A significant portion of mitochondrial proton leak in intact thymocytes depends on expression of UCP2 - PubMed (original) (raw)

A significant portion of mitochondrial proton leak in intact thymocytes depends on expression of UCP2

Stefan Krauss et al. Proc Natl Acad Sci U S A. 2002.

Abstract

The uncoupling protein homologue UCP2 is expressed in a variety of mammalian cells. It is thought to be an uncoupler of oxidative phosphorylation. Uncoupling proteins previously have been shown to be capable of translocating protons across phospholipid bilayers in proteoliposome systems. Furthermore, studies in mitochondria from yeast overexpressing the proteins have led to suggestions that they may act as uncouplers in cells. However, this issue is controversial, and to date, definitive experimental evidence is lacking as to whether UCP2 mediates part or all of the basal mitochondrial proton leak in mammalian cells in situ. In the present study, by using thymocytes isolated from UCP2-deficient and wild-type (WT) mice, we addressed the question whether UCP2 is directly involved in catalyzing proton leak in intact cells. Over a range of mitochondrial membrane potentials (DeltaPsi(m)), proton leak activity was lower in thymocytes from UCP2-deficient mice compared with WT mice. At physiological levels of DeltaPsi(m), a significant portion (50%) of basal proton leak in resting cells depended on UCP2. Of note, proton leak in whole cells from WT mice, but not UCP2-deficient mice, responded to stimulation by 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)-1-propenyl]benzoic acid (TTNPB), a known activator of UCP2 activity. Consistent with the observed changes in proton leak, DeltaPsi(m) and ATP levels were increased in untreated thymocytes from UCP2-deficient mice. Interestingly, resting respiration was unaltered, suggesting that UCP2 function in resting cells may be concerned with the control of ATP production rather than substrate oxidation. This study establishes that UCP2, expressed at endogenous levels, mediates proton leak in intact cells.

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Figures

Figure 1

Figure 1

(a) RNA was extracted from thymocytes, and Northern blot analysis was performed with a full-length rat Ucp2 cDNA hybridization probe. (b) Mitochondria were isolated from thymus and immunoblotted for UCP2 protein.

Figure 2

Figure 2

FACS analysis of thymocytes from (a) WT and (b) UCP2-deficient mice. (c) Quantitative assessment of CD4/CD8 distributions shown in a and_b_.

Figure 3

Figure 3

Proton leak kinetics in thymocytes from WT and UCP2-deficient mice. Titrations were performed as described in Materials and Methods. State 4 respiration (i.e., the cells do not synthesize ATP) was induced by incubation with 160 ng/ml oligomycin (top right-hand points of each curve). ΔΨm was altered by titrations with different concentrations of myxothiazol (40, 80, and 480 nM). Data are means ± SEM (n = 6 and_n_ = 5 cell preparations for WT and UCP2-deficient thymocytes, respectively). At steady state, the number of protons pumped by the complexes of the electron-transport chain equals the number of protons that leak back into the mitochondrial matrix. Because proton pumping and oxygen consumption are stoichiometrically linked, the oxygen consumption rates at any given potential reflect proton leak across the inner mitochondrial membrane.

Figure 4

Figure 4

Comparison of ΔΨm, total cell ATP content, and oxygen consumption in resting thymocytes. ATP data are means ± SEM for 9- and 13-cell preparations from WT and UCP2-deficient mice, respectively. Respiration and ΔΨm data are means ± SEM for 7- and 5-cell preparations from WT and UCP2-deficient mice, respectively. *, P < 0.05.

Figure 5

Figure 5

Effect of TTNPB on state 4 (i.e., nonphosphorylating) respiration in intact thymocytes. Thymocytes (at ≈7 × 107 cells per ml) were incubated with 160 ng/ml oligomycin, and respiration was measured. TTNPB was added in 0.5 μM increments, and respiration was measured after steady state was established.

Figure 6

Figure 6

Model of UCP2 function in resting thymocytes (see_Discussion_ for details). Protons are pumped into the mitochondrial intermembrane space by complexes of the electron transport chain during oxidation of substrates. UCP2 mediates a significant portion of mitochondrial proton leak in resting thymocytes, thus providing one of three possible routes for protons to re-enter the matrix (the other two being ATP synthase and UCP2-independent leak). UCP2 deficiency causes an increase in ΔΨm. This increase may either put backpressure on the proton pumps in the electron-transport chain (1), thus inhibiting substrate oxidation and oxygen consumption rates, or increase ATP production (2).

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