The twin-arginine translocation (Tat) pathway guides fully folded proteins across membranes of bacteria, archaea and plant chloroplasts. In Escherichia coli, Tat-specific transport is executed in a still largely unknown manner by three functionally diverse membrane proteins, termed TatA, TatB, and TatC. In order to follow the intracellular distribution of… (More)
Figure 2. The TatA-GFP fusion is stable and functionally active. (A) TatA-GFP was expressed in E. coli BL21(DE3) cells from a pBAD33 vector (pPR1) following 2 h of induction by the indicated amounts of arabinose. Whole cell proteins were precipitated with trichloroacetic acid and proteins were separated by SDS-PAGE. Shown is an immunoblot decorated with antibodies against TatA. TatABC wild type cells (wt) show a TatA signal at about 15 kDa (arrow) that is absent from tatABC deletion cells (Δtat) and enhanced in inner membrane vesicles (INV), which had been prepared from a TatABC-overexpressing strain. Cells induced with arabinose display a TatA-GFP band at about 36 kDa. Expression of TatA-GFP was slightly reduced by the co-expression of TorAmCherry from plasmid pPJ3 (lanes 8 and 10). * non-specific band. (B–D) Expression of TorA-mCherry-SsrA from plasmid pPR8 in JARV16 (ΔtatAE) E. coli cells and the indicated transformants thereof. In the absence of TatAE, the Tat substrate remains cytosolic and cell separation is impaired (B). Transport of TorA-mCherry to the periplasm (arrows) and cell separation are restored by coexpression of TatA from plasmid pPR4 (C) and of TatA-GFP from plasmid pPR1 (D, inset shows cells from a different picture) demonstrating functionality of the TatA-GFP fusion. (E) Cells as in (D) now showing distribution of TatA-GFP. TatA-GFP forms similar clusters as when co-expressed with TatA (cf. Figure 3). (F) Growth of cell colonies on LB agar plates containing 2% SDS and 0.1% arabinose. Cells grown in LB liquid media were serially diluted and 5µl were each applied to the agar plate. Both TatA and TatA-GFP complement the growth defect of the ΔtatAE mutant strain JARV16 on 2% SDS. (G) Immunoblot of subcellular fractions decorated with antibodies against SufI. The strains MC4100 (wt), JARV16 (ΔtatAE) and JARV16/pPR1 (ΔtatAE+pBAD33TatA-GFP) were transformed with plasmid pPJ9 expressing the native Tat-substrate SufI. Cells were grown at 37° C and 180 rpm to an OD600 = 0.3 in LB-medium, supplemented with 0.1% L-arabinose for 1.5 h to induce synthesis of TatA-GFP and subsequently with 50 ng/mL anhydro-tetracycline for an additional 1.5 h to induce synthesis of SufI. Fractionation was carried out as described in Materials and Methods. Periplasmic fractions (P) and cytoplasm/membrane fractions (C/M) were loaded in a ratio 40:1 onto an 8% SDS-gel.