The Mia40 substrate Mix17 exposes its N-terminus to the cytosolic side of the mitochondrial outer membrane

Abstract

Mitochondrial architecture and the contacts between the mitochondrial outer and the inner membranes depend on the mitochondrial contact site and cristae-organizing system (MICOS) that is highly conserved from yeast to human. Variants in the mammalian MICOS subunit Mic14 (also known as CHCHD10) have been linked to amyotrophic lateral sclerosis and frontotemporal dementia, indicating the importance of this protein. Mic14 has a yeast ortholog, Mix17, a protein of unknown function, which has not been shown to interact with MICOS so far. As a first step to elucidate the function of Mix17 and its orthologs, we analyzed its interactions, biogenesis and mitochondrial sublocation. We report that Mix17 is not a stable MICOS subunit in yeast. Our data suggest that Mix17 is the first Mia40 substrate in the mitochondrial outer membrane. Unlike all other Mia40 substrates, Mix17 spans the mitochondrial outer membrane and exposes its N-terminus to the cytosol. The insertion of Mix17 into the mitochondrial outer membrane is likely to be mediated by its interaction with Tom40, the pore of the TOM complex. Moreover, we show that the exposure of Mix17 to the cytosolic side of the mitochondrial membrane depends on its N-terminus.

Keywords: CHCHD10; Mia40; Mic14; Mix17; Protein import; Tom40.

Fig. 1.

Mix17 is not a MICOS subunit. (A) Mitochondria of wild-type yeast or yeast strains expressing Mic10–3×HA or Mic60–3×HA were lysed in digitonin-containing buffer (1% w/v). Lysates were subjected to immunoprecipitation using anti-HA affinity agarose. Samples were analyzed by SDS-PAGE and immunoblotting. T, total lysate (4%); UB, unbound protein (4%); B, bound protein (100%). l, long isoform of Mgm1; s, short isoform of Mgm1. Arrowhead, cross reaction of the anti-Mic60 antibody. (B) Wild-type mitochondria were treated as in A and subjected to immunoprecipitation using a Mix17-specific antibody or pre-immune serum bound to protein A sepharose. Samples were analyzed by SDS-PAGE and immunoblotting. T, total lysate (4%); UB, unbound protein (4%); B, bound protein (100%). PI, pre-immune serum; bracket, degradation products of Mix17; arrowhead, cross reaction of the anti-Mic60 antibody. Immunoblots are representative of three repeats.

Fig. 2.

Mix17 has the ability to span the mitochondrial outer membrane with an N_out–C_in topology. (A) Mix17 exposes its N-terminus to the cytosol. Mitochondria isolated from a Mix17–3×Myc-expressing strain were subjected to protease and alkaline treatments. Left: mitochondria were left at isotonic conditions, osmotically swollen (SW) or lysed in Triton X-100-containing buffer (TX). Proteinase K (PK) was added as indicated. Right: mitochondria were treated with alkaline buffer to separate soluble (S) and membrane proteins (M). Samples were analyzed by SDS-PAGE and immunoblotting. f1, f2, fragments generated by PK. Arrowhead, cross reaction of the anti-Tom70 antibody. Blots are representative of at least three repeats. (B) Wild-type Mix17 shows the same topology as Mix17–3×Myc. Yeast wild-type mitochondria were subjected to PK treatment as described in A. Blots are representative of at least three repeats. (C) The N-terminus of Mix17 is sensitive to trypsin. Wild-type mitochondria were subjected to protease treatment using trypsin. Samples were analyzed as in A. f, fragment generated by trypsin. Blots are representative of at least three repeats. (D) In vitro imported Mix17 adopts the same topology as the endogenous protein. Mix17 protein was synthesized in vitro in the presence of ³⁵S-methionine and incubated with isolated mitochondria. Mitochondria were re-isolated and treated as in C. The samples were analyzed by SDS-PAGE and autoradiography. The autoradiograph is representative of at least three repeats. The full-length protein (Mix17) or the fragment generated by trypsin (f) are indicated. 10% input, 10% of the amount of radiolabeled protein added to each lane.

