Search

International audience; The distribution of the pro-apoptotic protein Bax in the outer mi-tochondrial membrane (OMM) is a central point of regulation of apoptosis. It is now widely recognized that parts of the endoplasmic reticulum (ER) are closely associated to the OMM, and are actively involved in different signaling processes. We addressed a possible role of these domains, called Mitochon-dria-Associated Membranes (MAMs) in Bax localization and function, by ex-pressing the human protein in a yeast mutant deleted of MDM34, a ERMES (ER-Mitochondria Encounter Structure) component. By affecting MAMs stabil-ity, the deletion of MDM34 altered Bax mitochondrial localization, and de-creased its capacity to release cytochrome c. Furthermore, the deletion of MDM34 decreased the size of an incompletely released, MAMs-associated pool of cytochrome c.

International audience; BCL-2 family members are major regulators of apoptotic cell death in mammals. They form an intricate regulatory network that ultimately regulates the release of apoptogenic factors from mitochondria to the cytosol. The ectopic expression of mammalian BCL-2 family members in the yeast Saccharomyces cerevisiae, which lacks BCL-2 homologs, has been long established as a useful addition to the available models to study their function and regulation. In yeast, individual proteins can be studied independently from the whole interaction network, thus providing insight into the molecular mechanisms underlying their function in a living context. Furthermore, one can take advantage of the powerful tools available in yeast to probe intracellular trafficking processes such as mitochondrial sorting and interactions/exchanges between mitochondria and other compartments, such as the endoplasmic reticulum that are largely conserved between yeast and mammals. Yeast molecular genetics thus allows the investigation of the role of these processes on the dynamic equilibrium of BCL-2 family members between mitochondria and extramitochondrial compartments. Here we propose a model of dynamic regulation of BCL-2 family member localization, based on available evidence from ectopic expression in yeast.

"Available online 07 January 2021", Lactoferrin (Lf) is a bioactive milk-derived protein with remarkable wide-spectrum antifungal activity. To deepen our understanding of the molecular mechanisms underlying Lf cytotoxicity, the role of plasma membrane ergosterol- and sphingolipid-rich lipid rafts and their association with the proton pump Pma1p was explored. Pma1p was previously identified as a Lf-binding protein. Results showed that bovine Lf (bLf) perturbs sterol-rich lipid rafts organization by inducing intracellular accumulation of ergosterol. Using yeast mutant strains lacking lipid rafts-associated proteins or enzymes involved in the synthesis of ergosterol and sphingolipids, we found that perturbations in the composition of these membrane domains increase resistance to bLf-induced yeast cell death. Also, when Pma1p-lipid rafts association is compromised in the Pma110 mutant and in the absence of the Pma1p-binding protein Ast1p, the bLf killing activity is impaired. Altogether, results showed that the perturbation of lipid rafts and the inhibition of both Pma1p and V-ATPase activities mediate the antifungal activity of bLf. Since it is suggested that the combination of conventional antifungals with lipid rafts-disrupting compounds is a powerful antifungal approach, our data will help to pave the way for the use of bLf alone or in combination for the treatment/eradication of clinically and agronomically relevant yeast pathogens/fungi., This work was supported by national funds through the Portuguese Foundation for Science and Technology (FCT I.P.) under the scope of the strategic funding of “Contrato-Programa” UIDB/04050/2020 and UIDB/ 04469/2020 unit; by the BioTecNorte operation (NORTE-01-0145- FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte; and by the Servicio para el Control de la Esterilización, Laboratorio de Microbiología Oral (CN-16-036). Cátia Santos-Pereira acknowledges the PhD fellowship PD/BD/128032/2016 funded by FCT under the scope of the doctoral programme in Applied and Environmental Microbiology (DP_AEM)., info:eu-repo/semantics/publishedVersion

Bax is a major player in the apoptotic process, being at the core of the mitochondria permeabilization events. In spite of the major recent advances in the knowledge of Bax organization within the membrane, the precise behavior of the C-terminal helix α9 remains elusive, since it was absent from the resolved structure of active Bax. The Proline 168 (P168) residue, located in the short loop between α8 and α9, has been the target of site-directed mutagenesis experiments, with conflicting results. We have produced and purified a recombinant mutant Bax-P168A, and we have compared its behavior with that of wild-type Bax in a series of tests on Large Unilamellar Vesicles (LUVs) and isolated mitochondria. We conclude that Bax-P168A had a greater ability to oligomerize and bind to membranes. Bax-P168A was not more efficient than wild-type Bax to permeabilize liposomes to small molecules but was more prone to release cytochrome c from mitochondria.

Bcl-2 family members form a network of protein-protein interactions that regulate apoptosis through permeabilization of the mitochondrial outer membrane. Deciphering this intricate network requires streamlined experimental models, including the heterologous expression in yeast. This approach had previously enabled researchers to identify domains and residues that underlie the conformational changes driving the translocation, the insertion and the oligomerization of the pro-apoptotic protein Bax at the level of the mitochondrial outer membrane. Recent studies that combine experiments in yeast and in mammalian cells have shown the unexpected effect of the anti-apoptotic protein Bcl-xL on the priming of Bax. As demonstrated with the BH3-mimetic molecule ABT-737, this property of Bcl-xL, and of Bcl-2, is crucial to elaborate about how apoptosis could be reactivated in tumoral cells.

BFP - Equipe Mollicutes; The heterologous expression of Bax, and other Bcl-2 family members, in the yeast Saccharomyces cerevisiae, has proved to be a valuable reporter system to investigate the molecular mechanisms underlying their interaction with mitochondria. By combining the co-expression of Bax and Bcl-xL mutants with analyzes of their localization and interaction in mitochondria and post-mitochondrial supernatants, we showed that the ability of Bax and Bcl-xL to interact is dependent both on Bax phosphorylation - mimicked by a substitution S184D - and by Bax and Bcl-xL localization. This, and previous data, provide the molecular basis for a model of dynamic equilibrium for Bax localization and activation, regulated both by phosphorylation and Bcl-xL.

