement of long-chain fatty acids across the outer/inner mitochondrial membranes. While our understanding of the basic mechanisms regulating this process remains incomplete, carnitine palmitoyltransferase-I (CPT-I) and CD36 play key roles. While CPT-I is required mitochondrial membrane lipid transport, we aim to establish if cellular stresses can alter the sensitivity of CPT-I to various substrates, how CPT-I is regulated and what the biological consequence is to changes in catalytic flux through this enzyme. We plan to address these knowledge gaps by determining potential post-translational modifications of CPT-I during increased cellular stress (e.g. acute exercise). We anticipate that various serine residues will be phosphorylated during exercise (i.e. S330, S572, S401, S402 and S610) in association with changes in the biochemical properties of CPT-I, and therefore plan to transiently express mutated CPT-I constructs (i.e. alanine substitutions for serine to prevent phosphorylation) in a muscle specific knock out animal to study the impact on various mitochondrial and cellular parameters. This approach will enable us to determine cause-and-effect relationships between alterations in CPT-I phosphorylation, enzymatic flux, mitochondrial bioenergetics and cellular homeostasis. We have also identified that cellular stress can induce the movement of a protein (CD36) from an intracellular region to mitochondrial membranes. CD36 appears to be located upstream of CPT-I, where an accumulation influences the delivery of lipids to CPT-I to indirectly influence membrane transport, however, the molecular basis for this subcellular trafficking remains unknown. We plan to determine if the C-terminal YCACR motif of CD36 interacts with various signaling events to influence this cellular process. To achieve this, we will transiently transfect wild type and C-terminal mutants (C-terminal amino acids deleted) into the skeletal muscle of CD36 knock out mice. Thereafter we will provide metabolic challenges (e.g. muscle contraction) and determine the ability of CD36 to accumulate on mitochondrial membranes and the functional consequence. To determine the necessity of specific signalling events in mediating mitochondrial CD36 trafficking, similar experiments in genetically modified mice or with pharmacological inhibitors will be employed. Combined, the proposed studies will provide fundamental knowledge on the mechanisms regulating mitochondrial lipid transport, a process that has broad implications to understanding cellular homeostasis.