Preserving the membrane barrier for small molecules during bacterial protein translocation

Many proteins are translocated through the SecY channel in bacteria and archaea, and the related Sec61 channel in eukaryotes(1). The channel has an hourglass shape with a narrow constriction approximately halfway across the membrane, formed by a pore ring of amino acids(2). While the cytoplasmic cavity of the channel is empty, the extra-cellular cavity is filled with a short helix, the plug(2), which moves out of the way during protein translocation(3,4). The mechanism by which the channel transports large polypeptides and yet prevents the passage of small molecules, such as ions or metabolites, has been controversial(2,5–8). Here, we have addressed this issuein intact E. coli cells by testing the permeation of small molecules through wild-type and mutant SecY channels, which are either in the resting state or contain a defined translocating polypeptide chain. In the resting state, the channel is sealed by both the pore ring and the plug domain. During translocation the pore ring forms a gasket-like seal around the polypeptide chain, preventing the permeation of small molecules. The structural conservation of the channel in all organisms suggests a universal mechanism by which the membrane barrier is maintained during protein translocation.