The broad objective of the proposed research is to understand the enzymology of transport of proteins between cellular organelles of the secretory pathway of eucaryotic cells. The biochemical basis for transport of a plasma-membrane protein from the endoplasmic reticulum (ER) to the Golgi will be studied using a model system based on the G protein of vesicular stomatitis virus (VSV) in infected Chinese hamster ovary (CHO) cells. Transport will be studied using a newly developed in vitro assay which measures the export of the temperature-sensitive mutant VSV strain ts045 G protein from the ER to the cis compartment of the Golgi containing the oligosaccharide processing enzyme mannosidase I. Transport between the ER and Golgi requires ATP and a cytosol fraction (a high speed supernatant of crude cell homogenates). Transport may occur in two biochemically distinct steps which correspond to the budding and fusion of transport vesicles. The goals of the proposal are: (1) To characterize the requirements for transport of G protein between compartments in the crude cell homogenate. (2) To purify, and characterize the donor ER, and acceptor cis Golgi compartments from crude cell homogenates which are active in transfer of protein in vitro. (3) To purify the soluble cytoplasmic components which are required for transport of protein, and characterize their possible role in vitro in the budding and fusion of carrier vesicles. (4) To define the morphological basis for the events occurring during transport in vitro using electron microscopy (EM), and EM immunocytochemistry. Movement of proteins between cellular organelles must require both common transport components, and specific recognition signals. An integrated biochemical and morphological approach using a experimentally versatile model system will provide a foundation to directly relate the events observed in vitro to those in vivo. Many medically important diseases result from defects in intracellular transport including lysosomal storage diseases, familial hypercholesterolemia, and cystic fibrosis. Considering the integral role of the secretory pathway in synthesis and maintenance of cell surface determinants, it is important to understand the initial stages of membrane biogenesis in regulation of cell growth and differentiation in order to achieve an understanding of the events leading to loss of growth control during oncogenesis.
Showing the most recent 10 out of 68 publications