Bcl-xL is an oncogene of which the survival functions are finely tuned by post-translational modifications (PTM). Within the Bcl-2 family of proteins, Bcl-xL shows unique eligibility to deamidation, a time-related spontaneous reaction. Deamidation is still a largely overlooked PTM due to a lack of easy techniques to monitor Asn&rarr, Asp/IsoAsp conversions or Glu&rarr, Gln conversions. Being able to detect PTMs is essential to achieve a comprehensive description of all the regulatory mechanisms and functions a protein can carry out. Here, we report a gel composition improving the electrophoretic separation of deamidated forms of Bcl-xL generated either by mutagenesis or by alkaline treatment. Importantly, this new gel formulation proved efficient to provide the long-sought evidence that even doubly-deamidated Bcl-xL remains eligible for regulation by phosphorylation.

In the animal kingdom, proteins of the Bcl-2 family are widely recognized as regulators of mitochondrial outer membrane permeabilization (MOMP), leading to apoptotic cell death. These proteins were recently also shown to control IP3-dependent calcium fluxes at the level of the endoplasmic reticulum (ER). However, the origin and evolution of these pleiotropic functions remain elusive. Here, we molecularly characterized the four members of the Bcl-2 family (trBcl-2L1 to -2L4) in the most primitive metazoan, namely Trichoplax adhaerens. Primary structure and phylogenetic analyses demonstrated that all four trBcl-2 homologs belong to the multidomain Bcl-2 group and presented a conserved C-terminus transmembrane (TM) domain. TrBcl-2L1 and trBcl-2L2 are highly divergent proteins clustering with the anti-apoptotic Bcl-2 members, whereas trBcl-2L3 and trBcl-2L4 were homologous to the pro-apoptotic Bax (trBax) and Bak (trBak). Interestingly, at the functional level, trBak operates as a BH3 only sensitizer repressing the anti-apoptotic activities of trBcl-2L1 and trBcl-2L2, whereas trBax leads to MOMP, similarly to the well-known indirect model of Bax activation. Finally, we found that trBcl-2L1 had a dual ER and mitochondrial subcellular localization and was able to bind to IP3R. By generating two TM domain mutants we demonstrated that trBcl-2L1 targeted to the ER was able to control IP3-dependent calcium fluxes, whereas Mito-trBcl-2L1 represses trBax-dependent MOMP, suggesting that Bcl-2 pleiotropy appeared early and was conserved throughout metazoan evolution.

Mitophagy, the process that degrades mitochondria selectively through autophagy, is involved in the quality control of these organelles. In yeast, the presence of the Atg32 protein on the outer mitochondrial membrane allows for the recognition and targeting of superfluous or damaged mitochondria for degradation. Some posttranslational modifications, such as phosphorylation, are crucial for the execution of the mitophagy process. In our study, we showed that in the stationary phase of growth, and to a lesser extent during starvation, the Atg32 protein level decreases. The fact that a decline in Atg32 level can be prevented by inhibition of the proteolytic activity of proteasome may indicate that Atg32 is also ubiquitylated. In fact, mass spectrometry analysis of purified Atg32 protein showed ubiquitination of lysine residue in position 282. These different patterns of posttranslational modifications of Atg32 could allow cells to control the mitophagy process carefully.

International audience; Among the different models used to study the biochemical properties and function of proteins of the Bcl-2 family, their heterologous expression in the yeast Saccharomyces cerevisiae may look out of place. However, when grown under adequate conditions, yeast cells have mitochondria that have similar properties as those of mammalian cells, and are able to be targeted by mammalian Bcl-2 family members. Yeast thus provides a neutral cellular background to study how proteins of the Bcl-2 family interact with mitochondria, alone or as a couple (or a combination of more proteins). Most studies done in our laboratory has been done on the proapoptotic protein Bax and the antiapoptotic protein Bcl-xL, but yeast can bring about useful information about every protein of the family, in terms of their capacity to interact and to regulate the permeabilization of the outer mitochondrial membrane.

The distribution of the pro-apoptotic protein Bax in the outer mitochondrial membrane (OMM) is a central point of regulation of apoptosis. It is now widely recognized that parts of the endoplasmic reticulum (ER) are closely associated to the OMM, and are actively involved in different signalling processes. We adressed a possible role of these domains, called Mitochondria-Associated Membranes (MAMs) in Bax localization and fonction, by expressing the human protein in a yeast mutant deleted of MDM34, a ERMES component (ER-Mitochondria Encounter Structure). By affecting MAMs stability, the deletion of MDM34 altered Bax mitochondrial localization, and decreased its capacity to release cytochrome c. Furthermore, the deletion of MDM34 decreased the size of an uncompletely released, MAMs-associated pool of cytochrome c.

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cel-lular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the defi-nition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differ-ential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death rou-tines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the au-thors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the pro-gress of this vibrant field of research., info:eu-repo/semantics/published

Bax cytosol-to-mitochondria translocation is a central event of the intrinsic pathway of apoptosis. Bcl-xL is an important regulator of this event and was recently shown to promote the retrotranslocation of mitochondrial Bax to the cytosol. The present study identifies a new aspect of the regulation of Bax localization by Bcl-xL: in addition to its role in Bax inhibition and retrotranslocation, we found that, like with Bcl-2, an increase of Bcl-xL expression levels led to an increase of Bax mitochondrial content. This finding was substantiated both in pro-lymphocytic FL5.12 cells and a yeast reporting system. Bcl-xL-dependent increase of mitochondrial Bax is counterbalanced by retrotranslocation, as we observed that Bcl-xLΔC, which is unable to promote Bax retrotranslocation, was more efficient than the full-length protein in stimulating Bax relocation to mitochondria. Interestingly, cells overexpressing Bcl-xL were more sensitive to apoptosis upon treatment with the BH3-mimetic ABT-737, suggesting that despite its role in Bax inhibition, Bcl-xL also primes mitochondria to permeabilization and cytochrome c release.

