Akimaru J, Matsuyama S, Tokuda H, Mizushima S
Institute of Applied Microbiology, University of Tokyo, Japan.
Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6545-9. doi: 10.1073/pnas.88.15.6545.
Reconstitution of the translocation machinery for secretory proteins from purified constituents was performed. SecY was solubilized from SecY/SecE-overproducing Escherichia coli cells and purified by chromatography on ion-exchange and size-exclusion columns. Proteoliposomes active in protein translocation were reconstituted from the purified preparations of SecY and SecE. The reconstituted translocation activity was SecA- and ATP-dependent. Although the purified preparations of SecY and SecE were still contaminated with minute amounts of other proteins, the elution profiles of SecY and SecE on column chromatographies coincided with the elution profiles of reconstituted translocation activity, indicating that SecY and SecE are the indispensable components in these preparations. We conclude that SecY, SecE, and SecA are essential components of the protein secretion machinery and that translocation activity can be reconstituted from only these three proteins and phospholipids.
利用纯化成分对分泌蛋白转运机制进行了重构。SecY从过量表达SecY/SecE的大肠杆菌细胞中溶解出来，并通过离子交换和尺寸排阻柱层析进行纯化。从SecY和SecE的纯化制剂中重构了具有蛋白质转运活性的蛋白脂质体。重构的转运活性依赖于SecA和ATP。尽管SecY和SecE的纯化制剂仍被微量的其他蛋白质污染，但SecY和SecE在柱层析上的洗脱图谱与重构转运活性的洗脱图谱一致，表明SecY和SecE是这些制剂中不可或缺的成分。我们得出结论，SecY、SecE和SecA是蛋白质分泌机制的必需成分，并且仅从这三种蛋白质和磷脂就可以重构转运活性。
