Dr. Hubbard's long-term objective is to understand how epithelial cells establish and maintain their polarized surfaces. The post-transitional transport pathways of five well-characterized, endogenous, domain-specific integral glycoproteins of the rat hepatocyte plasma membrane (3 apical (AP) and 2 basolateral (BL) ) were followed in vivo. The results obtained suggest a mechanism for hepatocyte plasma membrane (PM) biogenesis in vivo in which all integral plasma membrane proteins are shipped first to the basolateral domain followed by the specific retrieval and transport of apical proteins to the apical domain at distinct rates. This newly-uncovered pathway can be perturbed in several ways. Bile duct ligation leads to the accumulation of the three apical proteins around the bile canalicular (apical) domain in putative transport structures. Low doses of colchicine in vivo lead to the accumulation of these same proteins in the basolateral membrane. A reversible colchicine analogue has been identified for use in the isolated perfused liver to accumulate all five membrane proteins in a late Golgi pre-basolateral compartment. These models will be exploited to accomplish the following: (1) identification of the compartment accumulating the molecules along the biogenetic pathway, using improved immuno-electron microscopic methods and subcellular fractionation in conjunction with metabolic labeling (amino acids, monosaccharides); (2) isolation of putative intermediates using antibodies directed against cytoplasmic domains of proven transmembrane proteins; and (3) biochemical, morphological and immunological characterization of the isolated structures. Kinetic experiments in combination with analytical subcellular fractionation will be conducted to trace the intracellular biogenetic pathway of alkaline phosphatase, an apical membrane protein that is anchored via glycocyl phosphatidylinositol. Dr. Hubbard will attempt to determine if it follows the same route as that of the three single-spanning apical proteins currently being studied. Finally, the uptake and metabolism of precursors of sphingomyelin and glycosyl-sphingolipids (fluorescent and radiolabeled) will be studied in the perfused liver with the goal of determining their transport kinetics and routes through the Golgi to the different plasma membrane domains.
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