Fatty acids (FA) are critical to the maintenance of physiological homeostasis. Transport of fatty acids (FA) between aqueous phases separated by a membrane is an essential part of most of the important physiologic roles played by FA. Defects in the transport process might affect the nutritional state and/or FA transport itself may play a role in pathologies, including the interaction between cytotoxic-T-lymphocytes and tumor cells. The long term goal of the proposed research is to determine the mechanism of FA transport across membranes. Two, quite divergent, views have developed about how physiologically significant transport of FA occurs across biological membranes. One is that FA transport is mediated by the lipid phase and the second is that a membrane protein is required. The problem is complicated by the fact that most studies indicate that FA can spontaneously transfer through lipid vesicles. On the other hand. increasing interest in this issue-has led in the past few years to the identification and, in several cases, to the cloning of membrane proteins believed to be responsible for FA transport in a number of cell systems. As described In this application. however, the nature of the FA transport assay used in these studies raises serious questions concerning the interpretation of these results. It is critical to this proposal that to resolve the ambiguities of such measurements it is essential that FA transport. the movement of FA from the aqueous phase on one side of a membrane to the aqueous phase on the other, be measured directly. Consequently, the most important tool in these studies is ADIFAB, the fluorescent probe of free FA (FFA). ADIFAB trapped within vesicles or cells will be used to monitor and quantitate the movement of unlabeled FA between aqueous phases separated by membranes. The design of the proposed studies is motivated by evidence obtained during the past two years of the grant period. indicating that the rate of transport across cell membranes is faster than through simple lipid vesicles and this faster transport may be mediated by a membrane protein. To extend these observations we will first determine the mechanism of FA transfer across lipid vesicles. The time course of FA transfer across lipid bilayers will be measured in vesicles of increasing size and varying composition since preliminary results indicate that transfer rates decrease with increasing vesicle size and with increasing cholesterol content. We will then determine if rapid FA transport observed in human erythrocytes is consistent with a membrane protein-mediated process and if so we will then identify, purify. and reconstitute this protein into simple lipid vesicles to further substantiate Its function. Once the transporter has been identified, and its characteristics understood in human erythrocytes we will extend our studies of FA transport to adipocytes and murine tumor cells by measuring transport across membrane vesicles and by measuring transport in whole cells using fluorescence microphotometry.
