| Summary: | Substructure of the <i>S. typhimurium </i>flagellum are built sequentially by the successive addition of structural subunits, which are delivered via a dedicated type III secretion system. By characterising export-defective variants of a representative early subunit (FlGD), it was found that the secretion signal contains two distinct, separable elements that act concurrently to promote secretion. Deletion of amino acids 36-40 prevented FlgD from being targeted to the membrane and abolished interactions between the export signal and machinery components <i>in vivo</i>. The sequence was important for FliI ATPase-dependent targeting but not in pathways lacking the ATPase complex. A similar amino acid motif was identified in all early subunits and was required for FliK and FlgE to localise to the membrane in a FliI-dependent way. The data are compatible with the view that FliI acts as a primary docking site for subunits and suggests that early export substrates are recognised on the basis of common motif. Removal of amino acids 2-5 of FlgD abolished secretion, but the resulting variant was not defective in FliI-dependent targeting. Instead, the variant stalled post-docking to the membrane and inhibited flagellar export. FliI was revealed to be essential, but not sufficient for formation of the membrane-associated stalled intermediate, which also required integral membrane export components. The role of these proteins in the cascade of subunit binding events that occur downstream of FliI is discussed. A key physiochemical property of the immediate <i>N</i>-terminal residues was identified by isolating suppressors of the truncated FlgD variant. These findings were extrapolated to other flagellar subunits and a model for FlgD recognition via a bipartite secretion signal is presented.
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