Abstract
The Sec translocon of bacterial plasma membranes mediates the linear translocation of secretory proteins as well as the lateral integration of membrane proteins. Integration of many membrane proteins occurs co-translationally via the signal recognition particle (SRP)-dependent targeting of ribosome-associated nascent chains to the Sec translocon. In contrast, translocation of classical secretory proteins across the Sec translocon is a post-translational event requiring no SRP but the motor protein SecA. Secretory proteins were, however, reported to utilize SRP in addition to SecA, if the hydrophobicity of their signal sequences exceeds a certain threshold value. Here we have analyzed transport of this subgroup of secretory proteins across the Sec translocon employing an entirely defined in vitro system. We thus found SecA to be both necessary and sufficient for translocation of secretory proteins with hydrophobic signal sequences, whereas SRP and its receptor improved translocation efficiency. This SRP-mediated boost of translocation is likely due to the early capture of the hydrophobic signal sequence by SRP as revealed by site-specific photo cross-linking of ribosome nascent chain complexes.
Highlights
The heterotrimeric SecYEG translocon located in the inner membrane of Gram-negative bacteria is used by two different groups of proteins to be exported from the cytoplasm of these organisms: secretory proteins destined for the periplasmic space or the outer membrane are translocated through the pore of the Sec translocon, whereas inner membrane proteins exit laterally from the Sec translocon into the lipid bilayer
Whilst SecA is sufficient for translocation of secretory proteins with hydrophobic signal sequences, signal recognition particle (SRP) and its receptor FtsY improve translocation efficiency by enabling co-translational membrane targeting
The DDM-solubilized SecYEG complex was reconstituted with E. coli phospholipids into small proteoliposomes by dialysis followed by sonication of the vesicles
Summary
The heterotrimeric SecYEG translocon located in the inner membrane of Gram-negative bacteria is used by two different groups of proteins to be exported from the cytoplasm of these organisms: secretory proteins destined for the periplasmic space or the outer membrane are translocated through the pore of the Sec translocon, whereas inner membrane proteins exit laterally from the Sec translocon into the lipid bilayer. Both classes of proteins carry characteristic targeting signals. In the case of secretory proteins these are classical N-terminal signal sequences consisting of a positively charged N-region, a hydrophobic core, and a polar C-region. For a recent comprehensive review on the structure and function of the Sec translocon, see ref. [1]
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