The Acute Regulation of Mitochondrial Proton Conductance in Cells and Mitochondria from the Brown Fat of Cold‐Adapted and Warm‐Adapted Guinea Pigs

Locke, Rebecca M.; Rial, Eduardo; Nicholls, David G.

doi:10.1111/j.1432-1033.1982.tb07061.x

Cited by 83 publications

(35 citation statements)

References 37 publications

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“…6). Moreover, the values of membrane potential measured in B5 mitochondria alone or with GDP were close to values reported for brown adipose tissue mitochondria incubated in the presence or absence of GDP (24).…”

Section: Resultssupporting

confidence: 58%

Stable expression of functional mitochondrial uncoupling protein in Chinese hamster ovary cells.

Casteilla

Blondel

Schimrigk

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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The mitochondrial uncoupling protein (UCP) is a membranous proton carrier exclusively synthesized in brown adipocytes. The cDNA for the rat UCP was placed in an expression vector and transfected into mammalian cells. Its expression was tested in transiently transfected CHO cells. In these cells the UCP was detected in mitochondria by using antibodies. Permanent expression of the UCP was achieved in stable transformed CHO cell lines. In these cells the UCP was characterized in mitochondrial membranes, by using antibodies and hydroxyapatite purification. The protein expressed in CHO cells displayed the functional characteristics of brown adipocyte UCP. It induced the uncoupling of respiration in isolated CHO mitochondria. The membrane potential of transformed mitochondria was also significantiy lowered, as a result of the proton translocating activity of the UCP. GDP is known to inhibit the proton pathway in brown fat mitochondria. Addition of GDP to CHO mitochondria containing UCP resuited in a recoupling of respiration and an increase in membrane potential. Thus we conclude that functional UCP is expressed in CHO cells and that the insertion of the UCP alone in any mitochondria is sufficient to induce the uncoupling of respiration. This approach should allow studies on the structure-function relationship of the UCP and of several other related mitochondrial carriers.In most eukaryotic cells, the energetic equilibrium results from processes that take place in the inner mitochondrial membrane. In this cell compartment, the synthesis of ATP is coupled to the oxidation process, so that respiration ceases when no ADP and phosphate are available. This is why two mitochondrial transporters, the ADP/ATP carrier and the phosphate carrier, play a significant role in the control of oxidative phosphorylation. These two carriers are present in any cell. The uncoupling protein (UCP) is a mitochondrial transporter (1, 2) that is expressed only in brown adipocytes of mammals, where it induces energy dissipation as heat. UCP acts as a natural uncoupler ofoxidative phosphorylation capable of collapsing the proton electrochemical gradient necessary for ADP phosphorylation (1). Its activity is inhibited by purine nucleotides (1, 3, 4) and its synthesis is essentially regulated at the level of transcription by the sympathetic nervous system acting through norepinephrine released at the surface of brown adipocytes (2, 5). Interestingly, several laboratories have reported that UCP, the ADP/ATP carrier, the mitochondrial phosphate carrier, and a recently identified component named hML-7 belong to the same protein family (6-10) and were derived from a common ancestor. Moreover UCP, the ADP/ATP carrier, and hML-7 have no N-terminal targeting sequence.Studies on UCP offer the possibility of investigating several fundamental questions such as the mechanisms of proton transport by membrane carriers, the organization of a regulatory nucleotide binding site, the topology ofa mitochondrial carrier folded inside the membrane, and lastly...

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Section: Resultssupporting

confidence: 58%

Stable expression of functional mitochondrial uncoupling protein in Chinese hamster ovary cells.

Casteilla

Blondel

Schimrigk

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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“…Unfortunately this experiment was performed in the absence of CAT and without looking at the proton leak kinetics meaning it is not possible to discount the contribution of inducible proton leak due to ANT, or differences in mitochondrial membrane potential resulting in differences in state 4 respiration observed. Earlier experiments performed on BAT mitochondria isolated from warm (low UCP1) or cold (high UCP1) adapted guinea pigs (Locke et al 1982;Rial and Nicholls 1984) or rats (Goubern et al 1990) showed no apparent difference in basal proton conductance in the presence of GDP. In none of these studies were differences in fatty acid stimulated leak through ANT accounted for.…”

Section: Discussionmentioning

confidence: 81%

Uncoupling protein-1 (UCP1) contributes to the basal proton conductance of brown adipose tissue mitochondria

Parker

Crichton

Vidal-Puig

et al. 2009

J Bioenerg Biomembr

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Proton leak pathways uncouple substrate oxidation from ATP synthesis in mitochondria. These pathways are classified as basal (not regulated) or inducible (activated and inhibited). Previously it was found that over half of the basal proton conductance of muscle mitochondria was catalyzed by the adenine nucleotide translocase (ANT), an abundant mitochondrial anion carrier protein. To determine whether ANT is the unique protein catalyst, or one of many proteins that catalyze basal proton conductance, we measured proton leak kinetics in mitochondria isolated from brown adipose tissue (BAT). BAT can express another mitochondrial anion carrier, UCP1, at concentrations similar to ANT. Basal proton conductance was measured under conditions where UCP1 and ANT were catalytically inactive and was found to be lower in mitochondria from UCP1 knockout mice compared to wild-type. Ablation of another abundant inner membrane protein, nicotinamide nucleotide transhydrogenase, had no effect on proton leak kinetics in mitochondria from liver, kidney or muscle, showing that basal proton conductance is not catalyzed by all membrane proteins. We identify UCP1 as a second protein propagating basal proton leak, lending support to the hypothesis that basal leak pathways are perpetrated by members of the mitochondrial anion carrier family but not by other mitochondrial inner membrane proteins.

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“…This has been performed previously with warm-and cold-acclimated animals because the brown fat mitochondria of the two appear only to differ in their UCP1 content (18,27,28). The recombinant expression of UCP1 in yeasts allows not only the comparison of mitochondria incorporating UCP1 with appropriate negative controls but also the use of mutants to further characterize transport functions.…”

Section: Resultsmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

The Uncoupling Protein UCP1 Does Not Increase the Proton Conductance of the Inner Mitochondrial Membrane by Functioning as a Fatty Acid Anion Transporter

González-Barroso¹,

Fleury²,

Bouillaud³

et al. 1998

Journal of Biological Chemistry

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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...

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The Acute Regulation of Mitochondrial Proton Conductance in Cells and Mitochondria from the Brown Fat of Cold‐Adapted and Warm‐Adapted Guinea Pigs

Cited by 83 publications

References 37 publications

Stable expression of functional mitochondrial uncoupling protein in Chinese hamster ovary cells.

Stable expression of functional mitochondrial uncoupling protein in Chinese hamster ovary cells.

Uncoupling protein-1 (UCP1) contributes to the basal proton conductance of brown adipose tissue mitochondria

The Uncoupling Protein UCP1 Does Not Increase the Proton Conductance of the Inner Mitochondrial Membrane by Functioning as a Fatty Acid Anion Transporter

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