Genetic and biochemical studies of the fatty acid transport proteins (FATP)

Fatty acids play important roles in a wide variety of cellular processes. The mechanisms for fatty acid transport across the plasma membrane are yet to be clarified. A mechanism involving both simple diffusion and membrane-associated proteins has been proposed. In support of this model, several distinct proteins involved in fatty acid uptake have been identified in prokaryotic and eukaryotic cells, one of which is the family of fatty acid transport proteins (FATPs). So far, six different isoforms of FATP have been identified in mammalian cells. Another protein, long chain fatty acyl-CoA synthetase (FACS) has also shown to be involved in long chain fatty acid uptake mediated through FATP family proteins. The yeast fatty acid transporter Fat1p is identified as the orthologue of mammalian FATP. Fat1p shows both long chain fatty acid uptake and very long chain fatty acyl-CoA synthetase (VLCS) activities. The current study was undertaken using yeast system to (1) explore the biological functions intrinsic in the FATP family proteins; (2) determine the relationship between the two functions intrinsic in these proteins, long chain fatty acid uptake and VLCS activities; and (3) define the functional and physical interaction of FATP family proteins and FACS.; First, alanine-scanning mutagenesis was used to determine the contribution of specific amino acid residues within Fat1p to the two biochemical activities intrinsic to this protein. The amino acid residue substitutions were chosen in the regions of Fat1p that are conserved within the FATP/VLCS family. The functional characterizations showed that for most fat1 mutants, there was a correlation between long chain fatty acid uptake, VLCS activities and altered fatty acid profiles. For several notable exceptions, the substitutions S258A D508A, F528A and L669R, the two functions fatty acid transport and very long-chain fatty acyl-CoA synthetase activities were distinguishable.; It is hypothesized that Fat1p is localized to the plasma membrane where it functions as a component of the fatty acid transport system. Both Fat1p and fatty acyl-CoA synthetases, Faa1p or Faa4p are required for the long chain fatty acid transport. Immunofluorescence on permeabilized yeast cell and plasma membrane fractionation showed that Fat1p, Faa1p and Faa4p were localized to plasma membrane, which supported the hypothesis that these proteins facilitate exogenous fatty acid transport across the cell membrane. The dominant negative effect of C-terminal Fat1p peptide on the long chain fatty acyl-CoA synthetase activities and the coimmunoprecipitation of Fat1p and Faa1p or Faa4p revealed a physical interaction between these proteins.; The functional studies of the murine FATP (mmFATP1–5) in yeast system brought new understanding to the mechanism by which the FATP family proteins facilitate long chain fatty acid uptake. Most of the FATP family members showed long chain fatty acid uptake and fatty acyl-CoA synthetase activities but the enzyme activities have different substrate specificities. There is a correlation between the long chain fatty acid uptake and very long chain fatty acyl-CoA synthetase activities, but in some cases the two biochemical functions were separable.; The findings from the present studies provided a platform to more completely understand the role of the FATP family of proteins in the linkage between fatty acid import and activation to CoA thioesters. This information will direct additional studies aimed at further understanding the mechanisms by which long chain fatty acid are transported through the plasma membranes.