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1 Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113-0033, Japan
2 Laboratory of Electron Microscopy, Open Research Center, Japan Women's University, Mejirodai, Tokyo, 112-8681, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresAssay of the kinase...References |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresAssay of the kinase...References |
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The discovery of a set of genes involved in autophagy (termed ATG) in the budding yeast Saccharomyces cerevisiae (Klionsky et al. 2003) has triggered extensive molecular genetic analyses of this process in this organism and in higher eukaryotes, where orthologs of many of the ATG genes have also been identified. Studies in different organisms have further revealed a conservation of the autophagic pathway that induces characteristic morphological changes and plays a crucial role in cells that experience a dramatic change in their fate (Levine & Klionsky 2004; Mizushima 2005). Extensive studies on the molecular mechanisms and regulatory systems underlying autophagy have also revealed in budding yeast that both the Ras/PKA pathway and the target of rapamycin (TOR) pathway are involved in the negative regulation of autophagy (Noda & Ohsumi 1998; Budovskaya et al. 2004).
Upon nutritional starvation, fission yeast cells exit the vegetative cell cycle and initiate sexual differentiation to produce spores, which are dormant progeny that can survive until the environmental conditions become more favorable for growth. The experimental depletion of nitrogen from the culture medium of this organism has also been shown to effectively induce G1 phase arrest and subsequent sexual differentiation. Recently, Nakashima et al. (2006) showed that bulk protein degradation is induced by nitrogen starvation and also observed morphological changes in cytoplasmic vacuoles, indicating that this may represent fission yeast autophagy.
In our present study, we show that fission yeast not only possesses autophagic activity, but also utilizes this response to initiate sexual differentiation in response to nitrogen depletion. In agreement with the previous report, we also demonstrate that low levels of nitrogen addition are sufficient to complement the sterile phenotype of the autophagy-defective mutants. This indicates that the major role of autophagy may be to supply a nutrition source in the absence of an environmental supply, rather than to dispose of excess proteins or organelles. Furthermore, we show that whereas environmental nitrogen levels regulate autophagy, depletion of environmental carbon or the ectopic induction of meiosis in the presence of nitrogen does not affect autophagy activity.
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Results |
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To elucidate the role of autophagy in fission yeast, we identified by computation (Thompson et al. 1994) orthologs of the budding yeast ATG1, ATG8 and ATG13 genes, which are essential for autophagy in this organism (Takeshige et al. 1992; Funakoshi et al. 1997; Matsuura et al. 1997; Kirisako et al. 1999). We hereafter designate these fission yeast orthologs as atg1, atg8 and atg13, and their coding products as SpAtg1, SpAtg8 and SpAtg13, respectively (Fig. S1). Recently, Nakashima et al. (2006) reported that nitrogen starvation (–N) induces bulk protein degradation activity, which is dependent upon the function of Isp6, an ortholog of the budding yeast vacuolar protease, Prb1 (Takeshige et al. 1992). To examine whether atg1, atg8 and atg13 are also involved in degradation activity induced by –N, we constructed deletion mutants of these genes and performed degradation assays.
Vegetatively growing fission yeast cells were transferred to the nitrogen-free medium MM-N and protein degradation was measured essentially as described previously (Tsukada & Ohsumi 1993). In response to –N conditions, bulk protein degradation occurred in wild-type (WT) cells and was blocked either by the addition of the protease inhibitor PMSF (Fig. 1A) or by deleting the isp6 gene as previously reported (Fig. 1B) (Nakashima et al. 2006). Significantly, in none of the atg gene deletion mutants could protein degradation be detected (Fig. 1B). These data suggest that the degradation activity induced by –N represents autophagy in fission yeast.
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Compared to budding yeast, in which a single conspicuous vacuole encompasses the ABs that are readily detectable under a light microscope, fission yeast carry multiple but less remarkable vacuoles (Takegawa et al. 2003).Thus, to enable detailed observations of the expected dynamic rearrangements of membrane structures that would accompany autophagy, we performed electron microscopic analyses employing high pressure freeze-substitution methods (Konomi et al. 2003). Electron microscopy of WT fission yeast cells revealed that although the number of vacuoles increased under –N conditions (compare Fig. 2A,D), the appearance of possible ABs within these cells was rare (Fig. 2D, arrows). In contrast, however, the isp6
mutant was found to harbor spherical bodies accumulating in the vacuoles under –N conditions (Fig. 2E, magnified image in Fig. 2G) that were highly reminiscent of the ABs observed in budding yeast (Takeshige et al. 1992). These findings are also in good agreement with the previous report of Nakashima and colleagues (Nakashima et al. 2006) that described the accumulation of vesicle-like particles in the vacuoles of an isp6 deletion mutant. It is noteworthy in this regard also that whereas our present fixation method of high pressure freeze-substitution allows for the better preservation of organelle structures, the imagery shown in the previous study of Nakashima was obtained by chemical fixation and highlights vacuoles that are packed with particles of electron-dense materials (Nakashima et al. 2006).
