Szczesna-Skorupa E, Kemper B
Department of Physiology and Biophysics, University of Illinois, Urbana 61801.
J Biol Chem. 1993 Jan 25;268(3):1757-62.
The mechanism of retention of cytochrome P450 in the endoplasmic reticulum is unknown, and the membrane topology of the N-terminal region remains controversial. To address these problems, a sequence of 29 amino acids encoding an internal N-glycosylation site of rabbit cytochrome P450 2C2 was attached to the N terminus of cytochrome P450 2C1. This protein is glycosylated at a single site in a cell-free translation system containing microsomal membranes, as indicated by gel mobility and sensitivity to endoglycosidase H. When expressed in COS1 cells, an immunoreactive species with the same gel mobility as the in vitro synthesized glycosylated product was detected. Treatment with endoglycosidase H changed its mobility to that of unglycosylated hybrid cytochrome P450 2C1. These results indicate that in intact cells, as in the cell-free system, the N terminus of cytochrome P450 is luminally oriented which is not consistent with a hairpin loop conformation. Sensitivity of the glycosylated protein to endoglycosidase H suggests that the protein does not reach the Golgi compartments. When transfected cells were incubated at low temperatures to inhibit retrograde transport from the intermediate pre-Golgi compartment into the endoplasmic reticulum, localization of cytochrome P450 was not changed, as assayed by subcellular fractionation and immunofluorescent staining. These observations suggest that cytochrome P450 is restricted to the endoplasmic reticulum membrane by a mechanism different from recycling through the intermediate compartment, which is a pathway utilized by soluble endoplasmic reticulum proteins.
细胞色素P450保留在内质网中的机制尚不清楚，其N端区域的膜拓扑结构仍存在争议。为了解决这些问题，将一段编码兔细胞色素P450 2C2内部N-糖基化位点的29个氨基酸序列连接到细胞色素P450 2C1的N端。在含有微粒体膜的无细胞翻译系统中，该蛋白在单个位点发生糖基化，这通过凝胶迁移率和对内切糖苷酶H的敏感性得以表明。当在COS1细胞中表达时，检测到一种免疫反应性物质，其凝胶迁移率与体外合成的糖基化产物相同。用内切糖苷酶H处理后，其迁移率变为未糖基化的杂合细胞色素P450 2C1的迁移率。这些结果表明，在完整细胞中，如同在无细胞系统中一样，细胞色素P450的N端是朝向内质网腔的，这与发夹环构象不一致。糖基化蛋白对内切糖苷酶H的敏感性表明该蛋白未到达高尔基体区室。当转染细胞在低温下孵育以抑制从高尔基前体中间区室向内质网的逆行转运时，通过亚细胞分级分离和免疫荧光染色检测，细胞色素P450的定位没有改变。这些观察结果表明，细胞色素P450通过一种不同于通过中间区室循环的机制被限制在内质网膜上，中间区室循环是可溶性内质网蛋白所利用的一条途径。
