CONTROL OF CONNEXIN TRANSPORT AND ASSEMBLY

描述：(逐字摘自申请者摘要)这项计划的长期目标 项目是了解细胞如何调节GAP的运输和组装 交汇点。新合成的连接蛋白在体内组装成六聚体 细胞内室前形成缝隙连接通道。我们 发现ROS细胞内源性地不同地组装和排序两个 表达缝隙连接蛋白CX43和CX46。ROS细胞将Cx43组装成 六聚体被运输到质膜上。相比之下，CX46 仍然是单体，仅限于反式高尔基网络(TGN)。目标是 了解CX43和CX46是如何区别的 受这些细胞的调节。为了识别控制连接蛋白靶向的结构域， 我们将制作包含Cx43和Cx43结构域的表位标记的连接蛋白嵌合体 CX46。这些细胞将被导入细胞系，然后进行分析 免疫荧光显微镜，用于转运到质膜和/或TGN。 然后将通过蔗糖梯度分馏来分析这些构建物 Triton X-100增溶细胞提取液确定所有TGN是否定位 连接蛋白以单体形式保留。然后我们将确定Cx43和 Cx46可以形成混合间隙结通道。这是一个关键点-- 这些连接蛋白形成杂交通道的兼容性意味着 是一种主动机制，可以阻止它们在ROS细胞中共同组装。混血儿 Cx43+CX46通道是在体外翻译过程中或由Hela细胞形成的 这两种蛋白质都被运输到质膜上，并被溶解为 完整的六聚体，并通过共同免疫隔离进行鉴定。最后，我们会 确定Cx46单体是否与Cx43六聚体共存 细胞内的隔间。细胞将被处理以阻止连接蛋白的运输 在不同的步骤中，含有CX46的均质和完整的囊泡将被 免疫分离，然后分析Cx43六聚体的存在。 了解差分CX43和CX46排序的基本原理 汇编将提供对缝隙连接的调节的见解 并可能反映蛋白质组装的一般原则 在后高尔基时代的隔间里。

DESCRIPTION: (Verbatim from applicant's abstract) The long term goal of this project is to understand how cells regulate the transport and assembly of gap junctions. Newly synthesized connexins are assembled into hexamers in an intracellular compartment prior to the formation of gap junction channels. We found that ROS cells differentially assemble and sort two endogenously expressed gap junction proteins, CX43 and CX46. ROS cells assemble CX43 into hexamers which are transported to the plasma membrane. In contrast, CX46 remains a monomer and is restricted to the trans Golgi network (TGN). The goal of this proposal is to understand how CX43 and CX46 are differentially regulated by these cells. To identify domains that control connexin targeting, we will make epitope tagged connexin chimeras containing domains from CX43 and CX46. These will be transfected into cell lines and then analyzed by immunofluorescence microscopy for transport to the plasma membrane and/or TGN. These constructs will then be analyzed by sucrose gradient fractionation of Triton X-100 solubilized cell extracts to determine whether all TGN localized connexins are retained as monomers. We will then determine whether Cx43 and Cx46 can form hybrid gap junction channels. This is a key point-the compatibility of these connexins to form hybrid channels would imply that there is an active mechanism to keep them from co-assembling in ROS cells. Hybrid CX43+CX46 channels formed during in vitro translation or by Hela cells where both proteins are transported to the plasma membrane will be solubilized as intact hexamers and identified by co-immunoisolation. Finally, we will determine whether Cx46 monomers co-exist with Cx43 hexamers in the same intracellular compartments. Cells will be treated to arrest connexin transport at different steps, homogenized and intact vesicles containing CX46 will be immunoisolated and then analyzed for the presence of CX43 hexamers. Understanding the fundamentals of differential CX43 and CX46 sorting and assembly will provide insights into the regulation of gap junctional communication and is likely to reflect general principles of protein assembly in post-Golgi compartments.