The mechanism by which newly synthesized membrane and secretory proteins are selectively removed from the endoplasmic reticulum (ER) and are transported to the various membrane compartments remains a central question in cell biology. Recently evidence has accumulated that clathrin coated vesicles may play an important role in the trnasport of newly synthesized plasma membrane elycoproteins to the plasma membrane. (Rothman, J. and Fine, R. Proc. Natl. Acad. Sci., 77:780-784, 1980). These studies and subsequent ones studying specifically the transport of vesicular Stomatitis Virus G protein in Chinese hamster ovary cells led to the development of biochemical techniques to purify to near homogeneity coated vesicles from small numbers of cultured cells. During the next grant period we will apply these techniques as well as cytochemical ones to investigate three hyptheses. 1) Coated vesicles are intermediates in the transport of newly synthesized secretory proteins between the ER and the plasma membrane. 2) That the same population of coated vesicles transports both newly synthesized secretory and plasma membrane proteins. 3) That newly synthesized secretory proteins are selectively bound to a membrane receptor protein during intracellular transport. We will utilize two chick embryo cell types for these studies. The isolated chick embryo tendon fibroblast actively synthesizes and secretes large quantities of Type I procollagen. We will purify coated vesicles from these cells by agarose gel electrophoresis and identify procollagen by biochemical, radiochemical and immunochemical methods. We will also identify, localize and eventually purify a putative procollagen receptor from these cells, employing 125I-procollagen binding and chemical crosslinking as important probes. Cultured myotubes will be used to determine whether the same population of coated vesicles transport newly synthesized acetylecholine receptors, an intrinsic plasma membrane protein, and acetylcholinesterase, a secretory molecule, to the plasma membrane. 125(IAlpha)-bungarotoxin binding and cytochemical localization of acetylcholinesterase will be important tools in these studies. We will also employ anti-clathrin antibodies in an enzyme linked immunoassay to determine the quantitative distribution of clathrin in liver and other cell membranes.
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