The TOM Complex of Amoebozoans: the Cases of the Amoeba Acanthamoeba castellanii and the Slime Mold Dictyostelium discoideum.

Protein import into mitochondria requires a wide variety of proteins, forming complexes in both mitochondrial membranes. The TOM complex (translocase of the outer membrane) is responsible for decoding of targeting signals, translocation of imported proteins across or into the outer membrane, and their subsequent sorting. Thus the TOM complex is regarded as the main gate into mitochondria for imported proteins. Available data indicate that mitochondria of representative organisms from across the major phylogenetic lineages of eukaryotes differ in subunit organization of the TOM complex. The subunit organization of the TOM complex in the Amoebozoa is still elusive, so we decided to investigate its organization in the soil amoeba Acanthamoeba castellanii and the slime mold Dictyostelium discoideum. They represent two major subclades of the Amoebozoa: the Lobosa and Conosa, respectively. Our results confirm the presence of Tom70, Tom40 and Tom7 in the A. castellanii and D. discoideum TOM complex, while the presence of Tom22 and Tom20 is less supported. Interestingly, the Tom proteins display the highest similarity to Opisthokonta cognate proteins, with the exception of Tom40. Thus representatives of two major subclades of the Amoebozoa appear to be similar in organization of the TOM complex, despite differences in their lifestyle.

Phylogenetic analysis of mitochondrial outer membrane ß-barrel channels.

Transport of molecules across mitochondrial outer membrane is pivotal for a proper function of mitochondria. The transport pathways across the membrane are formed by ion channels that participate in metabolite exchange between mitochondria and cytoplasm (voltage-dependent anion-selective channel, VDAC) as well as in import of proteins encoded by nuclear genes (Tom40 and Sam50/Tob55). VDAC, Tom40, and Sam50/Tob55 are present in all eukaryotic organisms, encoded in the nuclear genome, and have ß-barrel topology. We have compiled data sets of these protein sequences and studied their phylogenetic relationships with a special focus on the position of Amoebozoa. Additionally, we identified these protein-coding genes in Acanthamoeba castellanii and Dictyostelium discoideum to complement our data set and verify the phylogenetic position of these model organisms. Our analysis show that mitochondrial ß-barrel channels from Archaeplastida (plants) and Opisthokonta (animals and fungi) experienced many duplication events that resulted in multiple paralogous isoforms and form well-defined monophyletic clades that match the current model of eukaryotic evolution. However, in representatives of Amoebozoa, Chromalveolata, and Excavata (former Protista), they do not form clearly distinguishable clades, although they locate basally to the plant and algae branches. In most cases, they do not posses paralogs and their sequences appear to have evolved quickly or degenerated. Consequently, the obtained phylogenies of mitochondrial outer membrane ß-channels do not entirely reflect the recent eukaryotic classification system involving the six supergroups: Chromalveolata, Excavata, Archaeplastida, Rhizaria, Amoebozoa, and Opisthokonta.

Communication between mitochondria and nucleus: putative role for VDAC in reduction/oxidation mechanism.

Voltage dependent anion channel (VDAC) was identified in 1976 and since that time has been extensively studied. It is well known that VDAC transports metabolites across the outer mitochondrial membrane. The simple transport function is indispensable for proper mitochondria functions and, consequently for cell activity, and makes VDAC crucial for a range of cellular processes including ATP rationing, Ca2+ homeostasis and apoptosis execution. Here, we review recent data obtained for Saccharomyces cerevisiae cells used as a model system concerning the putative role of VDAC in communication between mitochondria and the nucleus. The S. cerevisiae VDAC isoform known as VDAC1 (termed here YVDAC) mediates the cytosol reduction/oxidation (redox) state that contributes to regulation of expression and activity of cellular proteins including proteins that participate in protein import into mitochondria and antioxidant enzymes. Simultaneously, copper-and-zinc-containing superoxide dismutase (CuZnSOD) plays an important role in controlling YVDAC activity and expression levels. Thus, it is proposed that VDAC constitutes an important component of a regulatory mechanism based on the cytosol redox state.

