Identification and characterization of a protozoan uncoupling protein in Acanthamoeba castellanii

An uncoupling protein (UCP) has been identified in mitochondria from Acanthamoeba castellanii, a nonphotosynthetic soil amoeboid protozoon that, in molecular phylogenesis, appears on a branch basal to the divergence points of plants, animals, and fungi. The existence of UCP in A. castellanii (AcUCP) has been revealed using antibodies raised against plant UCP, Its molecular mass (32,000 Da) was similar to those of plant and mammalian UCPs, The activity of AcUCP has been investigated in mitochondria depleted of free fatty acids. Additions of linoleic acid stimulated state 4 respiration and decreased transmembrane electrical potential (hly) in a manner expected from fatty acid cycling-linked H+ reuptake, The half-maximal stimulation by linoleic acid was reached at 8.1 +/- 0.4 mu M. Bovine serum albumin (fatty acid-free), which adsorbs linoleic acid, reversed the respiratory stimulation and correspondingly restored Delta Psi. AcUCP was only weakly inhibited by purine nucleotides like UCP in plants. A single force-flow relationship has been observed for state 4 respiration with increasing concentration of linoleic acid or of an uncoupler and for state 3 respiration with increasing concentration of oligomycin, indicating that linoleic acid has a pure protonophoric effect. The activity of AcUCP in state 3 has been evidenced by ADP/oxygen atom determination. The discovery of AcUCP indicates that UCPs emerged, as specialized proteins for H+ cycling, early during phylogenesis before the major radiation of phenotypic diversity in eukaryotes and could occur in the whole eukaryotic world.