Caenorhabditis elegans UCP4 protein controls complex II-mediated oxidative phosphorylation through succinate transport

Abstract

The novel uncoupling proteins (UCP2-5) are implicated in the mitochondrial control of oxidant production, insulin signaling, and aging. Attempts to understand their functions have been complicated by overlapping expression patterns in most organisms. Caenorhabditis elegans nematodes are unique because they express only one UCP ortholog, ceUCP4 (ucp4). Here, we performed detailed metabolic analyzes in genetically modified nematodes to define the function of the ceUCP4. The knock-out mutant ucp4 (ok195) exhibited sharply decreased mitochondrial succinate-driven (complex II) respiration. However, respiratory coupling and electron transport chain function were normal in ucp4 mitochondria. Surprisingly, isolated ucp4 mitochondria showed markedly decreased succinate uptake. Similarly, ceUCP4 inhibition blocked succinate respiration and import in wild type mitochondria. Genetic and pharmacologic inhibition of complex I function was selectively lethal to ucp4 worms, arguing that ceUCP4-regulated succinate transport is required for optimal complex II function in vivo. Additionally, ceUCP4 deficiency prolonged lifespan in the short-lived mev1 mutant that exhibits complex II-generated oxidant production. These results identify a novel function for ceUCP4 in the regulation of complex II-based metabolism through an unexpected mechanism involving succinate transport.

FIGURE 1.

ucp4 animals show hypometabolic phenotypes. A, respiration rates (mean ± S.E.) in age synchronized (L1) wild type (N2), ucp4, and N2 worms after ucp4 RNAi knockdown (n = 5) are shown. * indicates p < 0.05. B, shown is lipid granule staining (relative nile red fluorescence units (RFU)) of age synchronized, adult (L4) N2, ucp4, and N2 worms subjected to ucp4 RNAi (n = 4–6). * indicates p < 0.01. C, triglyceride (Tg) quantification (mean ± S.E.) in N2 and ucp4 worm homogenates (n = 3) is shown. * indicates p < 0.01. D, fatty acid methyl ester quantification (mean ± S.E.) in N2 and ucp4 worm homogenates (n = 3) is shown. *, **, and *** indicate significantly different (p < 0.05, 0.01, and 0.001, respectively) from N2 animals.

FIGURE 2.

ucp4 animals have a selective failure of complex II-mediated mitochondrial respiration. A–D, shown are maximal rates of state 3, state 4, and GTP-sensitive respiration (mean ± S.E.) in isolated mitochondria respiring on the following complex-specific substrates: malate for complex I (A), succinate for complex II (B), durohydroquinone for complex III (C), and tetramethylphenylenediamine/ascorbate for complex IV (D) (n = 3–6). * indicates significantly different (p < 0.01) from N2. E and F, comparisons of maximal state 3 respiration rates mediated by complex I (malate) (E) and complex II (succinate) (F) in isolated mitochondria from N2, ucp4, mev1 (complex II mutant), and gas11 (complex I mutant) worms (n = 3–6). * indicates significantly different (p < 0.05) from N2 mitochondria. G, palmitate-stimulated state 4 respiration in isolated mitochondria from N2 and ucp4 worms (n = 3) is shown. No significant (NS) differences were observed between genotypes.

FIGURE 3.

ceUCP4 regulates mitochondrial succinate uptake. A, shown is [¹⁴C]succinate and [¹⁴C]malate uptake (mean ± S.E.) in isolated mitochondria from N2 and ucp4 worms (n = 5–7). * indicates significantly different (p < 0.01) from N2. B, [¹⁴C]succinate uptake in isolated mitochondria from N2 and ucp4 worms alone (No Tx) or in the presence of the dicarboxylate carrier inhibitor BtM, the UCP inhibitor guanosine nucleotide (GDP), or both (BtM + GDP) is shown. * and ** indicate significantly different (p < 0.01 and p < 0.001, respectively) from the untreated N2 mitochondria. C, shown is the effect of preaddition of GDP on succinate-induced respiration in N2 worms (mean ± S.E., n = 3). D, [¹⁴C]succinate uptake in isolated mitochondria from N2 and ucp4 worms (n = 3–4) alone (No Tx) or in the presence of the mitochondrial uncoupler FCCP, the UCP inhibitor GDP, both (FCCP + GDP), or vehicle (DMSO) (n = 3) is shown. * indicates significantly different (p < 0.01) from untreated N2 mitochondria.

FIGURE 4.

ceUCP4 energizes complex II and shortens mev1 lifespan and fecundity and is required for survival in the absence of complex I function. A, shown is the percent survival (mean ± S.E.) in N2 and ucp4 L2 worms treated with the complex I inhibitor rotenone (48 h) (n = 3). * indicates significantly different (p < 0.001) from N2. B, shown are lifespan analyses in N2, ucp4, mev1 mutants along with ucp4; mev1 double mutant worms. C, no. of hatched eggs per adult in N2, ucp4, and mev1 mutants along with ucp4; mev1 double mutant worms (mean ± S.E., n = 3) is shown. * indicates significantly different from N2, p < 0.001. ** indicates significantly different from mev1, p < 0.01.

FIGURE 5.

Model of UCP4 function. ceUCP4-null mitochondria exhibit a striking decrease in complex II-driven OXPHOS without any other defects in other respiratory chain components, and these defects disappear when mitochondria are permeabilized, suggesting that UCP4 may regulate complex II by regulating mitochondrial succinate import. The main proposed mediator of dicarboxylate (succinate and malate) transport in mitochondria is via the BtM-inhibited DIC. Although malate respiration and transport are not regulated by UCP4 (Figs. 1A and 2A, respectively), maximal rates of succinate respiration and uptake into mitochondria are strongly decreased in the absence of ucp4 (Figs. 1B and 2B, respectively). Moreover, the UCP inhibitor GDP significantly inhibits succinate influx into N2 mitochondria (Fig. 3A). Importantly, intact ucp4 nematodes are far less sensitive compared with wild type to loss of complex I function, which argues that UCP4-mediated succinate uptake from the cytoplasm is required for optimal complex II function in vivo. cyt c, cytochrome c.