Cu,Zn-superoxide dismutase is necessary for proper function of VDAC in Saccharomyces cerevisiae cells.

Available data suggest that a copper-and zinc-containing dismutase (CuZnSOD) plays a significant role in protecting eukaryotic cells against oxidative modifications which may contribute to cell aging. Here we demonstrated that depletion of CuZnSOD in Saccharomyces cerevisiae cells (Deltasod1 cells) affected distinctly channel activity of VDAC (voltage dependent anion selective channel) and resulted in a moderate reduction in VDAC levels as well as in levels of protein crucial for VDAC import into mitochondria, namely Tob55/Sam50 and Tom40. The observed alterations may result in mitochondriopathy and subsequently in the shortening of the replicative life span observed for S. cerevisiaeDeltasod1 cells.

Response of Acanthamoeba castellanii mitochondria to oxidative stress.

The purpose of this study was to examine the effects of oxidative stress caused by hydroperoxide (H(2)O(2)) in the presence of iron ions (Fe(2+)) on mitochondria of the amoeba Acanthamoeba castellanii. We used isolated mitochondria of A. castellanii and exposed them to four levels of H(2)O(2) concentration: 0.5, 5, 15, and 25 mM. We measured basic energetics of mitochondria: oxygen consumption in phosphorylation state (state 3) and resting state (state 4), respiratory coefficient rates (RC), ADP/O ratios, membrane potential (DeltaPsi(m)), ability to accumulate Ca(2+) , and cytochrome c release. Our results show that the increasing concentrations of H(2)O(2) stimulates respiration in states 3 and 4. The highest concentration of H(2)O(2) caused a 3-fold increase in respiration in state 3 compared to the control. Respiratory coefficients and ADP/O ratios decreased with increasing stress conditions. Membrane potential significantly collapsed with increasing hydroperoxide concentration. The ability to accumulate Ca(2+) also decreased with the increasing stress treatment. The lowest stress treatment (0.5 mM H(2)O(2)) significantly decreased oxygen consumption in state 3 and 4, RC, and membrane potential. The ADP/O ratio decreased significantly under 5 mM H(2)O(2) treatment, while Ca(2+) accumulation rate decreased significantly at 15 mM H(2)O(2). We also observed cytochrome c release under increasing stress conditions. However, this release was not linear. These results indicate that as low as 0.5 mM H(2)O(2) with Fe(2+) damage the basic energetics of mitochondria of the unicellular eukaryotic organism Acanthamoeba castellanii.

Effects of VDAC isoforms on CuZn-superoxide dismutase activity in the intermembrane space of Saccharomyces cerevisiae mitochondria.

Copper and zinc containing superoxide dismutase (CuZnSOD) is located primarily in the cytosol but a small amount of the enzyme has also been identified in the intermembrane space of mitochondria (termed here IMS CuZnSOD). Using Saccharomyces cerevisiae mutants depleted of either isoform of VDAC (voltage-dependent anion-selective channel), we have shown that the activity of IMS CuZnSOD coincides with the presence of a given VDAC isoform and changes in a growth phase dependent way. Moreover, the IMS CuZnSOD activity correlates with the levels of O2*- release from mitochondria and the cytosol redox state. The latter in turn seems to influence the levels of the mitochondrial outer membrane channel protein other than VDAC. Thus, we conclude that in the case of S. cerevisiae both VDAC isoforms influence the IMS CuZnSOD activity and subsequently the expression levels of some mitochondrial proteins.

[The VDAC channel as the mitochondria function regulator]. / Kanal VDAC jako regulator funkcji mitochondriów.