The pro-apoptotic Bax protein is the main effector of mitochondrial permeabilization during apoptosis. Bax is controlled at several levels, including post-translational modifications such as phosphorylation and S-palmitoylation. However, little is known about the contribution of other protein modifications to Bax activity. Here, we used heterologous expression of human Bax in yeast to study the involvement of N-terminal acetylation by yNaa20p (yNatB) on Bax function. We found that human Bax is N-terminal (Nt-)acetylated by yNaa20p and that Nt-acetylation of Bax is essential to maintain Bax in an inactive conformation in the cytosol of yeast and Mouse Embryonic Fibroblast (MEF) cells. Bax accumulates in the mitochondria of yeast naa20Δ and Naa25-/- MEF cells, but does not promote cytochrome c release, suggesting that an additional step is required for full activation of Bax. Altogether, our results show that Bax N-terminal acetylation by NatB is involved in its mitochondrial targeting.

Summary Mitochondria are essential organelles producing most of the energy required for the cell. A selective autophagic process called mitophagy removes damaged mitochondria, which is critical for proper cellular homeostasis; dysfunctional mitochondria can generate excess reactive oxygen species that can further damage the organelle as well as other cellular components. Although proper cell physiology requires the maintenance of a healthy pool of mitochondria, little is known about the mechanism underlying the recognition and selection of damaged organelles. In this study, we investigated the cellular fate of mitochondria damaged by the action of respiratory inhibitors (antimycin A, myxothiazol, KCN) that act on mitochondrial respiratory complexes III and IV, but have different effects with regard to the production of reactive oxygen species and increased levels of reduced cytochromes. Antimycin A and potassium cyanide effectively induced nonspecific autophagy, but not mitophagy, in a wild-type strain of Saccharomyces cerevisiae; however, low or no autophagic activity was measured in strains deficient for genes that encode proteins involved in mitophagy, including ATG32, ATG11 and BCK1. These results provide evidence for a major role of specific mitophagy factors in the control of a general autophagic cellular response induced by mitochondrial alteration. Moreover, increased levels of reduced cytochrome b, one of the components of the respiratory chain, could be the first signal of this induction pathway.

The role of the mitochondrial protein receptor Tom22p in the interaction of pro-apoptotic protein Bax with yeast mitochondria was investigated. Co-immunoprecipitation assays showed that human Bax interacted with different TOM subunits, including Tom22p. Expression of the cytosolic receptor domain of human Tom22 increased Bax mitochondrial localization, but decreased the proportion of active Bax. BN–PAGE showed that the cytosolic domain of Tom22 interfered with the oligomerization of Bax. These data suggest that the interaction with the cytosolic domain of Tom22 helps Bax to acquire a conformation able to interact with the outer mitochondrial membrane. Structured summary of protein interactions BAX and BAX physically interact with TOM22 by anti bait coimmunoprecipitation ( View interaction ) BAX physically interacts with TOM70 by anti bait coimmunoprecipitation ( View interaction ) BAX physically interacts with TOM22 , TOM70 and TOM40 by anti bait coimmunoprecipitation ( View interaction ) BAX physically interacts with TOM22 by anti bait coimmunoprecipitation ( View interaction ) BAX and BAX bind by blue native page ( View interaction ) BAX physically interacts with TOM40 by anti bait coimmunoprecipitation ( View interaction )

We have previously shown that acetic acid activates a mitochondria-dependent death process in Saccharomyces cerevisiae and that the ADP/ATP carrier (AAC) is required for mitochondrial outer membrane permeabilization and cytochrome c release. Mitochondrial fragmentation and degradation have also been shown in response to this death stimulus. Herein, we show that autophagy is not active in cells undergoing acetic acid-induced apoptosis and is therefore not responsible for mitochondrial degradation. Furthermore, we found that the vacuolar protease Pep4p and the AAC proteins have a role in mitochondrial degradation using yeast genetic approaches. Depletion and overexpression of Pep4p, an orthologue of human cathepsin D, delays and enhances mitochondrial degradation respectively. Moreover, Pep4p is released from the vacuole into the cytosol in response to acetic acid treatment. AAC-deleted cells also show a decrease in mitochondrial degradation in response to acetic acid and are not defective in Pep4p release. Therefore, AAC proteins seem to affect mitochondrial degradation at a step subsequent to Pep4p release, possibly triggering degradation through their involvement in mitochondrial permeabilization. The finding that both mitochondrial AAC proteins and the vacuolar Pep4p interfere with mitochondrial degradation suggests a complex regulation and interplay between mitochondria and the vacuole in yeast programmed cell death.

It is still unclear whether the BH3-only protein Puma (p53 up-regulated modulator of apoptosis) can prime cells to death and render antiapoptotic BH3-binding Bcl-2 homologues necessary for survival through its ability to directly interact with proapoptotic Bax and activate it. In this study, we provide further evidence, using cell-free assays, that the BH3 domain of Puma binds Bax at an activation site that comprises the first helix of Bax. We also show that, in yeast, Puma interacts with Bax and triggers its killing activity when Bcl-2 homologues are absent but not when Bcl-xL is expressed. Finally, endogenous Puma is involved in the apoptotic response of human colorectal cancer cells to the Bcl-2/Bcl-xL inhibitor ABT-737, even in conditions where the expression of Mcl-1 is down-regulated. Thus, Puma is competent to trigger Bax activity by itself, thereby promoting cellular dependence on prosurvival Bcl-2 family members.

During apoptosis, the pro-apoptotic protein Bax relocalizes from the cytosol to the mitochondrial outer membrane. This relocalization is associated to major conformational changes, namely at the N- and C-terminal ends of the protein. Substitution of residues located at critical positions within the protein potentially stimulates or inhibits this process. In the present study, we investigated the hypothesis that phosphorylation of serine residues might trigger these conformational changes, with a focus on Ser(163) and Ser(184), which have been shown to be phosphorylatable by protein kinases GSK3beta and Akt/PKB, respectively, and on Ser(60), which is located in a consensus target sequence for PKA. Substitutions of these serine residues by alanine or aspartate were done in wild type or previously characterized Bax mutants, and the capacity of the resulting proteins to interact with mitochondria and to release cytochrome c was assayed in yeast, which provides a tool to study the function of Bax, independently of the rest of the apoptotic network. We conclude that sequential phosphorylation of these serine residues might participate in the triggering of the different conformational changes associated with Bax activation during apoptosis.

