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J. Biol. Chem., Vol. 279, Issue 37, 39068-39074, September 10, 2004
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From the Unité Mixte de Recherche 5095 CNRS, Université de Bordeaux 2, 33077 Bordeaux, France
Received for publication, June 22, 2004 , and in revised form, July 9, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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uth1 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 uth1 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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although the normal function of autophagy is thought to provide amino acids to starved cells, a fair amount of evidence suggests that it could also be required for the elimination of selected organelles, namely mitochondria, under peculiar conditions. In mammalian cells, autophagic removal of mitochondria has been shown to be triggered following a process of induction/blockade of apoptosis (6, 7). Also, coregulation of apoptosis and autophagy by the Akt/protein kinase B-signaling pathway has been evidenced (8, 9). Finally, molecular data suggest that autophagy-specific proteins might also be involved in the regulation of apoptosis (10). Because mitochondrial events are now regarded as a crucial step in apoptosis, this idea indicates that damaged mitochondria (following apoptotic induction) could control their own degradation by an autophagic process (11). This possibility is also supported by the observation that the overexpression of anti-apoptotic proteins, such as Bcl-2, is able to block autophagy (12) and that down-regulation of Bcl-2 induces autophagy (13). Also, the opening of the mitochondrial permeability transition pore, often considered to be a crucial event regulating the initial steps of apoptosis, induces the colocalization of mitochondrial and lysosomal markers, which is typical of an autophagic process in mammalian cells (14).
In yeast, a study reported the presence of mitochondria in autophagosomes following autophagy induction by nitrogen starvation (15). Under natural conditions, yeast often faces environmental changes requiring a modulation of its mitochondrial content (reviewed in Refs. 16, 17, and 18). For example, the shift from a respiratory carbon source to a fermentative carbon source or, more drastically, the shift from aerobic to anaerobic conditions is accompanied by a decrease of both the amount and the enzymatic equipment of mitochondria. Clearly, the inhibition of mitochondrial biogenesis is likely to play an important role in these changes (19, 20) but might not be enough to explain the rapid disappearance of cohorts of mitochondria. One study, based on a mutant inactivated in the mitochondrial protease Yme1p, suggests that alterations of mitochondrial biogenesis are able to trigger mitochondrial autophagy (21, 22). But hypotheses about the role of mitochondria in their own degradation remain highly speculative, because a molecular support for these hypotheses is still lacking (2).
In the present study, we used the autophagy-inducing drug rapamycin or physiological autophagy induced by nitrogen starvation to provide evidence that mitochondria are early targets of autophagic degradation in yeast, and we identified for the first time that a mitochondrial protein, Uth1p, is involved in this process.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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uth1 strain was constructed from W303-1B as described previously (23). The atg5 strain was constructed by disruption of the ATG5 gene with the KanMX4 cassette in the W303-1B strain. pep4, derived from the wild-type strain BY4742 (MATa, his3, leu2, ura3, met15), was obtained from Euroscarf (Frankfurt, Germany). For the measurement of ALP1 activity, the PHO8 locus was replaced with PHO860 in each strain by transformation with an HindIII fragment of the plasmid pTN9 (a gift from Y. Ohsumi, National Institute for Basic Biology, Okazaki, Japan) bearing PHO860 as described previously (24). For fluorescent microscopy experiments the cells were transformed with the plasmid pGAL-CLbGFP containing the presequence of mitochondrial citrate synthase fused to GFP under the control of a GAL1/10 promoter, as described (25), which was provided by J. P. di Rago (Institut de Biochimie et Génétique Cellulaires, Bordeaux, France).
Yeast cells were grown aerobically at 28 °C in a complete medium (1% yeast extract, 0.1% potassium phosphate, and 0.12% ammonium sulfate) or a minimal YNB medium (yeast nitrogen base without amino acids and with 0.175% ammonium sulfate, 0.5% ammonium sulfate, 0.1% potassium phosphate, 0.2% Drop-Mix, and 0.01% auxotrophic requirements, pH 5.5) supplemented with 2% glucose or 2% lactate as a carbon source.
