Structural and functional analysis of the C-terminal cytoplasmic domain of FIhA, an integral membrane component of the type III flagellar protein export apparatus in Salmonella

FlhA is an integral membrane component of the Salmonella type III flagellar protein export apparatus. It consists of 692 amino acid residues and has two domains: the N-terminal transmembrane domain consisting of the first 327 amino acid residues, and the C-terminal cytoplasmic domain (FlhA(C)) comprising the remainder. Here, we have investigated the structure and function of FlhA(C). DNA sequence analysis revealed that temperature-sensitive flhA mutations, which abolish flagellar protein export at the restrictive temperature, lie in FlhA(C), indicating that FlhA(C) plays an, important role in the protein export process. Limited proteolysis of purified His-FlhA(C) by trypsin and V8 showed that only a small part of FlhA(C) near its N terminus (residues 328-351) is sensitive to proteolysis. FlhA(C)38K, the smallest fragment produced by V8 proteolysis, is monomeric and has a spherical shape as judged by analytical gel filtration chromatography and analytical ultracentrifugation. The far-UV CD spectrum of FlhA(C)38K showed that it contains considerable amounts of secondary structure. FlhA(Delta328-351) missing residues 328-351 failed to complement the flhA mutant, indicating that the proteolytically sensitive region of FlhA is important for its function. FlhA(Delta328-351) was inserted into the cytoplasmic membrane, and exerted a strong dominant negative effect on wild-type cells, suggesting that it retains the ability to interact with other export components within the cytoplasmic membrane. Overproduced FlhA(C)38K inhibited both motility and flagellar protein export of wild-type cells to some degree, suggesting that FlhA(C)38K is directly involved in the translocation reaction. Amino acid residues 328-351 of FlhA appear to be a relatively flexible linker between the transmembrane domain and FlhA(C)38K. (C) 2004 Elsevier Ltd. All rights reserved.