The twin-arginine translocation (Tat) machinery transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. It has been inferred that the Tat translocation site is assembled on demand by substrate-induced association of the protein TatA. We tested this model by imaging YFP-tagged TatA expressed at native levels in living Escherichia coli cells in the presence of low levels of the TatA paralogue TatE. Under these conditions the TatA-YFP fusion supports full physiological Tat transport activity. In agreement with the TatA association model, raising the number of transport-competent substrate proteins within the cell leads to an increase in the number of large TatA complexes present. Formation of these complexes requires both a functional TatBC substrate receptor and the transmembrane proton motive force (PMF). Removing the PMF causes TatA complexes to dissociate, except in strains with impaired Tat transport activity. Based on these observations we propose that TatA assembly reaches a critical point at which oligomerization can be reversed only by substrate transport. In contrast to TatA-YFP, the oligomeric states of TatB-YFP and TatC-YFP fusions are not affected by substrate or the PMF, although TatB-YFP oligomerization does require TatC.

The twin-arginine protein translocation system (Tat) transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membranes of plant chloroplasts. The Tat transporter is assembled from multiple copies of the membrane proteins TatA, TatB, and TatC. We combine sequence co-evolution analysis, molecular simulations, and experimentation to define the interactions between the Tat proteins of Escherichia coli at molecular-level resolution. In the TatBC receptor complex the transmembrane helix of each TatB molecule is sandwiched between two TatC molecules, with one of the inter-subunit interfaces incorporating a functionally important cluster of interacting polar residues. Unexpectedly, we find that TatA also associates with TatC at the polar cluster site. Our data provide a structural model for assembly of the active Tat translocase in which substrate binding triggers replacement of TatB by TatA at the polar cluster site. Our work demonstrates the power of co-evolution analysis to predict protein interfaces in multi-subunit complexes.DOI: http://dx.doi.org/10.7554/eLife.20718.001

The mitochondrial inner and outer membranes are composed of a variety of integral membrane proteins, assembled into the membranes posttranslationally. The small translocase of the inner mitochondrial membranes (TIMs) are a group of approximately 10 kDa proteins that function as chaperones to ferry the imported proteins across the mitochondrial intermembrane space to the outer and inner membranes. In yeast, there are 5 small TIM proteins: Tim8, Tim9, Tim10, Tim12, and Tim13, with equivalent proteins reported in humans. Using hidden Markov models, we find that many eukaryotes have proteins equivalent to the Tim8 and Tim13 and the Tim9 and Tim10 subunits. Some eukaryotes provide "snapshots" of evolution, with a single protein showing the features of both Tim8 and Tim13, suggesting that a single progenitor gene has given rise to each of the small TIMs through duplication and modification. We show that no "Tim12" family of proteins exist, but rather that variant forms of the cognate small TIMs have been recently duplicated and modified to provide new functions: the yeast Tim12 is a modified form of Tim10, whereas in humans and some protists variant forms of Tim9, Tim8, and Tim13 are found instead. Sequence motif analysis reveals acidic residues conserved in the Tim10 substrate-binding tentacles, whereas more hydrophobic residues are found in the equivalent substrate-binding region of Tim13. The substrate-binding region of Tim10 and Tim13 represent structurally independent domains: when the acidic domain from Tim10 is attached to Tim13, the Tim8-Tim13(10) complex becomes essential and the Tim9-Tim10 complex becomes dispensable. The conserved features in the Tim10 and Tim13 subunits provide distinct binding surfaces to accommodate the broad range of substrate proteins delivered to the mitochondrial inner and outer membranes.

Three classes of ion-driven protein motors have been identified to date: ATP synthase, the bacterial flagellar motor, and a proton-driven motor that powers gliding motility and the Type 9 protein secretion system (T9SS) in Bacteroidetes bacteria. Here, we present cryo-EM structures of the gliding motility/T9SS motors GldLM from Flavobacterium johnsoniae and PorLM from Porphyromonas gingivalis . The motor is an asymmetric inner membrane protein complex in which the single transmembrane helices of two periplasm-spanning GldM/PorM proteins are positioned inside a ring of five GldL/PorL proteins. Mutagenesis and single-molecule tracking identifies protonatable amino acid residues in the transmembrane domain of the complex that are important for motor function. Our data provide evidence for a mechanism in which proton flow results in rotation of the periplasm-spanning GldM/PorM dimer inside the intra-membrane GldL/PorL ring to drive processes at the bacterial outer membrane.