Figure 2

The trivalent arsenical of GSAO and PENAO reacts with cysteines 57 and 257 of human ANT1. A. Localisation of human ANT1-GFP in S. cerevisiae. Yeast cells expressing ANT1-GFP and the cysteine mutants were analysed by fluorescence microscopy to determine cellular localisation. Cells were also stained with DAPI to identify mitochondrial and nuclear DNA. Wild-type and ANT1 cysteine mutants co-localized with mitochondrial DNA. The C160A and C257A ANT1 mutants were also found in the cytoplasm, which likely represents incomplete or inefficient incorporation into mitochondria. White arrows indicate the mitochondria (M) or the nucleus (N). B. Mitochondria from S. cerevisiae expressing ANT1-GFP and the cysteine mutants were isolated and incubated with biotin-tagged GSAO or PENAO. The labelled proteins were collected on avidin-coated beads and bound ANT1 was detected by Western blot. GSAO- and PENAO-biotin bound to wild-type and C160A mutant ANT1 but not to C57A and C257A mutants. The human ANT1 and yeast Por1p in the mitochondrial preparations was blotted to show the input protein in the binding assay. The sizes the proteins in kDa is indicated at left. The results from two separate experiments are shown.