SecY-mediated quality control prevents the translocation of non-gated porins

OmpC and OmpF are among the most abundant outer membrane proteins in $\text it {E. coli}$ and serve as hydrophilic channels to mediate uptake of small molecules including antibiotics. Influx selectivity is controlled by the so-called constriction zone or eyelet of the channel. Mutations in the loop domain forming the eyelet can disrupt transport selectivity and thereby interfere with bacterial viability. In this study we show that a highly conserved motif of five negatively charged amino acids in the eyelet, which is critical to regulate pore selectivity, is also required for SecY-mediated transport of OmpC and OmpF into the periplasm. Variants with a deleted or mutated motif were expressed in the cytosol and translocation was initiated. However, after signal peptide cleavage, import into the periplasm was aborted and the mutated proteins were redirected to the cytosol. Strikingly, reducing the proof-reading capacity of SecY by introducing the PrlA4 substitutions restored transport of OmpC with a mutated channel domain into the periplasm. Our study identified a SecY-mediated quality control pathway to restrict transport of outer membrane porin proteins with a deregulated channel activity into the periplasm

Posttranslational insertion of small membrane proteins by the bacterial signal recognition particle

Small membrane proteins represent a largely unexplored yet abundant class of proteins in pro- and eukaryotes. They essentially consist of a single transmembrane domain and are associated with stress response mechanisms in bacteria. How these proteins are inserted into the bacterial membrane is unknown. Our study revealed that in Escherichia coli, the 27-amino-acid-long model protein YohP is recognized by the signal recognition particle (SRP), as indicated by in vivo and in vitro site-directed cross-linking. Cross-links to SRP were also observed for a second small membrane protein, the 33-amino-acid-long YkgR. However, in contrast to the canonical cotranslational recognition by SRP, SRP was found to bind to YohP posttranslationally. In vitro protein transport assays in the presence of a SecY inhibitor and proteoliposome studies demonstrated that SRP and its receptor FtsY are essential for the posttranslational membrane insertion of YohP by either the SecYEG translocon or by the YidC insertase. Furthermore, our data showed that the yohP mRNA localized preferentially and translation-independently to the bacterial membrane in vivo. In summary, our data revealed that YohP engages an unique SRP-dependent posttranslational insertion pathway that is likely preceded by an mRNA targeting step. This further highlights the enormous plasticity of bacterial protein transport machineries