The Dep1 protein: A new regulator of mitophagy in yeast.

Mitochondria play a crucial role in most eukaryotic cells. Mitophagy is a process that controls their quality and quantity within the cells. The outer mitochondrial membrane protein, Atg32, serves as the mitophagic receptor. It interacts with the Atg11 protein to initiate mitophagy and with the Atg8 protein to ensure the engulfment of mitochondria into the autophagosomes for elimination. The Atg32 protein is regulated at the transcriptional level but also by posttranslational modifications. In this study, we described a new regulator of mitophagy, the protein Dep1, identified as a part of the Rpd3L histone deacetylase complex. We showed that the Dep1 protein is localized in the nucleus and associated with mitochondria. This protein is needed for mitophagy and to regulate the transcription and expression of the Atg32 protein. The absence of this protein affects the mitophagy process induced by either starvation for nitrogen or the stationary phase of growth.

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

Mitophagy, 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.

The SEACIT complex is involved in the maintenance of vacuole-mitochondria contact sites and controls mitophagy.

The major signaling pathway that regulates cell growth and metabolism is under the control of the target of rapamycin complex 1 (TORC1). In Saccharomyces cerevisiae the SEA complex is one of the TORC1 upstream regulators involved in amino acid sensing and autophagy. Here, we performed analysis of the expression, interactions and localization of SEA complex proteins under different conditions, varying parameters such as sugar source, nitrogen availability and growth phase. Our results show that the SEA complex promotes mitochondria degradation either by mitophagy or by general autophagy. In addition, the SEACIT subcomplex is involved in the maintenance of the vacuole-mitochondria contact sites. Thus, the SEA complex appears to be an important link between the TORC1 pathway and regulation of mitochondria quality control.

Increased fatty acid synthesis inhibits nitrogen starvation-induced autophagy in lipid droplet-deficient yeast.

Macroautophagy is a degradative pathway whereby cells encapsulate and degrade cytoplasmic material within endogenously-built membranes. Previous studies have suggested that autophagosome membranes originate from lipid droplets. However, it was recently shown that rapamycin could induce autophagy in cells lacking these organelles. Here we show that lipid droplet-deprived cells are unable to perform autophagy in response to nitrogen-starvation because of an accelerated lipid synthesis that is not observed with rapamycin. Using cerulenin, a potent inhibitor of fatty acid synthase, and exogenous addition of palmitic acid we could restore nitrogen-starvation induced autophagy in the absence of lipid droplets.

Increased levels of reduced cytochrome b and mitophagy components are required to trigger nonspecific autophagy following induced mitochondrial dysfunction.

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.

Mlg1, a yeast acyltransferase located in ER membranes associated with mitochondria (MAMs), is involved in de novo synthesis and remodelling of phospholipids.

In cells, phospholipids contain acyl chains of variable lengths and saturation, features that affect their functions. Their de novo synthesis in the endoplasmic reticulum takes place via the cytidine diphosphate diacylglycerol (CDP-DAG) and Kennedy pathways, which are conserved in eukaryotes. PA is a key intermediate for all phospholipids (PI, PIPs, PS, PE, PC, PG and CL). The de novo synthesis of PA occurs by acylation of glycerophosphate leading to the synthesis of 1-acyl lysoPA and subsequent acylation of 1-acyl lysoPA at the sn-2 position. Using membranes from Escherichia coli overexpressing MLG1, we showed that the yeast gene MLG1 encodes an acyltransferase, leading specifically to the synthesis of PA from 1-acyl lysoPA. Moreover, after their de novo synthesis, phospholipids can be remodelled by acyl exchange with one and/or two acyl chains exchanged at the sn-1 and/or sn-2 position. Based on shotgun lipidomics of the reference and mlg1Δ strains, as well as biochemical assays for acyltransferase activities, we identified an additional remodelling activity for Mlg1p, namely, incorporation of palmitic acid into the sn-1 position of PS and PE. By using confocal microscopy and subcellular fractionation, we also found that this acyltransferase is located in ER membranes associated with mitochondria, a finding that highlights the importance of these organelles in the global cellular metabolism of lipids.

