Identification and characterization of respirasomes in potato mitochondria

Abstract

Plant mitochondria were previously shown to comprise respiratory supercomplexes containing cytochrome c reductase (complex III) and NADH dehydrogenase (complex I) of I(1)III(2) and I(2)III(4) composition. Here we report the discovery of additional supercomplexes in potato (Solanum tuberosum) mitochondria, which are of lower abundance and include cytochrome c oxidase (complex IV). Highly active mitochondria were isolated from potato tubers and stems, solubilized by digitonin, and subsequently analyzed by Blue-native (BN) polyacrylamide gel electrophoresis (PAGE). Visualization of supercomplexes by in-gel activity stains for complex IV revealed five novel supercomplexes of 850, 1,200, 1,850, 2,200, and 3,000 kD in potato tuber mitochondria. These supercomplexes have III(2)IV(1), III(2)IV(2), I(1)III(2)IV(1), I(1)III(2)IV(2), and I(1)III(2)IV(4) compositions as shown by two-dimensional BN/sodium dodecyl sulfate (SDS)-PAGE and BN/BN-PAGE in combination with activity stains for cytochrome c oxidase. Potato stem mitochondria include similar supercomplexes, but complex IV is partially present in a smaller version that lacks the Cox6b protein and possibly other subunits. However, in mitochondria from potato tubers and stems, about 90% of complex IV was present in monomeric form. It was suggested that the I(1)III(2)IV(4) supercomplex represents a basic unit for respiration in mammalian mitochondria termed respirasome. Respirasomes also occur in potato mitochondria but were of low concentrations under all conditions applied. We speculate that respirasomes are more abundant under in vivo conditions.

Figure 1.

Identification of complex IV-containing supercomplexes in potato tuber (T) and stem (S) mitochondria. Protein complexes were solubilized by 5 g digitonin per g protein, separated by 1D BN-PAGE and either visualized by Coomassie staining (left gel strips) or by in-gel activity staining for cytochrome c oxidase (right gel strips). Activity stains are given in false-color mode to increase color contrast (red, Coomassie; black, enzyme activity). Molecular masses and identities of known protein complexes are indicated on the left side of the gels in Roman numerals (I, NADH dehydrogenase; II, succinate dehydrogenase; III, cytochrome c reductase; IVa and IVb, large and small form of cytochrome c oxidase; V, ATP synthase; I + III₂ and I₂ + III₄, supercomplexes of complexes I and III). Additional supercomplexes exhibiting cytochrome c oxidase activity are indicated by arrows.

Figure 2.

Oxygen consumption of isolated mitochondria from potato tubers and stems. Values are based on three independent mitochondrial preparations.

Figure 3.

Resolution of mitochondrial protein complexes from potato mitochondria after solubilization using varying digitonin to protein ratios. Protein complexes were separated by 1D BN-PAGE and visualized by Coomassie staining. Detergent to protein ratios are given in g detergent per g mitochondrial protein. The OXPHOS complexes are designated by Roman numerals (see legend of Fig. 1). Unknown protein complexes are indicated by arrows.

Figure 4.

Two-dimensional resolution of mitochondrial protein complexes from potato tubers (A) and potato stems (B) by BN/SDS-PAGE. Mitochondrial proteins were solubilized by 5 g digitonin per g protein. Gels were Coomassie stained. Strips of corresponding 1D BN gels and identities of protein complexes and supercomplexes are given above the 2D gels. The numbers on the left indicate the molecular masses of standard proteins. Subunit II of cytochrome c oxidase is marked by arrows.

Figure 5.

Two-dimensional resolution of mitochondrial protein complexes from potato tubers (A) and stems (B) by BN/BN-PAGE. Mitochondrial proteins were solubilized by 5 g digitonin per g protein. Corresponding strips of 1D BN gels are shown above the 2D gels. Identities of the resolved protein complexes and supercomplexes are given above and to the left of the gels in Roman numerals.

Figure 6.

Identification of cytochrome c oxidase-containing supercomplexes of potato stem mitochondria by in-gel activity staining on 2D BN/BN gels. Mitochondrial proteins were solubilized by 5 g digitonin per g protein. A corresponding stripe of an activity-stained 1D BN gel is shown above the 2D gel. Identities of protein complexes and supercomplexes of the OXPHOS system from potato are given in Roman numerals. The activity stain is given in false-color mode to increase color-contrast (red, Coomassie; black, enzyme activity).

Figure 7.

Identities of protein complexes and supercomplexes of the OXPHOS system in potato tubers (T) and stems (S) after separation by 1D BN-PAGE. Proteins were solubilized by 5 g digitonin per g protein. The gels were Coomassie stained. Identities of the protein complexes and supercomplexes are given by Roman numerals.

Figure 8.

Structure and function of the respirasome in mitochondria. [M], Matrix; [IMS], mitochondrial intermembrane space.