Olgierd Stobienia

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 (Δ sod1 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. cerevisiae Δ sod1 cells.

Phylogenetic Analysis of Mitochondrial Outer Membrane β-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 β-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 β-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 β-channels do not entirely reflect the recent eukaryotic classification system involving the six supergroups: Chromalveolata, Excavata, Archaeplastida, Rhizaria, Amoebozoa, and Opisthokonta.

Under Conditions of Insufficient Permeability of VDAC1, External NADH May Use the TOM Complex Channel to Cross the Outer Membrane of Saccharomyces cerevisiae Mitochondria

Thus far, only three channel-forming activities have been identified in the outer membrane of the yeast Saccharomyces cerevisiae mitochondria. Two of them, namely the TOM complex channel (translocase of the outer membrane) and the PSC (peptide-sensitive channel) participate in protein translocation and are probably identical, whereas a channel-forming protein called VDAC (voltage-dependent anion channel) serves as the major pathway for metabolites. The VDAC is present in two isoforms (VDAC1 and VDAC2) of which only VDAC1 has been shown to display channel-forming activity. Moreover, the permeability of VDAC1 has been reported to be limited in uncoupled mitochondria of S. cerevisiae. The presented data indicate that in S. cerevisiae-uncoupled mitochondria, external NADH, applied at higher concentrations (above 50 nmoles per 0.1 mg of mitochondrial protein), may use the TOM complex channel, besides VDAC1, to cross the outer membrane. Thus, the permeability of VDAC1 could be a limiting step in transport of external NADH across the outer membrane and might be supplemented by the TOM complex channel.

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.

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.