COATED VESICLES: MEMBRANE TRANSPORT IN MUSCLE, BRAIN

这项拨款的主要研究重点是阐明 笼状蛋白包被小泡在胞外转运中的作用(S) 新合成的钙调素(CSQ)和乙酰胆碱酯酶 (AChE)到发育中骨骼的肌浆网(SR) 肌肉和新合成的蛋白质的转移 将轴突快速输送到中枢神经末梢 神经系统。我们最近已经证明了联系服务队列和 乙酰胆碱酯酶(AChE)是两种N-连接的糖蛋白，存在于血管和肌浆网中 其形式可被内切糖苷酶H和 小麦胚芽凝集素的沉淀。它们的动力学 通过简历和上述数据与简历一致 早期中间高尔基体之间的介导性移植 和SR。在下一个授权期内，我们将准确地 确定CSQ上发现的低聚糖并搜索 终末SR池和中枢神经CSQ的可能受体 CVS。我们还将定位CSQ和另外两个主要的蛋白质 肌浆网、钙ATPase和高甘露糖糖蛋白， 使用免疫细胞化学技术。 关于快速运输进程，我们最近 分离出高度丰富的快速运输囊泡种群 从兔视神经注射35S-蛋氨酸后 兔眼。我们将进一步描述这些小泡的特征，并 确定特定的蛋白质，如P物质、葡萄糖转运体、 AChE，Kinesin，这将有助于识别快速运输 来自牛视神经和胆碱能轴突的囊泡。 一旦我们分离出大量的快速运输囊泡 我们将描述它们与激动素的相互作用 微管。我们还将免疫隔离标记的包被囊泡 视网膜的亚型，希望能证实这一假说 包被囊泡是快速运输囊泡的前驱物质。 我们还将进一步描述我们已有的类似CSQ的蛋白质 最近在大脑中发现，通过生化和 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 胞内钙离子的调控和胆碱能酶的转运 加深我们对阿尔茨海默氏症的根本原因的了解 疾病。最近有几份报告表明，钙离子 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.