Expression and regulation of mitochondrial uncoupling protein 1 from brown adipose tissue in Leishmania major promastigotes.
Rat uncoupling protein 1 (UCP1) was successfully translated in transfected Leishmania major promastigotes. Immune electron microscopy revealed that the protein was exclusively in the mitochondria. UCP1 expression was about 350,000 copies per promastigote, accounting for 4.7% of the total mitochondrial protein. In intact parasites, expression of UCP1 induced a slight increase in respiratory rate and a modest decrease in mitochondrial membrane potential (delta psi(m)). In contrast, in digitonin-permeabilized parasites, a significantly lower value both in delta psi(m) (57 +/- 10 vs 153 +/- 12 mV) and respiratory control ratio (0.99 vs 1.54) were observed for UCP1 versus control parasites, although when UCP1 activity was inhibited by bovine serum albumin (BSA) and GDP, control values were restored. Therefore, a fully functional UCP1 was present and only partially inhibited in vivo by endogenous purine nucleotides. However, neither ATP levels, growth rate nor mitochondrial protein import differed significantly between both types of parasites. Expression of the pore-like mutant UCP1 delta 9 was deleterious to the organism. Consequently, Leishmania was capable of expressing and importing into mitochondria proteins from higher eukaryotes lacking an N-terminal targeting pre-sequence as UCP1. As described previously, parasite metabolism had only a limited tolerance to mitochondrial disfunction. Transfection of Leishmania with foreign proteins which play an important regulatory role in metabolism is a useful tool to study both parasite metabolism in general, and alternative pathways involved in maintaining internal homeostasis.
Alvarez-Fortes
E
E
Centro de Investigaciones Biológicas CSIC, Madrid, Spain.
Ruiz-Pérez
L M
LM
Bouillaud
F
F
Rial
E
E
Rivas
L
L
D002001:Adipose Tissue, Brown / Q000737:chemistry; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D018320:Leishmania major / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins; D010101:Oxygen Consumption; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D000071256:Uncoupling Protein 1
D002352:Carrier Proteins; D007473:Ion Channels; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; C000604373:Ucp1 protein, rat; D000071256:Uncoupling Protein 1; D006153:Guanosine Diphosphate; D007521:Isocitrate Dehydrogenase