Probing the role of positive residues in the ADP/ATP carrier from yeast. The effect of six arginine mutations on transport and the four ATP versus ADP exchange modes.
Mutagenesis of three intrahelical arginines, R96, R204, or R294, and of each member of the arginine triplet R252, R253, R254 into neutral residues had resulted in a strong suppression of oxidative phosphorylation in cells and isolated mitochondria [Müller, V., Basset, G., Nelson, D. R., & Klingenberg, M. (1996) Biochemistry 35, 16132-16143]. Here we determine the transport activity of wild-type and mutant AAC in reconstituted proteoliposomes using a new rapid removal-stop method without relying on the inhibitor stop which can be compromised by mutations. The basic electroneutral ADP/ADP exchange activity is strongly or totally suppressed in six out of seven of these mutations, with the exception of R294A, which retains nearly wild-type activity. Carboxyatractylate (CAT) inhibits the ADP/ATP exchange rate only to 3-10% in wild type and R294A and up to 40% in other mutants, whereas bongkrekic acid (BKA) inhibits 50% (wild type and R294A) and 90% (other mutants). Consequently, AAC is preferentially reconstituted with the matrix surface outside. All these mutations drastically change activity distribution among the four exchange modes ADP/ADP, ADP/ATP, ATP/ADP, and ATP/ATP. Whereas in wild-type AAC the homo ATP/ATP exchange is twice as high as the ADP/ADP exchange, in mutants it is 10 to 15 times lower. Similarly, the hetero ATP/ADP exchange in wild-type AAC is higher than the ADP/ ATP exchange, but in mutants it is several times lower. Thus, these mutations afflict the ATP-linked modes, in particular those linked to external ATP. The inhibition of oxidative phosphorylation is thus explained by the suppression of ATP export versus ADP import mode. The "extra"-inhibition of oxidative phosphorylation in mutant cells is explained by the extreme shift in mutants in favour of ATP import versus ADP export. Besides structural changes, the mutant effects indicate electrostatic interactions of these arginines with the anionic substrates. The loss of one positive charge raises the translocation barrier the more negative the substrate, i.e. more for ATP4- than for ADP3-. In none of these arginine mutants was the binding of CAT or BKA abolished.
Heidkämper
D
D
Institute for Physical Biochemistry, University of Munich, Germany.
Müller
V
V
Nelson
D R
DR
Klingenberg
M
M
D001120:Arginine; D001278:Atractyloside / Q000031:analogs & derivatives / Q000494:pharmacology; D001665:Binding Sites; D007700:Kinetics; D008081:Liposomes; D008928:Mitochondria / Q000378:metabolism*; D000226:Mitochondrial ADP, ATP Translocases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D008081:Liposomes; D011994:Recombinant Proteins; D001278:Atractyloside; D000226:Mitochondrial ADP, ATP Translocases; D001120:Arginine; C003853:carboxyatractyloside