The long term goal of this program is to understand the hepatocellular uptake of albumin-bound organic anions such as free fatty acids. This proposal examines how mAspAT functions as an FFA transporter. There are three specific aims: 1) How mAspAT binds FFA will be studied by molecular modeling of a possible FFA binding site. Covalent labeling with (3H)-FFA analogs followed by peptide mapping will confirm its location. A structure: function analysis will employ site directed mutagenesis and the baculo-virus expression system to prepare mutant mAspATs in which specific residues within the binding site are altered. The effects of these mutations on FFA binding will be determined. 2) mAspAT is translated as a pre-protein with a 5' signal peptide which is absent in the mature protein of both mitochondria and plasma membranes. Its sorting to the plasma membrane is not the result of differential RNA splicing. To determine its route to the plasma membrane and the site of post-translational removal of the signal peptide, the organelles involved in its intracellular trafficking will be identified by immunoelectron microscopy, and by laser confocal scanning microscopy with organelle-specific fluorescent dyes and fluorescent antibodies. Its intracellular movement will be tracked with pulse-chase studies, using subcellular fractionation and selective immunoprecipitation after in vitro labeling with (35S)-amino acids. cDNAs encoding rat mAspATs mutated in their intracellular trafficking and/or FFA binding properties will be transfected into HeLa cells. The distribution of expressed mutants will be studied with species-specific anti-peptide antibodies that distinguish the endogenous human and transfected rat mAspATs. Changes in FFA uptake will be compared with expression on the plasma membrane of binding-competent mAspAT mutants, and with their ability to enter mitochondria and other relevant organelles. 3) Finally, they will examine how approximately 20 percent of membrane bound mAspAT intercalates into the lipid bilayer, and whether it is this component that mediates FFA transport, using freeze-fracture immunoelectron microscopy of plasma membranes and a computer model of the interactions between membranes and the mAspAT dimer. These studies will help to explain how a mitochondrial matrix enzyme can serve as a plasma membrane transporter.
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