Berthoud Viviana M, Minogue Peter J, Guo Jun, Williamson Edward K, Xu Xiaorong, Ebihara Lisa, Beyer Eric C
Department of Pediatrics, Section of Hematology/Oncology, University of Chicago, Chicago, IL 60637, USA.
Eur J Cell Biol. 2003 May;82(5):209-21. doi: 10.1078/0171-9335-00316.
A mutant human connexin50 (hCx50), hCx50P88S, has been linked to cataracts inherited as an autosomal dominant trait. The functional, biochemical and cellular behavior of wild-type and mutant hCx50 were examined in transfected cells. hCx50P88S was unable to induce gap junctional currents by itself, and it abolished gap junctional currents when co-expressed with wild-type (wt) hCx50. Cells transfected with hCx50P88S showed cytoplasmic accumulations of Cx50 immunoreactivity in addition to staining at appositional membranes; these accumulations did not significantly co-localize with markers for the endoplasmic reticulum, Golgi apparatus, lysosomes, endosomes or vimentin filaments. Immunoelectron microscopy studies localized hCx50P88S to cytoplasmic membrane stacks in close vicinity to the endoplasmic reticulum. In contrast, aggresome-like accumulations were induced by treatment of wt hCx50-transfected cells with proteasomal inhibitors. The formation of hCx50P88S accumulations in transiently transfected cells was not blocked by treatment with Brefeldin A suggesting that they form before Cx50 transits through the Golgi apparatus to the plasma membrane. Treatment of HeLa-hCx50P88S cells with cycloheximide demonstrated the presence of a very stable pool of hCx50P88S. Taken together, these results suggest that the P to S mutation at amino acid residue 88 causes a defect that leads to decreased degradation and subsequent accumulation of hCx50P88S in a cellular structure different from aggresomes.
一种突变的人类连接蛋白50（hCx50），即hCx50P88S，与作为常染色体显性性状遗传的白内障有关。在转染细胞中检测了野生型和突变型hCx50的功能、生化及细胞行为。hCx50P88S自身无法诱导间隙连接电流，当与野生型（wt）hCx50共表达时，它会消除间隙连接电流。转染了hCx50P88S的细胞除了在相邻细胞膜处有染色外，还显示出Cx50免疫反应性在细胞质中的积累；这些积累物与内质网、高尔基体、溶酶体、内体或波形蛋白丝的标记物没有明显的共定位。免疫电子显微镜研究将hCx50P88S定位在内质网附近的细胞质膜堆叠处。相比之下，蛋白酶体抑制剂处理野生型hCx50转染细胞会诱导类似聚集体的积累。用布雷菲德菌素A处理瞬时转染细胞中hCx50P88S积累物的形成未被阻断，这表明它们在Cx50通过高尔基体转运到质膜之前就已形成。用环己酰亚胺处理HeLa-hCx50P88S细胞证明存在非常稳定的hCx50P88S池。综上所述，这些结果表明，氨基酸残基88处的P到S突变导致了一种缺陷，导致hCx50P88S的降解减少并随后在不同于聚集体的细胞结构中积累。
