Abstract
The bacterial twin-arginine translocation (Tat) pathway is well known to translocate correctly folded monomeric and dimeric proteins across the tightly sealed cytoplasmic membrane. We identified a naturally occurring heterotrimer, the Escherichia coli aldehyde oxidoreductase PaoABC, that is co-translocated by the Tat translocase according to a ternary “hitchhiker” mechanism. Specifically, the PaoB and PaoC subunits, each devoid of export signals, are escorted to the periplasm in a piggyback fashion by the Tat signal peptide-containing subunit PaoA. Moreover, export of PaoA was blocked when either PaoB or PaoC was absent, revealing a surprising interdependence for export that is not seen for classical secretory proteins. Inspired by this observation, we created a bacterial three-hybrid selection system that links the formation of ternary protein complexes with antibiotic resistance. As proof-of-concept, a bispecific antibody was employed as an adaptor that physically crosslinked one antigen fused to a Tat export signal with a second antigen fused to TEM-1 β-lactamase (Bla). The resulting non-covalent heterotrimer was exported in a Tat-dependent manner, delivering Bla to the periplasm where it hydrolyzed β-lactam antibiotics. Collectively, these results highlight the remarkable flexibility of the Tat system and its potential for studying and engineering ternary protein interactions in living bacteria.
Highlights
The bacterial twin-arginine translocation (Tat) pathway is well known to translocate correctly folded monomeric and dimeric proteins across the tightly sealed cytoplasmic membrane
We hypothesized that PaoABC might be exported out of the cytoplasm by the Tat translocase according to a hitchhiker mechanism whereby PaoB and PaoC are ferried to the periplasm by the signal peptide-bearing PaoA subunit
Following expression of the synthetic paoABC operon in wild-type MC4100 cells, all three PaoABC subunits were detected in the periplasm, with a comparable amount of each subunit detected in the cytoplasm (Fig. 1)
Summary
The bacterial twin-arginine translocation (Tat) pathway is well known to translocate correctly folded monomeric and dimeric proteins across the tightly sealed cytoplasmic membrane. The HybOC heterodimer is exported to the periplasm by virtue of the Tat signal peptide on the HybO subunit[9] This mode of export, whereby one substrate protein devoid of any known export signal is co-translocated in a complex with its signal peptide-bearing partner, is referred to as ‘‘hitchhiker’’ co-translocation[9]. Hitchhiker-mimetic genetic assays for monitoring and engineering pairwise protein interactions have been reported[14,15] In these assays, the test protein (i.e., bait or receptor) to be screened is engineered with an N-terminal Tat signal peptide, whereas the known or www.nature.com/scientificreports putative partner protein (e.g., prey or ligand) is fused to a reporter enzyme whose co-translocation to the periplasm gives rise to a distinct and quantifiable phenotype. Akin to HybOC, there is an interdependence between the small, signalpeptide bearing PaoA subunit and the larger PaoB and PaoC subunits for productive membrane translocation of PaoABC
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