CONTROL OF CONNEXIN TRANSPORT AND ASSEMBLY

描述：（申请人摘要中的逐字逐句）本项目的长期目标 该项目旨在了解细胞如何调节间隙的运输和组装 交叉点新合成的连接蛋白在一个细胞中组装成六聚体。 在间隙连接通道形成之前，细胞内区室的结构。我们 发现活性氧细胞差异组装和排序两个内源性 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。相比之下，CX 46 仍然是一个单体，并限于反式高尔基体网络（TGN）。目标 本提案的目的是了解CX43和CX46在 由这些细胞控制。为了鉴定控制连接蛋白靶向的结构域， 我们将制备含有来自CX 43的结构域的表位标记的连接蛋白嵌合体， CX 46。这些将被转染到细胞系中，然后通过 免疫荧光显微镜检查用于运输到质膜和/或TGN。 然后通过蔗糖梯度分级分离分析这些构建体。 Triton X-100溶解的细胞提取物，以确定是否所有TGN定位 连接蛋白作为单体保留。然后我们将确定Cx43和 Cx46可以形成混合间隙连接通道。这是一个关键点- 这些连接蛋白形成混合通道的相容性意味着， 是阻止它们在活性氧细胞中共组装的一种主动机制。混合 CX43+CX46通道在体外翻译期间或由Hela细胞形成，其中 这两种蛋白质都被转运到质膜， 完整的六聚体并通过共免疫分离鉴定。最后我们将 确定Cx46单体是否与Cx43六聚体共存于相同的细胞中， 细胞内区室将处理细胞以阻止连接蛋白转运 在不同的步骤中，将含有CX 46的均质化和完整的囊泡 免疫分离，然后分析CX43六聚体的存在。 了解差异CX43和CX46排序的基本原理， 组装将提供对缝隙连接调节的见解， 这可能反映了蛋白质组装的一般原则 在高尔基体后的隔室中。

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.