Nitrogen starvation medium (SD-N) contained 0.175% yeast nitrogen base without amino acids and ammonium sulfate and 2% glucose or 2% lactate, respectively. To induce pGAL-CLbGFP expression, cells were grown overnight in an appropriate medium supplemented with 2% galactose. Rapamycin treatments were done with 0.2 µg/ml rapamycin by using a stock solution of 100 µg/ml rapamycin (Calbiochem) in 90% ethanol and 10% Triton X-100.
Plating Efficiency—The viability of cells was determined as plating efficiency. The cells were counted at indicates times and diluted, and aliquots with identical numbers of cells (5 x 102) were plated on solid complete YPD (1% yeast extract, 2% peptone, and 2% dextrose) or YPL (1% yeast extract, 2% peptone, and 2% lactate) medium, respectively. The number of colonies was scored after 2-3 days of growth at 28 °C. The viability of cells in each sample was expressed as the percentage of colonies produced by the plating of the same aliquots of culture at time 0.
Western Blot Analyses—Protein samples preparation, SDS-PAGE, and Western blots were done as described previously (26). The primary antibodies used were mouse monoclonal anti-yeast Cox2p (1/2000e; Molecular Probes), mouse monoclonal anti-yeast porin (1/2000e; Molecular Probes), mouse monoclonal anti-yeast phosphoglycerate kinase (1/2000e; Molecular Probes), goat polyclonal anti-aminopeptidase I (1/100e; Santa Cruz Biotechnology), goat polyclonal anti-yeast Atg8p (1/250e; Santa Cruz Biotechnology), and rabbit polyclonal anti-yeast actin (1/1000e; Santa Cruz Biotechnology). Secondary anti-mouse, anti-rabbit, and anti-goat IgG antibodies coupled to horseradish peroxidase (Jackson Laboratories) were used at 1/10000e. An ECL+ kit (Amersham Biosciences) was used for protein detection. Quantification of protein amounts was done using the ImageJ program (NCBI).
Alkaline Phosphatase Assay—ALP activity assays using 
-naphthyl phosphate (Sigma) as a substrate were performed on cells that had been treated with rapamycin (0.2 µg/ml) for 2 h or nitrogen-starved for 15 h according to the method described (27). Fluorescence intensity was measured at 472 nm (excitation at 345 nm) in a Safas spectrofluorometer. Protein concentration was measured with the Lowry method (28). ALP activities were expressed as arbitrary fluorescence units/min/mg protein.
Fluorescence Microscopy Measurements—Vacuoles were stained with N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM4-64; final concentration of 40 µM) according to a modified method as described previously (29). mtGFP-expressing cells stained with FM4-64 were visualized on a Leica microscope. The images were acquired with an SIS camera and processed with Corel Draw 9.0 suite software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Physiological autophagy induced by nitrogen starvation does not lead to cell death. On the contrary, strains impaired for autophagy, such as the atg5 mutant, do not survive nitrogen starvation (30). But overactivation of autophagy by rapamycin treatment induces growth arrest. As shown in Fig. 1, A and B, the growth of the uth1 mutant exhibits rapamycin resistance when cells were spotted on a solid medium. This resistance is more marked than that of the autophagy-deficient atg5 mutant.
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atg5 strains, but the uth1 strain exhibited a strong resistance. It is noteworthy that this resistance was more marked on lactate-supplemented medium (Fig. 1D) where yeast cells develop large and fully active mitochondria, because this carbon source is exclusively oxidized by mitochondrial metabolic pathways (31). Whatever the carbon source, under nitrogen starvation conditions, a better survival rate was observed for the uth1 strain in comparison with the wild-type, the autophagy-deficient atg5 mutant, and the pep4 mutant, which is deficient in the major vacuolar hydrolase, proteinase A (Fig. 2).
