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

Mitochondrial degradation in acetic acid-induced yeastapoptosis: the role of Pep4 and the ADP/ATP carriermmi_7122 1398..1410

Clara Pereira,1 Susana Chaves,1 Sara Alves,1

Bénédict Salin,2 Nadine Camougrand,2

Stéphen Manon,2 Maria João Sousa1 andManuela Côrte-Real1*1CBMA (Centre of Molecular and EnvironmentalBiology)/Department of Biology, University of Minho,Campus de Gualtar, 4710-057 Braga, Portugal.2CNRS and Université de Bordeaux 2, UMR5095,Bordeaux, France.

Summary

We have previously shown that acetic acid activates amitochondria-dependent death process in Saccharo-myces cerevisiae and that the ADP/ATP carrier (AAC)is required for mitochondrial outer membrane perme-abilization and cytochrome c release. Mitochondrialfragmentation and degradation have also been shownin response to this death stimulus. Herein, we showthat autophagy is not active in cells undergoing aceticacid-induced apoptosis and is therefore not respon-sible for mitochondrial degradation. Furthermore, wefound that the vacuolar protease Pep4p and the AACproteins have a role in mitochondrial degradationusing yeast genetic approaches. Depletion andoverexpression of Pep4p, an orthologue of humancathepsin D, delays and enhances mitochondrial deg-radation respectively. Moreover, Pep4p is releasedfrom the vacuole into the cytosol in response toacetic acid treatment. AAC-deleted cells also show adecrease in mitochondrial degradation in response toacetic acid and are not defective in Pep4p release.Therefore, AAC proteins seem to affect mitochondrialdegradation at a step subsequent to Pep4p release,possibly triggering degradation through their involve-ment in mitochondrial permeabilization. The findingthat both mitochondrial AAC proteins and the vacu-olar Pep4p interfere with mitochondrial degradationsuggests a complex regulation and interplay betweenmitochondria and the vacuole in yeast programmedcell death.

Introduction

Yeast mitochondria undergo both structural and functionalchanges and play a key role in the apoptotic death processactivated by acetic acid (Ludovico et al., 2002; 2003).Acetic acid induces different ultrastructural mitochondrialalterations in yeast, including mitochondrial swelling,reduction in cristae number and formation of myelinicbodies. Additionally, Saccharomyces cerevisiae cells com-mitted to apoptosis in response to acetic acid displaymitochondrial ROS accumulation, a transient hyperpolar-ization followed by depolarization, and a decrease in theactivity of COX affecting mitochondrial respiration. Mito-chondria become permeabilized in response to acetic acid,allowing the release of lethal factors like cytochrome c(Ludovico et al., 2002) and yeast AIF (Wissing et al.,2004), thus contributing to the death process. Other mito-chondrial proteins also affect the final cell fate, includingthose involved in fission/fusion (Fannjiang et al., 2004) andhomologues of the components of mammalian permeabil-ity transition pore (Pereira et al., 2007).

Mitochondrial degradation following induction of apop-tosis is a common feature in mammalian cells (reviewed inTolkovsky et al., 2002). This event usually occurs throughan autophagic process that shows selectivity for mito-chondria, termed mitophagy (Lemasters, 2005). It is notknown how the damaged mitochondria in a cell are selec-tively removed, but recent evidence supports the view thatmitochondria permeability transition (MPT) could be thetrigger for this removal (Rodriguez-Enriquez et al., 2006;Kim et al., 2007). The role of mitochondrial degradation incell death, either by a selective or non-selective autoph-agic process, is still unclear. It has been proposed it has acytoprotective function, because it allows the removal ofdeleterious ROS-producing mitochondria and hinders therelease of pro-apoptotic proteins to the cytosol. On theother hand, the catabolism of dysfunctional organellescould facilitate the apoptotic process by preventing anenergy drain caused by defective chemiosmotic coupling(when the MPT pore opens, ions equilibrate between thematrix and cytosol), while at the same time providing asource of ATP generation (Wallace, 2005; Maiuri et al.,2007; Terman et al., 2007).

The accumulation of damaged mitochondria is consid-ered to underlie a wide range of age-related disorders,

Accepted 2 March, 2010. *For correspondence. E-mail [email protected]; Tel. (+351) 253 604 314; Fax (+351) 253 678 980.

Molecular Microbiology (2010) 76(6), 1398–1410 � doi:10.1111/j.1365-2958.2010.07122.xFirst published online 6 April 2010

© 2010 Blackwell Publishing Ltd

various forms of cancer and late-onset genetic diseases(Zeviani and Carelli, 2007; Zhang and Qi, 2008). Thusunderstanding how mitochondria are degraded mightunravel attractive targets for the development of noveltherapeutics for different diseases.

Mitochondrial degradation in yeast cells undergoingapoptosis has been previously described (Fannjianget al., 2004). In yeast, selective removal of mitochondriawas reported following heterologous expression of Bax(Kissova et al., 2006), mitochondrial dysfunctions (Priaultet al., 2005), osmotic swelling (Nowikovsky et al., 2007)and in stationary phase cells (Tal et al., 2007). Removal ofmitochondria is not always dependent on the autophagicmachinery (Kissova et al., 2006), and the outcome interms of survival obtained by blocking mitochondrial deg-radation varies with the stimulus used (recently reviewedin Tolkovsky, 2009).

