Musil L S, Goodenough D A
Department of Anatomy and Cellular Biology, Harvard Medical School, Boston, Massachusetts 02115.
J Cell Biol. 1991 Dec;115(5):1357-74. doi: 10.1083/jcb.115.5.1357.
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.
我们之前证明，间隙连接蛋白连接蛋白43被翻译为一种42-kD的蛋白（连接蛋白43-NP），在具有间隙连接通讯能力的细胞中，该蛋白能有效地磷酸化为一种46,000-Mr的蛋白（连接蛋白43-P2），而在通讯缺陷的细胞中则不能。在本研究中，我们结合代谢性放射性标记和免疫沉淀法，研究连接蛋白43装配到间隙连接中的过程以及该事件与连接蛋白43磷酸化的关系。对具有通讯能力的NRK细胞中连接蛋白43的去污剂溶解性进行检测，结果显示，连接蛋白43加工为P2形式伴随着获得对1% Triton X-100溶解的抗性。对经Triton处理的NRK细胞进行连接蛋白43的免疫组织化学定位表明，连接蛋白43-P2（Triton不溶性）集中在间隙连接斑中，而连接蛋白43-NP（Triton可溶性）主要位于细胞内。使用20℃的细胞内运输阻断或细胞表面蛋白生物素化方法，我们确定连接蛋白43以Triton可溶性连接蛋白43-NP的形式运输到质膜。细胞表面生物素化的连接蛋白43-NP在37℃时加工为Triton不溶性的连接蛋白43-P2。连接蛋白43-NP也运输到通讯缺陷、间隙连接缺陷的S180和L929细胞的质膜，但未加工为Triton不溶性的连接蛋白43-P2。综上所述，这些结果表明，间隙连接装配在连接蛋白43到达质膜后受到调控，并且在时间上与获得对Triton X-100的不溶性以及磷酸化为连接蛋白43-P2形式相关。