Mitochondrial degradation in acetic acid-induced yeast apoptosis: the role of Pep4 and the ADP/ATP carrier.

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.

Mitophagy in Yeast: Decades of Research.

Mitophagy, the selective degradation of mitochondria by autophagy, is one of the most important mechanisms of mitochondrial quality control, and its proper functioning is essential for cellular homeostasis. In this review, we describe the most important milestones achieved during almost 2 decades of research on yeasts, which shed light on the molecular mechanisms, regulation, and role of the Atg32 receptor in this process. We analyze the role of ROS in mitophagy and discuss the physiological roles of mitophagy in unicellular organisms, such as yeast; these roles are very different from those in mammals. Additionally, we discuss some of the different tools available for studying mitophagy.

Mitophagy in yeast: actors and physiological roles.

Mitochondria are essential for oxidative energy production in aerobic eukaryotic cells, where they are also required for multiple biosynthetic pathways to take place. Mitochondria also monitor and evaluate complex information from the environment and intracellular milieu, including the presence or absence of growth factors, oxygen, reactive oxygen species, and DNA damage. It follows that disturbances of the integrity of mitochondrial function lead to the disruption of cell function, expressed as disease, aging, or cell death. It has been assumed that the degradation of damaged mitochondria by an autophagy-related pathway specific to mitochondria (mitophagy), recently found to be strictly regulated, is a fundamental process essential for cell homeostasis. Until now, the main role of mitophagy has been tentatively defined as a 'house-cleaning' pathway that allows to eliminate altered mitochondria, but mitophagy may also play a role in the adaptation of the number and quality of mitochondria to new environmental conditions. In yeast, recent data defined two categories of mitophagy actors: ones constitutively required for mitophagy and those with mitophagy-regulatory functions. Situations were also uncovered in normal physiology in which cells utilize mitophagy to eliminate damaged, dysfunctional, and superfluous mitochondria to adjust to changing physiological demands.

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.

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 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.

Dietary methionine deficiency affects oxidative status, mitochondrial integrity and mitophagy in the liver of rainbow trout (Oncorhynchus mykiss).

The low levels of methionine in vegetable raw materials represent a limit to their use in aquafeed. Methionine is considered as an important factor in the control of oxidative status. However, restriction of dietary methionine has been shown to reduce generation of mitochondrial oxygen radicals and thus oxidative damage in liver. Here, we aim to evaluate the effect of dietary methionine deficiency in hepatic oxidative status in rainbow trout and identify the underlying mechanisms. Fish were fed for 6 weeks diets containing two different methionine concentrations: deficient (MD, Methionine Deficient diet) or adequate (CTL, control diet). At the end of the experiment, fish fed the MD diet showed a significantly lower body weight and feed efficiency compared to fish fed the CTL diet. Growth reduction of the MD group was associated to a general mitochondrial defect and a concomitant decrease of the oxidative status in the liver. The obtained results also revealed a sharp increase of mitochondrial degradation through mitophagy in these conditions and emphasized the involvement of the PINK1/PARKIN axis in this event. Collectively, these results provide a broader understanding of the mechanisms at play in the reduction of oxidant status upon dietary methionine deficiency.

[Role of mitophagy in the mitochondrial quality control]. / Mitophagie et contrôle qualité des mitochondries.

Mitochondria are highly dynamic organelles that provide essential metabolic functions and represent the major bioenergetic hub of eukaryotic cells. Mitochondrial dysfunctions are implicated in numerous diseases. Therefore, maintenance of a healthy pool of mitochondria is required for cellular function and survival. Mitochondrial quality control is achieved through several mechanisms that act at different levels: proteases and chaperones, the Ubiquitin-Proteasome-System (UPS) and mitophagy. Multiple mitophagy-involved programs operate independently or undergo crosstalk, and require modulated receptor activities at the outer membranes of mitochondria. In mammals, different mitophagy effectors have been characterized such as the receptors NIX, BNIP3, FUNDC1, BCL2L13, cardiolipin and the PINK1/Parkin pathway. Here we discuss the different molecular mechanisms of these mitophagy involved pathways.

Lipid oxidation and autophagy in yeast.

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.

