Specificity of signal peptide recognition in Tat-dependent bacterial protein translocation

The bacterial twin arginine translocation (Tat) pathway translocates across the cytoplasmic membrane folded proteins which, in most cases, contain a t ightly bound cofactor. Specific amino-terminal signal peptides that exhibit a conserved amino acid consensus motif, SIT-R-R-X-F-L-K, direct these prot eins to the Tat translocon. The glucose-fructose oxidoreductase (GFOR) of Z ymomonas mobilis is a periplasmic enzyme with tightly bound NADP as a cofac tor, It is synthesized as a cytoplasmic precursor with an amino-terminal si gnal peptide that shows all of the characteristics of a typical twin argini ne signal peptide. However, GFOR is not exported to the periplasm when expr essed in the heterologous host Escherichia coli, and enzymatically active p re-GFOR is found in the cytoplasm, A precise replacement of the pre-GFOR si gnal peptide by an authentic E. coli Tat signal peptide, which is derived f rom pre-trimethylamine N-oxide (TMAO) reductase (TorA), allowed export of G FOR, together with its bound cofactor, to the E. coli periplasm, This expor t was inhibited by carbonyl cyanide m-chlorophenylhydrazone, but not by sod ium azide, and was blocked in E. coli tatC and tatAE mutant strains, Showin g that membrane translocation of the TorA-GFOR fusion protein occurred via the Tat pathway and not via the Sec pathway. Furthermore, tight cofactor bi nding (and therefore correct folding) was found to be a prerequisite for pr oper translocation of the fusion protein. These results strongly suggest th at Tat signal peptides are not universally recognized by different Tat tran slocases, implying that the signal peptides of Tat-dependent precursor prot eins are optimally adapted only to their cognate export apparatus. Such a s ituation is in marked contrast to the situation that is known to exist for Sec-dependent protein translocation.