Svyatoslav S. Sokolov

Unexpected link between anaphase promoting complex and the toxicity of expanded polyglutamines expressed in yeast

Protein aggregation is intimately linked to a number of neurodegenerative diseases. Expansion of the huntingtin polyglutamine-rich domain causes protein aggregation and neuronal degeneration. Recently we found that, similar to neurons, yeast expressing the expanded domain show markers of programmed cell death. Here we showed that deletion of yeast metacaspase gene YCA1 partly rescues the toxic effect of the domain overexpression. We also performed genetic screen for other genes deletions alleviating the toxic effect and found ASE1. Ase1 is a substrate of the Cdh1 form of anaphase promoting complex, APC/Cdh1. We tested Cdh1 overexpression and the deletion of CLB2 (mitotic cyclin, substrate of APC/Cdh1) and found that both mutations had a rescuing effect on the expanded polyglutamine toxicity. Our data suggest that the toxic effect of aggregated proteins is partly indirect. We speculate that cellular attempt to degrade the aggregates overloads the proteasome, and this leads to pathological accumulation of APC substrates.

Protein Aggregation and Neurodegeneration: Clues from a Yeast Model of Huntington's Disease

A number of neurodegenerative diseases are accompanied by the appearance of intracellular protein aggregates. Huntington’s disease (HD) is caused by a mutation in a gene encoding huntingtin. The mutation causes the expansion of the polyglutamine (polyQ) domain and consequently polyQ-containing aggregates accumulate and neurons in the striatum die. The role of the aggregates is still not clear: they may be the cause of cytotoxicity or a manifestation of the cellular attempt to remove the misfolded proteins. There is accumulating evidence that the main cause of HD is the interaction of the mutated huntingtin with other polyQ-containing proteins and molecular chaperones and most studies based on a yeast model of HD support this point of view. Data obtained using yeasts suggest pathological consequences of polyQ-proteasomal interaction: proteasomal overload by polyQs may interfere with functions of the cell cycle-regulating proteins.

Mitochondrial signaling in Saccharomyces cerevisiae pseudohyphae formation induced by butanol.

Yeasts growing limited for nitrogen source or treated with fusel alcohols form elongated cells--pseudohyphae. Absence of mitochondrial DNA or anaerobic conditions inhibits this process, but the precise role of mitochondria is not clear. We found that a significant percentage of pseudohyphal cells contained mitochondria with different levels of membrane potential within one cell. An uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), but not the ATP-synthase inhibitor oligomycin D, prevented pseudohyphal growth. Interestingly, repression of the MIH1 gene encoding phosphatase activator of the G2/M transition partially restores the ability of yeast to form pseudohyphal cells in the presence of FCCP or in the absence of mitochondrial DNA. At the same time, retrograde signaling (the one triggered by dysfunctional mitochondria) appeared to be a positive regulator of butanol-induced pseudohyphae formation: the deletion of any of the retrograde signaling genes (RTG1, RTG2, or RTG3) partially suppressed pseudohyphal growth. Together, our data suggest that two subpopulations of mitochondria are required for filamentous growth: one with high and another with low transmembrane potential. These mitochondria-activated signaling pathways appear to converge at Mih1p level.

Roles of Mitochondrial Dynamics under Stressful and Normal Conditions in Yeast Cells

Eukaryotic cells contain dynamic mitochondrial filaments: they fuse and divide. Here we summarize data on the protein machinery driving mitochondrial dynamics in yeast and also discuss the factors that affect the fusion-fission balance. Fission is a general stress response of cells, and in the case of yeast this response appears to be prosurvival. At the same time, even under normal conditions yeast mitochondria undergo continuous cycles of fusion and fission. This seems to be a futile cycle and also expensive from the energy point of view. Why does it exist? Benefits might be the same as in the case of sexual reproduction. Indeed, mixing and separating of mitochondrial content allows mitochondrial DNA to segregate and recombine randomly, leading to high variation in the numbers of mutations per individual mitochondrion. This opens a possibility for effective purifying selection-elimination of mitochondria highly contaminated by deleterious mutations. The beneficial action presumes a mechanism for removal of defective mitochondria. We argue that selective mitochondrial autophagy or asymmetrical distribution of mitochondria during cell division could be at the core of such mechanism.

Mitochondrial matrix fragmentation as a protection mechanism of yeast Saccharomyces cerevisiae

It was shown that separate fragments of the inner mitochondrial compartment (mitoplasts) can exist under a single non-fragmented outer membrane. Here we asked whether fragmentation of the inner mitochondria could prevent rupturing of the outer membrane and release of pro-apoptotic molecules from the mitochondrial intermembrane space into the cytoplasm during mitochondrial swelling. First, we showed that in Saccharomyces cerevisiae yeast addition of amiodarone causes formation of electrically separate compartments within mitochondrial filaments. Moreover, amiodarone treatment of Δysp2 mutant produced a higher proportion of cells with electrically discontinuous mitochondria than in the wild type, which correlated with the survival of cells. We confirmed the existence of separated mitoplasts under a single outer membrane using electron microscopy. Mitochondria with fragmented matrixes were also detected in cells of the stationary phase. Our data suggest that such fragmentation acts as a cellular protective mechanism against stress.

