Our current studies focus on protein targeting to and translocation across the E. coli IM. Most of the major factors that promote the targeting and translocation reactions are extremely highly conserved, and E. coli has proven to be an outstanding model organism in which to study these conserved factors. In addition, obtaining a better understanding of protein translocation across the IM is of great interest to the biotechnology industry, which has sought to optimize the production of secreted proteins in E. coli. Finally, it may ultimately be possible to exploit small differences in the protein targeting and translocation machinery in bacteria and eukaryotic cells to develop novel antimicrobials. Recent studies by other groups have shown that cytoplasmic proteins are exported efficiently in E. coli only if they are attached to signal peptides that are recognized by the signal recognition particle (SRP) and are thereby targeted to the SecYEG complex cotranslationally. These studies suggest that the entry of these proteins into the secretory pathway at an early stage of translation is necessary to prevent them from folding into a translocation-incompetent conformation. We found, however, that several glycolytic enzymes attached to signal peptides that are recognized by SRP are exported inefficiently. Based on previous studies of post-translational export, we hypothesized that the export block was due to the presence of basic residues at the extreme N-terminus of each enzyme. Consistent with our hypothesis, we found that the introduction of negatively charged residues into this segment increased the efficiency of export. Export efficiency was sensitive to the number, position and sequence context of charged residues. The importance of charge for efficient export was underscored by an in silico analysis that revealed a conserved negative charge bias at the N-terminus of the mature region of bacterial presecretory proteins. Our results demonstrate that cotranslational targeting of a protein to the E. coli SecYEG complex does not ensure its export, but that export also depends on a subsequent event (most likely the initiation of translocation) that involves sequences both within and just beyond the signal peptide.

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