Mitochondria are essential to oxidative energy production in aerobic eukaryotic cells, where they are also required for multiple biosynthetic pathways to take place. Mitochondrial homeostasis also plays a crucial role in ageing and programmed cell death, and recent data have suggested that mitochondria degradation is a strictly regulated process. Autophagy is an evolutionary conserved mechanism that provides cells with a mechanism for the continuous turnover of damaged and obsolete macromolecules and organelles. In this work, we investigated mitochondria degradation by autophagy. Electron microscopy observations of yeast cells submitted to nitrogen starvation after growth on different carbon sources provided evidence that microautophagy, rather than macroautophagy, preferentially occurred in cells grown under nonfermentable conditions. The observation of mitochondria degradation showed that both a selective process and a nonselective process of mitochondria autophagy occurred successively. In a yeast strain inactivated for the gene UTH1, the selective process was not observed.

Bcl-xL is a member of the Bcl-2 family, playing a critical role in the survival of tumor cells. Here, we show that Bcl-xL oncogenic function can be uncoupled from its anti-apoptotic activity when it is regulated by the post-translational deamidation of its Asn52. Bcl-xL activity can be regulated by post-translational modifications: deamidation of Asn52 and 66 into Asp residues was reported to occur exclusively in response to DNA damage, and to cripple its anti-apoptotic activity. Our work reports for the first time the spontaneous occurrence of monodeamidated Asp52Bcl-xL in control conditions, in vivo and in vitro. In the normal and cancer cell lines tested, no less than 30% and up to 56% of Bcl-xL was singly deamidated on Asn52. Functional analyses revealed that singly deamidated Bcl-xL retains anti-apoptotic functions, and exhibits enhanced autophagic activity while harboring impaired clonogenic and tumorigenic properties compared to native Bcl-xL. Additionally, Asp52Bcl-xL remains phosphorylatable, and thus is still an eligible target of anti-neoplasic agents. Altogether our results complement the existing data on Bcl-xL deamidation: they challenge the common acceptance that Asn52 and Asn66 are equally eligible for deamidation, and provide a valuable improvement of our knowledge on the regulation of Bcl-xLoncogenic functions by deamidation.

We found recently that, in yeast cells, the heterologous expression of Bax induces a loss of plating efficiency different from that induced by acute stress because it is associated with the maintenance of plasma membrane integrity (Camougrand, N., Grelaud-Coq, A., Marza, E., Priault, M., Bessoule, J. J., and Manon, S. (2003) Mol. Microbiol. 47, 495-506). Bax effects were neither dependent on the presence of the yeast metacaspase Yca1p and the apoptosis-inducing factor homolog nor associated with the appearance of typical apoptotic markers such as metacaspase activation, annexin V binding, and DNA cleavage. Yeast cells expressing Bax instead displayed autophagic features, including increased accumulation of Atg8p, activation of vacuolar alkaline phosphatase, and the presence of autophagosomes and autophagic bodies. However, the inactivation of autophagy did not prevent and actually slightly accelerated Bax-induced loss of plating efficiency. On the other hand, Bax expression induced a fragmentation of the mitochondrial network, which retained, however, some level of organization in wild-type cells. However, when expressed in cells inactivated for the gene UTH1, previously shown to be involved in mitophagy, Bax induced a complete disorganization of the mitochondrial network. Interestingly, although mitochondrially targeted green fluorescent protein was slowly degraded in the wild-type strain, it remained unaffected in the mutant. Furthermore, the slow loss of plating efficiency in the mutant strain correlated with a loss of plasma membrane integrity. These data suggest that Bax-induced loss of growth capacity is associated with maintenance of plasma membrane integrity dependent on UTH1, suggesting that selective degradation of altered mitochondria is required for a regulated loss of growth capacity.

During apoptosis, engagement of the mitochondrial pathway involves the permeabilization of the outer mitochondrial membrane (OMM), which leads to the release of cytochrome c and other apoptogenic proteins such as Smac/DIABLO, AIF, EndoG, Omi/HtraA2 and DDP/TIMM8a. OMM permeabilization depends on activation, translocation and oligomerization of multidomain Bcl-2 family proteins such as Bax or Bak. Factors involved in Bax conformational change and the function(s) of the distinct domains controlling the addressing and the insertion of Bax into mitochondria are described in this review. We also discuss our current knowledge on Bax oligomerization and on the molecular mechanisms underlying the different models accounting for OMM permeabilization during apoptosis.

UTH1 is a yeast aging gene that has been identified on the basis of stress resistance and longer life span of mutants. It was also shown to participate in mitochondrial biogenesis. The absence of Uth1p was found to trigger resistance to autophagy induced by rapamycin. Uth1p is therefore the first mitochondrial protein proven to be required for the autophagic degradation of mitochondria. Since this protein is also involved in yeast cell death induced by heterologous expression of the pro-apoptotic protein Bax, the results are discussed in the light of evidence suggesting a co-regulation of apoptosis and autophagy in mammalian cells.

The absence of the outer mitochondrial membrane protein Uth1p was found to induce resistance to rapamycin treatment and starvation, two conditions that induce the autophagic process. Biochemical studies showed the onset of a fully active autophagic activity both in wild-type and Deltauth1 strains. On the other hand, the disorganization of the mitochondrial network induced by rapamycin treatment or 15 h of nitrogen starvation was followed in cells expressing mitochondria-targeted green fluorescent protein; a rapid colocalization of green fluorescent protein fluorescence with vacuole-selective FM4-64 labeling was observed in the wild-type but not in the Deltauth1 strain. Degradation of mitochondrial proteins, followed by Western blot analysis, did not occur in mutant strains carrying null mutations of the vacuolar protease Pep4p, the autophagy-specific protein Atg5p, and Uth1p. These data show that, although the autophagic machinery was fully functional in the absence of Uth1p, this protein is involved in the autophagic degradation of mitochondria.

The yeast Saccharomyces cerevisiae is a powerful tool to investigate several aspects of the molecular mechanisms underlying programmed cell death, namely, the events involving mitochondria. Yeast has allowed new information to be gained about crucial aspects of the interaction between proapoptotic proteins Bax and Bid and mitochondria, namely, their addressing and insertion into the mitochondrial outer membrane and their ability to induce the relocalization of cytochrome c. Going one step further, the prooxidant effect of Bax can also be studied in yeast. Bax expression induces both the accumulation of reactive oxygen species and lipid oxidation. Lipid oxidation is involved in Bax-induced cell death and may be required for optimal insertion of Bax in mitochondria. The easy modulation of lipid composition in yeast is a powerful tool to investigate this process further, and studies can be extended to other regulators of apoptosis, such as proapoptotic Bid or antiapoptotic Bcl-x(L). Also, yeast is a model for the study of other types of cell death, such as autophagy-related forms of death, for which a role of lipid oxidation has also been evidenced.