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isp6 double mutant, the accumulation of ABs does not occur (Fig. 2F), indicating that the formation of the AB-like structures is dependent on SpAtg1 function. Together with the results presented in Fig. 1, these observations unambiguously indicate the presence of an autophagic pathway in fission yeast which is activated by nitrogen depletion and is dependant upon conserved autophagy-associated components. The fission yeast Atg8 protein forms a PAS-like structure
In budding yeast, the mature Atg8 protein localizes to the pre-autophagosomal structure (PAS, a scaffold for autophagosome formation (Suzuki et al. 2001)), autophagosomes and ABs (Kirisako et al. 1999). LC3, the mammalian counterpart of Atg8, also localizes to the autophagosomes and acts as a representative marker for their formation (Kabeya et al. 2000; Mizushima et al. 2004). To determine the localization of SpAtg8, we constructed an atg8.NGFP allele, which expresses an N-terminal tagged GFP-SpAtg8 under the control of the endogenous atg8 promoter. GFP-SpAtg8 was found to be fully functional as the atg8.NGFP cells did not show any obvious deficiencies in either sexual differentiation or protein degradation activity (data not shown). GFP-SpAtg8 was also constantly expressed regardless of the levels of nitrogen present in the medium (Fig. S2A,B). Analysis by fluorescence microscopy revealed that cytoplasmic GFP-SpAtg8, the expression of which can be observed during the vegetative cell cycle, dramatically re-localizes to form a few bright dots after 3 h incubation in –N conditions (Fig. S2C). These dots may represent the PAS in fission yeast. In the isp6 background however, the appearance of the GFP-SpAtg8 signals was somewhat different whereby their number increased but displayed a much weaker intensity (Fig. S2D). As the isp6 cells were found to accumulate autophagic body-like spherical bodies in their vacuoles (Fig. 2E), we speculated that the GFP-SpAtg8 signals in the isp6
atg8.NGFP cells might be representative of accumulating ABs in the vacuoles.
Nitrogen and not carbon regulates autophagy in fission yeast
The cAMP-PKA cascade plays a central role in monitoring environmental glucose (carbon source) in both fission and budding yeast (Isshiki et al. 1992; Welton & Hoffman 2000). In budding yeast, autophagy is induced by carbon starvation as well as by –N (Takeshige et al. 1992), and a high level of cAMP represses the induction of autophagy (Noda & Ohsumi 1998). However, we observed in our current experiments that protein degradation was undetectable upon carbon starvation in fission yeast (Fig. 3A), even when the transcription of the glucose-repressible fbp1 gene was fully derepressed (Hoffman & Winston 1990) (Fig. 3A, right panel). We then examined the effects of supplying cAMP in fission yeast. As shown in Fig. 5B, –N induced both the transcription of the mei2 gene (Shimoda et al. 1987) and autophagic protein degradation. In contrast, although the addition of cAMP completely abolished mei2 transcription, as shown previously (Watanabe et al. 1988), it did not affect the induction of autophagy (Fig. 3C). Taken together, we conclude from these data that the carbon-sensing machinery and its downstream cAMP-PKA pathway are unlikely to play a major role in the regulation of autophagy in fission yeast. Our results are also consistent with the previous observation that Pka1 is not involved in the induction of autophagy (Nakashima et al. 2006).
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SpAtg13 and SpAtg1 are dephosphorylated under depletion of nitrogen
We wished to examine whether the observed nitrogen-dependent switching mechanism in fission yeast involved SpAtg13, as is likely for the orthologous gene in budding yeast where in nutrient rich conditions, Atg13 is detectable in a hyper-phosphorylated form and does not interact with Atg1. Shifting these budding yeast cells to starvation conditions brings about dephosphorylation of Atg13 and its association with Atg1, and this coincides with activation of autophagy (Kamada et al. 2000). Hence, in budding yeast, it has been proposed that the nutrient conditions determine the phosphorylation status of Atg13, which may then regulate the "on/off" state of the autophagic response.
