New Insights into Amino-Terminal Translocation as Revealed by the Use of YidC and Sec Depletion Strains

Different attributes of membrane protein substrates have been proposed and characterized as translocation-pathway determinants. However, several gaps in our understanding of the mechanism of targeting, insertion, and assembly of inner-membrane proteins exist. Specifically, the role played by hydrophilic N-terminal tails in pathway selection is unclear. In this study, we have evaluated length and charge density as translocase determinants using model proteins. Strikingly, the 36-residue N-tail of 2Pf3-Lep translocates independent of YidC-Sec. This is the longest known substrate of this pathway. We confirmed this using a newly constructed YidC-Sec double-depletion strain. Increasing its N-tail length with uncharged spacer peptides led to YidC dependence and eventually YidC-Sec dependence, hence establishing that length has a linear effect on translocase dependence. Tails longer than 60 residues were not inserted; however, an MBP-2Pf3-Lep fusion protein could be ranslocated. This suggests that longer N-tails can be translocated if it can engage SecA. In addition, we have examined how the positioning of charges within the translocated N-tail affects the insertion pathway. Additional charges can be translocated by the Lep TM when the charges are distributed across a longer N-tail. We tested charge density as a translocase determinant and confirmed that the addition of positive or negatives charges led to a greater dependence on YidC-Sec when they were placed close to each other than away. Findings from this work make an important advance in our existing knowledge about the different insertion mechanisms of membrane proteins in Escherichia coli.