Type 3 secretion translocators spontaneously assemble a hexadecameric transmembrane complex

Atype 3 secretion system is used by many bacterial pathogens to inject proteins into eukaryotic cells. Pathogens insert a translocon complex into the target eukaryotic membrane by secreting two proteins known as translocators.Howthese translocators form a translocon in the lipid bilayer and why both proteins are required remains elusive. Pseudomonas aeruginosa translocators PopB and PopD insert pores into membranes forming homo- or hetero-complexes of undetermined stoichiometry. Single-molecule fluorescence photobleaching experiments revealed that PopD formed mostly hexameric structures in membranes, whereas PopB displayed a bi-modal distribution with 6 and 12 subunits peaks. However, individually the proteins are not functional for effector translocation.Wehave found that when added together, the translocators formed distinct heterocomplexes containing 8 PopB and 8 PopD molecules. Thus, the interaction between PopB and PopD guide the assembly of a unique hetero-oligomer in membranes.