In this work, we proposed to study the mechanismresponsible for mitochondrial degradation during aceticacid-induced apoptosis and to assess the role of Pep4pand the ADP/ATP carrier (AAC) proteins/mitochondrialpermeabilization in this process. We found that autophagyis not active during acetic acid-induced apoptosis. Alter-natively, the vacuolar protease Pep4p is translocated tothe cytosol and, together with the AAC proteins, plays animportant role in mitochondrial degradation. We proposethat removal of mitochondria can occur in yeast undergo-ing apoptosis through an autophagy-independentpathway involving the vacuolar protease Pep4p and themitochondrial AAC proteins. Moreover, we propose thatthe AAC proteins might relay a signal of mitochondrialdysfunction, targeting their destruction.

Results

Autophagy is not activated during aceticacid-induced apoptosis

Mitochondria play a key role in yeast apoptotic deathactivated by different lethal stimuli (reviewed in Eisen-berg et al., 2007; Pereira et al., 2008), in particular byacetic acid (Ludovico et al., 2002), and are fragmentedand subsequently removed in a late phase of the deathprocess (Fannjiang et al., 2004). It was our purpose toassess the contribution of autophagy to the process ofmitochondrial degradation that occurs during acetic acid-induced apoptosis. To this end, we used an establishedbiochemical method that follows the delivery of the vacu-olar Pho8D60p, a truncated form of the yeast vacuolaralkaline phosphatase (ALP) lacking the N-terminal 60amino acids (Noda et al., 1995). This truncated proteinaccumulates in the cytosol as a zymogen and is deliv-ered to the vacuole exclusively by an autophagicprocess, where the active form of the enzyme is gener-

ated by proteolytic cleavage (Noda et al., 1995). There-fore, in cells only expressing this truncated form of theenzyme, the level of ALP activity reflects the level ofautophagy.

A S. cerevisiae W303 strain in which the chromosomalPHO8 gene was replaced with pho8D60, was incubatedin the presence of 180 mM acetic acid and ALP activitydetermined after 0, 1 and 3 h treatment. We observedthat there was no increase in ALP activity following aceticacid treatment, indicating there was no transport of mate-rial into the vacuole (Fig. 1A). Nitrogen-starved cellswere used as a positive control for autophagy inductionand displayed a more than twofold increase in ALP activ-ity (Fig. 1A). Atg5p belongs to the autophagic pathway inyeast, where it takes part of a cytosolic complex essen-tial for the autophagosome formation/completion, and itsdisruption results in defective macroautophagy (Kame-taka et al., 1996; George et al., 2000). We therefore usedthe atg5D strain as a negative control for autophagyinduction. As expected, there was no activation of ALP innitrogen-starved cells without ATG5. The basal level ofALP activity found in control cells (t0) of both the W303and atg5D strains is probably due to Pho13p, a cytosolicvariant of ALP that is constitutively active and sensitive toacid pH (Tuleva et al., 1998). Consistently, acetic acidleads to a reduction of this activity after 3 h of treatment(Fig. 1A).

We also assessed autophagy induction by monitoringthe amount of Atg8p. This protein is essential for autopha-gosome formation and its increase in most of the cellularcontexts is a diagnostic marker of autophagy (Kirisakoet al., 1999). In accordance, we observed an increase inthe amount of Atg8p in nitrogen-starved cells used as apositive control (Fig. 1B). In contrast, we did not observean increase in Atg8p during acetic acid treatment(Fig. 1B), consistent with the lack of ALP activity. Cellviability (clonogenicity) in response to acetic acid was alsonot affected by an ATG5 deletion further indicating thatautophagy is not involved (Fig. 1C).

A typical, although not exclusive, morphological alter-ation of mitochondria during apoptosis, either in highereukaryotes or yeast, is the change from a tubular networkto a punctuated pattern. We observed a rapid fragmenta-tion of the network into small round units in response toacetic acid, using W303 and atg5D cells expressing aGFP fused downstream of the targeting sequence ofmitochondrial citrate synthase (Okamoto et al., 1998)(Fig. 2A). These fragmented mitochondria persisted forsome time until the fluorescence started to diffusethroughout the cell. No discrete vacuolar localization fol-lowing acetic acid treatment was ever observed for bothstrains. In contrast, during nitrogen starvation, W303 cellsexhibited an evident green fluorescence inside the vacu-oles, pointing to the internalization of mitochondria due to

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© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 76, 1398–1410

autophagy activation (Fig. 2B) as described (Kissovaet al., 2004). Analysis of acetic acid treated cells by trans-mission electron microscopy confirmed that mitochondriawere not internalized by the vacuole (Fig. S1).

Pep4p translocates from the vacuole to the cytosol inresponse to acetic acid

Saccharomyces cerevisiae Pep4p, or proteinase A, is apepsin-like aspartic protease found in the yeast vacuolewith homology to human cathepsin D. Pep4p was shownto translocate from the yeast vacuole to the cytosol duringhydrogen peroxide- (Mason et al., 2005) and actin cytosk-eleton stabilization-induced apoptosis (Gourlay andAyscough, 2006). This was achieved using a Pep4–EGFPfusion (Mason et al., 2005), which allowed visualization ofthe protein distribution in live cells. To address whetheracetic acid could also trigger the release of vacuolarPep4p we used the same Pep4–EGFP fusion. Asexpected, the Pep4–EGFP fusion protein localized spe-cifically to the vacuole in non-treated healthy cells (Fig. 3).After 60 min treatment with 180 mM acetic acid, an extra-vacuolar fluorescence started to be observed in somecells, indicative of the presence of Pep4p in the cytosol(Fig. 3). Transmission electron microscopy analysisshowed that vacuolar membrane integrity was preserved(Fig. S1). In addition, the Pep4p release was observed incells which maintained plasma membrane integrity asassessed by propidium iodide (PI) co-staining (Fig. 3).Together, these observations are consistent with dataobtained with mammalian cells showing that there is apartial release of cathepsins from the lysosomes in the

