Université de Paris-Sud, Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, Orsay, France.
J Bacteriol. 2014 Jan;196(1):121-8. doi: 10.1128/JB.00750-13. Epub 2013 Oct 18.
Proteins called secretins form large multimeric complexes that are essential for macromolecular transit across the outer membrane of Gram-negative bacteria. Evidence suggests that the channels formed by some secretin complexes are not tightly closed, but their permeability properties have not been well characterized. Here, we used cell-free synthesis coupled with spontaneous insertion into liposomes to investigate the permeability of the secretin PulD. Leakage assays using preloaded liposomes indicated that PulD allows the efflux of small fluorescent molecules with a permeation cutoff similar to that of general porins. Other secretins were also found to form similar pores. To define the polypeptide region involved in determining the pore size, we analyzed a collection of PulD variants and studied the roles of gates 1 and 2, which were previously reported to affect the pore size of filamentous phage f1 secretin pIV, in assembly and pore formation. Liposome leakage and a novel in vivo assay showed that replacement of the conserved proline residue at position 443 in PulD by leucine increased the apparent size of the pore. The in vitro approach described here could be used to study the pore properties of membrane proteins whose production in vivo is toxic.
被称为分泌蛋白的蛋白质形成大型多聚体复合物，对于革兰氏阴性细菌外膜的大分子跨膜运输是必不可少的。有证据表明，一些分泌蛋白复合物形成的通道不是紧密关闭的，但它们的渗透性特性尚未得到很好的描述。在这里，我们使用无细胞合成并自发插入脂质体来研究分泌蛋白 PulD 的渗透性。使用预加载脂质体的泄漏测定表明，PulD 允许小分子荧光分子的流出，其渗透率截止值类似于一般孔蛋白。还发现其他分泌蛋白形成类似的孔。为了确定决定孔径的多肽区域，我们分析了一组 PulD 变体，并研究了先前报道影响丝状噬菌体 f1 分泌蛋白 pIV 孔径的门 1 和门 2 在组装和孔形成中的作用。脂质体泄漏和新的体内测定表明，用亮氨酸取代 PulD 中位置 443 处保守的脯氨酸残基会增加孔的表观尺寸。这里描述的体外方法可用于研究体内产生毒性的膜蛋白的孔特性。
