Proteolytic analysis of the FliH/FliI complex, the ATPase component of the type III flagellar export apparatus of Salmonella.

The ATPase FliI of the Salmonella type III flagellar protein export apparatus is a 456 amino acid residue cytoplasmic protein consisting of two regions, an N-terminal flagellum-specific region and a C-terminal ATPase region. It forms a complex with a regulatory protein FliH in the cytoplasm. Multi-angle light-scattering studies indicate that FliH forms a homodimer, (FliH)2, and that FliH and FliI together form a heterotrimer, (FliH)2FliI. Mobility upon gel-filtration chromatography gives much higher apparent molecular masses for both species, whereas the mobility of FliI is normal. Sedimentation velocity measurements indicate that both (FliH)2 and the FliH/FliI complex are quite elongated. We have analyzed FliH, FliI and the FliH/FliI complex for proteolytic sensitivity. FliI was degraded by clostripain into two stable fragments, one of 48 kDa (FliI(CL48), missing the first seven amino acid residues) and the other of 46 kDa (FliI(CL46), missing the first 26 residues). Small amounts of two closely spaced 38 kDa fragments (FliI(CL38), missing the first 93 and 97 residues, respectively) were also detected. The FliH homodimer was insensitive to clostripain proteolysis and provided protection to FliI within the FliH/FliI complex. Neither FliI(CL48) nor FliI(CL46) could form a complex with FliH, demonstrating that the N terminus of FliI is essential for the interaction. ATP, AMP-PNP, and ADP bound forms of FliI within the FliH/FliI complex regained sensitivity to clostripain cleavage. Also, the sensitivity of the two FliI(CL38) cleavage sites was much greater in the ATP and AMP-PNP bound forms than in either the ADP bound form or nucleotide-free FliI. The ATPase domain itself was insensitive to clostripain cleavage. We suggest that the N-terminal flagellum-specific region of FliI is flexible and changes its conformation during the ATP hydrolysis cycle.