Co- and posttranslational translocation mechanisms direct cystic fibrosis transmembrane conductance regulator N terminus transmembrane assembly
Co- and posttranslational translocation mechanisms direct cystic fibrosis transmembrane conductance regulator N terminus transmembrane assembly
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DOI：
10.1074/jbc.273.1.568
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发表时间：
1998-01-02
影响因子：
4.8
通讯作者：
Skach, WR
中科院分区：
文献类型：
--
作者：
Lu, Y;Xiong, XM;Skach, WR
大多数真核多聚体蛋白的跨膜拓扑结构是通过交替的信号和停止转移序列的作用在内质网膜上以共翻译的方式建立的。在这里，我们证明囊性纤维化跨膜电导调节因子(CFTR)通过涉及共翻译和翻译后易位事件的这一机制的变体来实现其N末端拓扑结构。使用在网织红细胞裂解物系统中表达的一系列已定义的嵌合和截断蛋白，我们已经鉴定了在CFTR的第一(TM1)和第二(TM2)跨膜片段中编码的两个拓扑性决定因素。每个序列独立地(I)定向内质网靶向，(Ii)移位适当的侧翼残基，和(Iii)实现其正确的跨膜定向。然而，由于位于TM1疏水核心内的两个带电残基Glu(92)和Lys(95)的存在，TM1的信号序列活性低下，因此TM1能够指导不到一半的新生CFTR链的正确拓扑。与TM1相比，TM2信号序列的活性既高效又特异。即使在缺乏功能性TM1信号序列的情况下，TM2也能够通过核糖体依赖的翻译后机制来指导CFTRN末端的拓扑结构。将带电残基Glu(92)和Lys(95)突变为丙氨酸可以提高TM1信号序列的活性，以及TM1独立指导CFTRN末端拓扑的能力，因此，第一或第二TM片段中的单个功能信号序列足以指导正确的CFTR拓扑。这些结果识别了CFTRN末端跨膜组装的两条不同和冗余的易位途径，并支持一个模型，在该模型中，TM2起作用，以确保无法通过TM1转运的CFTR链的正确拓扑。这种新的拓扑发生信息的排列为传统的共翻译途径提供了一种替代多发性蛋白生物发生的途径。
Transmembrane topology of most eukaryotic polytopic proteins is established cotranslationally at the endo plasmic reticulum membrane through the action of alternating signal and stop transfer sequences. Here we demonstrate that the cystic fibrosis transmembrane conductance regulator (CFTR) achieves its N terminus topology through a variation of this mechanism that involves both co-and posttranslational translocation events. Using a series of defined chimeric and truncated proteins expressed in a reticulocyte lysate system, we have identified two topogenic determinants encoded within the first (TM1) and second (TM2) membrane-spanning segments of CFTR. Each sequence independently (i) directed endoplasmic reticulum targeting, (ii) translocated appropriate flanking residues, and (iii) achieved its proper membrane-spanning orientation. Signal sequence activity of TM1, however, was inefficient due to the presence of two charged residues, Glu(92) and Lys(95), located within its hydrophobic core, As a result, TM1 was able to direct correct topology for less than half of nascent CFTR chains. In contrast to TM1, TM2 signal sequence activity was both efficient and specific. Even in the absence of a functional TM1 signal sequence, TM2 was able to direct CFTR N terminus topology through a ribosome dependent posttranslational mechanism. Mutating charged residues Glu(92) and Lys(95) to alanine improved TM1 signal sequence activity as well as the ability of TM1 to independently direct CFTR N terminus topology, Thus, a single functional signal sequence in either the first or second TM segment was sufficient for directing proper CFTR topology. These results identify two distinct and redundant translocation pathways for CFTR N terminus transmembrane assembly and support a model in which TM2 functions to ensure correct topology of CFTR chains that fail to translocate via TM1. This novel arrangement of topogenic information provides an alternative to conventional cotranslational pathways of polytopic protein biogenesis.