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.

Fig. 1.

Characterization of strains expressing TatA-YFP. (A) Growth of the indicated strains on LB/glycerol/TMAO medium under anoxic conditions. (B) Periplasmic TMAO reductase activity measured from intact cells of the indicated strains. ODU is cell concentration in units of OD_600nm. Error bars in A and B represent the SEM (n = 4). (C) Immunoblot of membranes isolated from the indicated strains probed with TatA antibodies (Upper) or antibodies against the outer membrane protein BamA as a loading control (Lower).

Fig. 2.

Fluorescence imaging of strains expressing TatA-YFP. (A) Fluorescence images of TatA-YFP in representative cells of the indicated strains. (Scale bar: 1 μm.) (B) The population distributions of the number of intense fluorescence spots per cell for the indicated strains. (C) The data in B presented as a box plot showing the median (thick black line), the interquartile range (IQR) (gray box), data within 1.5 IQR of the lower and upper quartiles (whiskers), and data points falling outside this range (●). The number of cells (N) examined in each population is shown above each distribution. The statistical significance of differences between the mean spot counts observed for selected pairs of strains was assessed by Welch’s t test with P < 0.05 taken as significant and marked with an asterisk. (D) Two-state mechanistic model for TatA oligomerization. In our experiments TatA is visualized in either a dispersed state (Left) or in an assembled state (Right). The interconversion between the states is controlled by binding of a substrate protein (orange) to the TatBC complex in the presence of the transmembrane PMF. Substrate protein is released from the TatABC complex after transport, leading to dissociation of the TatA oligomer. The numbers of TatA subunits and TatBC units shown in the assembled TatABC complex are arbitrary and were selected for illustrative convenience.

Fig. 3.

Substrate-induced assembly of TatA-YFP complexes. (A) Fluorescence images of TatA-YFP in representative cells of the strain AyBCE overproducing the plasmid-encoded Tat substrate proteins CueO (Upper Left) and HiPIP (Lower Left), or the functionally inactive CueO^KK (Upper Right) and HiPIP^KK (Lower Right) variants of these proteins in which the signal peptide consensus motif contains an RR-to-KK substitution. (Scale bar: 1 μm.) (B) Distribution of the number of TatA-YFP complexes per cell within each experimental population. The presentation of the distribution data and the statistical tests of the significance of differences between populations are as in Fig. 2C. (C) Fractionation of cells expressing either wild-type (WT) or KK variants of CueO and HiPIP. Total, periplasm, and spheroplast fractions were subject to immunoblot with antibodies against CueO (CueO WT/KK panels) or His₆ (HiPIP WT/KK panels). The lower panel in each pair shows the same samples blotted with the cytosolic marker DnaK to assess the quality of the fractionation. m, the transported form of the protein from which the signal peptide has been removed; p, precursor protein. (D) Time-lapse fluorescence microscopy of AyBCE cells after the induction of CueO expression. Midlog AyBCE cells carrying pQE80-CueO were immobilized on a tunnel slide. Fresh growth medium containing 1mM IPTG (Upper) or no IPTG (Lower) was introduced, and images of the same field were captured at the indicated time points. (Scale bar: 1 μm.) (E) (Upper) Immunoblot analysis of CueO induction in liquid culture over the same timescale as in the time-lapse experiment. (Lower) TatA levels were determined as a loading control.

Fig. 4.

Assembly of TatA-YFP complexes requires functional TatBC. (A) Representative fluorescence images of TatA-YFP in strain AyE producing, variously, the Tat substrate protein CueO from plasmid pQE80-CueO (pCueO), TatBC from plasmid p101C*TatBC (pBC), or an inactive TatBC variant from plasmid p101C*TatBC^FEA (pBC^FEA). (B) Distribution of the number of TatA-YFP complexes per cell within each experimental population. The presentation of the distribution data and the statistical tests of the significance of differences between populations are as in Fig. 2C. (C) Immunoblot showing production of TatB and TatC in membranes isolated from the indicated strains.

Fig. 5.

TatA Phe39 is not required for TatA association. (A) Growth of the indicated strains on LB/glycerol/TMAO medium under anoxic conditions. Error bars represent the SEM (n = 4). (B) Fluorescence images of TatA^F39A-YFP in representative cells of the indicated strains. (Scale bar: 1 μm.) (C) Distribution of the number of TatA-YFP complexes per cell within each experimental population. The presentation of the distribution data and the statistical tests of the significance of differences between populations are as in Fig. 2C.

Fig. 6.