Fig. 1. Acetic acid does not induceautophagy.A. ALP activity in S. cerevisiae W303 or atg5Dcells (negative control for autophagyinduction) carrying a pho8D60 mutation inreplacement of wild-type PHO8, aftertreatment with 180 mM acetic acid for 1 and3 h. Nitrogen-starved cells (N-) for 15 h wereused as a positive control for ALP activity.ALP activities are expressed as fluorescencearbitrary units·min-1 mg-1 protein. Values aremeans � SD of four independentexperiments. ***P < 0.001, **P < 0.01;unpaired t-test.B. The levels of Atg8p, a protein essential forautophagosome formation, are not increasedby acetic acid treatment (180 mM) as verifiedby Western blot. Increased accumulation ofAtg8p was observed for the nitrogen-starvedcells (N-, 15 h) used again as a positivecontrol.C. Cell survival of S. cerevisiae W303 andatg5D strains (percentage of cfu on YEPDAplates; 100% corresponds to the number ofcfu at time 0). Cells were incubated for200 min with 100–220 mM acetic acid. Valuesare means � SD of five independentexperiments; P > 0.05 (atg5D versus W303cells); two-way ANOVA.

Fig. 2. Acetic acid-induced mitochondrial fragmentation isindependent of the autophagic protein Atg5p and fragmentedmitochondria are not internalized by the vacuole in aceticacid-treated cells.A. W303 and atg5D cells expressing mitochondrial matrix-targetedGFP (mtGFP) were treated with 180 mM acetic acid for 30 min.B. W303-1B cells expressing mtGFP, under nitrogen starvationconditions for 10 h. Bar, 4 mm.

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early phase of apoptosis (Brunk et al., 1995; Bidere et al.,2003).

Pep4p is involved in mitochondrial degradation and hasa pro-survival role during acetic acid-induced apoptosis

To assess the role of Pep4p in mitochondrial degradation,we compared a strain disrupted in the gene PEP4(Marques et al., 2006) with the respective W303 parentalstrain, and a W303 strain transformed with pDP34 (emptyvector, multicopy) with the same strain transformed withpDP34-PEP4 (Rupp and Wolf, 1995) to overexpressPEP4. Mitochondrial degradation in these strains wasassessed by monitoring the loss of GFP fused down-stream of the targeting sequence of mitochondrial citratesynthase (Okamoto et al., 1998) by flow cytometry. Detec-tion of mitochondrial degradation through matrix-targetedGFP allows the assessment of protein abundance inwhole live cells, and reflects the balance between proteinsynthesis and degradation. Therefore, the observation ofa decrease of GFP fluorescence could be attributed to theinhibition of protein synthesis rather than mitochondrialdegradation. This hypothesis was precluded by theobserved maintenance of galactose inducible GFP cellfluorescence until 14 h after blockage of GFP synthesis byglucose (data not shown). Thus, we assumed that adecrease in total GFP fluorescence caused by acetic acidmainly reflects protein degradation. Additionally, decreaseof GFP fluorescence levels as a reliable measure of mito-chondrial degradation was corroborated by biochemicalevidence through Western blot analysis (Fig. S2). Fig-ure S3 shows a typical histogram obtained for each strain

after treatment with acetic acid, which allowed the quan-tification of the percentage of cells displaying loss of mito-chondrial fluorescence over time. We observed a delay inGFP disappearance in the PEP4-deleted strain and aslight acceleration in GFP loss when PEP4 was overex-pressed (Fig. 4), indicating an increase and a decreasein mitochondrial degradation respectively. We alsoassessed the effect of acetic acid (100–220 mM) on cellviability in the PEP4-deleted and PEP4-overexpressingstrains. We observed a decrease in the cell survival ofpep4D compared with the W303 strain, and an increase inthe W303 pDP34-PEP4 strain compared with W303pDP34 (Fig. 5). We therefore conclude that Pep4p andthe ensuing degradation of mitochondria have a pro-survival role during acetic acid-induced apoptosis. In addi-tion, changes in GFP disappearance in PEP4-deleted andoverexpressing strains were not the result of a slower andfaster death rate, respectively.

In order to assess if deletion of PEP4 affected the modeof cell death, we monitored cytochrome c release, super-oxide accumulation, chromatin condensation, and loss of

Fig. 3. Pep4p translocates from the vacuole to the cytosol duringacetic acid-induced apoptosis. Release of the protease Pep4p fromthe vacuole due to acetic acid treatment (AA; 180 mM, 60 min) wasassessed in living cells using a W303 strain expressing aPEP4–EGFP fusion protein. Upper panels show control cells withthe Pep4p–EGFP fluorescence localized in the vacuole (visible inthe differential interference contrast) and the lower panels showsome cells with the PEP4–EGFP fusion already in the cytosol(indicated by arrow heads). Release of Pep4p precedes loss ofplasma membrane integrity as assessed by propidium iodide (PI)staining. In the insert we can see a dead cell with a strong brightunspecific green staining that also stains with PI. Bar, 10 mm.

