Comparison of the protein-unfolding pathways between mitochondrial protein import and atomic-force microscopy measurements

Abstract

Many newly synthesized proteins have to become unfolded during translocation across biological membranes. We have analyzed the effects of various stabilization/destabilization mutations in the Ig-like module of the muscle protein titin upon its import from the N terminus or C terminus into mitochondria. The effects of mutations on the import of the titin module from the C terminus correlate well with those on forced mechanical unfolding in atomic-force microscopy (AFM) measurements. On the other hand, as long as turnover of the mitochondrial Hsp70 system is not rate-limiting for the import, import of the titin module from the N terminus is sensitive to mutations in the N-terminal region but not the ones in the C-terminal region that affect resistance to global unfolding in AFM experiments. We propose that the mitochondrial-import system can catalyze precursor-unfolding by reducing the stability of unfolding intermediates.

Fig. 1.

The I27 domain can be imported from both the N terminus and C terminus. (A) Cartoon diagram, showing the β-sandwich structure of the I27 molecule and the amino acid residues that were substituted in this work. The β-strands are named A, A′, and B–G. (B) Fusion proteins with the I27 domain used in this study. Blue boxes, parts of the cytochrome b₂ presequence; yellow boxes, the I27 domain; orange boxes, parts of the C-terminal targeting sequence of Hmi1p. (C) In vitro import of the radiolabeled I27 fusion proteins into mitochondria. The fusion proteins in B with the wild-type I27 domain were incubated with isolated mitochondria for 1 min (pb₂(80)-I27), 5 min (pb₂(35)-I27 and I27-H37C), or 20 min (I27-H105C) at 25°C. The mitochondria were treated with proteinase K and reisolated by centrifugation. The proteins were analyzed by SDS/PAGE and radioimaging. Valinomycin was added to 10 μg/ml to dissipate Δψ. f, full-length form; c, imported and processed form.

Fig. 2.

Effects of mutations in the I27 domain on the import of radiolabeled pb₂(80)-I27 and I27-H105C. pb₂(80)-I27 (A) or I27-H105C (B) fusion proteins with indicated mutations or without a mutation (wt) in the I27 domain were incubated with isolated mitochondria for various times at 25°C and imported. Proteinase-K-protected fractions were quantified, and import rates (initial slopes of the import reactions) were plotted. Values are means ± SD.

Fig. 3.

Effects of mutations in the I27 domain on the import of radiolabeled pb₂(35)-I27 and I27-H37C. pb₂(35)-I27 (A) or I27-H37C (B) fusion proteins with indicated mutations or without a mutation (wt) in the I27 domain were incubated with isolated mitochondria for various times at 25°C and imported. Proteinase-K-protected fractions were quantified, and import rates (initial slopes of the import reactions) were plotted. Values are means ± SD.

Fig. 4.

Models of unfolding pathways of the I27 domain in forced mechanical unfolding (A) and upon mitochondrial import directed by a short targeting sequence (B). N, native state; I, unfolding intermediate with the detached A strand (19); T, transition state with the detached A′ strand for global unfolding (19); U, unfolded state; F, applied force.