Conserved in situ arrangement of complex I and III2 in mitochondrial respiratory chain supercomplexes of mammals, yeast, and plants

Abstract

We used electron cryo-tomography and subtomogram averaging to investigate the structure of complex I and its supramolecular assemblies in the inner mitochondrial membrane of mammals, fungi, and plants. Tomographic volumes containing complex I were averaged at ∼4 nm resolution. Principal component analysis indicated that ∼60% of complex I formed a supercomplex with dimeric complex III, while ∼40% were not associated with other respiratory chain complexes. The mutual arrangement of complex I and III₂ was essentially conserved in all supercomplexes investigated. In addition, up to two copies of monomeric complex IV were associated with the complex I₁III₂ assembly in bovine heart and the yeast Yarrowia lipolytica, but their positions varied. No complex IV was detected in the respiratory supercomplex of the plant Asparagus officinalis Instead, an ∼4.5-nm globular protein density was observed on the matrix side of the complex I membrane arm, which we assign to γ-carbonic anhydrase. Our results demonstrate that respiratory chain supercomplexes in situ have a conserved core of complex I and III₂, but otherwise their stoichiometry and structure varies. The conserved features of supercomplex assemblies indicate an important role in respiratory electron transfer.

Keywords: cryo-electron tomography; mitochondria; respirasomes; respiratory chain supercomplexes; subtomogram averaging.

Fig. 1.

Distribution of complex I and ATP synthase. Tomographic slices of mitochondrial membranes from bovine heart (A and D), Y. lipolytica (B and E), and A. officinalis (C and F) indicating the organization and orientation of ATP synthase (A–C, yellow arrowheads) and the complex I matrix arm (D–F, red rectangles). The random distribution and orientation of complex I in the membrane indicates that there are no respiratory strings (58). (Scale bar: 5 nm.)

Fig. 2.

Respiratory chain supercomplexes in bovine heart mitochondria. Subvolumes centered on the matrix arm of complex I were aligned and averaged using a tight mask and then classified based on the presence of neighboring protein densities. Four classes were observed: (A) complex I alone (44%); (B) supercomplex I₁III₂IV_a (30%); (C) supercomplex I₁III₂ (16%); and (D) supercomplex I₁III₂IV_ab (10%). A further possible protein density (gray above IV_a) is observed in C and D but its identity is unknown. (Scale bar: 5 nm.) Blue, complex I [Protein Data Bank (PDB) ID code 4UQ8]; red/orange, complex III (PDB ID code 1BCC); green, complex IV (PDB ID code 1OCC).

Fig. 3.

Respiratory chain supercomplexes of the yeast Y. lipolytica. (A) Subtomogram average of the I₁III₂IV_cd supercomplex. Blue, complex I (PDB ID code 4WZ7); red/orange, complex III dimer (PDB ID code 1BCC); green, complex IV (PDB ID code 1OCC). (Scale bar: 5 nm.) (B) Composition and frequency of supercomplexes in the inner mitochondrial membrane.

Fig. 4.

The plant respiratory chain supercomplex. (A) Subtomogram average (transparent gray) of the A. officinalis I₁III₂ supercomplex with fitted atomic models. Red/orange, complex III₂ (PDB ID code 1BCC); blue, complex I (PDB ID code 4WZ7); yellow, γ-carbonic anhydrase (PDB ID code 1QRE). (B) Atomic model of complex I and γ-carbonic anhydrase (CA, yellow) and complex I subunits likely to interact with it. Subunits are labeled as in bovine complex I. (Scale bar: 5 nm.)

Fig. 5.

Comparison of supercomplexes from three eukaryotic lineages. (A) Subtomogram averages of bovine heart, Y. lipolytica, and A. officinalis supercomplexes were aligned on complex I volumes. (B) Schematic overlay of complex positions. The relative positions of complexes I and III₂ in all three species are similar, but III₂ is rotated clockwise by 12° in A. officinalis (A.o.) as seen from the matrix. Supercomplexes of A. officinalis contain no visible complex IV but a density consistent with γ-carbonic anhydrase (CA), a known component of plant complex I. Both bovine heart and Y. lipolytica supercomplexes contain up to two complex IV monomers. The position of IV_c in Y. lipolytica coincides with the less frequently observed IV_b position in bovine heart. Pink, Bos taurus (B.t.); blue, Y. lipolytica (Y.l.); green, A. officinalis (A.o.). (Scale bar: 5 nm.)