Membrane insertion kinetics of a protein domain in vivo -: The bacterioopsin N terminus inserts co-translationally
Membrane insertion kinetics of a protein domain in vivo -: The bacterioopsin N terminus inserts co-translationally
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DOI：
10.1074/jbc.274.32.22693
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发表时间：
1999-08-06
影响因子：
4.8
通讯作者：
Krebs, MP
中科院分区：
文献类型：
--
作者：
Dale, H;Krebs, MP
多位膜蛋白片段插入膜的途径尚未在体内得到解决。我们已经开发了一种体内动力学测定，以检查插入途径的多位蛋白质细菌视蛋白，盐生盐杆菌的脱辅基蛋白。细菌视紫红质构建表达细菌视紫红质突变体I4 C：H-6和T5 C：H-6的菌株，其在N-末端胞外结构域中携带独特的Cys，在C末端携带多组氨酸标签。易位的N-末端结构域检测使用膜不渗透性凝胶位移试剂衍生化新生放射性标记的分子的Cys残基。通过凝胶电泳对完全延长的放射性标记群体进行衍生化评估。移位和完全衍生化I4 C：H-6和T5 C：H-6的Cys残基所需的时间分别为46 +/- 9和61 +/- 6 s。这明显短于蛋白质的延伸时间，分别为114 +/- 26和169 +/- 16 s。这些结果确定了细菌视蛋白N末端的易位和第一跨膜区段的插入协同发生，并证实了使用该测定来监测体内多位膜蛋白插入的动力学。
The pathway by which segments of a polytopic membrane protein are inserted into the membrane has not been resolved in vivo. We have developed an in vivo kinetic assay to examine the insertion pathway of the polytopic protein bacterioopsin, the apoprotein of Halobacterium salinarum. bacteriorhodopsin. Strains were constructed that express the bacteriorhodopsin mutants I4C:H-6 and T5C:H-6, which carry a unique Cys in the N-terminal extracellular domain and a polyhistidine tag at the C terminus. Translocation of the N-terminal domain was detected using a membrane-impermeant gel shift reagent to derivatize the Cys residue of nascent radiolabeled molecules. Derivatization was assessed by gel electrophoresis of the fully elongated radiolabeled population. The time required to translocate and fully derivatize the Cys residues of I4C:H-6 and T5C:H-6 is 46 +/- 9 and 61 +/- 6 s, respectively. This is significantly shorter than the elongation times of the proteins, which are 114 +/- 26 and 169 +/- 16 s, respectively. These results establish that translocation of the bacterioopsin N terminus and insertion of the first transmembrane segment occur co translationally and confirm the use of the assay to monitor the kinetics of polytopic membrane protein insertion in vivo.