ApoE is found in a number of animal species and although it is synthesized by various cell types, most plasma apoE is produced by the liver. In the circulation, apoE participates in lipid transport by mediating lipoprotein binding to the B/E receptor. A key factor in plasma lipid transport is the capacity of apoE to exchange between lipoprotein surfaces; however, not all apoE is capable of transfer, and not all lipoprotein-associated apoE is receptor active. The factors which govern apoE transfer between lipoproteins and receptor binding capacity are unclear. Two lines of evidence demonstrate that apoE secreted by the liver may be modified by fatty acid acylation. Whether this acylation is related to the functional state of plasma apoE is not known. Recently it has been suggested that a portion of the apoE synthesized by hepatic cells is not secreted, but remains within the cell where it may participate in intracellular lipid transport. The signal which determines whether apoE is secreted or remains in the cell is undefined, however, in other systems, targeting of proteins to specific cellular sites is accomplished by fatty acid acylation. The possibility that fatty acid acylation of apoE might affect its functional capacities in the plasma and/or its trafficking within the cell is intriguing. The goal of this project is to understand the factors which affect apoE transferability and receptor binding capacity and control targeting of apoE to intra- and extracellular pools. This goal will be accomplished through four aims designed to examine the existence and origins of different physical states of apoE and to probe their influence on apoE functional capacity. First, the physical and physiological state of the apoE component of nascent VLDL (isolated from the liver) will be examined by determining the susceptibility of apoE to thrombin cleavage, the capacity of apoE to transfer off of VLDL, and the capacity of apoE to mediate binding of nascent VLDL to the B/E receptor. The second aim is to examine fatty acid acylation of apoE and determine the appearance of apoE acylation in differing cell types and animal species, the localization of acyl-apoE in a specific lipoprotein class, the fatty acid specificity and site(s) of acylation, and the physical and physiological consequences of apoE acylation. The third aim is to examine cellular aspects of apoE acylation and determine the role of acylation in targeting of apoE to intra- and extracellular pools, the temporal relationship of apoE acylation to protein synthesis and post-translational modifications, and factors which influence apoE acylation. Finally, site-directed mutagenesis will be used to modify the acylation site(s) of apoE, and the cDNA will be transfected into hepatocytes to examine the effect of the modifications on targeting of apoE to intra- and extracellular pools, association of apoE with specific lipoproteins, and apoE transferability and thrombin sensitivity.