Mitochondrial ferredoxin-like is essential for forming complex I-containing supercomplexes in Arabidopsis

Abstract

In eukaryotes, mitochondrial ATP is mainly produced by the oxidative phosphorylation (OXPHOS) system, which is composed of 5 multiprotein complexes (complexes I-V). Analyses of the OXPHOS system by native gel electrophoresis have revealed an organization of OXPHOS complexes into supercomplexes, but their roles and assembly pathways remain unclear. In this study, we characterized an atypical mitochondrial ferredoxin (mitochondrial ferredoxin-like, mFDX-like). This protein was previously found to be part of the bridge domain linking the matrix and membrane arms of the complex I. Phylogenetic analysis suggested that the Arabidopsis (Arabidopsis thaliana) mFDX-like evolved from classical mitochondrial ferredoxins (mFDXs) but lost one of the cysteines required for the coordination of the iron-sulfur (Fe-S) cluster, supposedly essential for the electron transfer function of FDXs. Accordingly, our biochemical study showed that AtmFDX-like does not bind an Fe-S cluster and is therefore unlikely to be involved in electron transfer reactions. To study the function of mFDX-like, we created deletion lines in Arabidopsis using a CRISPR/Cas9-based strategy. These lines did not show any abnormal phenotype under standard growth conditions. However, the characterization of the OXPHOS system demonstrated that mFDX-like is important for the assembly of complex I and essential for the formation of complex I-containing supercomplexes. We propose that mFDX-like and the bridge domain are required for the correct conformation of the membrane arm of complex I that is essential for the association of complex I with complex III2 to form supercomplexes.

Figure 1

The atypical mFDX-like proteins form a phylogenetic branch distinct from other eukaryotic FDXs. Protein sequences of FDXs were retrieved from a selection of eukaryotic organisms representing the different eukaryotic lineages (see Supplemental Table S1). Sequences were analyzed using MEGA (www.megasoftware.net), and a minimum evolution tree was built using default parameters. The optimal tree is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. Plastid FDXs are shaded in grey. The 3 FDXs described as complex I subunits are shown with boxes. Ac, Acanthamoeba castellanii; At, Arabidopsis thaliana; Cm, Cyanidioschyzon merolae; Cr, Chlamydomonas reinhardtii; Dd, Dictyostelium discoideum; Eh, Emiliania huxleyi; Ev, Euglena viridis; Gi, Giardia intestinalis; Gt, Guillardia theta; Hs, Homo sapiens; Ng, Nannochloropsis gaditana; Pi, Phytophthora infestans; Rf, Reticulomyxa filosa; Tb, Trypanosoma brucei; Tg, Toxoplasma gondii; Th, Thecamonas trahens; To, Thalassiosira oceanica; Tt, Tetrahymena thermophila; Yl, Yarrowia lipolytica. The UniProt accessions of all the proteins analyzed are given in Supplemental Table S1.

Figure 2

mFDX-like does not coordinate an Fe-S cluster. UV-visible absorption spectra of as-purified Arabidopsis thaliana his-tagged mFDX1 (A), mFDX2 (B), mFDX-like (C), and mFDX-like L85C variant (D) recombinantly expressed in E. coli.

Figure 3

The absence of mFDX-like does not affect plant growth. Two independent deletion lines were generated using the CRISPR-Cas9 genome editing technology. A, Mitochondria were isolated from the lines and mitochondrial proteins were analyzed by SDS-PAGE followed by immunoblot experiments using an anti-mFDX serum. Left panel: Coomassie staining of the membrane. Right panel: Signals obtained after incubation of the membrane using the anti-mFDX-like antibodies. The position of the mature form of mFDX-like is indicated by an arrowhead. The aspecific signals observed in the top part of the blot serve as loading control. B, Representation images of 20-day-old seedlings grown on soil under standard growth conditions.

