A. Domain architecture of VWA8a and VWA8b. Both isoforms have
N-terminal mitochondrial targeting sequences and putative ATPase domains.
B. Mouse AML12 hepatocytes were infected with adenoviral
vectors encoding VWA8a with an HA tag or empty vector (control). Cells were
stained with MitoTracker Red and a green fluorescent tagged anti-HA antibody.
The overlap shows VWA8a is targeted to mitochondria. C. AML12
hepatocytes were fractionated into cytoplasmic and mitochondrial fractions and
immunoblot analysis was used to detect endogenous VWA8a, GAPDH, adenine
nucleotide translocase 1(ANT1) and Cytochrome C oxidase (Cyto C). Endogenous
VWA8a was present only in the mitochondrial fraction. AML12 cells were
transiently transfected with a vector encoding a VWA8a mutant, tagged with HA,
and lacking the 1–34 N-terminal amino acid putative mitochondrial
targeting sequence, VWA8 (35-1095). Immunoblot analysis with anti-HA antibody
showed that this mutant was not targeted to mitochondria.