Each protomer of a dimeric YidC functions as a single membrane insertase.

The membrane insertase YidC catalyzes the entrance of newly synthesized proteins into the lipid bilayer. As an integral membrane protein itself, YidC can be found as a monomer, a dimer or also as a member of the holotranslocase SecYEGDF-YajC-YidC. To investigate whether the dimeric YidC is functional and whether two copies cooperate to insert a single substrate, we constructed a fusion protein where two copies of YidC are connected by a short linker peptide. The 120 kDa protein is stable and functional as it supports the membrane insertion of the M13 procoat protein, the C-tailed protein SciP and the fusion protein Pf3-Lep. Mutations that inhibit either protomer do not inactivate the insertase and rather keep it functional. When both protomers are defective, the substrate proteins accumulate in the cytoplasm. This suggests that the dimeric YidC operates as two insertases. Consistent with this, we show that the dimeric YidC can bind two substrate proteins simultaneously, suggesting that YidC indeed functions as a monomer.