Kuehn M J, Normark S, Hultgren S J
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110.
Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10586-90. doi: 10.1073/pnas.88.23.10586.
Molecular chaperones are found in the cytoplasm of bacteria and in various cellular compartments in eukaryotes to maintain proteins in nonnative conformations that permit their secretion across membranes or assembly into oligomeric structures. Virtually nothing, however, has been reported about a similar requirement for molecular chaperones in the periplasm of Gram-negative bacteria. We used the well-characterized P pilus biogenesis system in Escherichia coli as a model to elucidate the mechanism of action of a periplasmic chaperone, PapD, which is specifically required for P pilus biogenesis. PapD probably associates with at least six P pilus subunits after their secretion across the cytoplasmic membrane, but PapD is not incorporated into the pilus. We used purified periplasmic complex that PapD forms with the PapG adhesin to investigate the function of interactions between the chaperone and its targets. We demonstrated that PapD binds to PapG to form a stable, discrete bimolecular complex and that, unlike cytoplasmic chaperones, the periplasmic PapD chaperone maintained PapG in a native-like conformation. Bound PapD in the complex was displaced by free PapD in vitro; however, the in vivo release of subunits to the nascent pilus is probably driven by an ATP-independent mechanism involving the outer membrane protein PapC. In addition, the binding of PapD to PapG in vitro prevented aggregation of PapG. We propose that the function of PapD and other periplasmic pilus chaperones is to partition newly translocated pilus subunits into assembly-competent complexes and thereby prevent nonproductive aggregation of the subunits in the periplasm. These data provide important information for understanding the mechanism of action of this general class of chaperones that function in the periplasmic space.
分子伴侣存在于细菌的细胞质以及真核生物的各种细胞区室中，可维持蛋白质处于非天然构象，使它们能够跨膜分泌或组装成寡聚结构。然而，关于革兰氏阴性菌周质中对分子伴侣的类似需求，几乎没有相关报道。我们以大肠杆菌中特征明确的P菌毛生物合成系统为模型，来阐明周质伴侣蛋白PapD的作用机制，PapD是P菌毛生物合成所特需的。PapD可能在至少六个P菌毛亚基跨细胞质膜分泌后与其结合，但PapD并不掺入菌毛中。我们利用PapD与PapG粘附素形成的纯化周质复合物，来研究伴侣蛋白与其靶标之间相互作用的功能。我们证明，PapD与PapG结合形成稳定、离散的双分子复合物，并且与细胞质伴侣蛋白不同，周质PapD伴侣蛋白使PapG维持在类似天然的构象。复合物中结合的PapD在体外被游离的PapD取代；然而，亚基在体内向新生菌毛的释放可能由一种不依赖ATP的机制驱动，该机制涉及外膜蛋白PapC。此外，PapD在体外与PapG的结合可防止PapG聚集。我们提出，PapD和其他周质菌毛伴侣蛋白的功能是将新转运的菌毛亚基分配到有组装能力的复合物中，从而防止亚基在周质中发生无效聚集。这些数据为理解在周质空间发挥作用的这类一般伴侣蛋白的作用机制提供了重要信息。