Keeping a cytosolic redox balance is a prerequisite for living cells in order to maintain a metabolic activity and enable growth. During growth of Saccharomyces cerevisiae, an excess of NADH is generated in the cytosol. Aerobically, it has been shown that the external NADH dehydrogenase, Nde1p and Nde2p, as well as the glycerol-3-phosphate dehydrogenase shuttle, comprising the cytoplasmic glycerol-3-phosphate dehydrogenase, Gpdlp, and the mitochondrial glycerol-3-phosphate dehydrogenase, Gut2p, are the most important mechanisms for mitochondrial oxidation of cytosolic NADH. In this review we summarize the recent results showing (i) the contribution of each of the mechanisms involved in mitochondrial oxidation of the cytosolic NADH, under different physiological situations; (ii) the kinetic and structural properties of these metabolic pathways in order to channel NADH from cytosolic dehydrogenases to the inner mitochondrial membrane and (iii) the organization in supramolecular complexes and, the peculiar ensuing kinetic regulation of some of the enzymes (i.e. Gut2p inhibition by external NADH dehydrogenase activity) leading to a highly integrated functioning of enzymes having a similar physiological function. The cell physiological consequences of such an organized and regulated network are discussed.

Because of structural homology with the transmembrane domain of Bcl-2, the proapoptotic protein Bax has been proposed to be anchored to the outer membrane of mitochondria through its carboxy-terminal end (CT). We took advantage of the absence of Bcl-2 family members in yeast to further investigate the role of Bax CT in its mitochondrial association and function. The complete deletion or the addition of a C-terminal c-myc tag as well as the replacement of CT by a random coiled sequence enhanced membrane insertion of Bax. It has previously been suggested that conformational change in the N-terminal end of Bax would allow the C-terminal end to play its anchoring function. We found that a mutant truncated in both N- and C-termini still exhibited a strong binding activity to mitochondria. In mammals, Bax interaction with the caspase-8-generated truncated form of Bid (tc-Bid) is believed to promote a conformational change necessary for the insertion of Bax into mitochondria. We coexpressed Bax and tc-Bid in yeast and found that native Bax functions are not stimulated by tc-Bid, whereas functions of an active variant with a modified CT are. We propose that Bax CT has to undergo a conformational change to allow the insertion of Bax in mitochondria but, contrary to current views, is not a bona fide membrane anchor.

The translocation of Bax alpha, a pro-apoptotic member of the BCL-2 family from the cytosol to mitochondria, is a central event of the apoptotic program. We report here that the N-terminal (NT) end of Bax alpha, which contains its first alpha helix (Eta alpha 1), is a functional mitochondrial-addressing signal both in mammals and in yeast. Similar results were obtained with a newly described variant of Bax called Bax psi, which lacks the first 20 amino acids of Bax alpha and is constitutively associated with mitochondria. Deletion of Eta alpha 1 impairs the binding of Bax psi to mitochondria, whereas a fusion of the N terminus of Bax alpha, which contains Eta alpha 1 with a cytosolic protein, results in the binding of the chimeric proteins to mitochondria both in a cell-free assay and in vitro. More importantly, the mitochondria-bound chimeric proteins inhibit the interaction of Bax psi with mitochondria as well as Bax-apoptogenic properties. The mutations of the Eta alpha 1, which inhibit Bax alpha and Bax psi translocation to mitochondria, also block the subsequent activation of the execution phase of apoptosis. Conversely, a deletion of the C terminus does not appear to influence Bax alpha and Bax psi mitochondrial addressing. Taken together, our results suggest that Bax is targeted to mitochondria by its NT and thus through a pathway that is unique for a member of the BCL-2 family.

A yeast mutant was isolated that was resistant to Bax-induced cell death. It supports a mutation leading to decreased amounts of the protein Uth1p. A strain in which the UTH1 gene is disrupted also exhibits resistance to Bax expression. The absence of Uth1p does not change the mitochondrial localization of Bax, its insertion in the mitochondrial outer membrane or its cytochrome c-releasing activity. On the other hand, the absence of Uth1p does prevent the appearance of other hallmarks related to Bax expression in yeast, such as oxidation of mitochondrial lipid, production of reactive oxygen species and maintenance of plasma membrane properties after ethanol stress. The absence of Uth1p was also found to induce resistance to rapamycin, a specific inducer of autophagy. This resistance only appears when cells are grown under respiratory conditions, but not under fermentative conditions, suggesting that Uth1p acts in an autophagic pathway involving mitochondria, in accordance with its main localization in the outer mitochondrial membrane. Taken together, these data show that Bax is able to activate a death pathway related to autophagy in yeast, which also exhibits typical hallmarks of apoptosis, revealing a possible dual function of Bax in both types of death. This hypothesis is discussed in the light of observations suggesting a co-regulation of apoptosis and autophagy in mammalian cells.

The oxidant function of pro-apoptotic protein Bax was investigated through heterologous expression in yeast. Direct measurements of fatty acid content show that Bax-expression induces oxidation of mitochondrial lipids. This effect is prevented by the coexpression of Bcl-xL. The oxidation actually could be followed on isolated mitochondria as respiration-induced peroxidation of polyunsaturated cis-parinaric acid and on whole cells as the increase in the amount of thiobarbituric acid-reactive products. Treatments that increase the unsaturation ratio of lipids, making them more sensitive to oxidation, increase kinetics of Bax-induced death. Conversely, inhibitors of lipid oxidation and treatments that decrease the unsaturation ratio of fatty acids decrease kinetics of Bax-induced death. Taken together, these results show that Bax-induced mitochondrial lipid oxidation is relevant to Bax-induced cell death. Conversely, lipid oxidation is poorly related to the massive Bax-induced superoxide and hydrogen peroxide accumulation, which occurs at the same time, as chemical or enzymatic scavenging of ROS does not prevent lipid oxidation nor has any effects on kinetics of Bax-induced cell death. Whatever the origin of mitochondrial lipid oxidation, these data show that it represents a major step in the cascade of events leading to Bax-induced cell death. These results are discussed in the light of the role of lipid oxidation both in mammalian apoptosis and in other forms of cell death in other organisms.

