None of the mutants bearing the non-mutated yNhANT4 allele could support growth on nonfermentable carbon sources. Psychosine from bovine brain Therefore, we concluded that the A30V, P95S and S202L amino acid substitutions were sufficient for functional expression in yeast. Since mitochondrial ADP/ATP exhange acitivity in yeast is not essential during fermentative growth but is essential for growth using nonfermentable carbon sources, it was possible to introduce and biochemically analyze nonfunctional or sub-functional ANTs. Consequently, we were able to knock-out all three native AAC genes and insert heterologous genes corresponding to hANT1, 2, 3, and 4 at the yeast AAC2 locus. There are some limitations of this heterologous expression method. Several previous reports as well as data presented here demonstrated that the N-terminal sequence greatly influences the functional localization of ANT proteins. Apparently the mammalian ANT proteins lack a necessary signal for compatibility with the yeast mitochondrial inner membrane protein transport machinery. Interestingly, hANT4 required additional mutations for functional expression in yeast mitochondria. These mutations were all missense mutations affecting A30, P95 or S202 in hANT4 protein and improved yeast mitochondrial expression. Without these modifications, hANT4 protein was unstable in yeast. Accumulation of excess unfolded protein could be the reason that over-production of the yNANT4 protein suppressed the yeast cell growth. It remained unclear how the specific amino acid substitution at the residues allowed functional expression in yeast mitochondria. Therefore, we mapped these amino acids onto a three-dimensional structure of hANT4, using the sequence alignments and the CP-101606 mesylate crystal structure of bovine ANT1 as a guide. Interestingly, all three mutation sites were located in transmembrane domains and are predicted to be located at similar levels with respect to the mitochondrial membrane. All three sites are oriented towards solvent, and two of the sites are located in positions that may permit interaction with lipid since these residues are in close proximity to the LAPAO detergent in the crystal structure of ANT1. Indeed, both substitutions of A30V and S202L increase hydrophobicity of these sites.