Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA.
Mol Biol Cell. 2011 Dec;22(23):4461-71. doi: 10.1091/mbc.E11-05-0396. Epub 2011 Oct 12.
Many missense mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR) result in its misfolding, endoplasmic reticulum (ER) accumulation, and, thus, cystic fibrosis. A number of these mutations are located in the predicted CFTR transmembrane (TM) spans and have been projected to alter span integration. However, the boundaries of the spans have not been precisely defined experimentally. In this study, the ER luminal integration profiles of TM1 and TM2 were determined using the ER glycosylation machinery, and the effects of the CF-causing mutations G85E and G91R thereon were assessed. The mutations either destabilize the integrated conformation or alter the TM1 ER integration profile. G85E misfolding is based in TM1 destabilization by glutamic acid and loss of glycine and correlates with the temperature-insensitive ER accumulation of immature full-length CFTR harboring the mutation. By contrast, temperature-dependent misfolding owing to the G91R mutation depends on the introduction of the basic side chain rather than the loss of the glycine. This work demonstrates that CF-causing mutations predicted to have similar effects on CFTR structure actually result in disparate molecular perturbations that underlie ER accumulation and the pathology of CF.
许多囊性纤维化跨膜电导调节蛋白（CFTR）中的错义突变导致其错误折叠、内质网（ER）积累，从而导致囊性纤维化。这些突变中的许多位于预测的 CFTR 跨膜（TM）跨度中，并被预测会改变跨度整合。然而，这些跨度的边界尚未在实验中精确定义。在这项研究中，使用 ER 糖基化机制确定了 TM1 和 TM2 的 ER 腔整合谱，并评估了导致 CF 的突变 G85E 和 G91R 对其的影响。这些突变要么使整合构象不稳定，要么改变 TM1 ER 整合谱。G85E 错误折叠基于谷氨酸引起的 TM1 不稳定和甘氨酸的丢失，并与携带该突变的不成熟全长 CFTR 的温度不敏感 ER 积累相关。相比之下，由于 G91R 突变引起的温度依赖性错误折叠取决于碱性侧链的引入，而不是甘氨酸的丢失。这项工作表明，预测对 CFTR 结构具有相似影响的导致 CF 的突变实际上导致了导致 ER 积累和 CF 病理学的不同分子扰动。