Expression of the pro-apoptotic protein Bax in yeast Saccharomyces cerevisiae induces a release of cytochrome c accompanied by a decrease of the amount of cytochrome c oxidase. Here we show that the decrease of cytochrome c oxidase is due to the activation of mitochondrial protease Yme1p, of which cytochrome c oxidase subunit 2 (Cox2p) is a substrate. The absence of Yme1p slightly delays Bax-induced cell death, suggesting a role of this protease in yeast cell death and thus of its mammalian homologue in apoptosis.

During apoptosis, proapoptotic factors are released from mitochondria by as yet undefined mechanisms. Patch-clamping of mitochondria and proteoliposomes formed from mitochondrial outer membranes of mammalian (FL5.12) cells has uncovered a novel ion channel whose activity correlates with onset of apoptosis. The pore diameter inferred from the largest conductance state of this channel is ∼4 nm, sufficient to allow diffusion of cytochrome c and even larger proteins. The activity of the channel is affected by Bcl-2 family proteins in a manner consistent with their pro- or antiapoptotic properties. Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2. Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax. These findings implicate this channel, named mitochondrial apoptosis–induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.

A new system for bax-expression in yeast has been devised to investigate bax’s effect under fermentative and respiro-fermentative conditions. This has allowed us to show unambiguously that the ability of bax to kill yeast is higher under respiratory conditions than under purely fermentative conditions. The extent of killing under respiro-fermentative conditions (non-repressive sugars) is intermediate. It has been proposed that the two proteins adenine nucleotides carrier (ANC) and cytochrome c play a crucial role in bax-induced cell death. We have investigated the effects of deletion of the genes encoding the two proteins on the toxicity induced by bax, using this new system. The absence of ANC did not modify bax-induced lethality in any way. Moreover, the absence of cytochrome c also did not prevent bax-induced death. Only the kinetics of lethality were altered. All these effects are prevented by co-expression of bcl-xL.

Large and unselective permeabilities through the inner membrane of yeast mitochondria have been observed for more than 20 years, but the characterization of these permeabilities, leading to hypothesize the existence of a large-conductance unselective channel in yeast inner mitochondrial membrane, was done only recently by several groups. This channel has been tentatively identified as a yeast counterpart to the mammalian permeability transition pore, the crucial role of which is now well-documented in physiopathological phenomena, such as Ca2+ homeostasis, ischemic damages, or programmed cell death. The aim of this review is to make a point on the known characteristics of this yeast mitochondrial unselective channel (YMUC) and to analyze whether or not it can be considered as a “yeast permeability transition pore.”

The modulation of the ATP-induced K(+)-transport pathway of the yeast inner mitochondrial membrane by delta pH was investigated in two ways. First, the inhibitory effect of phosphate was compared to the effect of other permeant acids, demonstrating that a part of the effect of phosphate was linked to its electroneutral transport down delta pH. However, an additional effect specific for phosphate also occurred inside the matrix. Second, the stimulation of the respiration by ATP in the presence of K+ was compared to the effects of the protonophore ClCCP2 and of the K(+)-ionophore valinomycin. Quite unexpectedly, the effect of ATP looked more like the effect of ClCCP than the effect of valinomycin. This and previous results (Manon et al., Biochimica et Biophysica Acta 1231, 282-288 (1995)) show that the rate of electrophoretic K(+)-entry is limited by the rate of electroneutral K(+)-exit via the K+/H+ exchange and is therefore delta pH-dependent.

The effect of ATP and other nucleotides on the respiration of Saccharomyces cerevisiae mitochondria was investigated. It was observed that ATP induced a stimulation of the respiration rate only in the presence of a salt in mitochondria from the baker's yeast Yeast Foam, whereas an ATP-induced stimulation occurred even in the absence of salt in mitochondria from three different laboratory strains. In both cases, the stimulation was related to a collapse of the transmembrane potential, suggesting the opening of ion- and/or proton-conducting pathways. Not only ATP, but also GTP and CTP, induced these pathways. Moreover, a similar stimulation was obtained with GDP and its analog GDP-beta-S. The fact that, as opposed to NTPs, GDP did not induce any non-specific anion channel, allowed us to use it to demonstrate unambiguously that a proton-conducting pathway was opened through the inner mitochondrial membrane of laboratory strains but not of Yeast Foam. Three additional aspects of this nucleotide-induced permeability were investigated. (i) The proton-conducting pathway was insensitive to Mg2+, whereas the anion-conducting pathway was fully inhibited by 4 mM Mg2-. (ii) The proton-conducting pathway of mitochondria isolated from laboratory strains was opened by the action of nucleotides outside the mitochondrion, since it was fully insensitive to (carboxy)atractyloside, and fully active in mitochondria isolated from op1 and delta anc strains. On the other hand, the cation-conducting pathway of Yeast Foam mitochondria was partly sensitive to (carboxy)atractyloside and insensitive to bongkrekic acid, suggesting a role of the conformational state of ANC in this activity. (iii) Both the proton and cation-conducting pathways were inhibited by very low concentrations of vanadate, under conditions where this oxyanion was polymerized to decavanadate: a competitor to nucleotide-binding sites on some enzymes.

International audience; : It has been established that sphingolipids are engaged in the regulation of apoptosis both as direct executors and as signalling molecules. However, the peculiarities of this class of bioactive lipids, namely the interconnectivity of their metabolic pathways, the specific subcellular localization where they are generated and the transport mechanisms involved, introduce a considerably high level of complexity in deciphering their role in the signalling and regulation of programmed cell death. Although yeast is undeniably a simple model, the conservation of the sphingolipid metabolism and of the core machinery engaged in regulated cell death has already provided valuable clues to the understanding of metabolic pathways involved in distinct cellular processes, including apoptosis. It can be anticipated that studies using this model system will further unravel mechanisms underlying the regulation of apoptosis by sphingolipids and contribute to novel therapeutic strategies against serious human diseases associated with dysfunction of sphingolipid-dependent cell death programmes.

