Disulfide bridge formation between SecY and a translocating polypeptide localizes the translocation pore to the center of SecY

Evidence is provided that translocating polypeptides pass through the center of the SecY complex, and the strongest cross-links were observed with residues that would form a constriction in an hourglass-shaped pore, suggesting that the channel makes only limited contact with a translocatingpolypeptide, thus minimizing the energy required for translocation.

Abstract

During their biosynthesis, many proteins pass through the membrane via a hydrophilic channel formed by the heterotrimeric Sec61/SecY complex. Whether this channel forms at the interface of multiple copies of Sec61/SecY or is intrinsic to a monomeric complex, as suggested by the recently solved X-ray structure of the Methanococcus jannaschii SecY complex, is a matter of contention. By introducing a single cysteine at various positions in Escherichia coli SecY and testing its ability to form a disulfide bond with a single cysteine in a translocating chain, we provide evidence that translocating polypeptides pass through the center of the SecY complex. The strongest cross-links were observed with residues that would form a constriction in an hourglass-shaped pore. This suggests that the channel makes only limited contact with a translocating polypeptide, thus minimizing the energy required for translocation.