Department of Biochemistry and Biomedical Sciences and the Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario L8S 4K1.
Program in Molecular Structure & Function, The Hospital for Sick Children, and the Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1X8, Canada.
J Biol Chem. 2013 Apr 5;288(14):9721-9728. doi: 10.1074/jbc.M113.453506. Epub 2013 Feb 14.
A systematic genetic analysis was performed to identify the inner membrane proteins essential for type IV pilus (T4P) expression in Pseudomonas aeruginosa. By inactivating the retraction aspect of pilus function, genes essential for T4P assembly were discriminated. In contrast to previous studies in the T4P system of Neisseria spp., we found that components of the inner membrane subcomplex consisting of PilMNOP were not essential for surface pilus expression, whereas the highly conserved inner membrane protein PilC was essential. Here, we present data that PilC may coordinate the activity of cytoplasmic polymerization (PilB) and depolymerization (PilT) ATPases via their interactions with its two cytoplasmic domains. Using in vitro co-affinity purification, we show that PilB interacts with the N-terminal cytoplasmic domain of PilC. We hypothesized that PilT similarly interacts with the PilC C-terminal cytoplasmic domain. Overexpression of that domain in the wild-type protein reduced twitching motility by ∼50% compared with the vector control. Site-directed mutagenesis of conserved T4P-specific residues in the PilC C-terminal domain yielded mutant proteins that supported wild-type pilus assembly but had a reduced capacity to support twitching motility, suggesting impairment of putative PilC-PilT interactions. Taken together, our results show that PilC is an essential inner membrane component of the T4P system, controlling both pilus assembly and disassembly.
我们对铜绿假单胞菌的 IV 型菌毛（T4P）表达进行了系统的遗传分析，以鉴定其内膜蛋白。通过使菌毛功能的回缩方面失活，区分了 T4P 组装所必需的基因。与先前在奈瑟氏菌属的 T4P 系统中的研究不同，我们发现由 PilMNOP 组成的内膜亚基的组成部分对于表面菌毛表达不是必需的，而高度保守的内膜蛋白 PilC 是必需的。在这里，我们提供的数据表明 PilC 可能通过与两个细胞质结构域的相互作用来协调细胞质聚合（PilB）和解聚（PilT）ATPase 的活性。我们使用体外共亲和纯化，表明 PilB 与 PilC 的 N 端细胞质结构域相互作用。我们假设 PilT 同样与 PilC 的 C 端细胞质结构域相互作用。与载体对照相比，该结构域在野生型蛋白中的过表达将扭转型运动降低了约 50%。在 PilC C 端结构域中 T4P 特异性保守残基的定点突变产生了支持野生型菌毛组装但支持扭转型运动的能力降低的突变蛋白，这表明 PilC-PilT 相互作用受损。总之，我们的结果表明 PilC 是 T4P 系统的必需内膜成分，控制菌毛的组装和拆卸。
