The early interaction of the outer membrane protein PhoE with the periplasmic chaperone Skp occurs at the cytoplasmic membrane
The early interaction of the outer membrane protein PhoE with the periplasmic chaperone Skp occurs at the cytoplasmic membrane
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DOI：
10.1074/jbc.m011194200
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发表时间：
2001-06-01
影响因子：
4.8
通讯作者：
de Cock, H
中科院分区：
文献类型：
--
作者：
Harms, N;Koningstein, G;de Cock, H
球体被用来研究早期的相互作用，新合成的外膜蛋白PhoE与周质蛋白采用蛋白质交联的方法。新易位的PhoE蛋白可以在内膜的周质侧与周质伴侣Skp交联。为了研究这种相互作用的时间，构建了形成易位中间体的PhoE-二氢叶酸还原酶杂合蛋白，其具有存在于周质中的PhoE部分和在细胞质中紧密折叠的二氢叶酸还原酶部分。发现杂合蛋白与Skp交联，表明当蛋白质仍处于易位酶中的跨膜取向时，PhoE与伴侣蛋白密切相互作用。去除的N-末端部分的PhoE蛋白影响Skp的结合在一个累积的方式，在该地区的两个Skp结合位点的存在是一致的。相反，C-末端部分的缺失导致与Skp的可变相互作用，这表明Skp与N-末端区域的相互作用受到PhoE蛋白C末端部分的影响。可溶性和膜相关的Skp蛋白被发现与PhoE相互作用，后一种形式被提出参与与外膜蛋白的N-末端区域的初始相互作用。
Spheroplasts were used to study the early interactions of newly synthesized outer membrane protein PhoE with periplasmic proteins employing a protein cross-linking approach. Newly translocated PhoE protein could be cross-linked to the periplasmic chaperone Skp at the periplasmic side of the inner membrane. To study the timing of this interaction, a PhoE-dihydrofolate reductase hybrid protein was constructed that formed translocation intermediates, which had the PhoE moiety present in the periplasm and the dihydrofolate reductase moiety tightly folded in the cytoplasm, The hybrid protein was found to cross-link to Skp, indicating that PhoE closely interacts with the chaperone when the protein is still in a transmembrane orientation in the translocase. Removal of N-terminal parts of PhoE protein affected Skp binding in a cumulative manner, consistent with the presence of two Skp-binding sites in that region. In contrast, deletion of C-terminal parts resulted in variable interactions with Skp, suggesting that interaction of Skp with the N-terminal region is influenced by parts of the C terminus of PhoE protein. Both the soluble as well as the membrane-associated Skp protein were found to interact with PhoE, The latter form is proposed to be involved in the initial interaction with the N-terminal regions of the outer membrane protein.