Supramolecular organization of the respiratory chain in Neurospora crassa mitochondria

Abstract

The existence of specific respiratory supercomplexes in mitochondria of most organisms has gained much momentum. However, its functional significance is still poorly understood. The availability of many deletion mutants in complex I (NADH:ubiquinone oxidoreductase) of Neurospora crassa, distinctly affected in the assembly process, offers unique opportunities to analyze the biogenesis of respiratory supercomplexes. Herein, we describe the role of complex I in assembly of respiratory complexes and supercomplexes as suggested by blue and colorless native polyacrylamide gel electrophoresis and mass spectrometry analyses of mildly solubilized mitochondria from the wild type and eight deletion mutants. As an important refinement of the fungal respirasome model, we found that the standard respiratory chain of N. crassa comprises putative complex I dimers in addition to I-III-IV and III-IV supercomplexes. Three Neurospora mutants able to assemble a complete complex I, lacking only the disrupted subunit, have respiratory supercomplexes, in particular I-III-IV supercomplexes and complex I dimers, like the wild-type strain. Furthermore, we were able to detect the I-III-IV supercomplexes in the nuo51 mutant with no overall enzymatic activity, representing the first example of inactive respirasomes. In addition, III-IV supercomplexes were also present in strains lacking an assembled complex I, namely, in four membrane arm subunit mutants as well as in the peripheral arm nuo30.4 mutant. In membrane arm mutants, high-molecular-mass species of the 30.4-kDa peripheral arm subunit comigrating with III-IV supercomplexes and/or the prohibitin complex were detected. The data presented herein suggest that the biogenesis of complex I is linked with its assembly into supercomplexes.

FIG. 1.

Respiratory supercomplexes and ATP synthase dimers in N. crassa wild-type mitochondria. (A and B) Digitonin-solubilized crude mitochondria were analyzed by BN-PAGE (A) and CN-PAGE (B) in the first dimension. In both panels, results are shown for in-gel activity of COX (upper panels) and subsequent 2D SDS-PAGE (silver stained) to resolve the subunits of all OXPHOS complexes and their supercomplexes (middle panels) and 2D BN-PAGE (Coomassie stained) with 0.02% DDM in the cathode buffer to dissociate the OXPHOS supercomplexes into their individual complexes (lower panels). For mass calibration, digitonin-solubilized bovine heart mitochondria were used: individual complexes I to V (130 to 1,000 kDa) and supercomplexes a to e (I₁III₂IV_0-4; 1,500 to 2,300 kDa). Additionally, 31 subunits of all five OXPHOS complexes separated in 2D BN-SDS-PAGE (A) were verified by MALDI-MS, which are marked in detail in Fig. 2 for the sake of clarity. The OXPHOS supercomplexes were assigned according to their subunit compositions and apparent molecular masses. Besides ATP synthase monomers and dimers (V₁ and V₂), the individual respiratory complexes I to IV as well as the respiratory supercomplexes I_xIII_yIV_z, I₁III₂, I₁IV₁, III₂IV₂, III₂IV₁, and IV₂ are indicated. Additionally, a subunit of complex IV (spot 43; Table 1) in the 2D SDS-PAGE (A) as well as the separated complex IV monomers, complex III dimers, and complex I monomers, which constitute the supercomplexes IV₂, III₂IV₁, III₂IV₂, I₁IV₁, and I_xIII_yIV_z, in the 2D BN-PAGE (A and B) are marked by arrowheads. Note that supercomplexes I₁IV₁ and III₂IV₂ have very similar apparent masses. Similarly, the mobility of dimeric complex IV (IV₂) is only slightly higher than that of complex III.

FIG. 2.

Identification of proteins in a representative 2D BN-SDS gel of digitonin-solubilized N. crassa wild-type crude mitochondria. The same gel as displayed in Fig. 1A is shown. The proteins identified by MALDI-TOF-MS peptide mass fingerprint are labeled by circles and numbers in different colors indicating their function, as follows: OXPHOS, complex I (light blue); complex II (red); complex III (pink); complex IV (yellow); complex V (green); tricarboxylic acid cycle and glycolysis (white); lipid metabolism (light green); amino acid metabolism and urea cycle (orange); chaperones (violet); transport and carrier proteins (dark blue); other proteins (brown). The identified proteins are listed by their numbers in Table 1. All proteins described in the text are highlighted by arrows. The other markings correspond to Fig. 1A and were done as described in the legend of Fig. 1.

