Genetic toggling of alkaline phosphatase folding reveals signal peptides for all major modes of transport across the inner membrane of bacteria.
Prediction of export pathway specificity in prokaryotes is a challenging endeavor due to the similar overall architecture of N-terminal signal peptides for the Sec-, SRP- (signal recognition particle), and Tat (twin arginine translocation)-dependent pathways. Thus, we sought to create a facile experimental strategy for unbiased discovery of pathway specificity conferred by N-terminal signals. Using a limited collection of Escherichia coli strains that allow protein oxidation in the cytoplasm or, conversely, disable protein oxidation in the periplasm, we were able to discriminate the specific mode of export for PhoA (alkaline phosphatase) fusions to signal peptides for all of the major modes of transport across the inner membrane (Sec, SRP, or Tat). Based on these findings, we developed a mini-Tn5 phoA approach to isolate pathway-specific export signals from libraries of random fusions between exported proteins and the phoA gene. Interestingly, we observed that reduced PhoA was exported in a Tat-independent manner when targeted for Tat export in the absence of the essential translocon component TatC. This suggests that initial docking to TatC serves as a key specificity determinant for Tat-specific routing of PhoA, and in its absence, substrates can be rerouted to the Sec pathway, provided they remain compatible with the Sec export mechanism. Finally, the utility of our approach was demonstrated by experimental verification that four secreted proteins from Mycobacterium tuberculosis carrying putative Tat signals are bona fide Tat substrates and thus represent potential Tat-dependent virulence factors in this important human pathogen.
Marrichi
Matthew
M
School of Chemical and Biomolecular Engineering, Department of Microbiology and Immunology, Cornell University, Ithaca, New York 14853, USA.
Camacho
Luis
L
Russell
David G
DG
DeLisa
Matthew P
MP
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D017510:Protein Folding; D021382:Protein Sorting Signals; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; C461379:TatC protein, E coli; C408585:twin-arginine translocase complex, E coli; D000469:Alkaline Phosphatase; C114089:phoA protein, E coli; D010100:Oxygen