Figure 4

mFDX-like is essential for supercomplex formation. Blue native PAGE analysis of mitochondrial complexes. Mitochondria were solubilized using digitonin and the complexes separated on a native gel. After migration, the gel was stained with Coomassie blue. OXPHOS complexes I, III_2, and V and supercomplexes (I + III₂, I + III₂ + IV) are indicated on the right. C1 and C2 are the complemented lines of mutants mfdxl-4 and mfdxl-5, respectively.

Figure 5

mFDX-like is important for the assembly of complex I. Blue native PAGE analysis of mitochondrial complexes. Mitochondria were solubilized using digitonin and the complexes separated on a native gel. After migration, the complexes were transferred on a PVDF membrane which was used for immunoblot analyses. m4: mfdxl-4, m5: mfdxl-5. C1 and C2 are the complemented lines of the mfdxl-4 and mfdxl-5 mutants, respectively. A, Coomassie staining of the membrane after transfer, the position of the OXPHOS complexes and supercomplexes is indicated on the left (I: complex I, III₂: complex III dimer, IV: complex IV, V: complex V). B, Signals obtained after incubation of the membrane with the anti-CA2 antibodies. Complex I related complexes accumulated in the deletion mutants are indicated with an arrowhead. *: aspecific reaction of complex III₂ with the ECL reagent. C, Signals obtained after incubation of the membrane with the anti-GLDH antibodies. Complex I assembly intermediates are indicated with an arrowhead. Free GLDH running close to the bottom of the gel is indicated with a circle. D, Signals obtained after incubation of the membrane with the anti-RISP antibodies. The same membrane has been used to perform the 3 immunoblot analyses. E, Complex IV-activity staining of a replicate BN gel. x: unknown complex IV-containing supercomplex.

Figure 6

CA2 is important for the assembly of the P_D module. Blue native PAGE analysis of mitochondrial complexes. Mitochondria were solubilized using dodecyl-maltoside and the complexes separated on a native gel. After migration, the complexes were transferred on a PVDF membrane which was used for immunoblot analyses. Left panel: Coomassie staining of the membrane after transfer, the position of the OXPHOS complexes is indicated on the left (I: complex I, III₂: complex III dimer, V: complex V). Right panel: Signals obtained after incubation of the membrane with the anti-Nad6 antibodies. Complex I assembly intermediates accumulating in the mutants are indicated on the right. ca2: mutant lacking the subunit CA2, s4: mutant lacking the subunit NDUFS4, ca2s4 and s4ca2: double mutants lacking CA2 and NDUFS4.

Figure 7

NDUFA11 is essential for supercomplex formation but its association with complex I is not affected in mfdxl mutants. A. Mitochondria were isolated from the ndua11 and WT plants and mitochondrial proteins were separated by SDS-PAGE and analyzed by immunoblot analyses. Left panel: Coomassie staining of the membrane. Right panels: Signals obtained after incubation of the membrane with first the anti-NDUA11 antibodies and second the anti-VDAC antibodies. The detection of VDAC is used as a loading control. B. Image of 20-day-old seedlings grown on soil under standard growth conditions. C. Blue native PAGE analysis of mitochondrial complexes. Mitochondria were solubilized using digitonin and the complexes separated on a native gel. After migration, the complexes were transferred on a PVDF membrane which was used for immunoblot analyses. Left panel: Coomassie staining of the membrane after the transfer, the position of the OXPHOS complexes is indicated on the left (I + III₂: supercomplex I + III₂, I: complex I, III₂: complex III dimer, V: complex V). Right panel: signals obtained after incubation of the membrane with the anti-CA2 antibodies. *: aspecific reaction of complex III₂ with the ECL reagent. D. Blue native PAGE analysis of mitochondrial complexes. Mitochondria were solubilized using digitonin and the complexes separated on a native gel. After migration, the complexes were transferred on a PVDF membrane which was used for immunoblot analyses. Left panel: Coomassie staining of the membrane after the transfer, the position of the OXPHOS complexes is indicated on the left (I + III₂: supercomplex I + III₂, I: complex I, III₂: complex III dimer, V: complex V). Right panel: signals obtained after incubation of the membrane with the anti-NDUA11 antibodies. *: aspecific reaction of complex III₂ with the ECL reagent.