Fig. 4. Pep4p is involved in mitochondrial degradation triggeredby acetic acid. Quantification of the percentage of cells displayingGFP loss over treatment with acetic acid (180 mM) for strainsW303, pep4D, W303 pDP34 and W303 pDP34-PEP4 transformedwith a mitochondrial matrix-targeted GFP. Loss of GFPfluorescence was used as a measure of mitochondrial degradation.Values are means � SD of four independent experiments.P < 0.001 (pep4D versus W303), P < 0.05 (W303 pDP34-PEP4versus W303 pDP34); two-way ANOVA.



plasma membrane integrity. During acetic acid treatment(180 mM), the pep4D mutant, displayed cytochrome crelease (Fig. 6A) and superoxide accumulation similar tothe wild-type (Fig. S4). However, the mutant strain exhi-bited earlier emergence of chromatin condensation(Fig. 6B) and loss of plasma membrane integrity (Fig. 6C).These data indicate that the absence of Pep4p acceler-ates apoptosis and secondary necrosis.

Absence of AAC proteins is also associated with adecrease in the degradation of mitochondria

Because we have previously reported that the absence ofthe inner mitochondrial membrane AAC impairs mitochon-drial outer membrane permeabilization and cytochrome crelease (Pereira et al., 2007), we questioned whetherthese proteins could also have a role on the onset ofmitochondrial degradation. We therefore characterizedmitochondrial degradation in AAC-deleted cells uponacetic acid-induced apoptosis. Mitochondrial degradationin aac1/2/3D cells (lacking the three yeast isoforms of the

ATP/ADP carrier) was assessed as in the pep4D strain bymonitoring loss of GFP fluorescence by flow cytometry.We observed a delay in GFP disappearance in the AAC-deleted strain (Fig. 7A). Additionally, we also validated theuse of mitochondrial matrix-targeted GFP fluorescenceloss to monitor mitochondrial degradation by Westernblot, in the wild-type and aac1/2/3D strains (Fig. 7B). Forthis purpose we monitored the degradation of two outermitochondrial proteins, because we have previouslyshown that acetic acid triggers a selective and fasterdegradation of cytochrome c and the inner mitochondrialmembrane protein Atp2p in the aac1/2/3D mutant (Pereiraet al., 2007). Western blots of total protein extracts col-lected at increasing times following acetic acid treatmentshowed that the degradation of Por1p and Tom22p wasdelayed in the aac1/2/3D mutant, in comparison withW303 strain (Fig. 7B). These results support our interpre-tation that AAC proteins, besides Pep4p, are required forefficient mitochondrial degradation.

We next investigated whether the lower degradation ofaac1/2/3D mutant mitochondria could be due to a hin-drance in the release of Pep4p from the vacuole. Theaac1/2/3D strain was therefore transformed with thePep4p–EGFP fusion protein. In aac1/2/3D cells withoutacetic acid treatment, Pep4p–EGFP localizes correctly tothe vacuole (Fig. 7C). Upon acetic acid treatment, thePep4p–EGFP fusion protein translocates to the cytosol in asimilar extent as that observed for the wild-type (Fig. 7C).

Absence of AAC and Pep4 proteins is associated withthe formation of mitochondrial clusters during aceticacid-induced apoptosis

The possibility that the delayed mitochondrial degradationin both aac1/2/3D and pep4D strains could be associatedwith altered mitochondrial morphology was ascertained.Treatment of these mutant strains with acetic acid leads toa very fast (less than 30 min) disorganization of the typicaltubular network, similarly to the W303 strain. Notably, bothmutant strains displayed a high percentage of cells(almost 70% in the culture) with a clustered mitochondrialmorphology after treatment with acetic acid. Figure 8Aillustrates cells displaying a clustered mitochondrial mor-phology as well as their respective quantification. Accord-ingly, the percentage of clusters in the culture decreasedwhen PEP4 was overexpressed (Fig. 8A). Formation ofmitochondrial aggregates was also observed duringhydrogen peroxide-induced apoptosis (Fig. 8B), indicat-ing that this peculiar morphology is not an effect restrictedto acetic acid treatment. We decided to further investigateif this altered mitochondrial morphology corresponded todysfunctional mitochondria. We could observe that mito-chondrial clusters were still able to accumulate the mem-brane potential-sensitive probe DiOC6(3) or Mitotracker

Fig. 5. Pep4p has a pro-survival role during acetic acid-inducedapoptosis. Cell survival of S. cerevisiae strains W303 and pep4D(upper image) and W303 pDP34 and W303 pDP34-PEP4 (lowerimage) (percentage of cfu on YEPDA plates; 100% corresponds tothe number of cfu at time 0). Strains were incubated for 200 minwith 100–220 mM acetic acid. Values are means � SD of fourindependent experiments; P < 0.001 (pep4D versus W303 cells),P < 0.05 (W303 pDP34 versus W303 pDP34-PEP4); two-wayANOVA.



Red CMXRos in most acetic acid-treated cells, indicatingthat clustered mitochondria were still functional.Figure 8C illustrates the colocalization of green and redfluorescence in aac1/2/3D cells expressing mt-GFP andstained with Mitotracker Red CMXRos. Quantification ofmitochondrial membrane potential showed that for longeracid treatments all strains tested (W303, aac1/2/3D andpep4D) began to depolarize. However, while the W303strain, as reported by Ludovico et al. (2002), and thepep4D strain displayed an initial transient hyperpolariza-tion in response to acetic acid, the same did not occur forthe aac1/2/3D mutant (Fig. 8D). This observation raisesthe possibility that a causal relation may exist betweenhyperpolarization and cytochrome c release.

