Abstract
Newly synthesized mitochondrial precursor proteins have to become unfolded by the mitochondrial Hsp70 (mtHsp70) import motor to cross the mitochondrial membranes. To assess the mechanism of unfolding of precursor proteins by mtHsp70, we designed a system to measure step sizes of the mtHsp70 import motor, which are distances at which the motor system moves along polypeptide chains during a single turnover of ATP. We made a series of fusion proteins consisting of a mitochondrial presequence containing the first mtHsp70 binding site, a spacer sequence containing an Hsp70 avoidance segment followed by the second mtHsp70 binding site, and different folded mature domains. Analyses of the dependence of the import rates of those fusion proteins on the lengths of Hsp70 avoidance segments allowed us to estimate the step sizes, which differ for different mature domains and different lengths of the spacers. These results suggest that the mtHsp70 import motor functions at least as a molecular Brownian ratchet to unfold mitochondrial precursor proteins.
Highlights
Mitochondrial presequences penetrate through the import channels of the translocators in the outer and inner membranes to reach the matrix, where it is grasped by mtHsp[70] that is bound to the inner membrane translocator, the TIM23 complex, via its subunit Tim[44] (9, 10)
In the power stroke or lever arm model, mtHsp[70] tethered to the outlet of the import channel undergoes a conformational change upon ATP hydrolysis to exert a mechanical pulling force on the precursor protein, which drives unfolding of the mature domain (11)
Step sizes for the heme binding domain of cytochrome b2 (HBD) and barnase fusion proteins are different from those for the dihydrofolate reductase (DHFR) fusion proteins when compared for the same total spacer lengths, indicating that the Brownian ratchet, which predicts that step sizes differ for different mature domains, is a likely mechanism that contributes primarily to unfolding of the folded domain by mtHsp[70]
Summary
Assume that various lengths of the Hsp[70] avoidance segment (m-residues) are inserted between the first and second mtHsp[70] binding sites (10 residues each) in the presequence and following the spacer sequence (l residues), respectively, in front of the folded mature domain in model mitochondrial precursor proteins (Fig. 1A). After binding of mtHsp[70] to the first (N-terminal) binding site, the second mtHsp[70] binding site is mechanically pulled in (the lever arm and entropic pulling models) or is allowed to move forward by Brownian motions after spontaneous and transient unfolding of the folded domain outside mitochondria (the Brownian ratchet model) in the import channels by a distance of the step size (Fig. 1B, left panels).
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