Assessment of membrane-bound mammal mitochondrial adenine nucleotide translocase topography by experimental antibodies

To gain insight into the immunogenicity of mitochondrial adenine nucleotide translocase (ANT), we raised antibodies against purified bovine heart ANT by induction of ascitic fluid in male Balb/c mice. We identified the antigenic determinants detected by these antibodies by (1) immunodetection of GST-ANT fusion proteins and selected partial constructs of ANT, (2) immunodetection of chemically synthesized overlapping peptides on solid support, and (3) back-titration ELISA. Results revealed a short epitope spreading of the antibodies, resulting in a small number of antigenic determinants. Thus, each antibody detects one or two major epitopes located in the putative hydrophilic loops M2 and M3. No evidence for the antigenicity of the first 133 amino acids of ANT was obtained. These well-characterized antibodies were used to study the topography of the membrane-bound ANT by back-titration ELISA with mitochondrial membranes. We demonstrated that amino acids 145-150 and 230-237 are fully accessible to the antibodies in native ANT, whereas regions 133-140 and 244-251 are not. Furthermore, we used mitochondria devoid of the outer membrane (mitoplasts) and inside-out submitochondrial particles (SMP) to establish the matrix or cytosolic orientation of loops M2 and M3. The results clearly show that these loops have a matrix orientation and thus support the six transmembrane segment model of ANT topography in the inner mitochondrial membrane.