To discard the possibility that mitochondrial morphologyin the aac1/2/3D mutant could be a consequence of lowmitochondrial energetic levels (AAC proteins exchange

cytosolic ADP for mitochondrial synthesized ATP acrossthe mitochondrial inner membrane), we monitored mito-chondrial morphology changes accompanying deathinduced by acetic acid in an atp2D strain. This mutant isdeleted for subunit b of the ATPase complex. We couldconfirm that the spots originated from the fragmentation ofthe mitochondrial network in the atp2D strain were ofnormal size and similar to those observed in the W303strain (Fig. 8E).

Depolymerization of actin filaments disrupts themitochondrial clusters formed in the absence of AACand Pep4 proteins

In yeast, mitochondria associate with actin filaments, anddepolymerization of these filaments alters the normalmitochondrial morphology giving rise to short tubular or

Fig. 6. pep4D strain displays cytochrome crelease and an earlier emergence ofchromatin condensation and loss of plasmamembrane integrity than the wild-type strain.Cultures of W303 and pep4D were grown(until OD 1) and treated with 180 mM aceticacid in YEPGal for all the experiments.A. Cytochrome c detected by Western blot inmitochondria and post-mitochondrialsupernatants of pep4D cells obtained after200 min treatment. Tom22p from outermitochondrial membrane and cytosolicphosphoglycerate kinase (Pgk1p) were alsoassessed.B. Quantification of chromatin condensation inW303 and pep4D mutant. The insert shows aphotomicrograph of pep4D cells stained withDAPI as an example of chromatincondensation. Chromatin condensation in thepep4D mutant is significantly enhanced, whencompared with the W303 strain. P < 0.05(pep4D versus W303 cells); two-way ANOVA.At time 100 min, the percentage of cfu in theW303 and pep4D strains was 50% and 3.9%respectively.C. Percentage of cells displaying loss ofplasma membrane integrity assessed by flowcytometry after 200 and 500 min treatmentwith acetic acid Images show monoparametrichistograms of PI fluorescence (FL3 area(log)). Data represent one of four independentexperiments.



small spherical structures (Boldogh et al., 1998).Latrunculin-A (lat-A) is a drug used for rapid depolymer-ization of F-actin (Spector et al., 1983). The mitochondrialnetwork was broken within 30 min of treatment with0.25 mM lat-A (dissolved in DMSO) in W303 and pep4D(data not shown) as well as in aac1/2/3D mutant cells(Fig. 9A), whereas the same cells treated with an equalvolume of DMSO presented no alterations in the mito-chondrial network organization. Figure 9A illustrates theeffect of lat-A or DMSO addition to acetic acid-treatedaac1/2/3D cultures (180 mM, 30 min). The percentageof aac1/2/3D cells containing clustered mitochondriadecreased to about one half (46.4% decrease), while thenumber of cells containing small spots of mitochondriaincreased (Fig. 9A, lower panels). In addition, incubationof the cells with lat-A prior to acetic acid treatment alsoprevented formation of the clusters. Taken together, theseresults reinforce the idea that clusters are composed ofaggregates of mitochondria and show that actin filamentsare responsible for the formation and maintenance ofsuch mitochondrial clusters.

Formation of these clusters appears to be a reversibleevent. Removal of acetic acid after up to 30 min of treat-ment allowed an almost full recovery of the tubular shape of

the mitochondrial network both in the W303 and aac1/2/3Dstrains, though the recovery of the latter strain was slowerthan that of the W303 strain (Fig. 9B). For longer periods ofincubation with acetic acid, the ability of both strains torecover normal mitochondrial morphology declined, andthis was even more apparent in the aac1/2/3D mutant,likely due to its altered mitochondrial morphology. Theseobservations further strengthen the idea that the clusteredmitochondria induced by acetic acid treatment in aac1/2/3D and pep4D strains are not internalized by the vacuoleand most likely reside in the cytosol, hence allowing forrecovery of their filamentous shape.

Discussion

Mitochondrial degradation has been shown to occur in anumber of systems following apoptosis induction andusually involves an autophagic process. However, mito-chondrial degradation proceeded normally in a straininactivated for ATG5 during the lens and erythroid celldifferentiation (Matsui et al., 2006). Therefore, it has beensuggested that degradation system(s) other than autoph-agy may account for mitochondrial degradation in distinctcellular settings. Our results suggest that this also

Fig. 7. Mitochondrial degradation is impaired in the absence of AAC proteins.A. Quantification of mitochondrial degradation for W303 (full line) and aac1/2/3D (dotted line) mutant along treatment with 180 mM acetic acid,assessed by loss of GFP fluorescence by flow cytometry. Values are means � SD of five independent experiments. *P < 0.05; unpaired t-test.B. Western blots for Tom22p and Por1p from W303 and aac1/2/3D mutant cell lysates prepared at the indicated times after treatment with180 mM acetic acid as a measure of mitochondrial degradation. Quantification is shown below. Cytosolic phosphoglycerate kinase (Pgk1p)level was used to normalize protein amount loaded on the gel. Values are means � SD of three independent experiments; P < 0.001 forPorin1 and P < 0.05 for Tom22 (aac1/2/3D versus W303 cells); two-way ANOVA.C. Release of the Pep4p protease from the vacuole due to acetic acid treatment (AA; 180 mM, 60 min) was assessed in aac1/2/3D cellsexpressing a PEP4–EGFP fusion protein. Release of PEP4–EGFP to the cytosol (indicated by the arrow heads) was not affected by theabsence of AAC proteins. Bar, 10 mm.



