PROPOSED PROGRAM (Applicant?s abstract) This Program Project combines techniques from lipid photochemistry, biochemistry and molecular and cell biology to characterize the structure and properties of the complexes formed between free cholesterol (FC) and phospholipid (PL) with two lipid binding proteins. Caveolin is a major structural protein of cell surface caveolae. Apolipoprotein A-1 (apo A-1) is the major component of high density lipoprotein (HDL), the major atheroprotective lipoprotein of human plasma. Photoactivable FC and PL analogs modified with benzophenone groups at different points in their structure will be synthesized and incorporated into living cells and crosslinks to caveolin and apo A-1 identified. The identity of lipid binding sites will be confirmed using site-directed mutagenesis. In further studies on caveolae, the mechanism by which vanadate, an inhibitor of protein phosphotyrosine phosphatases, reduces FC efflux will be identified. The hypothesis will be explored that phosphorylation of caveolin displaces FC from its binding site, with effects on signal transduction from the cell surface that lead to suppression of caveolin transcription. How oxysterols inhibit FC efflux will also be determined, and in particular, whether these lipids displace FC from caveolin. In studies of apo A-1-PL complex formation, the mechanism by which the ABC1 transporter protein transfers phosphatidyl choline to lipid-free apo A-1 will be analyzed in detail. The origin and mechanism of incorporation of FC into these complexes will be determined, and in particular, whether FC binds directly to apo A-1 or only via PL. Finally, they will investigate whether FC within lipid-poor apo A- 1 /PL/FC complexes formed at the cell surface can be directly esterified by lecithin: cholesterol acyltransferase, and the esters transferred to other HDL particles. In spite of its significance in defining the properties of the cell membrane, there has been little investigation of protein-FC binding. As a result, the information to be obtained in this program will be both novel and highly relevant to understanding the structure and functions of caveolae, the molecular basis of both FC and PL efflux, and the structure of HDL.