Assembly of TatA-YFP complexes is PMF-dependent, but disassembly is PMF-independent. (A) (Top) Representative fluorescence images of TatA-YFP in cells of strain AyBCE overproducing the plasmid-encoded Tat substrate proteins CueO (Left) or HiPIP (Right). (Middle) Cells ∼30 min after the addition of 50 µM CCCP to dissipate the PMF. (Bottom) Cells ∼30 min after the addition of 0.05% β-mercaptoethanol (βME) to inactivate CCCP and allow restoration of the PMF. (B) Fluorescence images of TatA-YFP in cells of strain AyBCE pCueO. (Upper) Cells were imaged before and after treatment with 10 μg/mL colicin E1 (ColE1). (Lower) Outer-membrane permeabilized cells were imaged after treatment with mock buffer or buffer containing 50 μM nigericin, 36 μM valinomycin, and 0.4 M potassium acetate. (C) Distribution of the number of TatA-YFP complexes per cell within each experimental population and in the untreated strains. (D–F) Time-lapse fluorescence imaging of AyBCE cells carrying pQE80-CueO. (D) Immobilized cells on a tunnel slide were treated with 1 mM IPTG and incubated for 20 min. One cell was imaged (Left). Buffer containing 50 μM CCCP was introduced, and images were taken at the indicated times afterward (second and third panels). Buffer containing 0.05% βME was then introduced, and images were collected at the indicated times. (E) IPTG-induced cells were immobilized on a tunnel slide and imaged at 5-s intervals. Buffer containing 50 μM CCCP was added at 15 s (red arrow) to the cell in the upper panel. (F) IPTG-induced cells were immobilized on a tunnel slide and treated with 50 μM CCCP. Cells were then imaged at 20-s intervals. Buffer containing 0.05% βME was added at 10 s (black arrow) to the cells in the upper panel. (G) Representative fluorescence images of TatA-YFP in cells of the Ay^F39ABCE strain (Upper) or in AyBC cells (Lower) after exposure to 50 μM CCCP for ∼30 min. (H) Population distribution of spots per cell compared with untreated cells. In C and H the presentation of the distribution data and the statistical tests of the significance of differences between populations are as in Fig. 2C. (Scale bars: 1 μm.)

Fig. 7.

Fluorescence imaging of strains expressing TatB-YFP or TatC-YFP. (A) Representative fluorescence images of TatB-YFP produced from plasmid p101KBy in a ΔtatB host strain (ACE pBy) or a ΔtatBC host (AE pBy). (Lower Left) ACE pBy cells overproducing the plasmid-encoded Tat substrate protein CueO. (Lower Right) ACE pBy cells after treatment for 20 min with 50 μM CCCP. (B) Distribution of the number of TatB-YFP complexes per cell within each experimental population. (C) Representative fluorescence images of TatC-YFP produced from plasmid p101C*Cy in a ΔtatC background (ABE pCy) or a ΔtatBC background (AE pCy). The lower panels show the effects of CueO and CCCP treatments, as in A, on ABE pCy cells. Note that the scaling used to display the TatC-YFP images differs from that used with the other Tat-YFP fusions and uses 1,000 a.u. as the minimum (black) and 3,500 a.u. as the maximum (white). (D) Distribution of the number of TatC-YFP complexes per cell within each experimental population. In B and D, the presentation of the distribution data and the statistical tests of the significance of differences between populations are as in Fig. 2C. (Scale bars: 1 μm.)

Fig. 8.

Mechanistic model for changes in TatA oligomerization. Assembly of TatA occurs by stepwise recruitment to a substrate-activated TatBC complex. This process is driven in the forward direction by the transmembrane PMF. At a critical stage in the assembly process the TatABC–substrate assembly enters a metastable state (indicated by square brackets) from which it can be released only by the transport of the substrate protein. The TatABC complex without bound substrate is unstable, and TatA disassembles. Under conditions of high substrate availability it is possible that a new substrate molecule will be bound to the TatBC complex before TatA is able to dissociate. In this case the TatABC site could be either reused directly or recycled from a partially disassembled complex (dotted arrows). Experimental perturbations of the system are shown in red. Dissipation of the PMF (− PMF) reverses the direction of TatA assembly. Transport-defective strains AyBC and Ay^F39ABCE still can assemble TatA, are insensitive to the removal of the PMF, and are inferred to have a defect in progression from the metastable state. The number of steps shown for the TatA oligomerization processes and the number of TatA subunits and TatBC units in the assembled TatABC complexes are arbitrary and were selected for illustrative convenience.