Role of the C-terminal domain of Bax and Bcl-xL in their localization and function in yeast cells

AbstractIt has been suggested that the C-terminal domain of Bcl-2 family members may contain a signal anchor sequence that targets these proteins to the mitochondrial outer membrane. We have investigated the consequence of deleting this domain upon cytochrome c release in yeast strains that coexpress truncated forms of Bax (i.e. BaxΔ) and Bcl-xL (i.e. Bcl-xLΔ). We find that (i) BaxΔ is as efficient as full-length Bax in promoting cytochrome c release, but Bcl-xLΔ has remarkably reduced rescuing ability compared to full-length Bcl-xL; (ii) full-length Bcl-xL protein acts by relocalizing Bax from the mitochondrial fraction to the soluble cytosolic fraction; (iii) Bax undergoes N-terminal cleavage when expressed in yeast, which is prevented by coexpression of Bcl-xL, suggesting that Bcl-xL may mask the cleavage site of Bax through a direct physical interaction of the two proteins

Mitochondria-Associated Membranes (MAMs) are involved in Bax mitochondrial localization and cytochrome c release

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 signaling processes. We addressed a possible role of these domains, called Mitochondria-Associated Membranes (MAMs) in Bax localization and function, by expressing the human protein in a yeast mutant deleted of MDM34, a ERMES (ER-Mitochondria Encounter Structure) component. 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 incompletely released, MAMs-associated pool of cytochrome c

Vacuole-mitochondrial crosstalk during apoptosis induced by acetic acid in yeast

This work was supported by the FCT project PTDC/BIA-BCM/69448/200

What yeast can tell us about how cells commit suicide?

Multicellular organisms developed a complex system to balance cell proliferation and cell death in order to guarantee correct embryonic development and tissue homeostasis. Failure of cells to undergo programmed cell death (PCD) can potentially lead to severe diseases, including neural degeneration, autoimmunity and cancer. Identifying the molecules involved in PCD and understanding the regulation of the process are crucial for prevention and management of these diseases. Evidence of the enormous impact of PCD, of which apoptosis is the most frequent morphological phenotype, on human health makes it one of the today’s main research topics. Since PCD was initially considered specific of metazoans, biological models were first restricted to animal cells. Actually, based on the absence of known crucial PCD regulators, as indicated by plain homologies searches, as well as on the difficulty to explain the sense of cell suicide in a unicellular organism, it was not accepted that these organisms could possess a PDC mechanism. However, evidence has been reported in the last decade indicating that the process of self-destruction in different unicellular organisms, namely in yeast, can also take place. In the present communication, I will present the research we have been developing on PCD, based on the exploration/exploitation of yeast as a simple eukaryotic unicellular model system. Particular focus will be given to our more recent studies suggesting a complex regulation and interplay between mitochondria and the vacuole in acetic acid induced PCD. The validation in mammalian cell lines of the hypothesis postulated with the yeast model will be also discussed.Fundação para a Ciência e a Tecnologia (FCT

Mitophagie et contrôle qualité des mitochondries

Les mitochondries sont des organites très dynamiques qui jouent un rôle primordial dans les cellules et des dysfonctionnements mitochondriaux sont souvent associés à des pathologies. Le contrôle qualité des mitochondries s’effectue à différents niveaux : protéases et chaperonnes, système ubiquitine-protéasome et mitophagie. Il existe différents programmes de mitophagie qui fonctionnent de façon indépendante ou non et qui font appel à l’activité de récepteurs localisés dans la membrane externe de la mitochondrie. Chez les mammifères, des récepteurs spécifiques ont été identifiés comme NIX, BNIP3, FUNDC1, BCL2L13 et la cardiolipine, ainsi que la voie PINK1/Parkin. Dans cette revue, nous discutons des différents mécanismes moléculaires de ces voies impliquées dans la mitophagie chez les mammifères

The yeast mitophagy receptor Atg32 is ubiquitinated and degraded by the proteasome.

Mitophagy, the process that degrades mitochondria selectively through autophagy, is involved in the quality control of mitochondria in cells grown under respiratory conditions. 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. Post-translational modifications such as phosphorylation are crucial for the execution of mitophagy. In our study we monitor the stability of Atg32 protein in the yeast S. cerevisiae and show that Atg32 is degraded under normal growth conditions, upon starvation or rapamycin treatment. The Atg32 turnover can be prevented by inhibition of the proteasome activity, suggesting that Atg32 is also ubiquitinated. Mass spectrometry analysis of purified Atg32 protein revealed that at least lysine residue in position 282 is ubiquitinated. Interestingly, the replacement of lysine 282 with alanine impaired Atg32 degradation only partially in the course of cell growth, suggesting that additional lysine residues on Atg32 might also be ubiquitinated. Our results provide the foundation to further elucidate the physiological significance of Atg32 turnover and the interplay between mitophagy and the proteasome

The mitochondrial phosphatidylserine decarboxylase Psd1 is involved in nitrogen starvation-induced mitophagy in yeast

International audienceMitophagy, the selective degradation of mitochondria by autophagy, is a central process that is essential for the maintenance of cell homeostasis. It is implicated in the clearance of superfluous or damaged mitochondria and requires specific proteins and regulators to perform. In yeast, Atg32, an outer mitochondrial membrane protein, interacts with the ubiquitin-like Atg8 protein, promoting the recruitment of mitochondria to the phagophore and their sequestration within autophagosomes. Atg8 is anchored to the phagophore and autophagosome membranes thanks to a phosphatidylethanolamine tail. In Saccharomyces cerevisiae, several phosphatidylethanolamine synthesis pathways have been characterized, but their contribution to autophagy and mitophagy are unknown. Through different approaches, we show that Psd1, the mitochondrial phosphatidylserine decarboxylase, is involved in mitophagy induction only after nitrogen starvation, whereas Psd2, which is located in vacuole, Golgi and endosome membranes, is required preferentially for mitophagy induction in the stationary phase of growth but also to a lesser extent for nitrogen starvation-induced mitophagy. Our results suggest that the mitophagy defect observed in Δpsd1 yeast cells after nitrogen starvation may be due to a failure of Atg8 recruitment to mitochondria.This article has an associated First Person interview with the first author of the paper