Fig. 8. aac1/2/3D and pep4D strains show alteration from a tubular mitochondrial morphology to a clustered pattern upon acetic acidtreatment.A. Photomicrograph obtained by differential interference contrast and fluorescence microscopy illustrating the alteration of the tubularmitochondrial network to a clustered mitochondrial morphology after acetic acid treatment (180 mM, 60 min) and quantification of thepercentage of cells displaying the clustered morphology for strains W303, pep4D, aac1/2/3D, W303 pDP34 and W303 pDP34-PEP4. Valuesare means � SD of four independent experiments with at least 300 cells counted per sample. **P < 0.01; unpaired t-test.B. W303 and aac1/2/3D cells treated with H2O2 (3 mM, 30 min). H2O2 treatment also leads to formation of mitochondria aggregates inaac1/2/3D. Bar, 4 mm.C. Mitochondrial membrane potential in S. cerevisiae W303 and aac1/2/3D cells, non-treated and treated with acetic acid (AA; 180 mM,30 min), was examined by Mito-Tracker Red staining (middle panels) and compared with mitochondrial matrix-targeted GFP (left panels) in themerged images (right panels). Bar, 4 mm.D. Quantification of mitochondrial membrane potential (relative fluorescence values) for strains W303, aac1/2/3D and pep4D along treatmentwith acetic acid (180 mM) assessed by flow cytometry using the probe DiOC6(3). Fluorescence at t0 was considered as 100%. The resultspresented are the mean values of at least three independent experiments.E. atp2D strain non-treated and treated with acetic acid (AA; 180 mM, 30 min). atp2D cells do not form clusters. Bar, 4 mm.



happens during acetic acid treatment of yeast, becausethere is no uptake of proteins from the cytosol to thevacuole or an increase in Atg8p, two events typicallyobserved during autophagy. Recently, Pep4p was shownto leave the vacuole during hydrogen peroxide- (Mason

et al., 2005) or actin stabilization-induced apoptosis(Gourlay and Ayscough, 2006). In the first case, it wasalso shown that Pep4p, like its mammalian orthologuecathepsin D (Tang and Wong, 1987), is released withoutmajor vacuolar rupture and is involved in nucleoporindegradation (Mason et al., 2005).

We observe that Pep4p is also released from the vacuoleduring acetic acid-induced apoptosis and has an importantrole in mitochondrial degradation during the apoptoticprocess. Whether Pep4p acts directly on mitochondria oractivates other proteases remains to be elucidated. Incontrast to what we observe for acetic acid treatment, thepep4D strain shows a cell survival similar to the wild-typestrain when death is induced by H2O2 (Mason et al., 2005),whereas it exhibits a shortened lifespan during chronologi-cal ageing (Marques et al., 2006). We demonstrate thatdeletion of PEP4 and consequent impairment in mitochon-drial degradation enhances acetic acid-induced death,while the opposite phenotype is observed for PEP4 over-expression. Therefore, the process of removal of damagedmitochondria apparently has a protective role in aceticacid-treated cells, although it is likely not the only factoraffecting cell viability. We have previously described(Pereira et al., 2007) that aac1/2/3D glucose-grown cellsare more resistant to acetic acid than wild-type cells.However, when the aac1/2/3D mutant is grown in galactosemedium, the delay in mitochondrial degradation is associ-ated with a cell survival identical to the wild-type, ratherthan with a higher sensitivity as observed for the pep4Dmutant. This difference is probably due to the protectionafforded to the aac1/2/3D mutant by its defect in mitochon-drial permeabilization. A protective role for mitochondrialdegradation has also been suggested for cells in prolongedstationary phase cultures (Tal et al., 2007). In Bax-induceddeath, impairment of mitochondrial uptake by the vacuoleleads to a higher resistance (Kissova et al., 2006).However, this is due to the switch from a regulated to anecrotic death occurring at a slower rate.

Although the role of mitochondrial degradation in celldeath is still unclear, there is a consensus regarding theapparent beneficial effects of degradation of dysfunctionalmitochondria for cell survival (Wallace, 2005; Maiuri et al.,2007; Terman et al., 2007). In our model, this beneficialeffect is unlikely due to the prevention of the release ofpro-apoptotic proteins because significant mitochondrialdegradation occurs mainly after mitochondrial permeabili-zation and cytochrome c release. Removal of damagedmitochondria to avoid ATP consumption (Gellerich et al.,2004) and subsequent necrosis is also not supported byour results, because acetic acid treatment leads to a similardecrease in the ATP pool of both wild-type and PEP4-deleted and overexpressing strains (Fig. S5). Although inthe absence of Pep4p we did not observe a significantdifference in superoxide accumulation (Fig. S4) or in the

Fig. 9. Mitochondrial clusters in the Daac1/2/3 mutant aredisrupted by treatment with latrunculin-A (lat-A), anactin-depolimerizing agent.A. aac1/2/3D cells expressing a mitochondrial matrix-targeted GFPwere grown until mid exponential phase and non-treated or treatedwith acetic acid (180 mM, 30 min). DMSO alone (the vehicle) or0.25 mM lat-A were added to non treated (upper right panel) oracetic acid (AA) treated cells (lower right panel) and incubated for30 min at 37°C before visualization. Bar, 5 mm.B. At early times of treatment with acetic acid, aac1/2/3D clusteredmitochondria can be reversed to the original filamentous structuresafter acetic acid removal, although not as efficiently as the normalfragmented wild-type mitochondria. Cells were treated with 180 mMacetic acid for 30 or 200 min, washed once and incubated in theSC media without acetic acid. Samples were taken after 20 or40 min of recovery in media without acid. W303 is represented byblack lines (with squares) and aac1/2/3D by grey lines (withcircles); full lines correspond to 30 min and dotted lines correspondto 200 min of acetic acid treatment.



levels of oxidized proteins (Fig. S6), we could detect anearlier emergence of chromatin condensation and loss ofplasma membrane integrity in response to acetic acid.These observations suggest that the mitochondrialdegradation-resistant pep4D mutant commits to an accel-erated apoptosis associated with an earlier secondarynecrosis. Further studies are needed to elucidate themolecular mechanisms underlying the protective role ofPep4p during acetic acid-induced apoptosis.

