The mitochondrial oxidase assembly protein1 (Oxa1) insertase forms a membrane pore in lipid bilayers

Abstract

The inner membrane of mitochondria is especially protein-rich. To direct proteins into the inner membrane, translocases mediate transport and membrane insertion of precursor proteins. Although the majority of mitochondrial proteins are imported from the cytoplasm, core subunits of respiratory chain complexes are inserted into the inner membrane from the matrix. Oxa1, a conserved membrane protein, mediates the insertion of mitochondrion-encoded precursors into the inner mitochondrial membrane. The molecular mechanism by which Oxa1 mediates insertion of membrane spans, entailing the translocation of hydrophilic domains across the inner membrane, is still unknown. We investigated if Oxa1 could act as a protein-conducting channel for precursor transport. Using a biophysical approach, we show that Oxa1 can form a pore capable of accommodating a translocating protein segment. After purification and reconstitution, Oxa1 acts as a cation-selective channel that specifically responds to mitochondrial export signals. The aqueous pore formed by Oxa1 displays highly dynamic characteristics with a restriction zone diameter between 0.6 and 2 nm, which would suffice for polypeptide translocation across the membrane. Single channel analyses revealed four discrete channels per active unit, suggesting that the Oxa1 complex forms several cooperative hydrophilic pores in the inner membrane. Hence, Oxa1 behaves as a pore-forming translocase that is regulated in a membrane potential and substrate-dependent manner.

FIGURE 1.

Oxa1 purified from S. cerevisiae constitutes an ion channel. A, three-step isolation of Oxa1 from yeast mitochondria. Upper panel, schematic of Oxa1 affinity tags; lower panels, Western blot analysis of solubilized extracts from wild type mitochondria (strain with authentic nontagged Oxa1) and mitochondria containing Oxa1^ZZ/His-10, subjected to Ni-NTA purification (Eluate I) followed by IgG chromatography and TEV cleavage (Eluate II). Cleavage of Oxa1 results in a faster migrating Oxa1 cleavage product indicated by the arrow. (Total = 0.5%, Eluate I = 0.5%, and Eluate II = 5%.) Pam17, Rip1, and Cox1 are mitochondrial proteins serving as controls. Asterisk indicates proteolytic cleavage product. B, current recordings of a bilayer after fusion of an Oxa1-containing proteoliposome (membrane potential as indicated) under symmetrical buffer conditions (250 mm KCl, 20 mm Mops/Tris, pH 7.0). C, current voltage relationship calculated from >10,000 single gating events. Conductance states are indicated. Buffer conditions as in B. D, current-voltage ramp recorded under asymmetrical buffer conditions (cis, 250 mm KCl, 20 mm Mops/Tris, pH 7.0; trans, 20 mm KCl, 20 mm Mops/Tris, pH 7.0). Reversal potential is as indicated. E, current recordings of an Oxa1 containing bilayer before (black) and after the addition of anti-Oxa1 antibodies (red) and the statistical analysis of the mean Oxa1 channel current (n = 3).

FIGURE 2.

A, current recordings of a bilayer after fusion of an Oxa1-containing proteoliposome (membrane potential as indicated). Zoom plots show single gating events in main and subconductance states. B, conductance state histograms of Oxa1 at positive and negative applied holding potentials. C, current recordings of a bilayer after fusion of an Oxa1_rec-containing proteoliposome (left) and from a bilayer fused with mock-treated sample liposomes obtained from cells expressing nontagged Oxa1_rec that were added to the cis compartment (membrane potential of the voltage gate as indicated). D, current recordings of a bilayer after fusion of an Oxa1_rec-containing proteoliposome in the absence (left) and presence of Pam17 antiserum (membrane potential of the voltage gate as indicated).

FIGURE 3.

Recombinantly expressed Oxa1 displays ion channel activity with similar characteristics as the yeast protein. A, SDS-PAGE of purified Oxa1_rec. A BSA loading control for the estimation of protein concentrations is shown on the same gel. B, Western blot of a flotation assay of Oxa1_rec-containing proteoliposomes. 40% represents the bottom of the gradient after centrifugation where aggregated or not incorporated protein is found. The majority of proteoliposomes floats into the 5% fraction of the gradient. C, CD spectrum of Oxa1_rec in DDM. D, current recordings of a bilayer after the fusion of Oxa1_rec (membrane potential as indicated) under symmetrical buffer conditions (see Fig. 1B). Diagram to the right shows that full channel closure occurs in four main conductance state gating events. E, mean variance analysis of D, with upper current trace showing that four gating events with the main conductance state led to complete channel closure. F, current voltage relationship calculated from over 5,000 single gating events. Conductance states are indicated. Buffer conditions are as in Fig. 1B. G, current-voltage ramp recorded under asymmetrical buffer conditions (as in Fig. 1D). Reversal potential as indicated. H, voltage-dependent open probability of Oxa1_rec. Quantification was performed by comparing the mean current determined over a range of 1 min with the maximum current at a constant holding potential. I, current recordings of an Oxa1_rec-containing bilayer before (black) and after the addition of anti-Oxa1 antibodies (upper gray).

