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 tightly bound cofactor. Specific amino-terminal signal peptides that exhibit a conserved amino acid consensus motif, S/T-R-R-X-F-L-K, direct these proteins to the Tat translocon. The glucose-fructose oxidoreductase (GFOR) of Zymomonas mobilis is a periplasmic enzyme with tightly bound NADP as a cofactor. It is synthesized as a cytoplasmic precursor with an amino-terminal signal peptide that shows all of the characteristics of a typical twin arginine signal peptide. However, GFOR is not exported to the periplasm when expressed in the heterologous host Escherichia coli, and enzymatically active pre-GFOR is found in the cytoplasm. A precise replacement of the pre-GFOR signal peptide by an authentic E. coli Tat signal peptide, which is derived from pre-trimethylamine N-oxide (TMAO) reductase (TorA), allowed export of GFOR, together with its bound cofactor, to the E. coli periplasm. This export was inhibited by carbonyl cyanide m-chlorophenylhydrazone, but not by sodium azide, and was blocked in E. coli tatC and tatAE mutant strains, showing that membrane translocation of the TorA-GFOR fusion protein occurred via the Tat pathway and not via the Sec pathway. Furthermore, tight cofactor binding (and therefore correct folding) was found to be a prerequisite for proper translocation of the fusion protein. These results strongly suggest that Tat signal peptides are not universally recognized by different Tat translocases, implying that the signal peptides of Tat-dependent precursor proteins are optimally adapted only to their cognate export apparatus. Such a situation is in marked contrast to the situation that is known to exist for Sec-dependent protein translocation.
Blaudeck
N
N
Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany.
Sprenger
G A
GA
Freudl
R
R
Wiegert
T
T
D000595:Amino Acid Sequence; D001120:Arginine; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000378:metabolism*; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D021382:Protein Sorting Signals; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019810:Sodium Azide / Q000494:pharmacology; D013045:Species Specificity; D017355:Zymomonas / Q000378:metabolism*
D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D001120:Arginine; D019810:Sodium Azide; D010088:Oxidoreductases; C074291:glucose-fructose oxidoreductase; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase