MECHANISMS OF ENTEROCYTE CELL SURFACE POLARITY

本提案的长期目标是定义

The long term objectives of this proposal are to define the cellular mechanisms responsible for sorting of cell proteins that are destined to be expressed on separate domains of the enterocyte plasma membrane. The specific aims are (1) to define the biosynthesis of microvillar and basolateral membrane proteins in the enterocyte using a combination of in vivo biosynthetic studies and immunoelectron microscopy. Dual-labeled immunogold techniques will be used for the simultaneous localization of amino-oligopeptidase (a microvillar membrane protein) and secretory component (a basolateral membrane protein); (2) to determine if solubilization or phosphorylation of secretory component is important in directing its transport from the basolateral membrane to the microvillar membrane. Anti-SC immunoblots will be used to detect conversion of the membrane to soluble form of the enzyme. The location of the solubilizing enzyme will be determined by incubating subcellular fractions in the standard assay to detect solubilization. Upon identifying inhibitors of the in vitro solubilization assay, attempts will be made to inhibit solubilization in vivo in the isolated perfused rat liver. Inhibition of SC solubilization will be assessed by the failure of release and secretion of secretory component in the bile. The dependence of SC phosphorylation on ligand binding will be assessed in the isolated perfused rat liver as an indirect means of determining whether SC phosphorylation is important in directing the transport of secretory component from the basolateral membrane to the microvillar membrane; (3) to develop an in vitro system for the study of Golgi to plasma membrane transport: this transport system will be modeled after the in vitro transport systems which have been developed to study the transport of proteins between the Golgi stacks: SDS polyacrylamide gel electrophoresis and radiofluorography will be used to assess transport of radiolabeled proteins from the donor Golgi membrane to the non-radiolabeled acceptor brush border membrane. These studies are aimed at answering the basic question of how enterocytes which are morphologically and functionally polar establish and maintain their cell surface polarity.