The sensor protein KdpD inserts into the Escherichia coli membrane independent of the Sec translocase and YidC
The sensor protein KdpD inserts into the Escherichia coli membrane independent of the Sec translocase and YidC
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DOI：
10.1046/j.1432-1033.2003.03531.x
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发表时间：
2003-04-01
期刊：
影响因子：
--
通讯作者：
Kuhn, A
中科院分区：
文献类型：
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作者：
Facey, SJ;Kuhn, A
KdpD 是大肠杆菌内膜中的一种传感器激酶蛋白，包含四个跨膜区域。连接跨膜区域的周质环非常短，蛋白酶图谱使我们只能追踪第二个周质环的易位。结果表明，KdpD 第二个环的膜插入不需要 Sec 转位酶和 YidC 蛋白。为了研究第一个周质环的易位，将短 HA 表位标签通过基因方法引入该区域。结果表明，第一个环的易位也独立于 YidC 和 Sec 易位酶。我们得出结论，KdpD 类似于一类新的膜蛋白，无需已知转位酶的酶促协助即可插入膜中。当第二个周质环通过表位标签延伸至 27 个氨基酸残基时，KdpD 该环的膜插入依赖于 SecE 和 YidC。为了测试两个周质区域是否彼此独立地易位，将 KdpD 蛋白在螺旋 2 和 3 之间分成两个大致相等大小的片段。两个构建的片段均含有 KdpD-N（KdpD 残基 1-448）和 KdpD-C（KdpD 残基 444-894），很容易插入膜中。与表位标记的 KdpD 蛋白类似，只有 KdpD-C 依赖于 Sec 易位酶和 YidC 的存在。这证实了 KdpD 的四个跨膜螺旋是成对插入的，每个易位事件涉及两个跨膜螺旋和一个周质环。
KdpD is a sensor kinase protein in the inner membrane of Escherichia coli containing four transmembrane regions. The periplasmic loops connecting the transmembrane regions are intriguingly short and protease mapping allowed us to only follow the translocation of the second periplasmic loop. The results show that neither the Sec translocase nor the YidC protein are required for membrane insertion of the second loop of KdpD. To study the translocation of the first periplasmic loop a short HA epitope tag was genetically introduced into this region. The results show that also the first loop was translocated independently of YidC and the Sec translocase. We conclude that KdpD resembles a new class of membrane proteins that insert into the membrane without enzymatic assistance by the known translocases.When the second periplasmic loop was extended by an epitope tag to 27 amino acid residues, the membrane insertion of this loop of KdpD depended on SecE and YidC. To test whether the two periplasmic regions are translocated independently of each other, the KdpD protein was split between helix 2 and 3 into two approximately equal-sized fragments. Both constructed fragments, which contained KdpD-N (residues 1-448 of KdpD) and the KdpD-C (residues 444-894 of KdpD), readily inserted into the membrane. Similar to the epitope-tagged KdpD protein, only KdpD-C depended on the presence of the Sec translocase and YidC. This confirms that the four transmembrane helices of KdpD are inserted pairwise, each translocation event involving two transmembrane helices and a periplasmic loop.