International audience; Bax-induced mitochondrial outer membrane permeabilization (MOMP) is considered as one of the key control switches of apoptosis. MOMP requires Bax relocation to and insertion into the outer mitochondrial membrane to oligomerize and form pores allowing the release of apoptogenic factors such as cytochrome c. Even if these essential steps are now well-defined, it is necessary to better understand the molecular changes underlying the switch between inactive Bax and active (pore-forming) Bax. One of the ongoing issues is to determine whether Bax mitochondrial translocation is a critical step in the control of Bax activation or if this control is carried by latter regulatory steps. In this focus article we discuss recent data suggesting that although Bcl-2 and Bcl-x(L) block the MOMP, they can also regulate the mitochondrial Bax content. A new model in which Bax inhibition by Bcl-x(L) occurs at the immediate proximity of the outer mitochondrial membrane is also discussed. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

International audience; Bcl-2 proteins are acknowledged as key regulators of programmed cell death. However, increasing data suggest additional roles, including regulation of the cell cycle, metabolism and cytoskeletal dynamics. Here we report the discovery and characterization of a new Bcl-2-related multidomain apoptosis accelerator, Bcl-wav, found in fish and frogs. Genetic and molecular studies in zebrafish indicate that Bcl-wav and the recently identified mitochondrial calcium uniporter (MCU) contribute to the formation of the notochord axis by controlling blastomere convergence and extension movements during gastrulation. Furthermore, we found that Bcl-wav controls intracellular Ca(2+) trafficking by acting on the mitochondrial voltage-dependent anion channel, and possibly on MCU, with direct consequences on actin microfilament dynamics and blastomere migration guidance. Thus, from an evolutionary point of view, the original function of Bcl-2 proteins might have been to contribute in controlling the global positioning system of blastomeres during gastrulation, a critical step in metazoan development.

The Bcl-2 protein family plays a central role in mitochondrial membrane permeabilization. This event and the ensuing release of cytochrome c are decisive in the apoptotic cascade. Therefore, a better knowledge of these processes and their regulation will probably lead to the development of novel therapeutic strategies for treatment of apoptosis-related diseases. However, the mode of action of Bcl-2 protein family and its regulation are not completely understood. Yeast has proved to be a powerful tool to investigate the molecular aspects of several biological processes, including the steps of the apoptotic cascade involving mitochondria. The fact that yeast does not have obvious homologues of the mammalian Bcl-2 family proteins and that these proteins conserve some of their molecular and biochemical functions when expressed in yeast favours the use of this simpler model system to unravel some of the functions of this family. In this review we attempt to encompass the current knowledge regarding Bcl-2 family mode of action and regulation obtained using the yeast model system. Moreover, we discuss how this model system can be used in the future to gain new understanding about the intricate mechanisms of Bcl-2 family protein regulation, and highlight novel therapeutic targets revealed by this system. We believe that the studies here summarized also provide a proof of principle of yeast as an important tool to elucidate some of the complex mechanisms of apoptotic cell death in higher eukaryotes., R. D. Silva (SFRH/BD/23774/2005) have a fellowship from Fundação para a Ciência e Tecnologia, Portugal. This work was supported by a FCT funded project (PTDC/BIA-BCM/ 69448/2006).

The pro-apoptototic protein Bax (Bcl-2 Associated protein X) plays a central role in the mitochondria-dependent apoptotic pathway. In healthy mammalian cells, Bax is essentially cytosolic and inactive. Following a death signal, the protein is translocated to the outer mitochondrial membrane, where it promotes a permeabilization that favors the release of different apoptogenic factors, such as cytochrome c. The regulation of Bax translocation is associated to conformational changes that are under the control of different factors. The evidences showing the involvement of different Bax domains in its mitochondrial localization are presented. The interactions between Bax and its different partners are described in relation to their ability to promote (or prevent) Bax conformational changes leading to mitochondrial addressing and to the acquisition of the capacity to permeabilize the outer mitochondrial membrane.

Bcl-2 homologues (such as Bcl-x(L)) promote survival in part through sequestration of "activator" BH3-only proteins (such as Puma), preventing them from directly activating Bax. It is thus assumed that inhibition of interactions between activators and Bcl-x(L) is a prerequisite for small molecules to antagonize Bcl-x(L) and induce cell death. The biological properties, described here of a terphenyl-based alpha-helical peptidomimetic inhibitor of Bcl-x(L) attest that displacement of Bax from Bcl-x(L) is also critical. Terphenyl 14 triggers Bax-dependent but Puma-independent cell death, disrupting Bax/Bcl-x(L) interactions without affecting Puma/Bcl-x(L) interactions. In cell-free assays, binding of inactive Bax to Bcl-x(L), followed by its displacement from Bcl-x(L) by terphenyl 14, produces mitochondrially permeabilizing Bax molecules. Moreover, the peptidomimetic kills yeast cells that express Bax and Bcl-x(L), and it uses Bax-binding Bcl-x(L) to induce mammalian cell death. Likewise, ectopic expression of Bax in yeast and mammalian cells enhances sensitivity to another Bcl-x(L) inhibitor, ABT-737, when Bcl-x(L) is present. Thus, the interaction of Bcl-x(L) with Bax paradoxically primes Bax at the same time it keeps Bax activity in check, and displacement of Bax from Bcl-x(L) triggers an apoptotic signal by itself. This mechanism might contribute to the clinical efficiency of Bcl-x(L) inhibitors.

