The ATP synthase is involved in generating mitochondrial cristae morphology

Abstract

The inner membrane of the mitochondrion folds inwards, forming the cristae. This folding allows a greater amount of membrane to be packed into the mitochondrion. The data in this study demonstrate that subunits e and g of the mitochondrial ATP synthase are involved in generating mitochondrial cristae morphology. These two subunits are non-essential components of ATP synthase and are required for the dimerization and oligomerization of ATP synthase. Mitochondria of yeast cells deficient in either subunits e or g were found to have numerous digitations and onion-like structures that correspond to an uncontrolled biogenesis and/or folding of the inner mitochondrial membrane. The present data show that there is a link between dimerization of the mitochondrial ATP synthase and cristae morphology. A model is proposed of the assembly of ATP synthase dimers, taking into account the oligomerization of the yeast enzyme and earlier data on the ultrastructure of mitochondrial cristae, which suggests that the association of ATP synthase dimers is involved in the control of the biogenesis of the inner mitochondrial membrane.

Fig. 1. Dimerization of subunit 4. Mutant and wild-type mitochondria (50 µg of protein) were incubated with 40 mM NEM, dissolved in sample buffer containing SDS and submitted to western blot analysis using polyclonal antibodies against subunit 4 of the ATP synthase. (A) Mitochondria were isolated from wild-type yeast (lane 1), and from 4D54C (lane 2), ΔATP20-4D54C (lane 3) and ΔTIM11-4D54C (lane 4) mutant strains. (B and C) Mitochondria isolated from cysteine mutants of subunit 4 were analysed as above.

Fig. 2. The yeast ATP synthase exists in an oligomeric form. Wild-type, ΔATP18 and ΔATP20 mitochondria were solubilized with the indicated digitonin/protein ratios (g/g). After centrifugation, the mitochondrial complexes were separated by BN–PAGE and the gels were either stained with Coomassie Blue (A) or incubated with ATP-Mg²⁺ and Pb²⁺ to reveal the ATPase activity (B and C). The figures shown in (B) and (C) are the negatives of original gels. Bands in (B) revealed by the ATPase activity were cut and submitted to SDS–PAGE (D), and the slab gel was then silver stained. Lane 1, purified yeast wild-type ATP synthase; lane 2, oligomer (digitonin/protein ratio of 0.75 g/g); lanes 3 and 4, higher and lower bands of dimers, respectively (digitonin/protein ratio of 2 g/g); lane 5, monomer (digitonin/protein ratio of 2 g/g). Std, standard proteins; wt, wild type; %T, acrylamide concentration.

Fig. 3. Yeast cells devoid of either subunit e or g have abnormal mitochondria. Samples were prepared as described in Materials and methods. They were observed by transmission electron microscopy. The arrows indicate abnormal mitochondria. (A) Wild-type, (B) ΔATP18, (C and D) ΔATP20 and (E and F) ΔTIM11. Bars indicate 0.5 µm.

Fig. 4. ΔATP4 yeast cells have abnormal mitochondria. (A) Cells were grown with 2% galactose as carbon source, and observed by transmission electron microscopy. The arrow indicates abnormal mitochondria. Bar indicates 0.5 µm. (B) Wild-type (lane 1) and ΔATP4 mitochondria (lane 2) were dissociated and samples (50 µg of protein) were submitted to western blot analysis. Blots were probed with polyclonal antibodies raised against subunits γ, 4, g and e.

Fig. 5. Immunological detection in yeast cells of β-subunit of yeast ATP synthase Immunogold electron microscopy was carried as described in Materials and methods. The pictures are representative of experiments performed with wild-type (A) and ΔATP20 (B–D) cells. Bars indicate 0.5 µm.

Fig. 6. Immunological detection in yeast cells of yeast mitochondrial porin. Immunogold electron microscopy was carried out as described in Materials and methods. The images are representative of experiments performed with wild-type (A) and ΔATP20 (B) cells. Bars indicate 0.5 µm.

Fig. 7. Schematic representation of associations of F₁F₀ ATP synthase dimers. The grey circles and blocks represent the membranous parts of yeast F₀ components as observed from the intra-cristae space. Subunit 4 (subunit b) is represented by two dark grey circles (the two membrane-spanning segments) that are linked by a line corresponding to the inter-membrane hydrophilic loop (amino acid residues 46–56). The black dots represent the mutation 4D54C. This model displays two interfaces (black bars). One is mediated by subunit 4 and the other is mediated at least by subunits e and g.