Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003, USA.
Biochemistry. 2011 Aug 23;50(33):7117-31. doi: 10.1021/bi200905x. Epub 2011 Jul 26.
Translocation of bacterial toxins or effectors into host cells using the type III secretion (T3S) system is a conserved mechanism shared by many Gram-negative pathogens. Pseudomonas aeruginosa injects different proteins across the plasma membrane of target cells, altering the normal metabolism of the host. Protein translocation presumably occurs through a proteinaceous transmembrane pore formed by two T3S secreted protein translocators, PopB and PopD. Unfolded translocators are secreted through the T3S needle prior to insertion into the target membrane. Purified PopB and PopD form pores in model membranes. However, their tendency to form heterogeneous aggregates in solution had hampered the analysis of how these proteins undergo the transition from a denatured state to a membrane-inserted state. Translocators were purified as stable complexes with the cognate chaperone PcrH and isolated from the chaperone using 6 M urea. We report here the assembly of stable transmembrane pores by dilution of urea-denatured translocators in the presence of membranes. PopB and PopD spontaneously bound liposomes containing anionic phospholipids and cholesterol in a pH-dependent manner as observed by two independent assays, time-resolved Förster resonance energy transfer and sucrose-step gradient ultracentrifugation. Using Bodipy-labeled proteins, we found that PopB interacts with PopD on the membrane surface as determined by excitation energy migration and fluorescence quenching. Stable transmembrane pores are more efficiently assembled at pH <5.0, suggesting that acidic residues might be involved in the initial membrane binding and/or insertion. Altogether, the experimental setup described here represents an efficient method for the reconstitution and analysis of membrane-inserted translocators.
使用 III 型分泌系统（T3S）将细菌毒素或效应物转运到宿主细胞中是许多革兰氏阴性病原体共有的保守机制。铜绿假单胞菌将不同的蛋白质注入靶细胞的质膜中，改变宿主的正常代谢。蛋白质转运推测是通过由两个 T3S 分泌蛋白转运器 PopB 和 PopD 形成的蛋白质跨膜孔发生的。未折叠的转运器在插入靶膜之前通过 T3S 针状结构分泌。纯化的 PopB 和 PopD 在模型膜中形成孔。然而，它们在溶液中形成异质聚集体的趋势阻碍了分析这些蛋白质如何从变性状态过渡到插入膜状态。转运器与同源伴侣蛋白 PcrH 形成稳定的复合物，并使用 6 M 尿素从伴侣蛋白中分离出来。我们在这里报告了在膜存在的情况下通过稀释尿素变性转运器来组装稳定的跨膜孔。PopB 和 PopD 以 pH 依赖性方式自发结合含有阴离子磷脂和胆固醇的脂质体，如通过两种独立的测定方法、时间分辨Förster 共振能量转移和蔗糖步梯度超速离心观察到的那样。使用 Bodipy 标记的蛋白质，我们发现 PopB 在膜表面与 PopD 相互作用，如激发能量迁移和荧光猝灭所确定的那样。在 pH <5.0 时更有效地组装稳定的跨膜孔，这表明酸性残基可能参与初始膜结合和/或插入。总的来说，这里描述的实验设置代表了用于重建和分析插入膜的转运器的有效方法。