The nuclear mutant AB1-4A/8/100, a respiratory-competent strain altered in the regulation of ATP synthesis, has been shown to be modified in the relative stoichiometry of the mtDNA-encoded proteolipids of the F0 sector of ATP synthase: the ratios mutant/wild type of the proteolipids were equal to 0.4/0.7/1 for Su8/Su6/Su9, respectively. This defect results from the simultaneous presence of two nuclear genes which promote a cryosensitive phenotype on a nonfermentable carbon source. Measurements of mitochondrial protein synthesis carried out "in vivo" and "in organello" evidenced a specific defect in the synthesis of subunits 6 and 8. Measurements of the steady state levels of mitochondrial mRNA showed that the defect in subunits 6 and 8 was correlated with a modification of the expression of a cotranscript ATP8-ATP6. This cotranscript is matured at a unique site to give two cotranscripts of 4600 and 5200 bases in length. In mutant mitochondria, the ratio between both cotranscripts, 5200/4600, was lowered. In parallel, expression of the whole mitochondrial transcription unit supporting the genes COXI, ATP8, ATP6, and RF3 was enhanced. However, despite this over expression, the amount of the long cotranscript ATP8-ATP6 remained lower than in wild type mitochondria.

The antioxidant N-acetyl-l-cysteine prevented the autophagy-dependent delivery of mitochondria to the vacuoles, as examined by fluorescence microscopy of mitochondria-targeted green fluorescent protein, transmission electron microscopy, and Western blot analysis of mitochondrial proteins. The effect of N-acetyl-l-cysteine was specific to mitochondrial autophagy (mitophagy). Indeed, autophagy-dependent activation of alkaline phosphatase and the presence of hallmarks of non-selective microautophagy were not altered by N-acetyl-l-cysteine. The effect of N-acetyl-l-cysteine was not related to its scavenging properties, but rather to its fueling effect of the glutathione pool. As a matter of fact, the decrease of the glutathione pool induced by chemical or genetical manipulation did stimulate mitophagy but not general autophagy. Conversely, the addition of a cell-permeable form of glutathione inhibited mitophagy. Inhibition of glutathione synthesis had no effect in the strain Δuth1, which is deficient in selective mitochondrial degradation. These data show that mitophagy can be regulated independently of general autophagy, and that its implementation may depend on the cellular redox status.

Bax insertion into the mitochondrial outer membrane is essential for the implementation of apoptosis. However, little is known about the first stage of Bax integration into the mitochondrial outer membrane. We have recently shown that TOM22, a mitochondrial outer membrane receptor, is important for insertion, although other reports have suggested that only mitochondrial lipids are involved in this process. Here, we show that monomers, but not dimers, of Bax require the presence of TOM22 and TOM40 to integrate into mitochondria. In addition we show that once inserted into the membrane, Bax can act as a receptor for cytosolic Bax.

The mitochondrial apoptotic pathway is a highly regulated biological mechanism which determines cell fate. It is defined as a cascade of events, going from an apoptotic stimulus to the MOM permeabilization, resulting in the activation of the so-called executive phase. This pathway is very often altered in cancer cells. The mitochondrial permeabilization is under the control of the Bcl-2 family of proteins (pBcls). These proteins share one to four homology domains (designed BH1-4) with Bcl-2, and are susceptible of homo- and/or hetero-dimerization. In spite of a poor amino-acid sequence homology, these proteins exhibit very similar tertiary structures. Strikingly, while some of these proteins are anti-apoptotic, the others are pro-apoptotic. Pro-apoptotic proteins are further divided in two sub-classes: multi-domains proteins, among which Bax and Bak, which exhibit BH1-3 domains, and BH3-only proteins (or BOPs). Schematically, BOPs and anti-apoptotic proteins antagonistically regulate the activation of the multi-domain proteins Bax and Bak and their oligomerization in the MOM, the latter process being responsible for the apoptotic mitochondrial permeabilization. Considering the critical role of Bax in cancer cells apoptosis, we focus in this review on the molecular events of Bax activation through its interaction with the other proteins from the Bcl-2 family. The mechanism by which Bax triggers the MOM permeabilization once activated will be discussed in some other reviews in this special issue.

In yeast, Ca2+ and long chain alkylguanidines interact with mitochondria modulating the opening of the yeast mitochondrial unspecific channel. Mammalians possess a similar structure, the mitochondrial permeability transition pore. The composition of these pores is under debate. Among other components, the voltage-dependent anion channel has been proposed as a component of either pore. In yeast from an industrial strain, octylguanidine and calcium closed the yeast mitochondrial unspecific channel. Here, the effects of the cations Ca2+ or octylguanidine and the voltage-dependent anion channel effector decavanadate were evaluated in yeast mitochondria from either a wild type or a voltage-dependent anion channel deletion laboratory strain. It was observed that in the absence of voltage-dependent anion channel, the yeast mitochondrial unspecific channel was desensitized to Ca2+, octylguanidine or decavanadate but remained sensitive to phosphate. It is thus suggested that in yeast mitochondria, the voltage-dependent anion channel has a cation binding site where Ca2+ and octylguanidine interact, conferring cation sensitivity to the yeast mitochondrial unspecific channel.

The association of Bax with mitochondria is an essential step in the implementation of apoptosis. By using a bacterial two-hybrid assay and crosslinking strategies, we have identified TOM22, a component of the translocase of the outer mitochondrial membrane (TOM), as a mitochondrial receptor of Bax. Peptide mapping showed that the interaction of Bax with TOM22 involved the first alpha helix of Bax and possibly two central alpha helices, which are homologous to the pore forming domains of some toxins. Antibodies directed against TOM22 or an antisense knockdown of the expression of TOM22 specifically inhibited the association of Bax with mitochondria and prevented Bax-dependent apoptosis. In yeast, a haploid strain for TOM22 exhibited a decreased expression of TOM22 and mitochondrial association of ectopically expressed human Bax. Our data provide a new perspective on the mechanism of association of Bax with mitochondria as it involves a classical import pathway.

Autophagy, a process involved in the degradation and the recycling of long-lived proteins and organelles to survive nitrogen starvation, is generally non-selective. However, recent data suggest that selective forms of autophagy exist, that are able to specifically target several organelles, including mitochondria. Conversely, mitochondrial alterations could trigger autophagy. Such a selective form of autophagy might be involved in the elimination of damaged mitochondria. We reported previously that, mitochondria were early targets of rapamycin-induced autophagy. Here we report that rapamycin-induced autophagy is accompanied by the early production of reactive oxygen species and by the early oxidation of mitochondrial lipid. Inhibition of these oxidative effects by resveratrol largely impaired autophagy of both cytosolic proteins and mitochondria, and delayed subsequent cell death. These results support a role of mitochondrial oxidation events in the activation of autophagy.