Apolipoprotein B-100 (apo B) is essential for the hepatic secretion of triglyceride-rich very low density lipoproteins (VLDL). The positive correlation between plasma concentrations of apo B and the risk of premature coronary heart disease suggests that an understanding of the intracellular assembly and transport of VLDL may have important implications for the prevention, pathogenesis, and management of atherosclerosis. The overall goals of the proposed research are to characterize early events in the biogenesis of VLDL and to determine how these events may contribute to the regulation of VLDL secretion. The following specific questions will be addressed: 1. Is intracellular turnover of apo B the predominant mechanism for the regulation of apo B secretion? It is known that a large percentage of newly synthesized apo B is degraded intracellularly, most likely in the endoplasmic reticulum (ER). In some cases, alterations in intracellular apo B turnover rates are responsible for the regulation of apo B secretion. To establish the generality of this mechanism, pre- and post- trans Golgi pools of apo B in HepG2 cells will be quantitated using a lectin affinity binding assay. Changes in these apo B intracellular pool sizes in response to mediators of apo B secretion will reveal if the amount of apo B shunted to a degradative compartment can quantitatively account for regulation of secretion and, if so, the general intracellular site(s) at which degradation occurs. 2. What features of the apo B molecule are responsible for its transient or stable association with the ER membrane? The ability of apo B to transiently or permanently associate with the ER membrane may be necessary for both VLDL assembly and regulation of VLDL secretion. To identify domains of apo B which are responsible for its association with the ER membrane, defined segments of apo B will be fused to a soluble secretory protein and expressed in cells of both hepatic (apo B- producing) and non-hepatic (non-apo B-producing) origin. The ability of apo B domains to direct transient or permanent membrane association and/or ER retention and degradation will be assessed using both biochemical and immunocytochemical criteria. Once identified, the consequences of altering or deleting these sequences in an intact apo B protein will be assessed. 3. Do trans-acting factors interact with apo B during its assembly into presecretory VLDL particles? The folding, assembly, and transport of many proteins within the secretory pathway requires transient assembly factors. To identify both general and specific factors which may be involved in the biogenesis and regulation of apo B-48 CDNA will be subjected to chemical cross-linking. Proteins which form cross-links with apo B as a function of metabolic labeling times will be examined in cells of both hepatic and non-hepatic origin.