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uth1 Cells—We were interested in examining the cellular autophagic activity in the uth1 mutant to know if the observed resistance to rapamycin was due to a general blockage of the autophagic process. Several biochemical methods for assaying autophagy have been established. One improved method (24) is to follow the vacuolar delivery of the Pho860p, a cytosolic variant of the Pho8p alkaline phosphatase in which the membrane-spanning region and the vacuolar targeting sequence have been deleted. Such an N-terminal truncated Pho860p variant cannot be delivered to the vacuolar compartment via the normal secretory pathway and is only imported to the vacuole upon the induction of autophagy, when cells are shifted to nitrogen starvation conditions or treated with rapamycin. The vacuolar delivery of randomly sequestrated Pho860p in autophagic vesicles results in cleavage of the C-terminal propeptide in a proteinase B-dependent manner and activation of its enzyme activity. In our study, ALP activity was assayed in wild-type cells and uth1 and atg5 mutant strains grown on glucose-(Fig. 3A) or lactate-supplemented (Fig. 3B) minimal medium after a 1-h rapamycin treatment or 15 h of nitrogen starvation, respectively. The level of autophagy was indicated by the difference in enzymatic activity found at time 0 (control) and under these two conditions.
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atg5 strain, where the autophagic process was impaired and, consequently, Pho860p cannot be delivered to the vacuole, no increase of ALP after rapamycin treatment or under starvation conditions was observed. Under the same conditions, ALP activity was significantly increased both in the wild-type and the uth1 strains whatever the condition of induction. The highest autophagic activity was found in starved uth1 mutant cells, when ALP activity was increased five times.
Another method for measuring autophagic activity is to follow the processing of the vacuolar aminopeptidase I (API) in starved cells (33). API is synthesized on soluble ribosomes as a 61-kDa inactive precursor that is transported to the vacuole in nutrient-rich conditions by a constitutive non-classical vesicular transport mechanism, the cytoplasm-to-vacuole targeting pathway, and is submitted to proteolytic maturation leading to a 50-kDa form. Under starvation conditions or in presence of rapamycin, API is transported to the vacuole by macroautophagy. Total protein extracts were prepared from cultures of wild-type and uth1 strains grown on glucose-containing minimal medium and after 15 h of nitrogen starvation. An immunoblot with an antibody specific for API was done to monitor precursor API processing (Fig. 4). Under starvation conditions, only the mature form was visualized in wild-type and uth1 strains showing the activation of autophagy. Taken together, these two sets of experiments (ALP activity and API maturation) showed that the autophagy process was unaffected in the uth1 mutant.
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A construction expressing GFP fused downstream of the targeting sequence of mitochondrial citrate synthase (mtGFP) (25) was introduced in wild-type, uth1, and atg5 strains in order to follow the behavior of mitochondria in situ. The autofluorescent probe FM4-64, which specifically labels the vacuolar membrane in yeast cells (29), was used simultaneously. Under control conditions, wild-type cells grown on a lactate and galactose-supplemented medium exhibit a well differentiated mitochondrial network, and FM4-64 labeling shows the presence of one or two large vacuoles (Fig. 5). Rapamycin treatment of wild-type cells induced the disappearance of the mitochondrial network and the appearance of patches predominantly at the periphery of the cells. Most cells lost the classical vacuolar membrane FM4-64 staining, and only few cells showed faintly stained vacuoles. The proportion of cells in which FM4-64 fluorescence was lost from vacuolar membranes to give rise to diffused cytoplasmic staining corresponded with the proportion of dead cells (85%; see Fig. 1D). The remaining 15% of the cells, which were still viable after a 2-h rapamycin treatment (Fig. 1D), exhibited intensely punctuated FM4-64 staining, with some of them colocalizing with mtGFP as shown in Fig. 5. The same perturbation of vacuolar membrane staining after rapamycin treatment was observed for glucose-grown cells treated with rapamycin (data not shown).
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uth1 and atg5 mutants also revealed a normal mitochondrial network (Fig. 5). Opposite to the case with wild-type, the addition of rapamycin to both mutants did not induce any rapid disorganization of the mitochondrial network that remained intact after a 2-h treatment (Fig. 5), and the network was marginally affected after a 6-h treatment (not shown). No colocalization of mtGFP and FM4-64 was ever observed in these strains. Interestingly, FM4-64 staining of vacuolar membranes showed the presence of a large number of small fragmented vacuoles in uth1 as described in some mutants defective in vacuolar biogenesis and autophagy, such as in the atg5 strain (Fig. 5) (35).
