Two-dimensional diffusion of F1F0-ATP synthase and ADP/ATP translocator. Testing a hypothesis for ATP synthesis in the mitochondrial inner membrane.
We report here the first experimentally determined lateral diffusion coefficients of the F1F0-ATP synthase and the ADP/ATP translocator in isolated inner membranes of rat liver mitochondria. Rabbit IgG developed against the F1F0-ATP synthase isolated from rat liver mitochondria was determined to be immunospecific for the synthase subunits, notably the alpha-beta doublet, gamma and delta subunits of F1 and subunits two, three and four of F0. This IgG, conjugated with lissamine-rhodamine, was used as a fluorescent probe to monitor the diffusion of the synthase in the membrane. IgG to cytochrome bc1 complex, prepared and labeled similarly, was used as a fluorescent probe for diffusion of this redox component. Eosin maleimide was determined to specifically label the ADP/ATP translocator in the isolated inner membrane and was used as a specific probe for the diffusion of the translocator. Using fluorescence recovery after photobleaching, the experimental average lateral diffusion coefficient of the F1F0-ATP synthase was determined to be 8.4 x 10(-10) cm2/s or twice that of cytochrome bc1 complex while the diffusion coefficient of the ADP/ATP translocator was 1.7 x 10(-9) cm2/s or four times that of cytochrome bc1 complex suggesting that all three components are independent two-dimensional diffusants. Using these diffusion coefficients and applying a number of basic assumptions, we calculated the theoretical two-dimensional diffusion-controlled collision frequencies and derived collision efficiencies (protons transferred per collision) between each of the three proton-transferring redox complexes and both the F1F0-ATP synthase and ADP/ATP translocator by treating the redox components as proton donors and the synthase and translocator as proton acceptors. These collision efficiencies support the physical possibility of a diffusion-based, random collision process of proton transfer and ATP synthesis in the mitochondrial inner membrane.
Gupte
S S
SS
Department of Cell Biology and Anatomy, University of North Carolina School of Medicine, Chapel Hill 27599-7090.
Chazotte
B
B
Leesnitzer
M A
MA
Hackenbrock
C R
CR
D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D000818:Animals; D000918:Antibody Specificity; D004058:Diffusion; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives; D007074:Immunoglobulin G; D007425:Intracellular Membranes / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology / Q000378:metabolism*; D000226:Mitochondrial ADP, ATP Translocases / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D018160:Receptors, Cytoplasmic and Nuclear; D011983:Receptors, Purinergic / Q000378:metabolism*; D013379:Substrate Specificity
C037688:ADP-ATP carrier receptor; D007074:Immunoglobulin G; D018160:Receptors, Cytoplasmic and Nuclear; D011983:Receptors, Purinergic; C033531:eosin maleimide; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases; D006180:Proton-Translocating ATPases; D004801:Eosine Yellowish-(YS)