Fig. 3.

Mia40 is essential for the import of Mix17. (A) The import of Mix17 depends on Mia40. ³⁵S-labeled Mix17 protein was translated in vitro and incubated with mitochondria isolated from wild-type (WT) yeast or a mutant strain in which Mia40 was downregulated (Mia40↓). Samples were taken at the indicated time points and treated with trypsin as indicated. Mitochondria were re-isolated and the samples were analyzed by SDS-PAGE and autoradiography. f, fragment generated by trypsin. The autoradiograph of the experiment (left) and the quantification are shown (right). (B) Dithiothreitol (DTT) inhibits the import of Mix17. ³⁵S-labeled Mix17 protein was imported into wild-type mitochondria for 20 min in the presence of increasing concentrations of DTT. Samples were analyzed as in A. (C) The conserved CX₉C motif is essential for the import of Mix17. ³⁵S-labeled Mix17 protein and a mutant lacking the four highly conserved C-terminal cysteine residues (Mix17 C4S) were incubated with wild-type mitochondria. Samples were treated with trypsin and analyzed as in A. (D) The steady-state levels of endogenous Mix17 depend on Mia40. Wild-type and Mia40↓ mitochondria were isolated and 25 and 50 µg of mitochondrial proteins were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. Blots and autoradiographs are representative of at least two experiments.

Fig. 4.

The N-terminus of Mix17 supports the import efficiency of Mix17 into mitochondria and is essential for its insertion into the mitochondrial outer membrane. (A) Import of Mix17 does not depend on mitochondrial membrane potential. ³⁵S-labeled Mix17 protein was incubated with wild-type mitochondria, which were either left untreated (+ΔΨ) or were treated with valinomycin (0.5 µM), antimycin A (8 µM) and oligomycin (20 µM) to dissipate the membrane potential (−ΔΨ). Samples were taken at the indicated time points and treated with trypsin as indicated. Mitochondria were re-isolated and the samples were analyzed by SDS-PAGE and autoradiography. Upper panel: autoradiograph of one representative experiment. The full-length protein (Mix17) or the fragment generated by trypsin (f) are indicated. Lower panel: quantitative analysis of three independent experiments. Results are presented as means of the imported protein normalized to the maximal imported material of −ΔΨ, which was set to 100%. Error bars show the standard deviation. (B) The N-terminal 24 residues of Mix17 strongly increase the import efficiency of Mix17. ³⁵S-labeled full-length Mix17 and a mutant lacking the first 24 amino acids (Mix17 Δ1–24) were imported into wild-type mitochondria. Samples were treated as in A. Upper panel: autoradiograph of one representative experiment. Lower panel: quantitative analysis of three independently performed experiments. Results are presented as means of the imported protein as a percentage of the respective input and normalized to the maximal imported full-length Mix17, which was set to 100%. Error bars show the standard deviation. (C) The N-terminus of Mix17 is essential for the insertion into the mitochondrial outer membrane in vitro. The import of full-length Mix17 and Mix17 Δ1–24 was performed as in B, with the difference that PK was added, as indicated, instead of trypsin. The full-length protein (Mix17) or the fragments generated by PK (f1, f2) are indicated. The autoradiograph is representative of at least three repeats. (D) The N-terminus of Mix17 is essential for the insertion into the mitochondrial outer membrane in vivo. Mitochondria of Δmix17 yeast strains expressing full-length Mix17 WT–FLAG or Mix17 Δ1–24-FLAG were treated with PK or trypsin (T). Samples were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. The fragments generated by PK or trypsin are indicated. Blots are representative of three experiments.

Fig. 5.

