The protonophoretic function of uncoupling protein
(UCP) is activated by fatty acids. According to the docking site hypothesis
(Jezek, P., and Garlid, K. D., J. Biol. Chem. 265, 19303-19311, 1990), the
fatty acid binding site is identical with the anion channel of UCP. Skulachev
(Skulachev, V. P. (1991) FEBS Lett. 294, 158-162) extended this hypothesis
by suggesting that fatty acid anions are transported by UCP and that H+ are
delivered by back-diffusion of the protonated fatty acid through the lipid
bilayer. In this model, UCP does not transport H+ at all but rather enables
fatty acids to act as cycling protonophores. New evidence supports this mechanism
(Garlid, K. D., Orosz, D. E., Modriansky, M., Vassanelli, S., and Jezek, P.
(1996) J. Biol. Chem. 271, 2615-2620). To help elucidate these hypotheses,
we synthesized a photoreactive analog of dodecanoic acid, 12-(4-azido-2-nitrophenylamino)dodecanoic
acid (AzDA), and studied its effect on transport in mitochondria and proteoliposomes.
AzDA behaved in every respect like a typical fatty acid. In micromolar doses,
AzDA activated H+ translocation and inhibited Cl- and hexanesulfonate uniport
through UCP. After UV light exposure, however, activation of H+ transport
was inhibited, whereas inhibition of anion transport was preserved. These
effects were irreversible. Photolabeling of mitochondria with [3H]AzDA resulted
in a prominent 32 kDa band of UCP, and few other proteins were labeled. The
results indicate that AzDA can be ligated to the protein at or near the docking
site, causing irreversible inhibition of both H+ and anion transport. The
finding that fatty acid-induced H+ transport disappears along with anion transport
supports the fatty acid-protonophore mechanism of H+ transport by UCP.
Back to Publications.