FIGURE 4.

A, conductance state histograms of Oxa1_complex at positive and negative applied holding potentials. Gating frequency of the complexes as indicated (LF, low gating frequency; HF, high gating frequency). B, current voltage ramp of an Oxa1_complex with low or high gating frequency. C, His-tagged Oxa1 was expressed in E. coli and purified. The protein was incubated in the presence or absence of E. coli lipids. Liposomes were generated by removal of the detergent using BioBeads. The sample was treated with 0.1 m Na₂CO₃, adjusted to 1.6 m sucrose, placed on the bottom of a centrifugation tube, overlaid by 1.4 m sucrose, and centrifuged for 2 h at 485,000 × g. The samples were analyzed by Western blotting.

FIGURE 5.

A, current recordings of a bilayer after fusion of a recombinant Oxa1 channel (membrane potential as indicated). Zoom plots show single gating events in main and subconductance states. B, conductance state histograms of Oxa1 at positive and negative applied holding potentials. C, conductance state histograms of Oxa1 at positive and negative applied holding potentials. Oxa1_rec-containing proteoliposomes were incubated with Cox2 peptides prior to fusion. D, current recordings of a bilayer after fusion of an Oxa1_rec-containing proteoliposome in the absence (left panel) and presence of 1 μm Cox IV_1–23 (membrane potential of the voltage gate as indicated).

FIGURE 6.

Activation of Oxa1_rec by a substrate peptide. A, current recordings of Oxa1_rec containing bilayers in the absence (left panel) and in the presence (right panel) of Cox2 peptide. Oxa1 containing proteoliposomes were incubated prior to fusion with 500 μm Cox2 peptide. B, current voltage relationship of Oxa1_rec in the presence of Cox2, calculated from >5,000 single gating events. Conductance states are indicated and show no significant change to Oxa1_rec in the absence of Cox2 (Fig. 3F). C, current-voltage ramp of Oxa1_rec in the presence of Cox2 recorded under asymmetrical buffer conditions (as in Fig. 1D). Reversal potential as indicated shows no alteration to Oxa1_rec in the absence of Cox2. D, voltage-dependent open probability of Oxa1_rec in the presence of Cox2.

FIGURE 7.

Oxa1 complex displays two activity states. A, purification of Oxa1 complex from S. cerevisiae mitochondria. Asterisk indicates proteolytic cleavage product. Arrow indicates faster migrating cleavage product. B, blue-native PAGE of purified Oxa1 complexes. C, Western blot of a flotation assay of the Oxa1 complex containing proteoliposomes. D and E, current recordings of a bilayer after fusion of the Oxa1 complex containing liposomes. D shows ion channel activity with low gating frequency, whereas the channel depicted in E shows high gating frequency. F, current-voltage relationship of channels with low gating frequency (D). Conductance states are indicated. G, current-voltage relationship of channels with high gating frequency (E). Conductance states are indicated. H, current-voltage ramp of the Oxa1 complex with low gating frequency. Reversal potentials are indicated. I, voltage-dependent open probability of Oxa1 complexes with low gating frequency. J, current-voltage ramp of the Oxa1 complex with high gating frequency. Reversal potentials are indicated. K, voltage-dependent open probability of Oxa1 complexes with high gating frequency.

FIGURE 8.

Occupancies of conductance states in the Oxa1 complex with a high gating frequency resembles Oxa1_rec in the presence of substrate. A, conductance states of Oxa1 complexes with low gating frequency at negative (dark gray) and positive (light gray) holding potentials. B, relative occupancies of conductance states shown in A. C and D, conductance states and relative occupancies of conductance states of Oxa1 complexes with high gating frequency. E and F, conductance states and relative occupancies of conductance states of recombinant Oxa1. G and H, conductance states and relative occupancies of conductance states of recombinant Oxa1 that was incubated with Cox2 peptides prior to fusion. Comparison of D and H shows a clear correlation of the relative occupancies of conductance state 2 of Oxa1 complexes with a high gating frequency and Oxa1_rec in the presence of substrate.

FIGURE 9.

Oxa1 complex gating shows four pores of which two are coupled in each case. A, current recording of an Oxa1 complex containing bilayer at +120 mV. B, mean variance analysis of the current trace depicted in A (zoom plot). C, schematic representation of the gating transitions shown in B. B and C mainly show gating events of the main conductance state (4 to 3, 3 to 2, etc, but also gating events over two main conductance states (i.e. 4 to 2, 3 to 1, and 2 to fully closed) indicating two of the four pores are coupled in each case.