The mitochondrial ADP/ATP exhange acitivity is not essential for cell growth under fermentation culture conditions and becomes essential only under non-fermentation conditions. This unique system provided the means to knock-out all three native AAC genes and insert heterologous hANT genes. The function of ADP/ ATP exhange acitivity can be readily determined by following growth on non-fermentable carbon sources. Moreover, the hANT1, 2, 3 proteins have all been functionally expressed in yeast mitochondria. Here we expressed hANT4 Picrotoxin protein in AAC- deficient yeast mitochondria along with somatic hANTs for parallel comparison. Using a similar methodology to that required for functional hANT1, 2 and 3 expression in yeast, hANT4 failed to complement the respiratory defect of yeast lacking the endogenous AAC genes. Moreover, overexpression of hANT4 led to deleterious Dofetilide effects on yeast cell growth. However, mutant forms of hANT4 protein were isolated that facilitated proper mitochondrial localization and complementation of AAC-deficient yeast. The ADP/ATP exchange kinetics of those modified hANT4 proteins compared favorably to the kinetics of the somatic hANTs expressed and analyzed under identical experimental conditions. We determined that the mutations in yNhANT4 were indeed responsible for the growth complementation on YPEG by reintroducing the mutated yNhANT4 alleles back into the parental strain. All the four alleles supported yeast growth on YPEG. The A30V mutation of yNhANT4 proved the strongest allele by showing rapid growth on nonfermentable carbon sources comparable to that of yeast bearing the wild-type yeast allele AAC2. Although each amino acid mutation within yNhANT4 was sufficient to complement growth on nonfermentable carbon sources, the mutant yeasts that were originally recovered from EMS mutagenesis might have contained additional mutations outside of the yNhANT4 that also contributed to improved growth on nonfermentable carbon sources. To test this hypothesis, the mutant yNhANT4 locus for three of the isolates was first replaced by URA3, and then wild type yNhANT4 was reintroduced using 5- FOA selection as described above.