Rotational diffusion of the ADP/ATP translocator in the inner membrane of mitochondria and in proteoliposomes.
The ADP/ATP translocator was selectively labeled with the triplet probe eosin-5-maleimide (EMA) after pretreatment with N-ethylmaleimide in beef heart mitochondria, as reported previously for submitochondrial particles (Müller, M., Krebs, J. J. R., Cherry, R. J., and Kawato, S. (1982) J. Biol. Chem. 257, 1117-1120). The EMA binding was completely inhibited by carboxyatractylate. 0.7-1.1 molecules of EMA conjugated with 1 molecule of the dimeric translocator with Mr approximately 65,000. The EMA binding decreased [14C]ADP uptake by about approximately 25%. The EMA-labeled translocator bongkrekate complex was purified and reconstituted in liposomes by removing Triton X-100 with Amberlite XAD-2. The liposomes were composed of phosphatidylcholine/phosphatidylethanolamine/cardiolipin and the lipid to protein ratio by weight was (L/P) = 60. Rotational diffusion of the ADP/ATP translocator around the membrane normal was measured in reconstituted proteoliposomes and in the mitochondrial inner membranes by observing the flash-induced absorption anisotropy, r(t), of EMA. In proteoliposomes with L/P = 60, the translocator was rotating with an approximate average rotational relaxation time of phi congruent to 246 microseconds and a normalized time-independent anisotrophy [r3/rr(0)]min congruent to 0.55. In intact mitochondria, values of phi congruent to 405 microseconds and r3/rr(0) congruent to 0.79 were obtained. The higher value of r3/rr(0) in mitochondria compared with proteoliposomes indicates the co-existence of rotating and immobile translocator (phi greater than 20 ms) in the inner mitochondrial membrane. Based on the assumption that all the translocator is rotating in the lipid-rich proteoliposomes, the population of the mobile translocator at 20 degrees C was calculated to be approximately 47%. By removing the outer membrane, the mobile population was increased to approximately 70% in mitoplasts, while approximately 53% of the translocator was rotating in submitochondrial particles. The above results indicate a significant difference in protein-protein interactions of the ADP/ATP translocator in the different types of inner membranes of mitochondria. The immobile population of the translocator could be due to nonspecific protein aggregates caused by the very high concentration of proteins in the inner membrane of mitochondria (L/P approximately 0.4).
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D002250:Carbon Radioisotopes; D002417:Cattle; D004058:Diffusion; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D007425:Intracellular Membranes / Q000201:enzymology*; D008081:Liposomes; D008929:Mitochondria, Heart / Q000201:enzymology*; D000226:Mitochondrial ADP, ATP Translocases / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000378:metabolism*; D011510:Proteolipids; D051381:Rats
D002250:Carbon Radioisotopes; D008081:Liposomes; D011510:Proteolipids; C020485:proteoliposomes; D000244:Adenosine Diphosphate; C033531:eosin maleimide; D000226:Mitochondrial ADP, ATP Translocases; D009713:Nucleotidyltransferases; D005033:Ethylmaleimide; D004801:Eosine Yellowish-(YS)