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

doi:10.1074/jbc.m509438200

Hedda U Ferris, Yukio Furukawa + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m509438200

Copy DOI

Abstract

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.

Highlights

A large percentage of the bacterial flagellar structure lies outside of the cell envelope, requiring that the vast majority of the subunits that compose the flagellum be exported from the cytosol across both the inner and outer membranes
In an attempt to better understand the role of FlhB cleavage in substrate-specificity switching, we addressed the fundamental question: 2 The abbreviations used are: FlhBC, cytoplasmic domain of FlhB; FlhBC(P270A), slowcleaving variant of FlhBC; FlhBC(N269A), non-cleaving variant of FlhBC; FlhBCN, N-terminal subdomain of FlhBC; FlhBCC, C-terminal subdomain of FlhBC; MALDI-TOF, matrix-assisted laser desorption ionization-time-of-flight
We decided to assay for cleavage when FlhBC is overexpressed in S. cerevisiae, based on the assumption that it is highly unlikely that this heterologous host would have a protease that would consistently and cleave a bacterial type III flagellar export component

Summary

Introduction

A large percentage of the bacterial flagellar structure lies outside of the cell envelope, requiring that the vast majority of the subunits that compose the flagellum be exported from the cytosol across both the inner and outer membranes. When FlhBTMϩCN and FlhBCC are expressed from two different plasmids (resulting in an “already cleaved” FlhB) they are able to successfully complete both rod/hook and filament formation, not to wild-type levels [17] All these observations led to a proposal that the C-terminal domain of FlhB has two substrate-specificity states and that a conformational change, mediated by cleavage and the interaction between FlhBCN and FlhBCC, regulates the specificity-switching process. The results support autocleavage via a succinimide pathway in which the cleavage site itself is the catalytic domain, active only when FlhB is in the appropriate conformation The implications of this on current models of export substrate-specificity switching are discussed

Objectives

Methods

Results

Conclusion