Regulation of apical traffic in renal epithelial cells

描述（由申请人提供）：肾细胞功能需要维持具有不同蛋白质和脂质组成的极化质膜结构域。这在一定程度上是通过将新合成的膜蛋白以载体的方式传递到细胞的顶端和底外侧表面来完成的。这些蛋白质通过高尔基体一起运输，但在不同的携带者群体中从反式高尔基网络（TGN）中产生。令人惊讶的是，尽管与富含糖脂的脂质微结构域的关联被认为对一部分根尖蛋白的递送很重要，但对于根尖蛋白如何在TGN中被选择和包装知之甚少。此外，越来越多的证据表明，N-聚糖和o -聚糖都可以作为一些蛋白质的顶端靶向基序。本提案旨在解决以下问题：通过不同机制分类的根尖蛋白是否在共同载体中或在单独的囊泡中退出TGN ？2. 从TGN输出根尖蛋白需要哪些细胞质成分？和3。如何对需要n -聚糖进行顶端递送的蛋白质进行分类？我们的工作假设是，一些方面的顶端货物装载和囊泡形成将类似于沿生物合成和内吞途径操作的已知机制。我们将使用体外实验和活细胞成像来检查不同种类的根尖蛋白的运输需求：这些方法结合起来将提供一个更全面的视角，了解根尖蛋白如何从TGN出口受到调节。

DESCRIPTION (provided by applicant): Kidney cell function requires the maintenance of polarized plasma membrane domains with distinct protein and lipid compositions. This is accomplished in part by the vectorial delivery of newly synthesized membrane proteins to the apical and basolateral surface. These proteins traffic together through the Golgi, and but emerge from the trans-Golgi network (TGN) in distinct populations of carriers. Surprisingly little is known about how apically destined proteins are selected and packaged in the TGN, although association with glycolipid-enriched lipid microdomains is thought to be important for the delivery of a subset of apical proteins. In addition, there is accumulating evidence that both N- and O-glycans can function as apical targeting motifs on some proteins. This proposal is designed to address the following questions: 1. Do apical proteins that are sorted via distinct mechanisms exit the TGN in common carriers or in separate vesicles? 2. What cytoplasmic components are required for the export of apical proteins from the TGN? and 3. How is the sorting of proteins that require N-glycans for apical delivery accomplished? Our working hypothesis is that some aspects of apical cargo loading and vesicle formation will resemble known mechanisms that operate along the biosynthetic and endocytic pathways. We will use in vitro assays and live-cell imaging to examine the transport requirements for distinct classes of apical proteins: these approaches in combination will provide a more comprehensive view of how apical protein export from the TGN is regulated.