Analysis of Polypeptide Movement in the SecY Channel during SecA-mediated Protein Translocation

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.

Bacterial Sec Protein Transport Is Rate-limited by Precursor Length: A Single Turnover Study

“…Because 5 M NiCl 2 was present in both the gel-and fluorescence-based assays, the observed difference in transport rates cannot be explained by an effect of NiCl 2 on transport activity. Likely explanations include the loss of the bound Ni 2ϩ before complete precursor translocation (discussed in detail in the Supplementary Material and Discussion), and/or backsliding of the precursor through the translocation channel during work-up for gel analysis (Erlandson et al, 2008b). According to the gel-based assay, the presence of 5 M NiCl 2 caused a slight (ϳ27%) increase in translocation time compared with that observed in the absence of NiCl 2 (Supplemental Figure S3A).…”

Position-dependent Effects of Polylysine on Sec Protein Transport

“…If counter ions do indeed move with charged residues through the translocation channel, sliding of the precursor protein in both directions could be relatively facile. Backsliding has been demonstrated experimentally (51). Note that the total charge translocated does not have to be entirely compensated, on average, by comigrating counter ions.…”

Steric contribution of macromolecular crowding to the time and activation energy for preprotein translocation across the endoplasmic reticulum membrane

“…As indicated in the introduction, experiments with inverted membrane vesicles and proteoliposomes (in the presence of ATP and chaperones that promote translocation) have reported different values for the translocation rate, that is, the number of amino acids translocated per second. For forward translocation of proOmpA derivatives, the translocation rate was found as 0.8-1 aa/s in proteoliposomes and 2.5 aa/s in inverted membrane vesicles (IMVs) [5]. The translocation rate for in-tandem constructs of proOmpA, whose length range from 347 to ∼1400 amino acids, was 4.5 aa/s in IMVs [6].…”

“…For cotranslational cases, the in vivo translocation speed is limited by the ribosome synthesis rate: in prokaryotes, it is 15-20 amino acids (aa) per second, and in eukaryotes is 3-4 aa/s [4]. For posttranslational translocation, in vitro experiments using inverted membrane vesicles and proteoliposomes yield translocation rates of 0.8-8 aa/s [5][6][7]. However, since these experiments have been performed only in a diluted regime, with soluble protein concentrations on the order of a few millimoles per liter, the effect of macromolecular crowding on protein translocation has not been measured.…”