The substitution of P95S might impose flexibility to fit the yeast lipid environment. It should be noted that the substitution of P89L in AAC1 that corresponds to the P95L substitution in hANT4 has shown to alter functionality of the protein in the native phospholipid environment of yeast. A previous study demonstrated that modifications of amino acids of somatic hANT proteins near a putative cardiolipin interaction site improved yeast growth in reduced oxgen conditions, and suggested that differences of lipid composition PD 404,182 between the mitochondrial inner membranes of yeast and mammals might limit proper function. Since ANT4 is exclusively found in mammalian germ cells and sperm mitochondria, it is reasonable to speculate that hANT4 may have evolved to adapt to the lipid environment of those cell types. Interestingly, the relatively subtle amino acid changes isolated in hANT4 influenced the ADP/ATP exchange kinetics even though those changes occurred far from the substrate binding site. This may also suggest that subtle changes in lipid composition of inner membrane may alter hANT function. Indeed, cardiolipin is a critical component for exchange activity in both yeast and mammalian ANT. Levels of phospholipids and cholesterol have also been shown to affect the exchange function of mammalian ANTs. In the case of ANT4, oxidation or other damage to lipids could alter the exchange function of ANT4, and potentially affect male germ cell meiosis or sperm motility. It is technically challenging to determine the ADP/ATP exchange kinetics of ANT proteins, which are influenced by various factors depending on the methodology. Phentolamine hydrochloride Therefore, the kinetic values vary considerably in literature. The previously reported transport kinetics of hANT4 had higher KM values, 72 mM for ADP and 120 mM for ATP in a liposome reconstitution system using purified hANT4. In the present study, we found that the KM values were much lower in all three mutant hANT4 proteins that are compatible with those of somatic hANTs. Although our hANT4 peptides contain mutations required for proper assembly and stability of the proteins in yeast mitochondrial membrane, these mutation sites are distant from the ADP/ ATP binding pocket and unlikely to substantially change the substrate binding affinity per se.