FIG. 3.

Putative complex I dimers in wild-type mitochondria after solubilization with Triton X-100 or DDM. 2D BN-SDS-PAGE of crude mitochondria solubilized with Triton X-100 (A) or DDM (B) at a detergent/protein ratio of 1.5 g/g. The subunits of monomeric complex I (I₁) and putative complex I dimers (I₂) are marked by boxes. One subunit of the pyruvate dehydrogenase complex (PDC; spot 4 of Table 1) and two subunits of the NAD⁺-dependent isocitrate dehydrogenase (IDH; spots 41 and 42 of Table 1) are indicated as in Fig. 2. (C) 2D BN/BN-PAGE of crude mitochondria solubilized with Triton X-100 at a detergent/protein ratio of 1.5 g/g. The separated complex I monomers of individual complex I (I₁) and dimeric complex I (I₂) are marked by arrowheads.

FIG. 4.

In-gel activity assays of NADH dehydrogenase, cytochrome c oxidase, ATP hydrolase, and succinate dehydrogenase. BN-PAGE of digitonin-solubilized mitochondria from bovine heart (BHM) as a con-trol, N. crassa wild type (wt), and eight complex I deletion mutants (nuo9.8, nuo11.5, nuo14, nuo20.8, nuo21, nuo29.9, nuo30.4, and nuo51). (A) NADH dehydrogenase (purple bands), reactive bands of wt and nuo21 corresponding to complex I are marked by red bars, and the red box indicates the active band of the peripheral arm of complex I from the four membrane arm mutants. (B to D) Cytochrome c oxidase (brown-yellowish bands) (B), ATP hydrolase (black-white bands) (C), and succinate dehydrogenase (purple band) (C). In all panels, some OXPHOS complexes and supercomplexes, like a to e (I₁III₂IV_0-4) of bovine heart mitochondria, are marked on the left side.

FIG. 5.

Wild-type-like respiratory supercomplexes in peripheral arm mutants nuo21 and nuo51. The nuo51 mutant lacking the 51-kDa subunit forms stable respirasomes containing complexes I, III, and IV without NADH oxidase activity. Results shown are from 2D BN-SDS-PAGE of digitonin-solubilized crude mitochondria of nuo21 (A) and nuo51 (B). The markings are according to the legend of Fig. 1. Additionally, each one subunit of complex I (spot 12 in Table 1), complex III (spot 24 in Table 1), complex IV (spot 43 in Table 1), and complex V (spot 22 in Table 1) are indicated as described for Fig. 2.

FIG. 6.

High-molecular-mass complexes of the 30.4-kDa subunit in complex I-deficient mutants each lacking a membrane arm subunit. (A to F) Alignment between silver-stained 2D BN-SDS gels and corresponding immunoblots probed with antibodies against complex I subunits, as well as corresponding 1D BN gel strips tested for COX in-gel activity from digitonin-solubilized crude mitochondria of (A) wild type (wt) (A), nuo29.9 (B), nuo30.4 (C), nuo11.5 (D), nuo14 (E), and nuo20.8 (F). The employed antibodies against the 30.4-kDa subunit of the peripheral arm and the 9.8-, 11.5-, 14-, and 20.8-kDa subunits of the membrane arm are indicated on the left side of the immunoblots. The subunits of ATP synthase monomers (V₁) and dimers (V₂), complex IV dimers, and supercomplexes III₂IV₁ and III₂IV₂, as well as of complex I and its peripheral arm are indicated by red, black, green, and white continuous vertical lines. (A and B) The subunits of supercomplexes of complex I (I_xIII_yIV_z, inclusively I₁III₂ and I₁IV₁) are marked by boxes. (D to F) The membrane arm mutants display distinct high-molecular-mass species of the 30.4-kDa subunit which contain none of the tested membrane subunits and comigrate with supercomplexes IV₂, III₂IV₁, and III₂IV₂ as well as the prohibitin complex, whose subunits (spots 7 and 8 of Table 1) are encircled. Furthermore, the 30.4-kDa subunit of the peripheral arm and at least three distinct subcomplexes containing the 30.4-kDa subunit marked by arrows were immunodetected.