For expression of human ANT1 in
S. cerevisiae, the coding sequence of the first 26 amino acid residues from yeast Aac2p followed by the ANT1 coding sequence was PCR amplified from y2NHANT1/pRS314 [
20] and gateway cloned into the yeast expression vector pAG416GAL-ccdB-EGFP [
21]. In this system, ANT1 is expressed as a fusion protein with EGFP at the C-terminus under regulation of the
GAL1 promoter, which is repressed by glucose [
22]. The ANT1-EGFP construct has a calculated molecular weight of 62.5 kDa. The C57A, C160A and C257A mutants of ANT1 were made using the QuikChange XL Site-Directed Mutagenesis kit (Integrated Sciences) and their sequence was confirmed. The wild type ANT1 and mutant constructs were transformed into yeast strain BY4741 (
MAT a,
his3Δ, leu2Δ, ura3Δ, met15Δ) as described [
23]. Yeast transformants were selected in synthetic complete media lacking uracil (SC-ura: 20 g/L glucose, 5 g/L ammonium sulphate, 1.7 g/L yeast nitrogen base, 10 mg/L adenine, 50 mg/L arginine, 80 mg/L aspartate, 20 mg/L histidine, 50 mg/L isoleucine, 100 mg/L leucine, 50 mg/L lysine, 20 mg/L methionine, 50 mg/L phenylalanine, 100 mg/L threonine, 100 mg/L tryptophan, 50 mg/L tyrosine and 140 mg/L valine). ANT1-GFP protein was expressed by culturing yeast cells to an optical density of 0.8 at 600 nm in SC-ura media containing raffinose (20 g/L) instead of glucose [
24]. Cells were cultured for another 6 h with 0.2% galactose before imaging or harvesting the mitochondria.