How do yeast sense mitochondrial dysfunction

Apart from energy transformation, mitochondria play important signaling roles. In yeast, mitochondrial signaling relies on several molecular cascades. However, it is not clear how a cell detects a particular mitochondrial malfunction. The problem is that there are many possible manifestations of mitochondrial dysfunction. For example, exposure to the specific antibiotics can either decrease (inhibitors of respiratory chain) or increase (inhibitors of ATP-synthase) mitochondrial transmembrane potential. Moreover, even in the absence of the dysfunctions, a cell needs feedback from mitochondria to coordinate mitochondrial biogenesis and/or removal by mitophagy during the division cycle. To cope with the complexity, only a limited set of compounds is monitored by yeast cells to estimate mitochondrial functionality. The known examples of such compounds are ATP, reactive oxygen species, intermediates of amino acids synthesis, short peptides, Fe-S clusters and heme, and also the precursor proteins which fail to be imported by mitochondria. On one hand, the levels of these molecules depend not only on mitochondria. On the other hand, these substances are recognized by the cytosolic sensors which transmit the signals to the nucleus leading to general, as opposed to mitochondria-specific, transcriptional response. Therefore, we argue that both ways of mitochondria-to-nucleus communication in yeast are mostly (if not completely) unspecific, are mediated by the cytosolic signaling machinery and strongly depend on cellular metabolic state.

Negative feedback of glycolysis and oxidative phosphorylation: Mechanisms of and reasons for it

There are two main pathways of ATP biosynthesis: glycolysis and oxidative phosphorylation. As a rule, the two pathways are not fully active in a single cell. In this review, we discuss mechanisms of glycolytic inhibition of respiration (Warburg and Crabtree effects). What are the reasons for the existence of this negative feedback? It is known that maximal activation of both processes can cause generation of reactive oxygen species. Oxidative phosphorylation is more efficient from the energy point of view, while glycolysis is safer and favors biomass synthesis. This might be the reason why quiescent cells are mainly using oxidative phosphorylation, while the quickly proliferating ones — glycolysis.

Inactivation of Pmc1 vacuolar Ca(2+) ATPase causes G(2) cell cycle delay in Hansenula polymorpha.

The vacuolar Ca2+ ATPase Pmc1 is involved in maintenance of a low Ca2+ concentration in cytosol in yeast cells. Here we observed that increase of Ca2+ cytosolic concentration in yeast Hansenula polymorpha due to inactivation of Pmc1 resulted in sensitivity to sodium dodecyl sulfate (SDS). To elucidate the mechanisms of the observed effect, a screening for mutations suppressing SDS sensitivity of the H. polymorpha pmc1 mutant was performed. As a result, three genes were identified. Two of them, designated as their Saccharomyces cerevisiae orthologs CCH1 and HOG1 encoded the plasma membrane voltage-gated high-affinity calcium channel and the MAP kinase involved in osmoregulation, respectively. The third gene, designated as WEE1, coded for the ortholog of Wee1/Swe1 kinase involved in cell cycle regulation by inhibiting of the G2/M transition. Detailed analysis of this mutant demonstrated that suppression of pmc1 SDS sensitivity by the wee1 mutation depended on an accompanying chromosomal rearrangement, whereas inactivation of WEE1 in the absence of this rearrangement caused SDS sensitivity. Expression of a chimeric protein containing an N-terminal portion of Wee1 in the pmc1 mutant led to abnormal morphology characteristic of G2 delay. Our data indicate that cytosolic Ca2+ rise causes SDS sensitivity in H. polymorpha through the activation of the Wee1 kinase, which is mediated by the Hog1 kinase. Wee1 has a dual role in the manifestation of SDS sensitivity in the H. polymorpha pmc1 mutant. Mechanisms of influence of the obtained mutations on the G2/M transition are discussed.

Physiological scenarios of programmed loss of mitochondrial DNA function and death of yeast

Recently it was convincingly shown that the yeast Saccharomyces cerevisiae does possess the basic modules of programmed cell death machinery. As programmed cell death is suicide for a unicellular organism, it is reasonable to assume that they trigger the program when the death is beneficial for the rest of the population. Not surprisingly, most of the scenarios of physiological death of S. cerevisiae, i.e. cell death in stationary culture, during meiosis, during mating, and driven by viruses are dependent on quorum sensing, meaning that they depend on the cell density. Here we also discuss possible mechanisms that govern fitness decline during replicative aging of S. cerevisiae cells. We argue that loss of mitochondrial DNA function that occurs during replicative aging is programmed and adaptive. Indeed, yeast cells with nonfunctional mitochondrial DNA are known to be extremely stress-resistant, and also the presence of a subpopulation of such cells might protect the culture from degeneration by preventing the fixation of opportunistic mutations.

Dodecyltriphenylphosphonium inhibits multiple drug resistance in the yeast Saccharomyces cerevisiae.

Multiple drug resistance pumps are potential drug targets. Here we asked whether the lipophilic cation dodecyltriphenylphosphonium (C12TPP) can interfere with their functioning. First, we found that suppression of ABC transporter gene PDR5 increases the toxicity of C12TPP in yeast. Second, C12TPP appeared to prevent the efflux of rhodamine 6G - a fluorescent substrate of Pdr5p. Moreover, C12TPP increased the cytostatic effects of some other known Pdr5p substrates. The chemical nature of C12TPP suggests that after Pdr5p-driven extrusion the molecules return to the plasma membrane and then into the cytosol, thus effectively competing with other substrates of the pump.