Regulation of apical traffic in renal epithelial cells

描述（由申请方提供）：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。这部分是通过将新合成的膜蛋白载体递送到顶端和基底外侧表面来实现的。这些蛋白质通过高尔基体一起运输，但在不同的载体群体中从高尔基体网络（TGN）中出现。令人惊讶的是，人们对TGN中如何选择和包装顶端蛋白质知之甚少，尽管与富含糖脂的脂质微结构域的结合被认为对于顶端蛋白质子集的递送很重要。此外，越来越多的证据表明，N-和O-聚糖都可以作为某些蛋白质的顶端靶向基序。本建议旨在解决以下问题：1.通过不同机制分选的顶端蛋白质是以共同的载体还是以单独的囊泡离开TGN？2.从TGN输出顶端蛋白质需要哪些细胞质成分？和3.需要N-聚糖进行顶端递送的蛋白质的分选是如何完成的？我们的工作假设是，顶端货物装载和囊泡形成的某些方面将类似于已知的机制，操作沿着生物合成和内吞途径。我们将使用体外测定和活细胞成像来检查不同类别的顶端蛋白的运输要求：这些方法相结合将提供一个更全面的观点，顶端蛋白从TGN的出口是如何调节的。

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.