Intracellular trafficking pathways in the assembly of connexins into gap junctions
Intracellular trafficking pathways in the assembly of connexins into gap junctions
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DOI：
10.1074/jbc.274.13.8678
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发表时间：
1999-03-26
影响因子：
4.8
通讯作者：
Evans, WH
中科院分区：
文献类型：
--
作者：
George, CH;Kendall, JM;Evans, WH
利用表达一系列连接蛋白-aequorin(Cx-Aeq)嵌合体的活COS-7细胞研究了连接蛋白组装到缝隙连接的运输途径。通过测量Aequorin融合伙伴的化学发光，发现寡聚连接蛋白从细胞内储存物转移到质膜上的速度不同，这取决于连接蛋白的异构体。用布雷菲尔丁A处理表达Cx32-Aeq和Cx43-Aeq的COS-7细胞，可分别抑制这些嵌合体向质膜移动84+/-4和88+/-4%。诺康唑对表达Cx32-Aeq和Cx43-Aeq的细胞的抑制率分别为29+/-16和4+/-7%。相反，使用Cx26/43T-Aeq结构(Cx26/43T-Aeq)研究了Cx26到质膜的运输，其中Cx26的短胞质羧基末端被Cx43的扩展羧基末端取代，诺可达唑对Cx26到质膜的运输抑制了89+/-5%，细胞暴露于布雷菲尔丁A的影响最小(17+/-11%)。野生型Cx32、Cx43与相应的Cx32-Aeq和Cx43-Aeq嵌合体细胞之间的缝隙连接转移被诺可达唑处理减少，并被灯盏花素A处理取消。然而，表达野生型Cx26或Cx26/43T-Aeq嵌合体的细胞之间的染料偶联程度不受灯盏花素A处理的显著影响，但经诺可达唑处理后，染料向邻近细胞的转移大大减少。布雷菲尔丁A和诺可达唑对连接蛋白的运输性质和细胞间染料转移的这些对比效应被解释为两条途径有助于连接蛋白到缝隙连接的路线。
Trafficking pathways underlying the assembly of connexins into gap junctions were examined using living COS-7 cells expressing a range of connexin-aequorin (Cx-Aeq) chimeras. By measuring the chemiluminescence of the aequorin fusion partner, the translocation of oligomerized connexins from intracellular stores to the plasma membrane was shown to occur at different rates that depended on the connexin isoform. Treatment of COS-7 cells expressing Cx32-Aeq and Cx43-Aeq with brefeldin A inhibited the movement of these chimera to the plasma membrane by 84 +/- 4 and 88 +/- 4%, respectively. Nocodazole treatment of the cells expressing Cx32-Aeq and Cx43-Aeq produced 29 +/- 16 and 4 +/- 7% inhibition, respectively. in contrast, the transport of Cx26 to the plasma membrane, studied using a construct (Cx26/43T-Aeq) in which the short cytoplasmic carboxyl-terminal tail of Cx26 was replaced with the extended carboxyl terminus of Cx43, was inhibited 89 +/- 5% by nocodazole and was minimally affected by exposure of cells to brefeldin A (17 +/- 11%). The transfer of Lucifer yellow across gap junctions between cells expressing wild-type Cx32, Cx43, and the corresponding Cx32-Aeq and Cx43-Aeq chimeras was reduced by nocodazole treatment and abolished by brefeldin A treatment. However, the extent of dye coupling between cells expressing wild-type Cx26 or the Cx26/43T-Aeq chimeras was not significantly affected by brefeldin A treatment, but after nocodazole treatment, transfer of dye to neighboring cells was greatly reduced. These contrasting effects of brefeldin A and nocodazole on the trafficking properties and intercellular dye transfer are interpreted to suggest that two pathways contribute to the routing of connexins to the gap junction.