Dierks T, Volkmer J, Schlenstedt G, Jung C, Sandholzer U, Zachmann K, Schlotterhose P, Neifer K, Schmidt B, Zimmermann R
Institut für Biochemie und Molekulare Zellbiologie, Universität Göttingen, Germany.
EMBO J. 1996 Dec 16;15(24):6931-42.
Protein transport into the mammalian endoplasmic reticulum depends on nucleoside triphosphates. Photoaffinity labelling of microsomes with azido-ATP prevents protein transport at the level of association of precursor proteins with the components of the transport machinery, Sec61alpha and TRAM proteins. The same phenotype of inactivation was observed after depleting a microsomal detergent extract of ATP-binding proteins by passage through ATP-agarose and subsequent reconstitution of the pass-through into proteoliposomes. Transport was restored by co-reconstitution of the ATP eluate. This eluate showed eight distinct bands in SDS gels. We identified five lumenal proteins (Grp170, Grp94, BiP/Grp78, calreticulin and protein disulfide isomerase), one membrane protein (ribophorin I) and two ribosomal proteins (L4 and L5). In addition to BiP (Grp78), Grp170 was most efficiently retained on ATP-agarose. Purified BiP did not stimulate transport activity. Sequence analysis revealed a striking similarity of Grp170 and the yeast microsomal protein Lhs1p which was recently shown to be involved in protein transport into yeast microsomes. We suggest that Grp170 mediates efficient insertion of polypeptides into the microsomal membrane at the expense of nucleoside triphosphates.
蛋白质转运到哺乳动物内质网中依赖于核苷三磷酸。用叠氮 - ATP对微粒体进行光亲和标记可在转运前体蛋白与转运机制组分（Sec61α和TRAM蛋白）结合的水平上阻止蛋白质转运。在用ATP - 琼脂糖处理微粒体去污剂提取物以耗尽ATP结合蛋白，随后将处理后的提取物重建到蛋白脂质体中后，观察到了相同的失活表型。通过ATP洗脱液的共重建恢复了转运。该洗脱液在SDS凝胶中显示出八条不同的条带。我们鉴定出五种腔内蛋白（Grp170、Grp94、BiP/Grp78、钙网蛋白和蛋白质二硫键异构酶）、一种膜蛋白（核糖体结合蛋白I）和两种核糖体蛋白（L4和L5）。除了BiP（Grp78）外，Grp170最有效地保留在ATP - 琼脂糖上。纯化的BiP不刺激转运活性。序列分析显示Grp170与酵母微粒体蛋白Lhs1p有显著相似性，最近的研究表明Lhs1p参与蛋白质转运到酵母微粒体中。我们认为Grp170以核苷三磷酸为代价介导多肽高效插入微粒体膜。
