9506059 Black In all organisms, fatty acids (FA) and their derivatives are components of membranes, are sources of metabolic energy, and are effector molecules that regulate metabolism. This research is on the transport of long-chain fatty acids (C14-C18) into the cell, followed by their enzymatic conversion to coenzyme A thioesters prior to metabolism. These FAs traverse the cell envelope of Escherichia coli by a specific, energy-dependent process that requires the outer membrane-bound FA binding protein FadL and the inner membrane associated acyl CoA synthetase (ACS). ACS activates FAs concomitant with transport and results in net FA accumulation in the cell against a concentration gradient. Processes that govern FadL-mediated long-chain FA transport across the outer membrane will be determined by i evaluating the topology of FadL using limited proteolysis and protein modification and ii defining the FA binding pocket within FadL using the affinity labeled long-chain fatty acid 9-p-azidophenoxy nonanoic acid (3H-APNA). The contribution of acyl CoA synthetase to long-chain FA transport will be evaluated by i defining the ATP and FA binding domains within ACS using the affinity labeled ligands azido- (32P ATP and 3H-APNA, respectively, and ii mutagenesis of the fadD gene at specific sites involved in CoA and/or FA binding. Studies are also being conducted to define protein-protein interactions between the membrane-bound (FadL) and soluble protein components of this transport system using far Western analyses, and by performing experiments with glutathione S-transferase (GST) and histidine fusion proteins. Soluble protein components may interact with FadL. The H+/FA cotransporter in the inner cell membrane, and acyl CoA dehydrogenase and acyl CoA binding protein in the cell cytosol may bind specifically with ACS. %%% In living cells from bacteria to man, dietary fats (or fatty acids; FAs) are important sources of energy to support growth. This research project addresses the qu estion of how long-chain FAs are transported across the membrane into the cell to be used to produce energy. In the bacterium, Escherichia coli, long-chain FAs are taken up by an enzyme system that requires an outer membrane-bound FA binding and transport protein (FadL) and an inner membrane associated enzyme (CoA synthetase; ACS). The use of E. coli for these studies is ideal as basic questions can be answered concerning transport of long-chain FAs using biochemistry, molecular biology and molecular genetics. Work will define regions within FadL that are essential in FA transport, and will thus serve as a paradigm in understanding specific interactions between proteins and FAs. In addition, regions within acyl CoA synthetase that bind long-chain FAs and ATP will be defined, thus providing information on the role of this enzyme in FA transport. Lastly, studies are being conducted to define protein-protein interactions between the membrane-bound (FadL) and soluble protein components of the transport system. These studies will be important in further defining the transport system and by identifying specific cytosolic/membraneous protein associations that develop with FadL and with ACS for FA transport to occur. ***
