CONTROL OF VESICULAR CARRIER TRAFFIC IN HEPATOCYTES

该项目的重点是分离和表征 囊泡载体已知在真核细胞中在每个中继器中起作用 沿着内质网-质膜途径及其分支， 此外，在上皮细胞和腺细胞中。 囊泡 承运人是控制系统的一个不可分割的重要组成部分， 细胞内蛋白质和膜运输。 系统分布 在单个细胞区室中高效且准确地产生蛋白质 (the胞质溶胶）到二十多个位点（膜和隔室）， 最后的功能性住宅。 到ER的交通由蛋白质控制， 蛋白质，但从ER-高尔基体连接下游，蛋白质被分选 单独运输，并通过特定的囊泡 承运人的目的地不同，承运人是 由特定的飞行员指挥。 该项目的基本前提是， 分拣机和飞行员，即，交通控制系统的关键要素， 是囊泡载体的完整膜蛋白， 识别它们的第一步是隔离， 特定类别的囊泡载体的表征。 我们提出 使用完整大鼠的肝脏，或灌注的大鼠肝脏或原代大鼠肝脏， 大鼠肝细胞培养物，作为囊泡载体的方便来源 我们有一个可靠的标志物，聚合伊加受体，其形式和 峰标记的时间是每个主要类别的特征， 囊泡载体 我们最近通过免疫吸附分离出一种 转胞吞囊泡载体部分。 我们建议完成其 表征和重建转胞吞作用的最后一步， 无细胞系统，使用上述级分作为供体， 受体，质膜部分，包括适当的目标。 我们 建议提高抗体的完整膜蛋白的 并测试它们阻止与目标融合的能力。 同源抗原将作为推定的先导试验进行进一步研究。 抗独特型抗体将作为一种潜在的工具进行测试， 隔离分拣机。 我们打算利用胆汁淤积的肝脏 作为转胞载体的来源， 核聚变 我们打算进行分离和定性 囊泡载体在高尔基体-窦状结构域的运作， 质膜接力，以找出是否不同或相同的载体， 参与分泌和膜蛋白的运输。 由于这 工作集中在囊泡载体本身，它将 补充目前的工作所需的胞质因子的囊泡 运输 预计这两种方法之间的互动将进一步发展 我们对整个交通控制系统的理解。

This project is focused on the isolation and characterization of vesicular carriers known to operate in eukaryotic cells at each relay along the endoplasmic reticulum-plasmalemma pathway, its branches, and, in addition, across epithelial and glandular cells. The vesicular carriers are an integral and important part of the system that controls intracellular protein and membrane traffic. The system distributes efficiently and accurately proteins produced in a single cell compartment (the cytosol) to more than twenty sites (membranes and compartments) of final functional residence. Traffic to the ER is controlled protein by protein, but from the ER-Golgi junction downstream, proteins are sorted individually and transported in sorted batches by specific vesicular carriers to their different destinations to which the carriers are directed by specific pilots. The basic premise of the project is that sorters and pilots, i.e., the key elements of the traffic control system, are integral membrane proteins of the vesicular carriers and that the first step towards their identification is the isolation and characterization of specific classes of vesicular carriers. We propose the use the liver of the intact rat, or perfused rat livers or primary cultures of rat hepatocytes, as convenient sources of vesicular carriers and we have a reliable marker, the polymeric IgA receptor, whose form and time of peak labeling are characteristic for each major class of vesicular carriers. We have recently isolated, by immunoadsorption, a fraction of transcytotic vesicular carriers. We propose to complete its characterization and reconstitute the last step in transcytosis in a cell-free system using as donor the fraction mentioned above and, as acceptor, a plasmalemmal fraction that includes the proper target. We propose to raise antibodies to the integral membrane proteins of the carrier and test them for their ability to block fusion with the target. The cognate antigen will be further investigated as a putative pilot. Anti-idiotypic antibodies will be tested as a potential tool for isolating sorters. We intend to take advantage of the cholestatic liver as a source of transcytotic carriers defective in terminal vesicular fusion. We intend to proceed towards the isolation and characterization of vesicular carriers operating at the trans Golgi-sinusoidal domain of the plasmalemma relay to find out if different or the same carriers are involved in the transport of secretory and membrane proteins. Since this work concentrates on the vesicular carriers themselves, it will complement current work on cytosolic factors required for vesicular transport. Interaction between the two approaches is expected to advance our understanding of the overall traffic control system.