COATED VESICLES: MEMBRANE TRANSPORT IN MUSCLE, BRAIN

这项资助的主要研究重点是阐明 网格蛋白包被的囊泡（CV）在胞吐转移中的作用 新合成的钙螯合蛋白（CSQ）和乙酰胆碱酯酶 （AChE）的肌浆网（SR） 肌肉和新合成的蛋白质的转移， 用于快速轴突运输到中枢神经末梢 神经系统 我们最近证明了CSQ和 乙酰胆碱酯酶，两个N-连接的糖蛋白，发现在CV和SR 以易于被糖苷内切酶H裂解形式存在， 麦胚凝集素沉淀法。 他们的动力学 通过CV，上述数据与CV一致 介导的早期中间高尔基体之间的转移 和SR.在下一个赠款期间，我们将准确地 识别CSQ上发现的寡糖，并搜索 末端SR池和中CSQ的推定受体 栽培变种 我们还将定位CSQ和其他两个主要的蛋白质， SR、钙ATP酶和高甘露糖糖蛋白， 用免疫细胞化学技术。 关于快速运输过程，我们最近 分离出一个高度富集的快速运输囊泡群 35 S-蛋氨酸注入兔视神经后， 兔眼 我们将进一步表征这些囊泡， 鉴定特定蛋白质，例如P物质、葡萄糖转运蛋白， 乙酰胆碱酯酶，驱动蛋白，这将允许识别快速运输 来自牛视神经的囊泡和胆碱能轴突轨迹。 一旦我们分离出大量的快速运输囊泡 我们将描述它们与驱动蛋白的相互作用， 微管 我们还将免疫隔离标记的包被囊泡 视网膜上的亚型，希望能证实这一假设 包被囊泡是快速运输囊泡的前体。 我们还将进一步表征我们所拥有的CSQ样蛋白质， 最近通过生物化学和 免疫细胞化学方法。 我们的长期目标是将我们获得的知识应用于 控制胞质Ca++和胆碱能酶转运到 增加我们对老年痴呆症潜在原因的了解 疾病 最近有几份报告表明Ca++ 阿尔茨海默病患者的细胞缺陷。

The primary research focus of this grant is the elucidation of the role(s) of clathrin coated vesicles (CVs) in the exocytic transfer of newly synthesized calsequestrin (CSQ) and acetylcholinesterase (AChE) to the sarcoplasmic reticulum (SR) of developing skeletal muscle and in the transfer of newly synthesized proteins destined for rapid axonal transport to the nerve endings of the central nervous system. We have recently demonstrated that CSQ and AChE, both N-linked glycoproteins, are found in the CVs and SR in a form succeptible to cleavage by Endoglycosidase H and precipitation by Wheat Germ Agglutinin. The kinetics of their passage through CVs and the above data are consistent with a CV mediated transfer between the early intermediate Golgi apparatus and the SR. During the next grant period, we will precisely identify the oligosaccharide found on CSQ and search for a putative receptor for CSQ in the terminal SR cisternae and in CVs. We will also localize CSQ and two other major proteins of the SR, the calcium ATPase and the high mannose glycoprotein, using immunocytochemical techniques. With respect to the rapid transport process, we have recently isolated a highly enriched population of rapid transport vesicles from rabbit optic nerve after 35S-methionine injection into the rabbit eye. We will further characterize these vesicles and identify specific proteins e.g. substance P, glucose transporters, AChE, kinesin, which will allow identification of rapid transport vesicles from bovine optic nerve and cholinergic axon tracks. Once we have isolated rapid transport vesicles in large amounts we will characterize their interactions with kinesin and microtubules. We will also immunoisolate labelled coated vesicles subtypes from the retina to, hopefully, confirm the hypothesis that coated vesicles are precursors to rapid transport vesicles. We will also further characterize a CSQ like protein we have recently identified in brain, by biochemical and immunocytochemical methods. Our long term goal is to apply the knowledge we gain on the control of cytosolic Ca++ and transport of cholinergic enzymes to increase our understanding of the underlying cause of Alzheimer's disease. There are several recent reports suggesting a Ca++ deficit in cells from patients with Alzheimer's disease.