Schlenstedt G, Gudmundsson G H, Boman H G, Zimmermann R
Institut für Physiologische Chemie der Universität München, Federal Republic of Germany.
J Biol Chem. 1990 Aug 15;265(23):13960-8.
Translocation of large presecretory proteins into the mammalian endoplasmic reticulum requires the ribonucleoparticles, signal recognition particle, and ribosome and is tightly coupled to ongoing protein synthesis. We have shown previously that small presecretory proteins can translocate post-translationally in a reaction that does not require these ribonucleoparticles. We now report that one large protein, a synthetic hybrid between preprocecropin A and dihydrofolate reductase, translocates both cotranslationally (with the aid of signal recognition particle and ribosome) and post-translationally (without the involvement of these ribonucleoparticles) during its in vitro synthesis in the presence of dog pancreas microsomes. The distinction between these two modes of translocation was made possible by adding methotrexate to the translocation reaction. Methotrexate can only form a tight complex with those preprocecropin A-dihydrofolate reductase hybrid chains that have completed their synthesis and folded, but in forming this tight complex, this drug prevents translocation of the dihydrofolate reductase domain across the membrane.
大型前分泌蛋白转运到哺乳动物内质网需要核糖核蛋白颗粒、信号识别颗粒和核糖体，并且与正在进行的蛋白质合成紧密偶联。我们之前已经表明，小型前分泌蛋白可以在不需要这些核糖核蛋白颗粒的反应中进行翻译后转运。我们现在报告，一种大型蛋白，即前肽聚糖A和二氢叶酸还原酶之间的合成杂合体，在狗胰腺微粒体存在的情况下进行体外合成时，既能共翻译转运（借助信号识别颗粒和核糖体），也能翻译后转运（无需这些核糖核蛋白颗粒的参与）。通过在转运反应中添加甲氨蝶呤，使得区分这两种转运模式成为可能。甲氨蝶呤只能与那些已经完成合成并折叠的前肽聚糖A-二氢叶酸还原酶杂合链形成紧密复合物，但在形成这种紧密复合物时，这种药物会阻止二氢叶酸还原酶结构域跨膜转运。
