Nagamori S, Nishiyama K, Tokuda H
Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
J Biochem. 2000 Jul;128(1):129-37. doi: 10.1093/oxfordjournals.jbchem.a022723.
SecG, a membrane component of the protein translocation apparatus of Escherichia coli, undergoes membrane topology inversion, which is coupled to the membrane insertion and deinsertion cycle of SecA. Eighteen SecG derivatives possessing a single cysteine residue at various positions were constructed and expressed in a secG null mutant. All the SecG-Cys derivatives retained the SecG function, and stimulated protein translocation both in vivo and in vitro. Inverted membrane vesicles containing a SecG-Cys derivative were labeled with a membrane-permeable or -impermeable sulfhydryl reagent before or after solubilization with a detergent. The accessibility of these reagents to the cysteine residue of each derivative determined the topological arrangement of SecG in the membrane. Derivatives having the cysteine residue in the periplasmic region each existed as a homodimer crosslinked through disulfide bonds, indicating that two SecG molecules closely co-exist in a single translocation machinery. The crosslinking did not abolish the SecG function and the crosslinked SecG dimer underwent topology inversion upon protein translocation.
SecG是大肠杆菌蛋白质转运装置的一种膜成分，会经历膜拓扑结构反转，这与SecA的膜插入和去插入循环相关。构建了18种在不同位置含有单个半胱氨酸残基的SecG衍生物，并在secG缺失突变体中表达。所有SecG-半胱氨酸衍生物都保留了SecG功能，并在体内和体外刺激蛋白质转运。在用去污剂溶解之前或之后，用可透过膜或不可透过膜的巯基试剂标记含有SecG-半胱氨酸衍生物的反转膜囊泡。这些试剂对半胱氨酸残基的可及性决定了SecG在膜中的拓扑排列。在周质区域含有半胱氨酸残基的衍生物各自以通过二硫键交联的同型二聚体形式存在，这表明两个SecG分子在单个转运机制中紧密共存。交联并不消除SecG功能，并且交联的SecG二聚体在蛋白质转运时会发生拓扑结构反转。