We first analyzed the apparent molecular weight of SpAtg13 by Western blotting in isp6 cells carrying the pREP81-atg13 plasmid, as endogenous SpAtg13 expression was barely detectable. The pREP81-atg13 plasmid was constructed to express SpAtg13 under the control of a weak thiamine repressible nmt81 promoter. In addition, the protease deficient isp6 cells facilitate the stabilization of the expressed SpAtg13. The SpAtg13 protein extracted from vegetatively growing cells was found to migrate more slowly than from the same product in cells grown in –N conditions (Fig. 4A). Phosphatase treatment of +N and –N samples indicated that this mobility shift was due to phosphorylation (Fig. 4B). Importantly, dephosphorylation of SpAtg13 after a shift to –N conditions occurred after 60 min in tandem with the subsequent induction of autophagic degradation activity. Furthermore, the addition of a nitrogen source to the nitrogen free medium instantly affected the phosphorylation status of SpAtg13 (Fig. 4A) and inhibited this degradation activity (Fig. 3E).
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We measured the possible kinase activity of SpAtg1 using myelin basic protein as a substrate. To use as a negative control, we constructed a putative kinase-dead allele, atg1.K43ANPk, carrying a substitution of lysine at position 43 to alanine in the kinase domain, which was tagged N-terminally with the Pk epitope and expressed from the endogenous atg1 promoter. The atg1.K43ANPk mutant was fully viable but deficient in autophagy and sexual differentiation (data not shown), suggesting that the kinase activity of SpAtg1 is essential for its function. WT Pk-SpAtg1 and Pk-SpAtg1.K43A were immunoprecipitated from either vegetatively growing cells or cells shifted to –N conditions for 1 h. Immunocomplexes were then processed for in vitro kinase assays. SpAtg1 showed kinase activity that was elevated when cells were starved for nitrogen, whereas SpAtg1.K43A showed no activity (Fig. 4E). Altogether, our data indicate that environmental nutritional conditions cause an alteration in the molecular status of SpAtg1 and SpAtg13, leading to activation of SpAtg1 kinase, which may subsequently act as a determinant of autophagy activity.
Fission yeast autophagy contributes to the maintenance of cell viability under conditions of nitrogen-depletion
The autophagy pathway is required to maintain viability under limited nutrient conditions in both budding yeast and in higher eukaryotes (Tsukada & Ohsumi 1993; Kuma et al. 2004; Scott et al. 2004). In our current experiments, we examined whether a similar role might be attributed to autophagy in fission yeast. Although the atg1, atg8 and atg13 mutants were fully viable at 7 days after the transfer to –N, they displayed a decreased viability after 25 days when compared with WT cells that remained fully viable (Fig. 5A). This suggests that fission yeast autophagy is indeed required for cell survival in conditions of starvation.
In the case of mutants harboring the leu1.32 mutation, the phenotype became more dramatic resulting in almost 100% cell death within 12 h under –N conditions (Fig. 5B). Interestingly, each of the other auxotrophic mutations, such as arg1, ura4 or lys3, affected cell viability in different ways following nitrogen depletion (Fig. 5B). Whereas the leu1 mutation conferred the most severe phenotype upon autophagy defective mutants, the arg1 or lys3 mutation had relatively mild effects with ura4 causing an intermediate effect (Fig. 5B). These observations suggest that the requirement for different amino acids or nucleic acids for the adaptive response to conditions of starvation might be differently regulated and/or contributed by autophagy.
The isp6 mutant cells were found to lose viability at 4 days after the transfer to –N but showed increased survival when combined with atg1, atg8, or atg13 (Fig. 5C). One explanation for this phenomenon might be that the accumulation of ABs, which occurs only in isp6 cells (Fig. 2E), may cause toxic effects. Another explanation might be that the isp6 cells, suffering from a lack of nutrients because of their disrupted autophagic degradation pathways, might even accelerate the activation of autophagy. This might result in the excess generation of ABs encompassing cytoplasmic materials essential for survival in starvation conditions.
To see the effect of the isp6 mutation on the viability of the leu1.32 mutant, we constructed a leu1.32 isp6 double mutant and a leu1.32 isp6
atg1 triple mutant. These two mutants showed cell death within 12 h under –N, just like the atg1
leu1.32 mutant (Fig. 5D). Therefore, although the isp6 single mutant loses its viability earlier than other autophagy-defective mutants, this mutation appears to enhance the cell death under leucine deprivation only similarly to other autophagy-defective mutations.
