Two distinct membrane potential-dependent steps drive mitochondrial matrix protein translocation

Abstract

Two driving forces energize precursor translocation across the inner mitochondrial membrane. Although the membrane potential (Δψ) is considered to drive translocation of positively charged presequences through the TIM23 complex (presequence translocase), the activity of the Hsp70-powered import motor is crucial for the translocation of the mature protein portion into the matrix. In this study, we show that mitochondrial matrix proteins display surprisingly different dependencies on the Δψ. However, a precursor's hypersensitivity to a reduction of the Δψ is not linked to the respective presequence, but rather to the mature portion of the polypeptide chain. The presequence translocase constituent Pam17 is specifically recruited by the receptor Tim50 to promote the transport of hypersensitive precursors into the matrix. Our analyses show that two distinct Δψ-driven translocation steps energize precursor passage across the inner mitochondrial membrane. The Δψ- and Pam17-dependent import step identified in this study is positioned between the two known energy-dependent steps: Δψ-driven presequence translocation and adenosine triphosphate-driven import motor activity.

Figure 1.

Protein import is impaired in Tim50-depleted mitochondria. (A) Steady-state Western blot analysis of WT and Tim50-depleted mitochondria. (B) Δψ of isolated mitochondria was assessed using the Δψ-sensitive dye DiSC₃(5). Fluorescence was recorded before and after addition of valinomycin. (C–F) ³⁵S-labeled precursors were imported into isolated mitochondria, and import stopped at the indicated time points with antimycin A, valinomycin, and oligomycin (AVO). Samples were PK treated and analyzed by SDS-PAGE and autoradiography. Results are presented as mean ± SEM. n = 3. The longest import time of the WT sample was set to 100%. m, mature protein.

Figure 2.

pam17Δ mitochondria display similar import defects as mitochondria lacking Tim50. (A) WT and Tim50-depleted mitochondria were solubilized with digitonin and subjected to α-Tim23 immunoisolation. Samples were analyzed by Western blotting. Total, 10%; elution, 100%. (B) Quantification of mean red/green fluorescence intensities from WT and pam17Δ cells. For each condition, three independent clones were analyzed and 150–1,500 cells were quantified. Results are presented as mean ± SEM. n = 3. (C) Δψ of WT and pam17Δ mitochondria was assessed as described in Fig. 1 B. (D–G) ³⁵S-labeled precursors were imported as described in Fig. 1 (C–F). p, precursor; m, mature protein. (H) Comparison of import efficiency of indicated ³⁵S-labeled precursors into Tim50-depleted or pam17Δ mitochondria after 15 min (results from Fig. 1 [C–F] and D–G).

Figure 3.

Pam17 plays a motor-independent role in protein import. (A and B) The inward driving force of the motor was assessed using ³⁵S-labeled b₂(167)_Δ-DHFR (A and B) or b₂(220)-DHFR (B alone) in the presence of MTX. After an initial import reaction, membrane potential was dissipated with valinomycin. The precursor was chased in a second incubation step for indicated time points before PK was added. The amount of processed intermediate was quantified (100%: amount of processed intermediate without protease treatment). Results are presented as mean ± SEM. n = 3. (C) Schematic representation of F₁α, F₁β, pF₁α-F₁β, and pF₁β-F₁α. For pF₁α-F₁β, the first 38 aa of F₁α were fused to the mature part of F₁β (40–end). For pF₁β-F₁α, the first 45 aa of F₁β were fused to the mature part of F₁α (36–end). (D and E) ³⁵S-labeled pF₁α-F₁β and pF₁β-F₁α were imported into isolated mitochondria from indicated strains as described in Fig. 1. p, precursor; m, mature protein.

Figure 4.

Import of matrix proteins depends to different extents on membrane potential. (A–H) Isolated WT mitochondria were treated with the indicated amounts of CCCP for 5 min before import. After 15 min of import, reactions were stopped with AVO, and import was analyzed by SDS-PAGE and digital autoradiography. Results are presented as mean ± SEM. n = 3. p, precursor; m, mature protein. (E and F) Overlay of results from CCCP titration experiments with F₁α, F₁β, Tim44, and b₂(167)_Δ-DHFR. (H) Overlay of results from CCCP titration experiments with F₁α, F₁β, pF₁α-F₁β, and pF₁β-F₁α.