Functional Characterization of a Drosophila Mitochondrial Uncoupling Protein

Abstract

Sequence alignment of conserved signature motifs predicts the existence of the uncoupling protein 5 (UCP5)/brain mitochondrial carrier protein (BMCP1) homologue in Drosophila melanogaster. Here we demonstrate the functional characterization of the Drosophila melanogaster UCP5 protein (DmUCP5) in the heterologous yeast system, the first insect UCP reported to date. We show that physiological levels of DmUCP5 expression are responsible for an increase in state 4 respiration rates and a decrease in mitochondrial membrane potential. Furthermore, similar to UCP1, UCP2, and UCP3, the uncoupling activity of DmUCP5 is augmented by fatty acids and inhibited by the purine nucleotide GDP. Thus, DmUCP5 shares the mechanisms known to regulate the UCPs characterized to date. A lack of growth inhibition observed in DmUCP5 expressing yeast is consistent with the notion that physiological uncoupling has a minimal effect on cell growth. Finally, semiquantitative RT-PCR analysis shows a distinctive pattern of DmUCP5 expression predominantly localized in the adult head, similar to the expression pattern of its mammalian homologues. The conserved regulation of the expression of this gene from mammals to fruit flies suggests a role for UCP5 in the brain.

Identification and characterization of uncoupling protein 4 in fat body and muscle mitochondria from the cockroach Gromphadorhina cocquereliana

“…(amino acid residues belonging to the same class). Abbreviations, species and GenBank accession numbers are as follows: hUCP4is1, human (H. sapiens) UCP4, NP_004268.3; rUCP4, rat (R. norvegicus) UCP4, NP_445952.1; DmUCP4A, Drosophila melanogaster UCP4, isoform A, NP_573246.1; predicted TcUCP4A, Tribolium castaneum UCP4, isoform A, XP_969929.1; PhcUCP, predicted Pediculus humanus corporis UCP, XP_002429177.1; AedesUCP, Aedes aegypti UCP, EAT41275.1 melanogaster UCP5 expressed in a yeast system (Fridell et al 2004) indicate that insect UCPs conserve this regulatory mechanism similar to mammals, plants and unicellular organisms (Fridell et al 2004;Vercesi et al 2006;Echtay 2007;Jarmuszkiewicz et al 2010). In phosphorylating cockroach mitochondria, PA-induced GTP-sensitive GcUCP activity decreased the ADP/O ratio and, thus, diverted energy from oxidative phosphorylation (Table 1).…”

“…As the first insect UCP, D. melanogaster UCP5 (DmUCP5) was functionally characterized in the heterologous yeast system where its expression reduced mitochondrial membrane potential and increased the respiration rate (Fridell et al 2004). Similar to other UCPs, the uncoupling activity of DmUCP5 is augmented by fatty acids and inhibited by purine nucleotides.…”

“…The functional characterization of D. melanogaster UCP in the heterologous yeast system (Fridell et al 2004) and the loss-of-function investigations using UCP5 knockout flies (Sanchez-Blanco et al 2006) are the only studies on function and the metabolic importance of UCPs in insects. Thus, the aim of the present study was to identify molecularly and to characterize functionally Gromphadprhina coquereliana UCP (GcUCP).…”

Involvement of Drosophila Uncoupling Protein 5 in Metabolism and Aging

“…Additionally, homozygous control lines were established from individual males resulting from the Bmcp BG02446 /TM6 3 TM3/TM6B cross. To create transgenic ucp5 lines, the full-length ucp5 cDNA fragment fused at the 39-end to the FLAG epitope tag (FT) was subcloned from the pRS426 vector (Fridell et al 2004) into the pUAST transformation vector. The pUAST-UCP5TFT construct was injected into the germline of w; D 2,3 /TM3 flies.…”

“…Drosophila UCP5 uncoupling activity has been functionally characterized in the heterologous yeast system, where UCP5 expression reduces mitochondrial membrane potential and increases respiration rate. UCP5 action is governed by the mechanisms known to regulate the UCPs characterized to date, including fatty acid stimulation and GDP inhibition (Fridell et al 2004). ucp5 is expressed throughout Drosophila development but at higher levels in adults, where it is expressed most abundantly in the head (Fridell et al 2004).…”