Oligomeric State of Membrane-Assembled Type III Secretion Translocators PopB and PopD Determined by Single-Molecule Photobleaching

Abstract

P. aeruginosa and other Gram-negative pathogens use a Type 3 Secretion (T3S) system to secrete and inject proteins into eukaryotic cells. The T3S system resembles a “nanosyringe” formed by a basal body that spans both bacterial membranes and a needle structure through which proteins are secreted. The secreted proteins translocate across the target cell membrane presumably through a proteinaceous pore or translocon assembled by two T3S proteins, the translocators PopB and PopD. We have recently shown that PopB and PopD efficiently bind liposomal membranes, homo or hetero-oligomerize individually or in combination, and form discrete size pores in membranes. (Romano et al, 2011 Biochemistry. 50 (33), 7117-31). However, it remains unknown how these complexes assemble and what is their specific composition. We studied the order of protein-protein and protein-membrane interactions preceding complex assembly by time-resolved FRET. Consequently, membrane inserted complexes were studied using single-molecule fluorescence photobleaching on supported lipid bilayers.Time-resolved FRET studies revealed unfolded translocators bind to the membrane first, then oligomerize and form discrete transmembrane pores. Single-molecule analysis indicated translocators form similar oligomeric structures when assembled individually, and larger hetero-complexes when assembled together. Our data provides a first report on the stoichiometric arrangement of membrane inserted T3S translocators.