Because we have previously shown that AAC proteinsare required for mitochondrial outer membrane permeabi-lization and cytochrome c release induced by acetic acid(Pereira et al., 2007), we addressed the role of AAC pro-teins in mitochondrial degradation. The aac1/2/3D straindisplays a mitochondrial aggregation pattern associatedwith a decrease in degradation of mitochondria similar tothe pep4D mutant. Taking into consideration that overex-pression of PEP4 results in a decrease in aggregationassociated with an increase in mitochondrial degradation,the results indicate that mitochondrial aggregation is aconsequence of the diminished degradation. Moreover,the clustering of mitochondria in the aac1/2/3D and pep4Dmutants seems to be dependent on actin, because disrupt-ing actin filaments also partly disrupts clusters. Addition-ally, Pep4p is still released from the vacuole inAAC-deletedcells, indicating that the delay in mitochondrial degradationobserved in the aac1/2/3D mutant is not due to a blockagein the release of this protease, but is associated with asubsequent event. Taken together, these observationssuggest that vacuole and mitochondria destabilization,measured by Pep4p and cytochrome c release, respec-tively, are events in the cell death cascade. Althoughcathepsin D, the mammalian orthologue of Pep4p, wasshown to have a role in cell death by triggering mitochon-drial dysfunction and subsequent release of mitochondrialproteins, some studies showed an inhibitory role for cathe-psin D in apoptosis (Roberg et al., 1999; Bidere et al.,2003; Boya et al., 2003; Jaattela et al., 2004; Chwieralskiet al., 2006; Liaudet-Coopman et al., 2006; Minarowskaet al., 2007). Nonetheless, a role for cathepsin D in mito-chondrial degradation in mammals has not been assessedso far.

Lysosomes, the mammalian organelle akin to the yeastvacuole, were considered to be only involved in necroticcell death until recently. Increasing evidence indicates thatlysosomes communicate with mitochondria throughselective leakage of proteins and activate some forms ofapoptotic cell death, while necrosis follows only withmassive lysosomal permeabilization (for reviews seeKroemer et al., 2007; Terman et al., 2007). Herein, wedemonstrate that the vacuole communicates with mito-chondria in yeast cells undergoing apoptosis. The cross-talk between these two organelles when autophagy isinhibited seems to involve a partial release of Pep4p

engaged in the proteolytic degradation of mitochondria,rather than an extensive vacuolar permeabilization. Deg-radation of damaged mitochondria also appears todepend on the mitochondrial AAC proteins through a stepdownstream of Pep4p release.

In summary, while relevant differences in cellular pro-cesses exist between yeast and mammalian cells, ourstudy identifying a novel role for Pep4p and AAC proteinsopens new perspectives for an enhanced understandingof the role of vacuole and mitochondria in apoptosis.Because the importance of lysosomes in mammalian celldeath is increasingly apparent, our study further rein-forces the use of yeast as a valuable model of pro-grammed cell death. Finally, as the accumulation ofdamaged mitochondria is associated with different humandiseases, a deeper understanding of how mitochondrialdegradation is activated and regulated during apoptosiscan unravel novel attractive targets for the development ofapoptosis-based therapies.

Experimental procedures

Strains and plasmids

The following S. cerevisiae strains were used in this study:W303 (MATa, ade2, his3, leu2, trp1, ura3, can1); W303PHO8D60 (Kissova et al., 2004); W303 atg5D::KanMX4(Kissova et al., 2004); W303 atg5D::KanMX4 PHO8D60(Kissova et al., 2004); W303 atp2D::KanMX4 (Pereira et al.,2007), JL1-3D2D3 (W303; aac1::LEU2, Daac2::HIS3,Daac3::URA3) (Postis et al., 2005) and pep4D::HIS3(Marques et al., 2006). The plasmids used were: pDP34 andpDP34-PEP4 (Rupp and Wolf, 1995) for PEP4 overexpres-sion; pGAL-CLbGFP (Okamoto et al., 1998) and YX232-mtGFP (Westermann and Neupert, 2000) for mitochondriavisualization; p416ADH and p416ADH-PEP4–EGFP (Masonet al., 2005) for Pep4p tagging.

Whenever strains were transformed the lithium acetatemethod (Ito et al., 1983) was used and the resulting transfor-mants were grown in selective media lacking the appropriateamino acids.

Growth conditions and cell death assays

Yeast cells were grown in rich medium [YEPGal; 1% (w/v)yeast extract, 2% (w/v) bactopeptone, 2% (w/v) galactose] orsynthetic complete medium [SC; 0.67% (w/v) Bacto-yeastnitrogen base w/o amino acids, 2% (w/v) galactose and 0.2%(w/v) Dropout mix] lacking the appropriate amino acids untilexponential phase in an orbital shaker, at 26°C, 160 r.p.m.The strains were harvested and suspended (107 cells ml-1) inthe treatment medium consisting of YEPGal or SC at pH 3.0(set with HCl), containing the appropriate amounts of aceticacid or H2O2 and incubated as described for the growthconditions. For nitrogen starvation assays cells were grown inrich medium overnight, washed twice with distilled sterilewater and transferred to nitrogen starvation media. Nitrogenstarvation media consisted on 0.67% (w/v) Bacto-yeast nitro-



gen base w/o amino acids and ammonium sulphate (Difco)and 2% (w/v) glucose. Cells were then incubated at 26°C,160 r.p.m., for 15 h.

