The uncoupling protein UCP1 does not increase the proton conductance of the inner mitochondrial membrane by functioning as a fatty acid anion transporter

Abstract

The activity of the brown fat uncoupling protein (UCP1) is regulated by purine nucleotides and fatty acids. Although the inhibition by nucleotides is well established, the activation by fatty acids is still controversial. It has been reported that the ADP/ATP carrier, and possibly other members of the mitochondrial carrier family, mediate fatty acid uncoupling of mitochondria from a variety of sources by facilitating the transbilayer movement of the fatty acid anion. Brown fat mitochondria are known to be more sensitive to fatty acid uncoupling, a property that has been assigned to the presence of UCP1. We have analyzed the transport properties of UCP1 and conclude that fatty acids are not essential for UCP1 function, although they increase its uncoupling activity. In order to establish the difference between the proposed carrier-mediated uncoupling and that exerted through UCP1, we have studied the facility with which fatty acids uncouple respiration in mitochondria from control yeast and strains expressing UCP1 or the mutant Cys-304 → Gly. The concentration of free palmitate required for half-maximal activation of respiration in UCP1-expressing mitochondria is 80 or 40 nm for the mutant protein. These concentrations have virtually no effect on the respiration of mitochondria from control yeast and are nearly 3 orders of magnitude lower than those reported for carrier-mediated uncoupling. We propose that there exist two modes of fatty acid-mediated uncoupling; nanomolar concentrations activate proton transport through UCP1, but only if their concentrations rise to the micromolar range do they become substrates for nonspecific carrier-mediated uncoupling.

The ability of long-chain fatty acids to uncouple mitochondrial respiration from ATP synthesis (see Ref. 1; reviewed in Ref. 2) has been generally assumed to resemble that of other weak acids, which can permeate through the lipid bilayer in both protonated and unprotonated forms. However, recent data have established that fatty acid anion flip-flop across the phospholipid bilayer occurs far too slowly (t > 1 min) (3) to allow the anion to complete a classic protonated/deprotonated cycle as for classical uncouplers. The protonophoric activity observed when extremely high fatty acid concentrations (above 0.1 mm) are employed is probably caused by the disruption of the lipid bilayer in a detergent-like mode (1, 4, 5), inasmuch as, for example, effects are not reversed by albumin. The situation is less clear when lower fatty acid concentrations are employed and detergent effects are negligible. It has been shown recently that, under these conditions, fatty acid uncoupling becomes sensitive to inhibitors such as carboxyatractylate, and consequently it has been proposed that permeation of the fatty acid anion is mediated by the ADP/ATP carrier (6-9) and possibly by other anion carriers of the mitochondrial inner membrane (10, 11).

Brown fat is a specialized tissue, the function of which is to produce heat. The thermogenic mechanism is centered around the brown fat uncoupling protein (UCP1),which allows dissipation of the proton electrochemical potential gradient and therefore uncouples respiration from ATP synthesis (12, 13). Recently, three new uncoupling proteins have been identified with homology to UCP1 and termed UCP2 (14), UCP3 (15), and StUCP (16). Free fatty acids, liberated by the noradrenergic stimulation of lipolysis, are the substrate for brown fat thermogenesis and also act as the cytosolic second messengers by which noradrenaline activates UCP1 (17, 18).

The mechanism by which fatty acids activate UCP1 is a matter of debate. Two main hypothesis are current. The first is an extension of the observation of carboxyatractylate-sensitive mitochondrial fatty acid uncoupling, and proposes that UCP1 and other mitochondrial anion carriers (19, 20) can catalyze the transport of the fatty acid anion (21, 22). The second model proposes that fatty acids act as a prosthetic group in UCP1 delivering protons to a site from which they are translocated to the other side of the membrane (23). The anion transporting activity would have little physiological importance and thus would be a vestige of its evolutionary relationship with the rest of mitochondrial metabolite carriers.

The present report will examine the characteristics of fatty acid-mediated uncoupling of yeast mitochondria expressing recombinant UCP1. Two questions are addressed. First, does UCP1 function in the absence of both nucleotides and fatty acids? Second, what is the difference between UCP1-mediated uncoupling and that mediated by other carriers? We conclude that UCP1 retains the capacity to transport H+(OH−) in the absence of fatty acids and that fatty acids activate UCP1 at much lower concentrations than are required for other mitochondrial carriers