Figure 5

Heterologously expressed TatA proteins are located exclusively in the membrane. A. Strains MC4100 (E. coli parental strain; tat⁺; WT) and JARV16-P (as MC4100, ΔtatA/ΔtatE, pcnB; ΔtatA/E) were grown aerobically to late exponential phase in LB medium prior to harvesting and fractionation into membrane (M) and soluble (C) fractions. Samples were separated by SDS PAGE, electroblotted and probed with anti E. coli TatA antiserum. B. Strain JARV16-P (ΔtatA/E) or JARV16-P carrying pUniprom-SA encoding S. coelicolor TatA (ScoA) were cultured, fractionated and blotted as above, and probed with anti S. coelicolor TatA peptide antiserum. C. Strain M15 [pREP4] harboring either pQEAQ1 (AaeA1) or pQEAQ2 (AaeA2) encoding A. aeolicus TatA1 or TatA2 with C-terminal histags, respectively. Cells were cultured in LB medium until OD600 of 0.4 was reached, after which expression of the TatA protein was induced by addition of 1 mM isopropyl-β-D-galactopyranoside (IPTG) for a further 2 hours. Cells were fractionated and blotted as above, and probed with anti pentahis antiserum. For each panel, membrane and soluble material from an equivalent amount of cells was loaded.