To verify that rapamycin treatment did not induce any side effects by affecting mtGFP synthesis or import, cells were incubated in the presence of cycloheximide (an inhibitor of protein synthesis) or carbonyl cyanide m-chlorophenylhydrazone (CCCP; an uncoupler dissipating transmembrane electrical potential). For the time of the experiment, the mitochondrial network remained unaffected by these treatments in both the wild-type (Fig. 6) and the uth1 strains (not shown).
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atg5 mutant cells under the same conditions (Fig. 7). In the culture of a uth1 strain starved for 15 h, the following two distinct behaviors were observed. (i) About half of the cells exhibited normal FM4-64 staining and an intact mitochondrial network like that of the atg5 mutant. (ii) The other half exhibited some hallmarks of a weak autophagy process, with patches of mtGFP at the periphery of the cells and vacuolar delivery of mtGFP but also the visible presence of a residual mitochondrial network (Fig. 7).
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To verify that the rapamycin-induced decrease of the two mitochondrial proteins was actually caused by vacuolar proteolysis and not by a blockade of their synthesis, the experiment was reproduced in a pep4 strain (Fig. 8). The amounts of Por1p and Cox2p did not decrease after a 6-h rapamycin treatment, whereas in fact a slight increase was actually observed.
This lack of degradation in pep4 cells confirms that the protein degradation observed in wild-type cells is executed in the vacuoles.
Rapamycin treatment of the autophagy-deficient atg5 strain grown on lactate induced a higher increase of Atg8p in wild-type (Fig. 8); this is in accordance with the fact that the process of autophagosome formation and the subsequent vacuolar degradation of Atg8p is blocked in this mutant. Concerning mitochondrial proteins, no decrease in the amounts of Por1p and Cox2p was observed, but, as in the pep4 mutant, a slight increase was recorded instead.
From this set of experiments it was seen that rapamycin treatment of cells grown under strict respiratory conditions induces the early degradation of mitochondrial proteins by a process that requires the following: (i) the formation of autophagosomes; and (ii) vacuolar proteolysis. During the short course of the experiment cytosolic proteins remain poorly affected, suggesting that fully differentiated organelles like mitochondria are early targets of the rapamycin-induced process. Also, Por1p and Cox2p, which are localized on different membranes, are degraded together, suggesting that whole mitochondria are targeted by the process.
In accordance with the resistance of the uth1 strain to rapamycin (Fig. 1), degradation of the two mitochondrial proteins was largely impaired in this strain (Fig. 8). On other hand, the product of ATG8 increased up to a level similar to that of the wild-type strain (Fig. 8), confirming the conclusion drawn from the measurement of ALP activity (Fig. 3) and API processing (Fig. 4) that the autophagic process is still active. This supports the view that the resistance of the uth1 strain is not caused by a general defect in the autophagic machinery but that the mitochondrial degradation targeted by this machinery is delayed.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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atg5 mutant. Opposite to the case with the atg5 mutant, the autophagic machinery remains fully active in uth1 mutant, but mitochondria degradation by this process is delayed. These results showed that the mitochondrial protein Uth1p is involved in autophagic mitochondrial degradation. This protein, Uth1p, has been initially found in a genetic screen aiming to identify proteins involved in the regulation of the yeast life span (38). Furthermore, it was found to be a member of the so-called "SUN" family, which includes four proteins having a very high degree of identity (23, 39). The product of UTH1 exhibits some remarkable properties. It is mainly localized in the mitochondrial outer membrane (34) but was also found in the cell wall (34, 40). This alternative cell wall localization is likely to have a physiological meaning, because it can be selectively suppressed by point mutations in the protein.2 The absence of Uth1p does not have dramatic consequences under normal growth conditions; it induced a 15-25% decrease of mitochondrial cytochromes as well as other mitochondrial enzymes and an increase of growth yield (23). This higher metabolic efficiency, linked to a general but slight slowing down of mitochondrial functions, is likely to explain the extended life span as well as the modified response to oxidative stress exhibited by this mutant (41).
