The long-term goal of this Project is to elucidate mechanisms of plasma lipoprotein assembly and secretion by hepatocytes. We have three specific aims. The first is to determine how nascent very low density lipoproteins (VLDL), recovered from highly purified Golgi fractions from rat hepatocytes, contribute to the formation of high density lipoproteins (HDL). Detailed chemical analysis of nascent VLDL from these fractions has shown that, as compared with plasma VLDL, nascent VLDL contain much more phospholipids, apo A-I in the pro form, and large amounts of apo E. Some of these surface constituents may therefore dissociate extracelllularly to form nascent discoidal HDL.
The second aim i s to elucidate the mechanism by which apo B is lipidated in the rough endoplasmic reticulum (RER). Because standard methods of isolating highly purified RER fractions from rat liver have proven inadequate, we have developed a rapid, simple technique of recovering very large amounts of morphologically intact and highly purified RER fractions from rat liver homogenates. Rupture of RER membranes, in the presence of suramin with the French pressure cell (a technique validated for releasing nascent VLDL from Golgi fractions) releases apo B, which sediments with other content proteins at a density of 1.006 g/ml; however, most apo B is recovered in an opalescent band in the middle of a metrizamide gradient that contains homogeneous discoidal particles (400-600 Angstroms in diameter). This fraction contains phospholipids, but little or no nonpolar lipids. The top fraction from the metrizamide gradient contains membrane fragments, together with small (250-300 Angstroms diameter) lipoprotein-like particles, apo B, and cholesteryl esters. We will characterize further the particles in these two fractions and investigate their relationships to early lipidation events in the assembly of nascent VLDL. The third specific aim is to generate an immunologic map of the subcellular distribution of the major proteins that participate in the assembly of nascent lipoproteins by rat hepatocytes. We have recently reported the distribution of apo E in rat liver in which some unexpected observations were made. The first requirement for success in this type of research is the development of highly specific, affinity-purified immunoglobulin probes. We now have such probes for apo B and apo A-I, and we are developing probes for the low density lipoprotein receptor- related protein (LRP) the microsomal triglyceride transfer protein and acyl CoA cholesterol acyltransferase (ACAT). The subcellular localization of these macromolecules, together with our cell fractionation studies, will provide new insights into the mechanisms of assembly of nascent plasma lipoproteins by hepatocytes.
