Structure-function relationships in the yeast adenine nucleotide carrier (AAC2) were probed by genetic selection techniques in Saccharomyces cerevisiae. As described in the preceding paper, yeast require a functional AAC2 (or AAC1) to grow on a non-fermentable carbon source. Mutants of AAC2 that could not grow on glycerol were subjected to selection for spontaneous suppressors. Ile mutants in the 100% conserved matrix Arg triplet R252 to R254 proved amenable to this approach, yielding colonies on glycerol plates at modest frequency. All mutants analyzed were single point mutations within the AAC2 gene at a different site than the Arg cluster. R254I gave the largest number (11) of unique revertants, while R253I gave only four and R252I gave none, thus, there was a gradient of effect in mutations of the Arg cluster. Unexpectedly, 14 of the 15 revertant mutations affected 13 different amino acids in a narrow sector of the AAC2 topological map, near the cytosolic surface. These mutants are proposed to be in, or very near, the membrane on the opposite side from the matrix Arg cluster. Helical wheel projections of the transmembrane segments show, with one exception, that mutations and charged residues fall within half of each helix. These mutants appear either to line the throat of the membrane channel, or to be involved in helix contacts near the cytosolic face of the inner mitochondrial membrane. These suppressors place further limitations on the organization of the nucleotide channel. We present a model of the AAC2 nucleotide channel based on these results. The region defined by these suppressors is dynamically linked to the Arg cluster through the function of the AAC2 protein. Discrete structures defined by multiple revertant mutations will likely be a common feature of similar regain-of-function schemes, especially when applied to membrane transport proteins. We propose these functionally mapped regions of proteins be named morphological units or morphs for short.