Ypr140wp, 'the yeast tafazzin', displays a mitochondrial lysophosphatidylcholine (lyso-PC) acyltransferase activity related to triacylglycerol and mitochondrial lipid synthesis.

When the yeast protein Ypr140w was expressed in Escherichia coli, a lyso-PC [lysophosphatidylcholine (1-acylglycerophosphorylcholine)] acyltransferase activity was found associated with the membranes of the bacteria. To our knowledge, this is the first identification of a protein capable of catalysing the acylation of lyso-PC molecules to form PC. Fluorescence microscopy analysis of living yeasts revealed that the fusion protein Ypr140w-green fluorescent protein is targeted to the mitochondria. Moreover, in contrast with wild-type cells, in the absence of acyl-CoA, the yeast mutant deleted for the YPR140w gene has no lyso-PC acyltransferase activity associated with the mitochondrial fraction. When yeast cells were grown in the presence of lactate, the mutant synthesized 2-fold more triacylglycerols when compared with the wild-type. Moreover, its mitochondrial membranes contained a lesser amount of PC and cardiolipin, and the fatty acid composition of these latter was greatly changed. These modifications were accompanied by a 2-fold increase in the respiration rates (states 3 and 4) of the mitochondria. The relationship between the deletion of the YPR140w gene and the lipid composition of the ypr140wDelta cells is discussed.

Regulation of Bax/mitochondria interaction by AKT.

Bax-dependent mitochondrial permeabilization during apoptosis is controlled by multiple factors, including the phosphorylation by the protein kinase AKT. We used the heterologous co-expression of human Bax and AKT1 in yeast to investigate how the kinase modulates the different steps underlying Bax activation. We found that AKT activated Bax and increased its cellular content. Both effects were dependent on Ser184, but a phosphorylation of this residue did not fully explain the effects of AKT. Additional experiments with mutants substituted on Ser184 suggested that the regulation of Bax dynamic equilibrium between the cytosol and mitochondria might be more tightly regulated by Bcl-xL when Bax is phosphorylated.

Looking at the metabolic consequences of the colchicine-based in vivo autophagic flux assay.

Monitoring autophagic flux in vivo or in organs remains limited and the ideal methods relative to the techniques possible with cell culture may not exist. Recently, a few papers have demonstrated the feasibility of measuring autophagic flux in vivo by intraperitoneal (IP) injection of pharmacological agents (chloroquine, leupeptin, vinblastine, and colchicine). However, the metabolic consequences of the administration of these drugs remain largely unknown. Here, we report that 0.8 mg/kg/day IP colchicine increased LC3-II protein levels in the liver of fasted trout, supporting the usefulness of this drug for studying autophagic flux in vivo in our model organism. This effect was accompanied by a decrease of plasma glucose concentration associated with a fall in the mRNA levels of gluconeogenesis-related genes. Concurrently, triglycerides and lipid droplets content in the liver increased. In contrast, transcript levels of ß-oxidation-related gene Cpt1a dropped significantly. Together, these results match with the reported role of autophagy in the regulation of glucose homeostasis and intracellular lipid stores, and highlight the importance of considering these effects when using colchicine as an in vivo "autophagometer."

Investigation of the role of the C-terminus of Bax and of tc-Bid on Bax interaction with yeast mitochondria.

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.

Insights into the relationship between the proteasome and autophagy in human and yeast cells.

In eukaryotes, the ubiquitin-proteasome system (UPS) and autophagy are two major intracellular protein degradation pathways. Several lines of evidence support the emerging concept of a coordinated and complementary relationship between these two processes, and a particularly interesting finding is that the inhibition of the proteasome induces autophagy. Yet, there is limited knowledge of the regulation of the UPS by autophagy. In this study, we show that the disruption of ATG5 and ATG32 genes in yeast cells under both nutrient-deficient conditions as well as stress that causes mitochondrial dysfunction leads to an activation of proteasome. The same scenario occurs after pharmacological inhibition of basal autophagy in cultured human cells. Our findings underline the view that the two processes are interconnected and tend to compensate, to some extent, for each other's functions.

Bcl-xL stimulates Bax relocation to mitochondria and primes cells to ABT-737.

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.