Abstract
In bacteria most secretory proteins are transported across the plasma membrane by the interplay of the ATPase SecA with the translocation channel formed by the SecY complex; SecA uses cycles of ATP hydrolysis to “push” consecutive segments of a polypeptide substrate through the channel. Here we have addressed the mechanism of this process by following the fate of stalled translocation intermediates. These were generated by using a polypeptide substrate containing a bulky disulfide-bonded loop, thus preventing the final residues from passing through the channel. Protease protection experiments showed that the intermediates were stable in the presence of ATP and could complete translocation once the block was removed. The translocation intermediate was also stable when SecA associated with ATPγS, a poorly hydrolyzable ATP analog, or ADP plus AlF4, which mimics the transition state during ATP hydrolysis. In contrast, when SecA was in its ADP-bound state, the translocating polypeptide moved back into the cytosol, as indicated by the disappearance of the protected fragment. Backsliding was not significantly altered by deletion of the plug domain, a short helix in the center of the SecY channel, but it was slowed down when changes were introduced into the pore ring, the constriction of the hourglass-shaped channel. In all cases, backsliding was significantly slower than forward translocation. Together, these data suggest that SecA binds the polypeptide chain in its ATP state and releases it in the ADP state. The channel itself does not bind the polypeptide chain but provides “friction” that minimizes backsliding when ADP-bound SecA resets to “grab” the next segment of the substrate.
Highlights
In bacteria most secretory proteins are transported posttranslationally across the plasma membrane
As expected [18], a protected ϳ18k-Da fragment was seen that corresponds to the ϳ150 N-terminal amino acids of the substrate (Fig. 1A, lane 2); this corresponds to the segment of the translocation intermediate that is located inside the vesicles when the disulfide loop reaches the channel
When DTT was added at the beginning of the incubation to reduce the disulfide-bonded loop, only the full-length polypeptide was seen after proteolysis (Fig. 1A, lane 1; the doublet is caused by cleavage of the signal sequence by signal peptidase in Inverted membrane vesicles (IMVs))
Summary
In bacteria most secretory proteins are transported posttranslationally across the plasma membrane (for reviews, see Refs. 1–3). As expected [18], a protected ϳ18k-Da fragment was seen that corresponds to the ϳ150 N-terminal amino acids of the substrate (Fig. 1A, lane 2); this corresponds to the segment of the translocation intermediate that is located inside the vesicles when the disulfide loop reaches the channel.
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