COATED VESICLES: MEMBRANE TRANSPORT IN MUSCLE, BRAIN

新合成的膜蛋白和分泌蛋白 选择性地从内质网（ER）中去除， 运输到不同的膜室仍然是一个中心问题 在细胞生物学中。 最近有证据表明，网格蛋白涂层 囊泡可能在新合成的蛋白质的转运中起重要作用。 质膜糖蛋白到质膜。 （Rothman，J. 好吧，R。Proc. Natl. Acad.科学，77：780-784，1980）。 这些研究和 随后专门研究水泡性口炎的运输 中国仓鼠卵巢细胞中的病毒G蛋白导致了 生物化学技术纯化到接近均匀的包被囊泡， 少量的培养细胞。 在下一个资助期内，我们将 应用这些技术以及细胞化学的调查三个 假设 1)包被囊泡是转运新的 在内质网和质膜之间合成分泌蛋白。 （二） 同一群被膜囊泡将两种新的 合成分泌和质膜蛋白。 3)新近 合成的分泌蛋白选择性地结合到膜受体上 蛋白质在细胞内运输。 我们将利用两个鸡胚 这些研究的细胞类型。 分离的鸡胚肌腱成纤维细胞 主动合成并分泌大量的I型前胶原。 我们将通过琼脂糖凝胶从这些细胞中纯化包被的囊泡 通过生化、放化、 免疫化学方法。 我们还将识别，本地化，并最终 从这些细胞中纯化推定的前胶原受体， 125 I-前胶原结合和化学交联作为重要的探针。 将使用培养的肌管来确定是否相同的肌管群。 包被囊泡转运新合成的乙酰胆碱受体， 内源性质膜蛋白和乙酰胆碱酯酶，一种分泌性 分子，到质膜。 125（I α）-银环蛇毒素结合和 乙酰胆碱酯酶的细胞化学定位将是重要的工具 在这些研究中。 我们还将采用抗网格蛋白抗体， 酶联免疫测定，以确定定量分布 肝脏和其他细胞膜中的网格蛋白。

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.