A, Representative immunoblot of subcellular compartments isolated by ultracentrifugation (12) revealed strong enrichments of ABCC6 in MAM but not in pure endoplasmic reticulum (ER) or mitochondria. B, Cell surface proteins were biotinylated and affinity-purified. ABCC6 associated with the intracellular, not cell surface fraction. PM indicates plasma membrane; VDAC1, voltage-dependent anion channel 1.

A, Representative immunoblot of subcellular compartments shows that ABCC6 cosediments with mitochondria in C3H and C3H-Abcc6-Tg livers. B, ABCC6 (S-20) antibody specifically identified predominant 160-kDa bands in C3H, C3H-Abcc6-Tg, B6-wildtype (WT), and Abcc6-knockout (KO) mouse liver lysate. Voltage-dependent anion channel 1 (VDAC1) and protein disulfide isomerase (PDI): loading controls. C, Immunofluorescence of purified mitochondrial fraction from kidney by confocal microscopy (representative images from 3 independent experiments). Mitotracker (left) stained mitochondria with intact membrane potential in both B6-WT (top) and KO (bottom) mice. ABCC6 (middle) signals are present in B6-WT but not KO mice. COXIV (immunostaining control) was present in virtually all mitochondria. White color in the upper right panel shows ABCC6 colocalizes with COXIV and mitochondria in B6-WT but not KO mice. D, ABCC6 clusters are present on mitochondria. Conventional confocal microscopy (green) was overlaid on STED (stimulated emission depletion) microscopy images (red) with eliminated scattered fluorescence. PM, indicates plasma membrane; ER, endoplasmic reticulum.

A, Representative electron micrograph of liver, kidney, and heart sections in B6-wildtype (WT) (top) and knockout (KO) (bottom) mice. Mitochondrial abnormalities of different types were observed in the 3 organs. In the liver, Abcc6 deletion attenuated the extensive network of ER membranes (arrowhead) associating with mitochondria (m). Many kidney mitochondria were virtually devoid of cristae (m*). In the heart, Abcc6-KO mice had disrupted cristae (arrowhead) and disordered mitochondria (m); sm=sarcomere. B, Abcc6 deletion decreased maximal mitochondrial respiration rates. Percentage increases in oxygen consumption rates (OCR) in response to FCCP were significantly lower in KO mice. Data were averaged from 3 independent experiments. Error bars: SEM.