Katz F N, Rothman J E, Lingappa V R, Blobel G, Lodish H F
Proc Natl Acad Sci U S A. 1977 Aug;74(8):3278-82. doi: 10.1073/pnas.74.8.3278.
Membrane assembly was observed to proceed in cell-free extracts. Specifically, the membrane glycoprotein of vesicular stomatitis virus was synthesized in crude extracts of wheat germ in the presence of membrane vesicles derived from pancreatic endoplasmic reticulum. The resulting glycoprotein spans the lipid bilayer asymmetrically, is glycosylated, and is indistinguishable in these respects from the form of the glycoprotein found in the rough endoplasmic reticulum of virus-infected cells. Both glycosylation and asymmetric transmembrane insertion of the glycoprotein into membranes in vitro require protein synthesis in the presence of membranes. The carboxyl-terminal 5% of the polypeptide chain is located on the external surface of vesicles, corresponding to the cytoplasmic surface of the endoplasmic reticulum in cells. Most, or all, of the amino-terminal portion of the glycoprotein, as well as the protein-bound carbohydrate, appears to be located within the lumen of the membrane vesicles. These findings demonstrate that insertion of this membrane protein occurs during or immediately after protein synthesis. The results are consistent with the concepts that the growing membrane protein is extruded across the endoplasmic reticulum membrane amino terminus first and that glycosylation is restricted to the lumenal surface of the membrane. The cell-free system reported here should prove valuable for studying these processes.
在无细胞提取物中观察到膜组装过程。具体而言，在存在源自胰腺内质网的膜泡的情况下，在小麦胚芽粗提取物中合成了水疱性口炎病毒的膜糖蛋白。所得糖蛋白不对称地跨越脂质双层，进行了糖基化，并且在这些方面与在病毒感染细胞的糙面内质网中发现的糖蛋白形式没有区别。糖蛋白在体外的糖基化和不对称跨膜插入到膜中都需要在有膜存在的情况下进行蛋白质合成。多肽链的羧基末端5%位于膜泡的外表面，对应于细胞内质网的细胞质表面。糖蛋白的大部分或全部氨基末端部分以及与蛋白质结合的碳水化合物似乎位于膜泡的腔内。这些发现表明这种膜蛋白的插入发生在蛋白质合成期间或之后立即发生。结果与以下概念一致，即正在生长的膜蛋白首先通过内质网膜从氨基末端挤出，并且糖基化仅限于膜的腔表面。此处报道的无细胞系统对于研究这些过程应该是有价值的。
