Ota K, Sakaguchi M, Hamasaki N, Mihara K
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan.
J Biol Chem. 1998 Oct 23;273(43):28286-91. doi: 10.1074/jbc.273.43.28286.
Band 3 protein is a typical multispanning membrane protein whose membrane topology has been extensively studied from various protein chemical approaches. To clarify the membrane topogenesis of this multispanning protein on the endoplasmic reticulum, the topogenic functions of the anticipated transmembrane segments were individually assessed in an in vitro system using two series of model proteins in which each segment was placed in either a "stop-transfer" context or a "translocation initiation" context. They were expressed in a cell-free system containing rough microsomal membranes, and their topologies were evaluated by taking advantage of either sensitivity to protease or accessibility to N-glycosylation. We found that some segments seem to possess insufficient topogenic functions for membrane integration: the second transmembrane segment (TM2) is insufficient for the stop-transfer sequence, and TM3, TM5, and TM7 are not sufficient for the translocation initiation. In contrast to these phenomena, we herein demonstrate that TM2 shows an efficient stop-transfer function when it is near the preceding TM1 and suggest that TM3, TM5, and TM7 are followed by TM segments with a strong topogenic function to form Nexo/Ccyt topology, via which the preceding segments are integrated into the membrane. From these results, we propose that the interactions between the TMs should be operative during membrane integration, and that the segments with a weak topogenic function are given a transmembrane orientation by their following TMs.
带3蛋白是一种典型的多次跨膜蛋白，其膜拓扑结构已通过各种蛋白质化学方法进行了广泛研究。为了阐明这种多次跨膜蛋白在内质网上的膜拓扑发生过程，在体外系统中使用了两个系列的模型蛋白，分别对预期跨膜片段的拓扑功能进行了单独评估，其中每个片段分别处于“终止转移”或“转运起始”环境中。它们在含有糙面微粒体膜的无细胞系统中表达，并利用对蛋白酶的敏感性或N-糖基化的可及性来评估其拓扑结构。我们发现，一些片段的膜整合拓扑功能似乎不足：第二个跨膜片段（TM2）作为终止转移序列是不足的，而TM3、TM5和TM7作为转运起始序列是不够的。与这些现象相反，我们在此证明，当TM2靠近前面的TM1时，它表现出有效的终止转移功能，并表明TM3、TM5和TM7后面跟着具有强大拓扑功能的TM片段，以形成Nexo/Ccyt拓扑结构，通过这种结构，前面的片段被整合到膜中。从这些结果中，我们提出跨膜片段之间的相互作用在膜整合过程中应该是起作用的，并且具有弱拓扑功能的片段由其后面的跨膜片段赋予跨膜方向。
