Structural cues involved in endoplasmic reticulum degradation of G85E and G91R mutant cystic fibrosis transmembrane conductance regulator.
Structural cues involved in endoplasmic reticulum degradation of G85E and G91R mutant cystic fibrosis transmembrane conductance regulator.
复制标题
参与 G85E 和 G91R 突变体囊性纤维化跨膜电导调节剂内质网降解的结构线索。
DOI：
10.1172/jci119618
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Skach,WR
中科院分区：
文献类型：
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作者：
Xiong,X;Bragin,A;Widdicombe,JH;Cohn,J;Skach,WR
突变型囊性纤维化跨膜传导调节因子（CFTR）的异常折叠和随后在内质网中的降解是大多数囊性纤维化病例的基础。然而，野生型（WT）和突变蛋白之间的结构差异仍然未知。在这里，我们研究了两个突变CFTR蛋白，G85 E和G91 R，其中每一个包含一个额外的带电残基内的第一个假定的跨膜螺旋（TM 1）的细胞内贩运，降解和跨膜拓扑结构。在显微注射非洲爪蟾卵母细胞，这些突变显着破坏CFTR质膜氯离子通道活性。G85 E和G91 R突变体（但不是保守突变体，G91 A）未能获得复杂的N-连接的碳水化合物，并在到达高尔基复合体之前迅速降解，因此表现出类似于DeltaF 508 CFTR的运输表型。拓扑学分析显示，G85 E和G91 R突变均不破坏CFTR NH 2末端跨膜拓扑学。相反，WT以及突变体TM 1跨越膜的预测C-反式（II型）的方向，和残基85 E和91 R被定位在或邻近的脂质双层的平面。为了理解这些带电残基如何为ER降解提供结构线索，我们检查了在密码子188、393、589或836处截短的WT、G85 E和G91 R CFTR蛋白的稳定性（分别在TM 2、TM 6、第一个核苷酸结合结构域或R结构域之后）。这些结果表明，G85 E和G91 R突变影响CFTR折叠，不是通过跨膜组装的总体破坏，而是通过在双层平面内插入带电残基，这反过来影响更高级的三级结构。
Abnormal folding of mutant cystic fibrosis transmembrane conductance regulator (CFTR) and subsequent degradation in the endoplasmic reticulum is the basis for most cases of cystic fibrosis. Structural differences between wild-type (WT) and mutant proteins, however, remain unknown. Here we examine the intracellular trafficking, degradation, and transmembrane topology of two mutant CFTR proteins, G85E and G91R, each of which contains an additional charged residue within the first putative transmembrane helix (TM1). In microinjected Xenopus laevis oocytes, these mutations markedly disrupted CFTR plasma membrane chloride channel activity. G85E and G91R mutants (but not a conservative mutant, G91A) failed to acquire complex N-linked carbohydrates, and were rapidly degraded before reaching the Golgi complex thus exhibiting a trafficking phenotype similar to DeltaF508 CFTR. Topologic analysis revealed that neither G85E nor G91R mutations disrupted CFTR NH2 terminus transmembrane topology. Instead, WT as well as mutant TM1 spanned the membrane in the predicted C-trans (type II) orientation, and residues 85E and 91R were localized within or adjacent to the plane of the lipid bilayer. To understand how these charged residues might provide structural cues for ER degradation, we examined the stability of WT, G85E, and G91R CFTR proteins truncated at codons 188, 393, 589, or 836 (after TM2, TM6, the first nucleotide binding domain, or the R domain, respectively). These results indicated that G85E and G91R mutations affected CFTR folding, not by gross disruption of transmembrane assembly, but rather through insertion of a charged residue within the plane of the bilayer, which in turn influenced higher order tertiary structure.