Studies of long chain fatty acid (LCFA) uptake in mammalian cells support the existence of a high affinity protein mediated transport mechanism at the cell surface that facilitates uptake at physiologic concentrations. Two proteins that play an important role in cellular import of LCFAs were identified by a functional assay for LCFA uptake. Fatty acid transport protein (FATP), a putative plasma membrane LCFA transporter, and a long chain fatty acyl CoA synthetase (FACS) both promote the uptake of LCFAs when expressed in mammalian cells. Both proteins are expressed in high levels in adipocytes and cardiac myocytes, where by they be essential for LCFA uptake for storage and energy metabolism, respectively. Furthermore, these proteins may serve as targets for regulatory mechanisms governing LCFA import. Alterations in LCFA uptake may contribute to the pathophysiology of cardiac hypertrophy and failure, diseases in which cardiac substrate utilization shifts from LCFAs to these proteins in LCFA uptake. Structure-function analysis of the FATP molecule will include site directed mutagenesis of highly conserved amino acid residues and definition of FATP's membrane topology using chimeric FATP-reporter protein constructs. The function of FATP will be studied in mammalian over-expression system and in isolated yeast secretory vesicles. Potential interactions of FATP and FACS will be studied in 3T3-L1 adipocytes by immunofluorescence co-localization and co- immunoprecipitation. Finally, to identify other proteins, which participate in LCFA transport, we will screen a CHO-K1 cell retroviral gene trap library for mutations affecting LCFA uptake. We will also use a yeast two-hybrid screen to identify proteins that interact with FATP. These studies will provide insight to human disorders, which result from abnormal LCFA metabolism, and illuminate the physiologic roles of FATP and FAC in fatty acid homeostasis.
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