Regulation of mitochondria physiology, indispensable for proper cell activity, requires an efficient exchange of molecules between mitochondria and cytoplasm at the level of the mitochondrial outer membrane. The common pathway for the metabolite exchange between mitochondria and cytoplasm is the VDAC channel (voltage dependent anion channel), known also as mitochondrial porin. The channel was identified for the first time in 1976 and since that time has been extensively studied. It has been recognized that the VDAC channel plays a crucial role in the regulation of metabolic and energetic functions of mitochondria. In this article we review the VDAC channel relevance to ATP rationing, Ca2+ homeostasis, protection against oxidative stress and apoptosis execution.

An inception report on the TOM complex of the Amoeba Acanthamoeba castellanii, a simple model protozoan in mitochondria studies.

It is suggested that in the course of the TOM complex evolution at least two lineages have appeared: the animal-fungal and green plant ones. The latter involves also the TOM complexes of algae and protozoans. The amoeba Acanthamoeba castellanii is a free-living non-photosynthetic soil protozoan, whose mitochondria share many bioenergetic properties with mitochondria of plants, animals and fungi. Here, we report that a protein complex, identified electrophysiologically as the A. castellanii TOM complex, contains a homologue of yeast/animal Tom 70. Further, molecular weight of the complex (about 500 kDa) also points to A. castellanii evolutionary relation with fungi and animal. Thus, the data indicates that the TOM complex of A. castellanii is not a typical example of the protozoan TOM complex.

Modulation of the voltage-dependent anion-selective channel by cytoplasmic proteins from wild type and the channel depleted cells of Saccharomyces cerevisiae.

It is well known that effective exchange of metabolites between mitochondria and the cytoplasm is essential for cell physiology. The key step of the exchange is transport across the mitochondrial outer membrane, which is supported by the voltage-dependent anion-selective channel (VDAC). Therefore, it is clear that the permeability of VDAC must be regulated to adjust its activity to the actual cell needs. VDAC-modulating activities, often referred to as the VDAC modulator, were identified in the intermembrane space of different organism mitochondria but the responsible protein(s) has not been identified as yet. Because the VDAC modulator was reported to act on VDAC of intact mitochondria when added to the cytoplasmic side it has been speculated that a similar modulating activity might be present in the cytoplasm. To check the speculation we used mitochondria of the yeast Saccharomyces cerevisiae as they constitute a perfect model to study VDAC modulation. The mitochondria contain only a single isoform of VDAC and it is possible to obtain viable mutants devoid of the channel (Deltapor1). Moreover, we have recently characterised a VDAC-modulating activity located in the intermembrane space of wild type and Deltapor1 S. cerevisiae mitochondria. Here, we report that the cytoplasm of wild type and Deltapor1 cells of S. cerevisiae contains a VDAC-modulating activity as measured in a reconstituted system and with intact mitochondria. Since quantitative differences were observed between the modulating fractions isolated from wild type and Deltapor1 cells when they were studied with intact wild type mitochondria as well as by protein electrophoresis it might be concluded that VDAC may influence the properties of the involved cytoplasmic proteins. Moreover, the VDAC-modulating activity in the cytoplasm differs distinctly from that reported for the mitochondrial intermembrane space. Nevertheless, both these activities may contribute efficiently to VDAC regulation. Thus, the identification of the proteins is very important.

The key role of the energized state of Saccharomyces cerevisiae mitochondria in modulations of the outer membrane channels by the intermembrane space proteins.

Mitochondria of the yeast Saccharomyces cerevisiae constitute a perfect model to study the outer membrane channel modulation as besides the TOM complex channel they contain only a single isoform of the VDAC channel and it is possible to obtain viable mutants devoid of the channel. Here, we report that the fraction of the intermembrane space isolated from wild type and the VDAC channel-depleted yeast mitochondria, except of the well-known VDAC channel modulator activity, displays also the TOM complex channel modulating activity as measured in the reconstituted system and with intact mitochondria. The important factor influencing the action of both modulating activities is the energized state of mitochondria. Moreover, the presence of the VDAC channel itself seems to be crucial to properties of the intermembrane space protein(s) able to modulate the outer membrane channels because in the case of intact mitochondria quantitative differences are observed between modulating capabilities of the fractions isolated from wild type and mutant mitochondria.