Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system

Abstract

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.

TatE as a Regular Constituent of Bacterial Twin-arginine Protein Translocases

“…1B, arrows). A similar occasional clustering has been described previously for a GFP fusion of TatA when expressed in wild-type E. coli cells (31,34), and it has been shown to represent Tat translocases that are engaged in the transport of the endogenous Tat substrates of the cells.…”

The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia coli upon protein substrate binding

“…The problem of these approaches is, that the dissociation of such TatA-XFP fusions from TatBC could be triggered by the ~26 kDa XFP tag attached to the 9.6 kDa TatA. In agreement with this, it has been observed that TatA-YFP was functionally inactive and activity could be only reestablished by co-production of non-tagged TatA or TatE (54). Substrate binding might influence the affinity of TatBC for TatA, as mutations that enhance the affinity of TatBC to Tat substrates show a constitutive TatA-YFP/TatBC interaction (55).…”

“…The initial experimental evidence for a transient recruitment of TatA to TatBC upon substrate binding to TatBC is based on cross-link data that may also have resulted from rearrangements of TatA at TatBC upon substrate binding (44,12,45). Other lines of evidence come from TatA that has been labeled with fluorescent protein (XFP) tags (53,54). The problem of these approaches is, that the dissociation of such TatA-XFP fusions from TatBC could be triggered by the ~26 kDa XFP tag attached to the 9.6 kDa TatA.…”

Assembly pathway of a bacterial complex iron sulfur molybdoenzyme

“…Substrate protein recognition by the TatBC complex subsequently results in the recruitment and oligomerization of TatA protomers to form the active translocon pore using energy from the proton motive force (86,87). As the purported protein-conducting channel, the TatA multi-protomer pore unit must be large enough to permit passage of large protein substrates possessing secondary, tertiary, and in many cases quaternary structure with sizes ranging from ~10 kDa to ~140 kDa (88)(89)(90).…”

“…As the purported protein-conducting channel, the TatA multi-protomer pore unit must be large enough to permit passage of large protein substrates possessing secondary, tertiary, and in many cases quaternary structure with sizes ranging from ~10 kDa to ~140 kDa (88)(89)(90). Following translocation initiation from the TatBC recognition unit, substrates are translocated through the TatA multimer pore across the cytoplasmic membrane utilizing the electrochemical gradient (86,88,(90)(91)(92)(93). Several studies have found that the substrate protein interacts with TatA and TatBC separately, suggesting that the translocon is not fully assembled until receiving a substrate (88)(89)(90)94).…”