Targeting of unfolded PhoA to the TAT translocon of Escherichia coli.
In Escherichia coli, the Tat system does not translocate Tat signal sequence fused PhoA (RR-PhoA), as it requires disulfide formation for folding. Here we show that such a RR-PhoA construct can be efficiently targeted to the Tat translocon, but the transport is not completed. RR-PhoA is detectable in a 580-kDa TatBC-containing complex, which is the first substrate-bound TatBC complex detected in a bacterial system so far. A second TatBC complex near 440 kDa comprises most of the TatB and TatC but is devoid of RR-PhoA. The targeting of PhoA to the Tat translocon depends on the twin-arginine motif and results in severe growth defects. This physiological effect is likely to be due to proton leakage at the cytoplasmic membrane. The results point to mechanistic incompatibilities of the Tat system with unfolded proteins such as RR-PhoA. There does not exist an intrinsic quality control at the TatBC complex itself, although correct folding is inevitable for Tat-dependent translocation.
Richter
Silke
S
Institute of Microbiology, University of Halle-Wittenberg, Kurt-Mothes-Strasse 3, D-06120 Halle, Germany.
Brüser
Thomas
T
D020816:Amino Acid Motifs; D001120:Arginine / Q000737:chemistry; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D021381:Protein Transport; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors
D004220:Disulfides; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011522:Protons; D011994:Recombinant Proteins; C499697:TatA protein, E coli; C473116:TatB protein, E coli; C461379:TatC protein, E coli; D001120:Arginine