The transmembrane arrangement of the ADP/ATP carrier as elucidated by the lysine reagent pyridoxal 5-phosphate.
The lysine reagent pyridoxal 5-phosphate was applied to the ADP/ATP carrier (AAC) in order to elucidate topological and functional properties of the numerous lysines within the primary structure. To establish appropriate labeling conditions, the influence of pyridoxal-P on transport and inhibitor binding to the AAC was examined. The ADP/ATP transport is sensitive to low concentrations of pyridoxal-P with a Ki = 0.4 mM. Binding of [3H]carboxyatracylate and [3H]bongkrekate is largely inhibited by pyridoxal-P treatment with Ki approximately 1 mM. [3H]Carboxyatractylate is not and [3H]bongkrekate weakly removed by pyridoxal-P, whereas [3H]atractylate is displaced to a large extent. Under optimized conditions of pyridoxal-P concentration, of pH and of time exposure, the AAC was exposed to [3H]pyridoxal-P in mitochondria, in submitochondrial particles and in the detergent-solubilized carrier. The [3H]pyridoxal-P-labeled AAC was isolated from mitochondria and particles. After citraconylation thermolysinolytic peptides were prepared. The pyridoxyl-lysine-containing peptides were purified and the pyridoxal-P incorporation to specific lysines was determined by sequencing. The pyridoxal-P incorporation into the AAC in various states was evaluated with regard to structural and functional aspects. First, by comparing pyridoxal-P incorporation in mitochondria and sonic particles, the segments of the polypeptide chain exposed to the cytosolic and matrix side of the membrane are detected. Second, the additional lysine incorporation into the isolated as compared to the membrane-bound carrier is attributed to the protein collar facing the phospholipid headgroups. Third, the difference between lysine incorporation into the carboxyatractylate-AAC and bongkrekate-AAC complexes reflect either conformational changes or lysines involved in the translocation channel through the protein. Fourth, the additional lysine labeled in the atractylate-carrier complex as compared to the carboxyatractylate-carrier complex is attributed to a cationic site in the binding center. These results are incorporated into a transmembrane folding model of the carrier.
D000818:Animals; D001483:Base Sequence; D001894:Borohydrides; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000378:metabolism; D003902:Detergents; D008239:Lysine / Q000032:analysis; D008929:Mitochondria, Heart / Q000378:metabolism; D000226:Mitochondrial ADP, ATP Translocases / Q000302:isolation & purification* / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000302:isolation & purification*; D010449:Peptide Mapping; D011485:Protein Binding / Q000187:drug effects; D011732:Pyridoxal Phosphate / Q000378:metabolism
D001894:Borohydrides; D003902:Detergents; D011732:Pyridoxal Phosphate; C025364:sodium borohydride; D000226:Mitochondrial ADP, ATP Translocases; D009713:Nucleotidyltransferases; D008239:Lysine