15N-ラベリング法を用いた枯草菌膜タンパク質局在化における SecAの機能解析

抄録

Bacillus subtilis is a Gram-positive bacterium having high levels of ability to secrete extracellular enzymes and its genome sequence was published at 1997 by Kunst et al. (1). B. subtilis possesses SecA protein, which is a prokaryote unique ATPase to plays a central role on the extracellular enzyme translocation across the cytoplasmic membrane. However, the relation between SecA function and the localization of membrane proteins in B. subtilis is studied very little. To resolve this subject, the ideal method, in which the de novo synthesized proteins were labeled with ¹⁵N and the relative amount of newly located proteins into the cell membrane were quantitively analyzed by MALDI-TOF MS after the separation of the proteins by 2D-PAGE, provided an efficient one. As results, the localization of 25 proteins in the tested 28 was inhibited by the absence of SecA. The dependency of the 28 proteins on SecA for their localization into the cell membrane could be classified into three groups by the strength of the inhibition. The first protein group including all lipoproteins and some membrane proteins was directly dependent on SecA, whose localization was immediately inhibited by the lack of SecA. In contrast, the second protein group including most of integral membrane proteins was indirectly dependent on SecA because the inhibition by the lack to SecA was delayed. Third group was independent from SecA because no inhibition of the localization was observed. These results suggested that several kinds of machinery relating to the membrane protein localization will be equipped in B. subtilis cells.
(1) Kunst, F. et al., Nature 390. 249-256 (1997).