Mitochondrial synthesis of coenzyme A is on the external surface

Synthesis of coenzyme A (CoA) in isolated rat heart mitochondria was studied. Mitochondria synthesized CoA from 4'phosphopantetheine (4'PP), a precursor of CoA which is synthesized from pantothenic acid in the cytosol. The synthesis was time dependent and was absolutely dependent on the presence of external ATP under the conditions used. The rate of synthesis was increased either by increasing the pH from 7.4 to 8.5 or by adding 0.01% deoxycholate to the incubation medium. To determine whether the synthesis was intra- or extramitochondrial, mitochondria were separated from the incubation mixture by centrifugation and assayed for CoA. The amount of CoA appearing in the supernatant after 30 mins of incubation increased with increasing concentrations of 4'PP while that in the mitochondrial pellet remained constant over the concentration range studied. Synthesis of CoA from 4'PP was not affected by the uncoupler 2,4-dinitrophenol, or by carboxyatractyloside, or by a combination of the two drugs. The combination was used in an effort to deplete intramitochondrial ATP and to prevent external ATP from entering the mitochondria, thus resulting in mitochondria devoid of matrix ATP. The absolute dependence of synthesis on external ATP, the appearance of newly synthesized CoA in the incubation buffer and the ability of mitochondria lacking matrix-ATP to synthesize CoA suggest that the mitochondrial enzymes responsible for synthesis of CoA from 4'PP are on the outer membrane or on the outer side of the inner membrane.