More recently, Uth1p was found to be involved in yeast cell death induced by the heterologous expression of the human proapoptotic protein Bax (26). As in mammalian cells, Bax targets the mitochondrial outer membrane, where it induces the formation of a large channel likely involved in the mitochondria-to-cytosol relocalization of cytochrome c (42-44). Bax-induced characteristics in yeast resemble apoptotic like hallmarks induced by H2O2 treatment (45) in which a caspase-like activity might be involved (46). However, cytochrome c release is not mandatory to Bax-induced cell death in yeast (47). In addition, because Uth1p is not involved in the mitochondrial targeting of Bax or in cytochrome c release activity (26), we proposed that Bax was able to activate an alternative death-inducing pathway involving Uth1p. As a matter of fact, Uth1p overexpression was found to induce cell death, and uth1 inactivation induced a resistance of growth to rapamycin (26). These observations suggested that Uth1p might be involved in a death pathway related to autophagy.
The observation reported in the present paper that Uth1p is involved in the autophagic degradation of mitochondria induced by rapamycin or nitrogen starvation supports this hypothesis. Clearly, Uth1p does not participate in the autophagic machinery, because the biochemical hallmarks related to this process (ALP activation and API processing) are as active as those in wild-type, in contrast to the behavior of the autophagy-deficient strain atg5. More likely, Uth1p might be involved in the recognition of mitochondria by the autophagic machinery. However, it should be noted that, both from viability and fluorescence microscopy experiments, about half of uth1 cells are still able to undergo normal mitochondria autophagy, suggesting that Uth1p may not be the only protein involved in the process.
The degradation process termed autophagy covers distinct, although related, phenomena. The best characterized, and also the less specific, is the macroautophagy of large parts of the cytoplasm including the organelles (reviewed in Ref. 2). However, more selective phenomena were also evidenced. As an example, microautophagy of the nucleus via the physical contact between nuclei and vacuoles was shown to involve selective protein-protein interactions such as Nvj1p on the nuclear envelope and Vac8p on the vacuolar membrane (5). This process does not involve the well established components of macroautophagy signaling but is still under the control of the TOR (target of rapamycin) kinase pathway. Another example of selective autophagy was observed in the methylotrophic yeast Pichia pastoris, where peroxisomes can be selectively degraded by vacuoles in a way independent from macroautophagy (3). The degradation of other organelles has not been investigated to date, but tools such as selectively targeted GFP and specific antibodies are now available for such studies.
Concerning yeast mitochondria, since the first report that they could be engulfed by the macroautophagy machinery (15), one study has suggested that a selective mechanism of mitochondria autophagy also occurred in yeast, i.e. inactivation of the mitochondrial AAA-type protease Yme1p leads to a vacuole-dependent mitochondria-to-nucleus transfer of genetic material (21, 22). However, the molecular basis of this transfer has not been identified to date.
The finding that Uth1p is a component involved in the early selective degradation of mitochondria is now a basis for the identification of the other components involved in the autophagic degradation of mitochondria. This might be of general interest, not only in yeast but also, given the recent evidence of connections between apoptosis and autophagy, in mammalian cells (48).
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FOOTNOTES |
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Permanent address: Faculty of Natural Sciences, Comenius University, 84215 Bratislava, Slovak Republic.
To whom correspondence should be addressed: Institut de Biochimie et Génétique Cellulaires/CNRS, 1 Rue Camille Saint-Saëns, F-33077 Bordeaux, France. Tel.: 33-556-99-90-45; Fax: 33-556-99-90-51; E-mail: n.camougrand{at}ibgc.u-bordeaux2.fr.
1 The abbreviations used are: ALP, alkaline phosphatase; API, aminopeptidase 1; CCCP, carbonyl cyanide m-chlorophenylhydrazone; FM4-64, N-[3-triethylammoniumpropyl]-4-[p-diethylaminophenyl-hexatrienyl] pyridinium dibromide; mtGFP, mitochondria-targeted green fluorescent protein.
2 N. Camougrand, I. Kissová, M. Deffieu, and S. Manon, unpublished data.
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REFERENCES |
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, wild-type; 
, 
, 
