β-barrel integral membrane proteins are present in the outer membranes of Gram-negative bacteria and of eukaryotic organelles such as mitochondria and chloroplasts. These proteins consists of amphipatic, antiparallel β strands that form cylindrical barrel-like structure. In bacteria, β-barrel membrane proteins perform a variety of functions, e.g. they are metabolite and protein transporters, receptors and enzymes. In the case of mitochondria the number of the identified β-barrel membrane proteins is distinctly lower and they are shown to be crucial to mitochondria biogenesis as well as morphology and distribution. Some of the known representatives of mitochondrial β-barrel membrane proteins are channel forming proteins. These are isoform of VDAC (voltage dependent anion selective channel), responsible for the solutes passage and Tom40 and Tob55 that form the protein-conducting channel of the protein import complexes, namely TOM complex (translocase of the mitochondrial outer membrane) and TOB complex (topogenesis of the mitochondrial outer membrane β-barrel), respectively.
The presence of β-barrel membrane proteins in mitochondria probably reflects the evolutionary origin of mitochondria from α-proteobacterium. However, homology to bacterial proteins on the basis of amino acid sequence has been observed only for Tob55 and the primary sequences of β-barrel is highly divergent. The amoeba Acathamoeba castellanii is a free-living nonphotosynthetic soil protozoan, whose mitochondria share many bioenergetics properties with mitochondria of plant, animal and fungi. Moreover, on the basis of ribosomal RNA analysis, A. castellanii is located in the molecular phylogenetic tree on a branch basal to the divergence points of the above kingdoms. Thus, β-barrel membrane proteins from A. castellanii mitochondria could be a very interesting subject of evolutionary analysis. This in turn requires an effective methods of these protein isolation and fractionation.
Our present studies concentrate on fractionation and purification of β-barrel membrane proteins of the outer membrane of A. castellanii mitochondria that display channel activities. Ion-exchange chromatography of Triton-X114 solubilised the chosen proteins allowed to obtain several fractions with distinct electrophysiological characteristics. At low salt concentration (25mM and 50 mM KCL) the prevailing protein in the studied fractions was VDAC. At higher salt concentration (100mM KCl) amount of VDAC considerably decreased but the amount of Tom40 increased significantly The fraction obtained in the presence of 500mM KCl contained two types of electrophysiological activity that refer to Tom40 and Tob55, respectively. At higher concentrations of KCl (1M) no channel activity was found. The electrophysiological characteristics of the obtained fractions constitute a starting point for sequencing analysis that should confer new data to functional and evolutionary studies of A. castellanii mitochondria.

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1. In Saccharomyces cerevisiae mitochondria the reduction/oxidation state is mediated by VDAC proteins.