Hortsch M, Avossa D, Meyer D I
J Biol Chem. 1985 Aug 5;260(16):9137-45.
Docking protein is a 73-kDa integral membrane protein of the rough endoplasmic reticulum. It is essential for translocation of nascent secretory proteins into the lumen of the endoplasmic reticulum. Monoclonal and polyclonal antibodies have been generated which, in conjunction with limited proteolysis, have been used to characterize several subspecies of docking protein. These proteolytic fragments have been analyzed with respect to the various functions ascribed to docking protein which can be assayed in vitro. Proteolytic digestion of membrane-associated or of affinity-purified intact docking protein showed that: elastase cleavage generates a 59-kDa soluble fragment and one of 14 kDa which contains the membrane anchoring domain; trypsin as well as endogenous proteolysis generates a 46-kDa fragment, leaving a 27-kDa domain containing the membrane anchor. This 27-kDa fragment can be reduced to a 13- and a 14-kDa piece by elastase digestion. The characteristics of these various subspecies were examined. The 59-kDa soluble fragment, which can reconstitute full translocation activity to docking protein-depleted microsomes (Meyer, D. I., and Dobberstein, B. (1980) J. Cell Biol. 87, 503-508) was capable of releasing a signal recognition particle-mediated translation arrest. The 46-kDa fragment was neither able to reassociate with nor to reconstitute the activity of docking protein-depleted microsomes. Moreover this fragment was unable to release a signal recognition particle-mediated arrest. This suggests that the 13-kDa fragment (the difference between 46 and 59 kDa) is both essential for association with the membrane, and for the release of translation arrests.
对接蛋白是一种存在于糙面内质网中的73 kDa整合膜蛋白。它对于新生分泌蛋白转运到内质网腔中至关重要。已经制备了单克隆抗体和多克隆抗体，这些抗体与有限的蛋白水解相结合，被用于表征对接蛋白的几个亚类。这些蛋白水解片段已针对对接蛋白的各种体外可检测功能进行了分析。对膜相关的或亲和纯化的完整对接蛋白进行蛋白水解消化显示：弹性蛋白酶切割产生一个59 kDa的可溶性片段和一个14 kDa的片段，后者包含膜锚定结构域；胰蛋白酶以及内源性蛋白水解产生一个46 kDa的片段，留下一个包含膜锚定的27 kDa结构域。通过弹性蛋白酶消化，这个27 kDa的片段可以被降解为一个13 kDa和一个14 kDa的片段。研究了这些不同亚类的特性。59 kDa的可溶性片段能够恢复对接蛋白缺失的微粒体的完全转运活性（迈耶，D. I.，和多伯斯坦，B.（1980年）《细胞生物学杂志》87卷，503 - 508页），并且能够解除信号识别颗粒介导的翻译停滞。46 kDa的片段既不能与对接蛋白缺失的微粒体重组，也不能恢复其活性。此外，这个片段无法解除信号识别颗粒介导的停滞。这表明13 kDa的片段（46 kDa和59 kDa之间的差异）对于与膜的结合以及解除翻译停滞都是必不可少的。
