MULTIPLE TOPOGENIC SEQUENCES IN BOVINE OPSIN
MULTIPLE TOPOGENIC SEQUENCES IN BOVINE OPSIN
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DOI：
10.1073/pnas.84.16.5783
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发表时间：
1987-08-01
影响因子：
11.1
通讯作者：
BLOBEL, G
中科院分区：
文献类型：
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作者：
AUDIGIER, Y;FRIEDLANDER, M;BLOBEL, G
牛视蛋白（Bovine opsin）是一种多位整合的膜蛋白，包含七个跨膜片段，其连接在膜的每一侧交替的八个亲水结构域。为了定位可能指定视蛋白在膜中的不同拓扑结构的拓扑序列，我们构建了各种视蛋白突变体，每个突变体仅包含一个跨膜片段。从这些突变体转录的信使RNA在补充有微粒体膜的无细胞系统中翻译。在分析的视蛋白的七个跨膜区段中的六个中，五个能够作为信号序列起作用，并且还表达可变强度的停止转移序列。通过在pH 11下的可提取性和蛋白酶敏感性的标准，与“强”停止转移序列组合的信号序列的存在产生了大多数链的脂质双层中的整合。然而，结合“wek”停止转移序列，我们观察到只有一些链整合到脂质双层中，其他链要么完全易位穿过膜，要么保留在膜中的水可进入的空间中。
Bovine opsin, a polytopic integral membrane protein, contains seven transmembrane segments connecting eight hydrophilic domains alternating on each side of the membrane. To localize topogenic sequences that might specify the distinct topology of opsin in the membrane, we constructed various opsin mutants, each containing only one transmembrane segment. Messenger RNAs transcribed from these mutants were translated in a cell-free system supplemented with microsomal membranes. Among six of the seven transmembrane segments of opsin that were analyzed, five were able to function as signal sequences and also expressed stop-transfer sequences of variable strength. By the criteria of extractability at pH 11 and protease sensitivity, the presence of a signal sequence in combination with a "strong" stop-transfer sequence yielded integration into the lipid bilayer of the majority of chains. However, in combination with a "wek" stop-transfer sequence, we observed integration into the lipid bilayer of only some chains, with the others either completely translocated across the membrane or retained in a water-accessible space in the membrane.