The small intestinal epithelial cell surface consists of two distinct domains, the microvillaus membrane (MVM) and the laterobasal membrane (LBM). Normal intestinal function is dependent on the maintenance of this strict surface polarity. The goal of this project is to define the biosynthetic pathways of glycoproteins that are destined to be expressed on separate domains of the enterocyte plasma membranes in vivo.
The specific aims of this project are (1) to determine if MVM proteins are transported directly from the Golgi to the MVM or are initially inserted into the LBM and subsequently redistributed to the MVM, and (2) to determine if MVM and LBM glycoproteins are transported to the cell surface in common or distinct subsets of transport vesicles.
The first aim will be accomplished by in vivo (3H)-Fucose pulse chase experiments in vivo in the rat followed by cell fractionation of the small intestinal epithelial cells to prepare highly purified MVM, LBM and Golgi membranes. Specific MVM (sucrase-Gamma dextrinase, aminooligopeptidase) and LBM (secretory component, transferrin receptor) glycoproteins will be immunoprecipitated from these membranes at intervals (5' to 180') after the pulse. Newly synthesized glycoproteins will be quantitated by scintillation counting and characterized by SDS polyacrylamide electrophoresis and radioflurography.
The second aim will be accomplished by purification of the enterocyte transport vesicle population by a combination of density gradient centrifugation and counter-current distribution separation. Double-labeled immunoelectron microscopy with two separate sized colloidal gold particles will be used to determine if specific MVM and LBM glycoproteins are found in the same or different subsets of transport vesicles.
| Hauft, S M; Kim, S H; Schmidt, G H et al. (1992) Expression of SV-40 T antigen in the small intestinal epithelium of transgenic mice results in proliferative changes in the crypt and reentry of villus-associated enterocytes into the cell cycle but has no apparent effect on cellular differentiation progr J Cell Biol 117:825-39 |
