Figure 4.
Oligomerization-deficient Mic10 restores physiological functions in mic10Δ cells
(A) Mic10-containing complexes of mitochondria from WT and mic10Δ cells as well as cells expressing the variant Mic10-G76A, analyzed by BN-PAGE.
(B) Radiolabeled Mic10 or the oligomerization-deficient mutant variants Mic10-G76A or Mic10-G74A were imported into Atp21-His mitochondria, followed by lysis with digitonin and isolation of Atp21-His and interacting proteins. The co-isolation efficiency relative to the levels in the load fraction was not impaired for the Mic10 mutants. Load, 2.5%; eluate, 100%.
(C) In organello crosslinking shows that the band reflecting a direct crosslink between Atp21 and Mic10 is similarly enhanced upon overexpression of Mic10 or Mic10-G76A. SMPB, succinimidyl 4-(p-maleimido-phenyl)butyrate.
(D) Analysis of oligomeric ATP synthase by BN-PAGE and in gel ATPase activity staining using very mild solubilization (0.9% digitonin).
(E) Growth of mic10Δ cells without or with expression of Mic10 (WT) or Mic10-G76A in selective media with 3% glycerol + 0.1% glucose. Data are represented as mean SEM; n = 4.
(F) The mitochondrial membrane potential was assessed in mitochondria isolated from the indicated strains. Data are represented as mean SEM; n = 5.
(G) Dual function of Mic10 at MICOS and the ATP synthase. The majority of Mic10 supports MICOS function and crista junction stability by membrane bending, which relies on Mic10 oligomerization. A distinct Mic10 population interacts with the ATP synthase at substoichiometric amounts and regulates its higher order assemblies. This regulatory role does not require Mic10 oligomerization and is crucial for the maintenance of a healthy membrane potential and mitochondrial network and consequently for efficient metabolic adaptation and respiration. BN-PAGE panels: western blot of Mic10-containing complexes in a Mic60-ProtA eluate (left panel; shown is a shorter exposure of lane 1 of Figure 2G) and autoradiograph of imported radiolabeled Mic10 in an Atp21-His eluate (right panel) are shown.
See also Figures S1 and S3.