Mix17 is able to insert into the mitochondrial outer membrane from the intermembrane space. ³⁵S-labeled full-length Mix17 or Mix17 fused to the N-terminus of Cytb₂ [Cytb₂(1–84) Mix17] was incubated with wild-type mitochondria. The import was stopped at the indicated time points and the samples were treated with trypsin as indicated. Mitochondria were re-isolated and the samples were analyzed by SDS-PAGE and autoradiography. f, fragment generated by trypsin. i-Cytb₂(1–84) Mix17, intermediate form of Cytb₂(1–84) Mix17. The autoradiograph is representative of at least three repeats.

Fig. 6.

The insertion of Mix17 into the mitochondrial outer membrane does not depend on the TOB/SAM or MIM complexes. (A) The steady-state level of Mix17 does not depend on the presence of Tob55. Mitochondria were isolated from wild-type yeast and a mutant strain in which Tob55 was downregulated (Tob55↓). Two different amounts of the mitochondria were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. (B) Tob55 is not important for the insertion of Mix17 into the mitochondrial outer membrane. Wild-type and Tob55↓ mitochondria were either left untreated or incubated with PK or trypsin (T) at isotonic conditions. Samples were analyzed by SDS-PAGE and immunoblotting. Asterisk, degradation product of Mix17 in untreated mitochondria. The fragments generated by PK or trypsin are indicated. (C) The absence of Mim1 does not affect the level of Mix17. Mitochondria isolated from wild-type yeast and the Δmim1 deletion mutant were analyzed as in A. (D) Mim1 is dispensable for the insertion of Mix17 into the mitochondrial outer membrane. Wild-type and Δmim1 mitochondria were analyzed as in B. Asterisk, degradation product of Mix17 in untreated mitochondria. The fragments generated by PK or trypsin are indicated. All blots are representative of three repeats.

Fig. 7.

Mix17 interacts with Tom40 to be exposed to the cytosol. (A) The conserved hydrophobic stretch of Mix17 is not required for the insertion into the mitochondrial outer membrane. ³⁵S-labeled Mix17 Δ53–80 protein was incubated with wild-type mitochondria. Samples were either left untreated or incubated with PK or trypsin (T). Re-isolated mitochondria were analyzed by SDS-PAGE and autoradiography. The fragments generated by PK or trypsin are indicated. Asterisks, potential degradation products of Mix17 in the untreated sample. The autoradiograph is representative of at least three repeats. (B,C) Mix17 interacts with Tom40 at the endogenous protein level. (B) DSP-crosslinked mitochondria of wild-type yeast and a yeast strain expressing Tom40–3×HA were lysed in digitonin-containing buffer (1% w/v). Lysates were subjected to immunoprecipitation using anti-HA affinity agarose. Samples were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. T, total lysate (4%); UB, unbound protein (4%); B, bound protein (100%). Bracket, degradation products of Mix17. (C) Wild-type mitochondria were treated as described in B and subjected to immunoprecipitation using a Mix17-specific antibody or pre-immune serum (PI) bound to protein A sepharose. Samples were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. T, total lysate (4%); UB, unbound protein (4%); B, bound protein (100%). Bracket, degradation products of Mix17. (D) The N-terminus of Mix17 is essential for its interaction with Tom40. Mitochondria isolated from yeast strains expressing Tom40–3×HA and either full-length Mix17–FLAG (WT) or Mix17 Δ1–24–FLAG were left untreated or DSP crosslinked and subjected to immunoprecipitation as described in B. Blots are representative of at least three repeats.

Fig. 8.

Working model of Mix17 biogenesis. (1) The N-terminus of Mix17 supports the import of the protein into the mitochondrial intermembrane space. (2) The MIA40 system is essential for the formation of the C-terminal disulfide bonds and thus the import of Mix17. (3a,b) The exposure of Mix17 to the cytosolic side of the mitochondrial outer membrane depends on its N-terminus, as well as its association with Tom40. Two possible locations of Mix17 in proximity to Tom40 are shown. Dotted double arrows indicate potential dynamic topologies of the Mix17 N-terminus. OM, outer membrane; IM, inner membrane.