Disulfide bridge formation between SecY and a translocating polypeptide localizes the translocation pore to the center of SecY
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.
© 2005 Rockefeller University Press. After the Initial Publication Period, RUP will grant to the public the non-exclusive right to copy, distribute, or display the Article under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/legalcode, or updates thereof. Submitted: 2 December 2004; Accepted: 4 March 2005. Thanks to Drs. Wendy Garrett, Tommy Kirchhausen, Andrew Osborne, and Pamela Wearsch for critical reading of the manuscript. We thank Andrew Osborne (T. Rapoport's laboratory) for the cysteine-free SecA mutant. This work was supported by a National Institutes of Health grant to T.A. Rapoport. K.S. Cannon is a Richard D. Frisbee III Foundation Fellow of the Leukemia & Lymphoma Society. W.M. Clemons Jr. has a fellowship from the Damon Runyon Cancer Research Foundation. T.A. Rapoport is a Howard Hughes Medical Institute Investigator.
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