The mitochondrial Hsp70-dependent import system actively unfolds preproteins and shortens the lag phase of translocation

Abstract

Unfolding is an essential process during translocation of preproteins into mitochondria; however, controversy exists as to whether mitochondria play an active role in unfolding. We have established an in vitro system with a kinetic saturation of the mitochondrial import machinery, yielding translocation rates comparable to in vivo import rates. Preproteins with short N-terminal segments in front of a folded domain show a characteristic delay of the onset of translocation (lag phase) although the maximal import rate is similar to that of longer preproteins. The lag phase is shortened by extending the N-terminal segment to improve the accessibility to matrix heat shock protein 70 and abolished by unfolding of the preprotein. A mutant mtHsp70 defective in binding to the inner membrane prolongs the lag phase and reduces the translocation activity. A direct comparison of the rate of spontaneous unfolding in solution with that during translocation demonstrates that unfolding by mitochondria is significantly faster, proving an active unfolding process. We conclude that access of mtHsp70 to N-terminal preprotein segments is critical for active unfolding and initiation of translocation.

Fig. 1. Maximal import rate of preproteins in vitro. (A) Left panel: schematic drawing of the fusion protein cytochrome b₂(107)_Δ-DHFR. White, presequence; light gray, mature cytochrome b₂; black, DHFR moiety. The cleavage site of the matrix processing peptidase (arrow) and the 19 amino acid deletion of the hydrophobic sorting signal (▵) are indicated. Right panel: expression and purification of b₂(107)_Δ-DHFR from E.coli cells. Lane 1, cell lysate of non-induced cells; lane 2, cell lysate after 2 h induction with 1 mM IPTG, lane 3, eluate from the MonoS column. The molecular mass of control proteins is indicated. (B) The import of b₂(107)_Δ-DHFR. Import was performed at 25°C with native (–Urea) or denatured (+Urea) preprotein for the indicated times in the presence (+Δψ) or absence (–Δψ) of an inner membrane potential. After the import, the reactions were divided and one half was treated with proteinase K (+PK). Imported preproteins were detected by western blotting using antibodies against DHFR: p, precursor form; i, matrix-targeted intermediate form. (C) Quantification of the import reaction. b₂(107)_Δ-DHFR was imported as described above and the absolute amount of imported preproteins in pmol/mg of mitochondrial protein was determined by a standardized western blot.

Fig. 2. Identification of an initial lag phase of the import reaction. (A) Schematic drawing of the cytochrome b₂-DHFR fusion proteins used. (B) Expression and purification of b₂-DHFR fusion proteins, for description see legend to Figure 1A. (C) Purified b₂-DHFR preproteins were imported into wild-type mitochondria for the indicated times at 25°C in the presence (+Δψ) or absence (–Δψ) of an inner membrane potential as described in Materials and methods: p, precursor form; i, matrix-targeted intermediate form. All samples were treated with proteinase K (100 µg/ml) after import. (D) The import reactions were quantified by standardized western blotting. Values reached for each preprotein after 15 min import were set to 100% (control).

Fig. 3. Folding states of recombinant preproteins. (A) b₂-DHFR fusion proteins contain a folded DHFR domain. The indicated preproteins (40 pmol) were treated with (+PK) 50 µg/ml proteinase K at 2°C for 15 min or left untreated (–PK). After inactivation of the protease by addition of 5 mM PMSF, the samples were subjected to precipitation with trichloroacetic acid and were analyzed by SDS–PAGE and western blotting using anti-DHFR antibodies. The PK-resistant folded DHFR domains and the full-length proteins are indicated. (B) Spontaneous unfolding rates of b₂-DHFR fusion proteins. Unfolding rates were determined by measuring the loss of enzymatic activity of the mouse DHFR in the presence of excess GroEL. At the start of the reaction, all preproteins showed full enzymatic activity like purified DHFR. The reduction of DHFR activity by 1 h incubation with GroEL was set to 100%. Filled circles, b₂(167)_Δ-DHFR; filled diamonds, b₂(107)_Δ-DHFR; open squares, b₂(47)-DHFR.

Fig. 4. Unfolding by mitochondria is faster than the spontaneous unfolding rate in solution. (A) Import of urea-denatured b₂(47)-DHFR into wild-type mitochondria. The import reactions were performed as described in Figure 1B. Where indicated, the preproteins were denatured by 8 M urea (+Urea) before import. The import reactions were stopped, treated with proteinase K and imported preproteins were detected by immunodecoration with anti-DHFR antibodies. The import values reached after 15 min import were set to 100% (control). The dotted line shows import of non-denatured b₂(47)-DHFR. (B) Import of urea-denatured b₂(167)_Δ-DHFR into wild-type mitochondria. The experiment was performed as described above. (C) Comparison of unfolding rates in solution and by mitochondria. The unfolding rates in solution were obtained by quantification of Figure 3B. The time for half-maximal unfolding during import into mitochondria was determined by subtraction of the values for the half-maximal import times of the folded preprotein from the values of half-maximal import of the urea-denatured preprotein. The values were calculated from the quantification of import reactions presented in (A) and (B) and Figure 1C.

Fig. 5. The lag phase of import is prolonged in ssc1-2 mutant mitochondria. (A) Denatured (+Urea) or non-denatured b₂(107)_Δ-DHFR (–Urea) was imported into ssc1-2 mitochondria under non-permissive conditions as described in Materials and methods. All samples were treated with proteinase K after import. (B) Import kinetics of b₂(107)_Δ-DHFR in ssc1-2 mutant mitochondria. Imported preproteins (i-form) were quantified with anti-DHFR antibodies after SDS–PAGE and western blotting. The amount of preprotein imported after 15 min was set to 100% (control). Filled squares, import of denatured b₂(107)_Δ-DHFR into ssc1-2 mitochondria (ssc1-2/+Urea); filled circles, import of non-denatured b₂(107)_Δ-DHFR into ssc1-2 mitochondria (ssc1-2/–Urea). The dotted line shows the import rate into corresponding wild-type mitochondria as shown in Figure 2D (WT/–Urea).

Fig. 6. Spontaneous unfolding of preproteins becomes rate-limiting in mtHsp70-defective mitochondria. (A) b₂(47)-DHFR was imported into ssc1-2 mutant mitochondria and detected by western blotting as described in Figure 5A. Either denatured (+Urea) or non-denatured (–Urea) preprotein was used. (B) Import kinetics of b₂(47)-DHFR in ssc1-2 mutant mitochondria. Import rates of non-denatured (ssc1-2/–Urea, open squares) and denatured b₂(47)-DHFR (ssc1-2/+Urea, filled squares) in ssc1-2 mutant mitochondria are indicated. The dotted line shows the import rate of non-denatured b₂(47)-DHFR in the corresponding wild-type mitochondria (WT/–Urea) as determined in Figure 2D. The value of imported native b₂(47)-DHFR reached after 15 min import into wild-type mitochondria was set to 100% (control).