Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques.
Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques.
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DOI：
10.1083/jcb.115.5.1357
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发表时间：
1991-12
影响因子：
7.8
通讯作者：
Goodenough, D A
中科院分区：
文献类型：
--
作者：
Musil, L S;Goodenough, D A
我们先前已经证明，缝隙连接蛋白Cx43被翻译为一种42kD的蛋白(Cx43-NP)，在具有缝隙连接通讯能力的细胞中被有效地磷酸化为46,000个MR物种(Cx43-P2)，而在通讯缺乏的细胞中则不被磷酸化。在这项研究中，我们使用代谢放射性标记和免疫沉淀相结合的方法来研究缝隙连接中连接蛋白43的组装，以及这一事件与连接蛋白43磷酸化的关系。对连接蛋白43在通讯功能NRK细胞中的洗涤剂溶解性的检测表明，连接蛋白43处理为P2形式的过程中伴随着对1%Triton X-100的增溶抗性的获得。连接蛋白43在Triton提取的NRK细胞中的免疫组织化学定位表明，连接蛋白43-P2(不溶于Triton)集中在缝隙连接斑块中，而连接蛋白43-NP(可溶于Triton)主要分布在细胞内。使用20℃的细胞内转运阻断或细胞表面蛋白生物素化，我们确定连接蛋白43以Triton可溶性连接蛋白43-NP的形式被运输到质膜。细胞表面生物素化的连接蛋白43-NP在37℃下被加工成Triton不溶的连接蛋白43-P2，在通讯缺陷、缝隙连接缺陷的S180和L929细胞中，连接蛋白43-NP也被转运到质膜上，但不被加工成Triton不溶的连接蛋白43-P2。综上所述，这些结果表明，缝隙连接组装在连接蛋白43到达质膜后受到调控，并与Triton X-100中不溶性的获得和连接蛋白43-P2形式的磷酸化有关。
We previously demonstrated that the gap junction protein connexin43 is translated as a 42-kD protein (connexin43-NP) that is efficiently phosphorylated to a 46,000-Mr species (connexin43-P2) in gap junctional communication-competent, but not in communication-deficient, cells. In this study, we used a combination of metabolic radiolabeling and immunoprecipitation to investigate the assembly of connexin43 into gap junctions and the relationship of this event to phosphorylation of connexin43. Examination of the detergent solubility of connexin43 in communication-competent NRK cells revealed that processing of connexin43 to the P2 form was accompanied by acquisition of resistance to solubilization in 1% Triton X-100. Immunohistochemical localization of connexin43 in Triton-extracted NRK cells demonstrated that connexin43-P2 (Triton-insoluble) was concentrated in gap junctional plaques, whereas connexin43-NP (Triton-soluble) was predominantly intracellular. Using either a 20 degrees C intracellular transport block or cell-surface protein biotinylation, we determined that connexin43 was transported to the plasma membrane in the Triton-soluble connexin43-NP form. Cell-surface biotinylated connexin43-NP was processed to Triton-insoluble connexin43-P2 at 37 degrees C. Connexin43- NP was also transported to the plasma membrane in communication defective, gap junction-deficient S180 and L929 cells but was not processed to Triton-insoluble connexin43-P2. Taken together, these results demonstrate that gap junction assembly is regulated after arrival of connexin43 at the plasma membrane and is temporally associated with acquisition of insolubility in Triton X-100 and phosphorylation to the connexin43-P2 form.