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 3 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 Ͼ 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 u...

The uncoupling protein homologues: UCP1, UCP2, UCP3, StUCP and AtUCP

“…When yeast mitochondria containing UCP1 were challenged with fatty acids, they showed a much higher sensitivity to the fatty acids than the control mitochondria [106]. The inhibition of both the basal and the fatty acid-induced uncoupling by nucleotides indicated that the proton transport was due to UCP1 [106]. As expected, addition of a higher concentration of fatty acids partially uncoupled control mitochondria without UCP1, but, in contrast, this uncoupling could not be inhibited by nucleotides [106].…”

“…The inhibition of both the basal and the fatty acid-induced uncoupling by nucleotides indicated that the proton transport was due to UCP1 [106]. As expected, addition of a higher concentration of fatty acids partially uncoupled control mitochondria without UCP1, but, in contrast, this uncoupling could not be inhibited by nucleotides [106]. Certain modified fatty acids with hydrophilic substitutions make the ' flip flop ' across the hydrophobic phase very unlikely.…”

“…Moreover, the fatty acid cycling model implicates a pool of fatty acids in equilibrium between UCP1 and the lipid phase of the membrane, and it is therefore difficult to understand why this fatty acid pool would not equilibrate with albumin. Finally, in the yeast recombinant expression system, the basal rate of proton leak catalysed by UCP1 in respiring mitochondria was not influenced by a mutation increasing UCP1 sensitivity towards fatty acids [106]. These observations suggest that in respiring mitochondria, where a high and stable membrane potential value was reached, UCP1 was able to catalyse a proton backflow, inducing a high uncoupled respiratory rate not dependent on fatty acid activation.…”

“…However, the observation of UCP1-mediated uncoupling in the absence of fatty acids would indicate that the proton pathway still operates when fatty acid cycling cannot take place. As indicated above, data obtained with respiring mitochondria showed that, even when fatty acids were trapped by albumin, mitochondria were still uncoupled, and lowering of the respiratory rate required the further addition of nucleotides [106]. The efficiency of fatty acid removal by albumin has been questioned, and therefore remaining non-exchangeable fatty acids have been proposed to explain the persistence of the uncoupling activity of UCP1 in BAT mitochondria [87].…”

“…The introduction of UCP1 either in CHO cell mitochondria, or in yeast mitochondria using recombinant DNA technology, induced a partially uncoupled state [81,105]. When yeast mitochondria containing UCP1 were challenged with fatty acids, they showed a much higher sensitivity to the fatty acids than the control mitochondria [106]. The inhibition of both the basal and the fatty acid-induced uncoupling by nucleotides indicated that the proton transport was due to UCP1 [106].…”