COATED VESICLES: MEMBRANE TRANSPORT IN MUSCLE, BRAIN

新合成的膜蛋白和分泌蛋白的作用机制 选择性地从内质网(ER)中移除，并被 运输到各个膜室仍然是一个中心问题 在细胞生物学中。最近，越来越多的证据表明，笼状蛋白涂层 囊泡可能在新合成的细胞转运过程中起重要作用 质膜蛋白向质膜转移。(Rothman，J.和 好吧，R·普罗克。娜塔莉。阿卡德。科学，77：780-784,1980)。这些研究和 随后专门研究水泡性口炎的转运 病毒G蛋白在中国仓鼠卵巢细胞中的表达 生物化学技术提纯近均一包被囊泡的研究 少量培养的细胞。在下一个授权期内，我们将 应用这些技术以及细胞化学技术来研究三个 假设。1)包被的囊泡是新陈代谢的中间产物。 内质网和质膜之间合成的分泌蛋白。2) 同样数量的包被囊泡将两种新的 合成分泌和质膜蛋白。3)新的 合成的分泌蛋白选择性地与膜受体结合 细胞内运输过程中的蛋白质。我们将利用两个小鸡胚胎 这些研究的细胞类型。鸡胚腱成纤维细胞的分离培养 主动合成和分泌大量I型前胶原。 我们将用琼脂糖凝胶从这些细胞中纯化包被的囊泡。 生化、放化、凝胶电泳法鉴定前胶原蛋白 免疫化学方法。我们还将确定、本地化并最终 从这些细胞中纯化一种推定的前胶原受体 125I-前胶原结合和化学交联是重要的探针。 培养的肌管将被用来确定相同数量的肌细胞 包被的囊泡运输新合成的乙酰胆碱受体，以及 内源性质膜蛋白和一种分泌物乙酰胆碱酯酶 分子，到质膜。125(IAlpha)-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。我们还将在一种 酶联免疫测定法测定人血清白蛋白定量分布 在肝脏和其他细胞膜中的网状蛋白。

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.