FlhB regulates ordered export of flagellar components via autocleavage mechanism

Hedda U. Ferris, Yukio Furukawa, Tohru Minamino, Mary B. Kroetz, May Kihara, Keiichi Namba, Robert M. Macnab

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Abstract

The bacterial flagellum is a predominantly cell-external supermacromolecular construction whose structural components are exported by a flagellum-specific export apparatus. One of the export apparatus proteins, FlhB, regulates the substrate specificity of the entire apparatus; i.e. it has a role in the ordered export of the two main groups of flagellar structural proteins such that the cell-proximal components (rod-/hook-type proteins) are exported before the cell-distal components (filament-type proteins). The controlled switch between these two export states is believed to be mediated by conformational changes in the structure of the C-terminal cytoplasmic domain of FlhB (FlhBC), which is consistently and specifically cleaved into two subdomains (FlhBCN and FlhBCC) that remain tightly associated with each other. The cleavage event has been shown to be physiologically significant for the switch. In this study, the mechanism of FlhB cleavage has been more directly analyzed. We demonstrate that cleavage occurs in a heterologous host, Saccharomyces cerevisiae, deficient in vacuolar proteinases A and B. In addition, we find that cleavage of a slow-cleaving variant, FlhBC(P270A), is stimulated in vitro at alkaline pH. We also show by analytical gel-filtration chromatography and analytical ultracentrifugation experiments that both FlhBC and FlhB C(P270A) are monomeric in solution, and therefore self-proteolysis is unlikely. Finally, we provide evidence via peptide analysis and FlhB cleavage variants that the tertiary structure of FlhB plays a significant role in cleavage. Based on these results, we propose that FlhB cleavage is an autocatalytic process.

Original languageEnglish (US)
Pages (from-to)41236-41242
Number of pages7
JournalJournal of Biological Chemistry
Volume280
Issue number50
DOIs
StatePublished - Dec 16 2005

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    Ferris, H. U., Furukawa, Y., Minamino, T., Kroetz, M. B., Kihara, M., Namba, K., & Macnab, R. M. (2005). FlhB regulates ordered export of flagellar components via autocleavage mechanism. Journal of Biological Chemistry, 280(50), 41236-41242. https://doi.org/10.1074/jbc.M509438200