EXPRESSION AND REGULATION OF MITOCHONDRIAL UNCOUPLING PROTEIN-1 FROM BROWN ADIPOSE-TISSUE IN LEISHMANIA-MAJOR PROMASTIGOTES

Rat uncoupling protein I (UCP1) was successfully translated in transfe cted Leishmania major promastigotes. Immune electron microscopy. revea led that the protein was exclusively in the mitochondria. UCP1 express ion was about 350000 copies per promastigote, accounting for 4.7% of t he total mitochondrial protein. In intact parasites, expression of UCP 1 induced a slight increase in respiratory rate and a modest decrease in mitochondrial membrane potential (Delta Psi(m)). In contrast, in di gitonin-permeabilized parasites, a significantly lower value both in D elta Psi(m), (57 +/- 10 vs 153 +/- 12 mV) and respiratory control rati o (0.99 vs 1.54) were observed for UCP1 versus control parasites, alth ough when UCP1 activity was inhibited by bovine serum albumin (BSA) an d GDP, control values were restored. Therefore, a fully functional UCP 1 was present and only partially inhibited in vivo by endogenous purin e nucleotides. However, neither ATP levels, growth rate nor mitochondr ial protein import differed significantly between both types of parasi tes. Expression of the pore-like mutant UCP1 Delta 9 was deleterious t o the organism. Consequently, Leishmania was capable of expressing and importing into mitochondria proteins from higher eukaryotes lacking a n N-terminal targeting pre-sequence as UCP1. As described previously, parasite metabolism had only a limited tolerance to mitochondrial disf unction. Transfection of Leishmania with foreign proteins which play a n important regulatory role in metabolism is a useful tool to study bo th parasite metabolism in general, and alternative pathways involved i n maintaining internal homeostasis. (C) 1998 Elsevier Science B.V. All rights reserved.