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J. Biol. Chem., Vol. 280, Issue 50, 41236-41242, December 16, 2005

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FlhB Regulates Ordered Export of Flagellar Components via Autocleavage Mechanism^*

Hedda U. Ferris1, Yukio Furukawa, Tohru Minamino¶, Mary B. Kroetz, May Kihara, Keiichi Namba¶, and Robert M. Macnab

From the Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, the Dynamic NanoMachine Project, International Cooperative Research Project, Japan Science and Technology Corporation, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan, and the ^¶Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan

The bacterial flagellum is a predominantly cell-external super-macromolecular 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 (FlhB_C), which is consistently and specifically cleaved into two subdomains (FlhB_CN and FlhB_CC) 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, FlhB_C(P270A), is stimulated in vitro at alkaline pH. We also show by analytical gel-filtration chromatography and analytical ultracentrifugation experiments that both FlhB_C 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.

Received for publication, August 26, 2005 , and in revised form, October 20, 2005.

^* This work was supported by USPHS Grant AI12202 (to R. M. M.), a graduate research fellowship from the National Science Foundation (to M. B. K.), and the MMPATH T32 AI007640 Training Grant (to H. U. F.). This report was taken from a dissertation submitted to fulfill in part the requirements for the degree of Doctor of Philosophy at Yale University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Robert M. Macnab passed away on September 7, 2003.

¹ To whom correspondence should be addressed: Dept. of Molecular Biophysics and Biochemistry, Yale University, P. O. Box 208114, New Haven, CT 06520-8114. Tel.: 203-432-5589; Fax: 203-432-9782; E-mail: hedda.ferris{at}yale.edu.

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