Mapping of the nucleotide-binding sites in the ADP/ATP carrier of beef heart mitochondria by photolabeling with 2-azido[.alpha.-32P]adenosine diphosphate

Abstract

2-Azido[alpha-32P]adenosine diphosphate (2-azido[alpha-32P]ADP) has been used to photolabel the ADP/ATP carrier in beef heart mitochondria. In reversible binding assays carried out in the dark, this photoprobe was found to inhibit ADP/ATP transport in beef heart mitochondria and to bind to two types of specific sites of the ADP/ATP carrier characterized by high-affinity binding (Kd = 20 microM) and low-affinity binding (Kd = 400 microM). In contrast, it was unable to bind to specific carrier sites in inverted submitochondrial particles. Upon photoirradiation of beef heart mitochondria in the presence of 2-azido[alpha-32P]ADP, the ADP/ATP carrier was covalently labeled. After purification, the photolabeled carrier protein was cleaved chemically by acidolysis or cyanogen bromide and enzymatically with the Staphylococcus aureus V8 protease. In the ADP/ATP carrier protein, which is 297 amino acid residues in length, two discrete regions extending from Phe-153 to Met-200 and from Tyr-250 to Met-281 were labeled by 2-azido[alpha-32P]ADP. The peptide fragments corresponding to these regions were sequenced, and the labeled amino acids were identified. As 2-azido-ADP is not transported into mitochondria and competes against transport of externally added ADP, it is concluded that the two regions of the carrier which are photolabeled are facing the cytosol. Whether the two photolabeled regions are located in a single peptide chain of the carrier or in different peptide chains of an oligomeric structure is discussed.

Photoaffinity labeling of mitochondrial proteins with 2‐azido [32P]palmitoyl CoA

“…Although more studies are indicated, an additional distinctive feature was that this photolabel could react with the AAC from both the cytosolic and matrix sides of the inner mitochondrial membrane. This important characteristic, which has not been detected with other photolabeling reagents including 2-azido ADP [24], supports previous results obtained by kinetic experiments with nucleotides and palmitoyl CoA on adenine nucleotide transport [5]. Thus, it has now been conclusively demonstrated that, whereas, there are asymmetric receptors for CAT and BKA on either side of the inner mitochondrial membrane, both binding sites recognize acyl CoA esters [25].…”

The ADP/ATP carrier from yeast (AAC‐2) is uniquely suited for the assignment of the binding center by photoaffinity labeling

“…By combining the localizations of 2-azido-ATP incorporation with the two cleavage procedures, one arrives, as shown in fig.4, at a clearly delimited assignment of the 2-azido-ATP incorporation to the segment between residues 172 and 210. There is no trace of incorporation near the C-terminal, as was described for the incorporation of 2-azido-ATP into bovine AAC [2].…”

“…In bovine heart mitochondria the incorporation of 2-azido-ADP was found to be localized in the homologous region between residues 153 to 200 [2]. Within these peptides a diffuse incorporation into the various residues was observed with minor excesses at Lys 162, Lys 165 and Ile 183.…”

“…3 ' -Arylazido-ATR was the first photoaffinty label of which an insertion was localized in a region between residues 154 and 200 in the bovine heart AAC [l]. With the advent of 2-azido-ADP, the binding was found in the same center region of bovine AAC, in addition to some incorporation at the C-terminal region [2]. In a different approach, using pyridoxal phosphate as a covalent probe for Correspondence address: M. Klingenberg, Institut fiir Physikalische Biochemie, Universitat Miinchen, Goethestrasse 33, 8000 Miinchen 2, FRG Abbreviations: CraEs, n-decylpentaoxyethylene; &Es, ndodecyloctaoxyethylene;…”

Mitochondrial carriers in the cytoplasmic state have a common substrate binding site

“…Second, mutation of residues corresponding to K22, R79, Y186, and R279 abolishes transport activity of the yeast ADP͞ATP carrier (4,9,10). Third, the photoaffinity analogue 2-azido[␣-32 P]adenosine diphosphate labels I183 of BtAAC1 preferentially (11). Fourth, the mutation of the equivalent G182 BtAAC1 in the proposed mitochondrial folate transporter͞carrier (12) has a detrimental phenotypic effect.…”