Stéphen Manon

Release of cytochrome c and decrease of cytochrome c oxidase in Bax-expressing yeast cells, and prevention of these effects by coexpression of Bcl-xL.

The characteristics of mitochondria of yeast cells expressing the pro‐apoptotic gene Bax or coexpressing Bax and the anti‐apoptotic gene Bcl‐xL have been investigated in whole cells, isolated mitochondria and permeabilized spheroplasts. It is found that Bax‐induced growth arrest of yeast cells is related to two defects in the respiratory chain: (i) a decrease in the amount of cytochrome c oxidase, the terminal enzyme of the respiratory chain, and (ii) a dramatic increase in the release of cytochrome c to the cytosol. Other components of the inner mitochondrial membrane (bc1 complex and F0F1‐ATPase) are unaffected. Coexpression of Bcl‐xL almost fully prevented the effect of Bax. Surprisingly, these results obtained in yeast parallel similar observations reported in mammalian cells.

A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast

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.

Bax activation and mitochondrial insertion during apoptosis.

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.

Selective and Non-Selective Autophagic Degradation of Mitochondria in Yeast

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 non-fermentable conditions. The observation of mitochondria degradation showed that both a selective process and a non-selective process of mitochondria autophagy occurred successively. In a yeast strain inactivated for the gene UTH1, the selective process was not observed.

ADP/ATP carrier is required for mitochondrial outer membrane permeabilization and cytochrome c release in yeast apoptosis.

Adenine nucleotide translocator (ANT) is a mitochondrial inner membrane protein involved in the ADP/ATP exchange and is a component of the mitochondrial permeability transition pore (PTP). In mammalian apoptosis, the PTP can mediate mitochondrial outer membrane permeabilization (MOMP), which is suspected to be responsible for the release of apoptogenic factors, including cytochrome c. Although release of cytochrome c in yeast apoptosis has previously been reported, it is not known how it occurs. Herein we used yeast genetics to investigate whether depletion of proteins putatively involved in MOMP and cytochrome c release affects these processes in yeast. While deletion of POR1 (yeast voltage‐dependent anion channel) enhances apoptosis triggered by acetic acid, H2O2 and diamide, CPR3 (mitochondrial cyclophilin) deletion had no effect. Absence of ADP/ATP carrier (AAC) proteins, yeast orthologues of ANT, protects cells exposed to acetic acid and diamide but not to H2O2. Expression of a mutated form of Aac2p (op1) exhibiting very low ADP/ATP translocase activity indicates that AACs pro‐death role does not require translocase activity. Absence of AAC proteins impairs MOMP and release of cytochrome c, which, together with other mitochondrial inner membrane proteins, is degraded. Our findings point to a crucial role of AAC in yeast apoptosis.

Bax activation by the BH3-only protein Puma promotes cell dependence on antiapoptotic Bcl-2 family members

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.

TOM22, a core component of the mitochondria outer membrane protein translocation pore, is a mitochondrial receptor for the proapoptotic protein Bax

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.

Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae

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, Gpd1p, 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.

Bax: Addressed to kill.

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.

Distinct Domains Control the Addressing and the Insertion of Bax into Mitochondria

The translocation of Bax from the cytosol into the mitochondrial outer membrane is a central event during apoptosis. We report that beyond the addressing step, which involves its first α-helix (hα1), the helices α5 and α6 (hα5α6) are responsible for the insertion of Bax into mitochondrial outer membrane bilayer. The translocation of Bax to mitochondria is associated with specific changes in the conformation of the protein that are under the control of two prolines: Pro-13, which controls the unfolding of hα1, and Pro-168, a proline located immediately before the hydrophobic carboxyl-terminal end (i.e. helix α9, hα9), which controls the disclosure of hα5α6. An additional step, the disruption of an electrostatic bond formed between Asp-33 (hα1) and Lys-64 (BH3), allows the mitochondria addressing of Bax. We conclude that, although the intramolecular interactions of hα1 with the BH3 region control the addressing of Bax to mitochondria, the Pro-168 is involved in the control of its membrane insertion through hα5α6.