Functional self-nonamerization of the Type III translocase chaperone/exported protein receptor

The Type III protein secretion (T3S) pathway is widespread in bacterial Gram-negative pathogens. It comprises the injectisome with a cytoplasm-facing inner membrane translocase and a surface-exposed needle. The translocase comprises a conical SctR5S4T1 export channel, decorated by SctU, and enveloped by SctV. The large cytoplasmic domain (C-domain) of SctV binds T3S chaperone/exported protein and forms a putative ante-chamber leading to the membrane translocase. Here we probed the mechanism of assembly and function of SctV. Using live cell imaging, SctV was shown to assemble in peripheral oligomeric clusters in both EPEC and a non-T3SS harbouring E.coli strain. Non-ionic detergents extracted SctV homo-nonamers from membranes of both strains. His-SctV9 reconstituted in peptidiscs revealed an elongated, tripartite particle of ~22nm with a membrane domain and a narrower linker connecting to a C-domain. The C-domain assembles in a hollow asymmetric ring with a 5-6 nm-wide inner opening. SctV9 is necessary and sufficient to act as a receptor for two different chaperone/exported protein pairs by binding them at distinct C-domain sites identified by immobilized peptide arrays. Binding sites are not only important for binding but also essential for secretion suggesting a close mechanistic link between the receptor and secretion activities. These findings advance structural understanding of injectisome assembly and reveal that chaperone/exported protein targeting is mechanistically uncoupled from the succeeding translocation step.