Klose M, Schimz K L, van der Wolk J, Driessen A J, Freudl R
Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, Germany.
J Biol Chem. 1993 Feb 25;268(6):4504-10.
The SecA protein is a major component of the cellular machinery that mediates the translocation of proteins across the Escherichia coli plasma membrane. The secA gene from Bacillus subtilis was cloned and expressed in E. coli under the control of the lac or trc promoter. The temperature-sensitive growth and secretion defects of various E. coli secA mutants were complemented by the B. subtilis SecA protein, provided the protein was expressed at moderate levels. Under overproduction conditions, no complementation was observed. One of the main features of the SecA protein is the translocation ATPase activity which, together with the protonmotive force, drives the movement of proteins across the plasma membrane. A putative ATP-binding motif can be identified in the SecA protein resembling the consensus Walker A type motif. Replacement of a lysine residue at position 106, which corresponds to an invariable amino acid residue, in the consensus motif by asparagine (K106N) resulted in the loss of the ability of the B. subtilis SecA protein to complement the growth and secretion defects of E. coli secA mutants. In addition, the presence of the K106N SecA protein interfered with protein translocation, most likely at an ATP-requiring step. We conclude that lysine 106 is part of the catalytic ATP-binding site of the B. subtilis SecA protein, which is required for protein translocation in vivo.
SecA蛋白是介导蛋白质穿过大肠杆菌质膜转运的细胞机制的主要组成部分。枯草芽孢杆菌的secA基因在lac或trc启动子的控制下在大肠杆菌中克隆并表达。只要该蛋白以适度水平表达，枯草芽孢杆菌SecA蛋白就能弥补各种大肠杆菌secA突变体的温度敏感生长和分泌缺陷。在过量表达条件下，未观察到互补作用。SecA蛋白的主要特征之一是转运ATP酶活性，它与质子动力一起驱动蛋白质穿过质膜的运动。在SecA蛋白中可以鉴定出一个假定的ATP结合基序，类似于一致的沃克A型基序。在一致基序中，将对应于一个不变氨基酸残基的第106位赖氨酸残基替换为天冬酰胺（K106N），导致枯草芽孢杆菌SecA蛋白丧失弥补大肠杆菌secA突变体生长和分泌缺陷的能力。此外，K106N SecA蛋白的存在干扰了蛋白质转运，最有可能是在一个需要ATP的步骤。我们得出结论，赖氨酸106是枯草芽孢杆菌SecA蛋白催化ATP结合位点的一部分，这是体内蛋白质转运所必需的。