Alkaline phosphatase assay

Alkaline phosphatase activity assays using a-naphthyl phos-phate (Sigma) as a substrate were performed as describedpreviously (Nothwehr et al., 1996). Fluorescence intensitywas measured at 472 nm (excitation at 345 nm) in a Xeniusspectrofluorimeter (Safas). Protein was quantified by theBiuret method and the ALP activities are expressed as fluo-rescence arbitrary units·min-1 mg-1 protein. ALP activity wasmeasured in non treated cells; cells treated with 180 mMacetic acid (1 or 3 h) and in nitrogen-starved cells (15 h).

Western blot analysis

Preparation of protein samples, SDS-PAGE and Western blotswere performed as described previously (Camougrand et al.,2003). The primary antibodies used were rabbit polyclonalanti-yeast cytochrome c (CYC1) antibody (1:1000, custom-made by Millegen), mouse monoclonal anti-yeast phospho-glycerate kinase (PGK1) antibody (1:5000, MolecularProbes), mouse monoclonal anti-yeast porin (POR1) antibody(1:5000, Molecular Probes), goat polyclonal anti-yeast Atg8p(1:250, Santa Cruz Biotechnology), and mouse polyclonalanti-yeast Tom22 (1:15 000, Millegen). Secondary antibodiesagainst mouse, goat IgGs, coupled to horseradish peroxidase(Jackson Laboratories), were used at 1:5000 and revealed bychemiluminescence (ECL, Amersham). Quantification ofprotein amounts was done using the ImageJ program (NCBI).

Fluorescence microscopy

Mitochondria were visualized using a matrix-targeted GFP,either pGAL-CLbGFP (Okamoto et al., 1998) for strainsW303, JL1-3D2D3, atg5D, atp2D and pep4D, or YX232-mtGFP (Westermann and Neupert, 2000) for strains W303pDP34 and W303 pDP34-PEP4.

For Pep4p labelling, cells expressing the constructionp416ADH–PEP4–EGFP were used. Cells were grown andtreated as described in the growth conditions section, andimmobilized in the slides by adding 0.5% (w/v) agar prior tomicroscopy.

Loss of plasma membrane integrity was monitored by PIstaining. Cells (5.106 cells ml-1 in PBS) were incubated with5 mg ml-1 of PI for 10 min at room temperature. When used,Mitotracker Red CMXRos (Molecular Probes) was added inculture medium at a concentration of 0.4 mg ml-1 and incu-bated for 20 min at 37°C.

Overnight cultures were analysed on a Leica MicrosystemsDM-5000B epifluorescence microscope with appropriate filtersettings using a 100¥ oil-immersion objective. Images wereacquired with a Leica DCF350FX digital camera and pro-cessed with LAS AF Leica Microsystems software.

Flow cytometry

Mitochondrial degradation was assessed by quantification ofloss of mitochondrial GFP fluorescence by flow cytometry in

strains W303, JL1-3D2D3, pep4D, W303 pDP34 and W303pDP34-PEP4 carrying the plasmids described in the previ-ous section. Cells with different relative volumes may showdifferent levels of GFP fluorescence that may not reflectchanges in mitochondrial content. To account for this issue,samples were normalized by calculating the ratio betweenthe green fluorescence (FL1, log) and the relative size (FS,log). For quantification of loss of plasma membrane integrity,5.106 cells ml-1 in PBS were processed as described in theprevious section. For assessment of mitochondrial potentialthe probe 3,3′dihexyloxacarbocyanine iodide (DiOC6(3)) wasused. Cells were collected and suspended at a concentra-tion of 5.106 cells ml-1 in 10 mM MES buffer containing0.1 mM MgCl2 and 2% (w/v) glucose. After addition of 1 nMDiOC6(3) cells were incubated for 30 minutes at 30°C in thedark.

Sample analysis was performed in an Epics® XL™(Beckman Coulter) flow cytometer, equipped with an argon-ion laser emitting a 488 nm beam at 15 mW. Monoparametricdetection of PI fluorescence was performed using FL-3 (488/620 nm) and detection of DiOC6(3) or GFP fluorescence wasperformed using Fl-1 (488/525 nm).

Thirty thousand cells were analysed per sample andexperiments were reproduced independently at least fourtimes. Data were analysed using WinMDI 2.8 software.

Acknowledgements

We thank V. Trézéguet and G. Lauquin (University of Bor-deaux) for providing strain JL1-3D2D3. We thank Dieter H.Wolf (University of Stuttgart) as well as D. S. Goldfarb (Uni-versity of Rochester) for generously providing pDP34/pDP34-PEP4 and p416ADH-PEP4–EGFP respectively. We thank B.Westermann (University of Bayreuth) for providing plasmidpYX232-mtGFP. We also thank Vítor Costa (University ofPorto) for providing W303 pep4D::HIS3. This work was partlysupported by Programa de Financiamento Plurianual (POCI2010, Fundação para a Ciência e Tecnologia, Portugal). Stayof C.P. at CNRS/Université de Bordeaux 2 was partially sup-ported by ‘Cooperação Luso/Francesa -Programa Pessoa2004’. C.P. was supported by a Fellowship BD/13497/2003from Fundação para a Ciência e Tecnologia, Portugal.

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