Autophagy supports the adaptation of fission yeast cells to the complete depletion of available nitrogen
To adapt to –N conditions, fission yeast cells commence sexual differentiation if they are in contact with their opposite mating types. We thus examined the possible role of autophagy in this process by combining almost equal amounts of WT h+ and h– cells in –N. These cells subsequently mated, initiated meiosis, and produced four spores in each ascus (Fig. 5E, top left panel). In contrast, our autophagy-defective mutants neither mated nor underwent sexual differentiation (Fig. 5E, lower four panels), suggesting that autophagy may indeed play an essential role in sexual development in fission yeast. The sterility of the autophagy-defective mutants did not appear to be due to a defect in G1 arrest, as is the case with the tor1 mutant which is defective for TOR kinase activity (Fig. 5F, rightmost panel) (Weisman & Choder 2001; Matsuo et al. 2003), because more than 20% of the autophagy-defective mutant cells showed G1 arrest in response to –N (Fig. 5F, boxed).
It was of further interest that when the culture medium was supplemented with 30 mM of ammonium chloride, all of our autophagy-defective mutants produced spores (Fig. 6A, lower four panels), consistent with the previous report for the isp6 mutant (Nakashima et al. 2006). Treatment with ammonium chloride could not rescue the phenotype of other sterile mutants such as tor1 or ste11 (Fig. 6A, upper panels), the latter of which encodes an HMG family transcription factor essential for the up-regulation of meiosis-specific genes (Sugimoto et al. 1991; Mata et al. 2002). In addition, the viability of the spores generated by the autophagy mutants was comparable to that of the WT (data not shown). These results lead us to speculate that autophagy is dispensable for sexual differentiation in fission yeast as long as an adequate nitrogen source is available. To further confirm this hypothesis, we used a temperature-sensitive pat1-114 mutant, which initiates meiosis at the restrictive temperature regardless of the nutritional conditions (Iino 1985; Nurse 1985). In the absence of nitrogen, h–/h– pat1-114/pat1-114 cells defective in one of the atg genes did not execute meiosis upon the temperature shift, but did so when a nitrogen source was present (Fig. 6B). These cells also executed meiosis when the medium contained a concentration of NH4Cl as low as 1 mM, but failed to do so at 0.1 mM NH4Cl (data not shown). The control h–/h– pat1-114/pat1-114 cells underwent meiosis in the absence of a nitrogen source (Fig. 6B). These results are in agreement with the previous observation that isp6
pat1-114 cells executed ectopic meiosis only when nitrogen was supplied (Nakashima et al. 2006). Thus, the major function of fission yeast autophagy, at least during the sexual development, is to provide a supply of nitrogen in conditions where this element is depleted, rather than to remove surplus proteins or organelles that would potentially interfere with crucial cellular activities.
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Our above observations suggested that sexual differentiation could proceed independently of autophagy in fission yeast if proper nutritional supplements are available. We therefore examined whether the sexual differentiation pathway might be involved in the regulation of autophagy in this organism. Fission yeast cells activate transcription of ste11 in response to –N, which in turn up-regulates many meiosis-specific genes (Sugimoto et al. 1991; Mata et al. 2002). This eventually results in the inactivation of Pat1 kinase and an increase in the levels of the unphosphorylated active form of Mei2, the master regulator of meiosis (Watanabe et al. 1997). To examine whether autophagy accompanied ectopic meiosis driven by the pat1 mutation, we shifted pat1-114 mutant cells to the restrictive temperature in the presence of nitrogen. No evidence of autophagy was observed; however, although ectopic meiosis was induced in more than 90% of the cells, as determined by the progression of nuclear divisions (Fig. 7A). Hence, activation of the sexual differentiation pathway does not appear to stimulate autophagy. We next monitored the occurrence of autophagy in ste11 mutant cells placed under –N conditions and full activation of the autophagic pathways was evident in these cells (compare Figs 7B and 1A), even though sexual differentiation was completely blocked. Taken together, we conclude that the induction of autophagy and initiation of sexual differentiation are mutually independent phenomena, despite the fact that both can be triggered by the depletion of nitrogen.
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Discussion |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresAssay of the kinase...References |
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In the fission yeast Gene Database (Sanger centre), putative orthologs were discernible for 12 out of 15 ATG genes that had been identified originally in a screen for budding yeast mutants that were defective in macroautophagy (Tsukada & Ohsumi 1993). These 12 genes are widely conserved, further indicating the preservation of the molecular process of autophagy among eukaryotic organisms.
Significance of autophagy in fission yeast
Autophagy-deficient fission yeast mutants were viable, with no obvious phenotype, during the vegetative cell cycle. However, these mutants lost their viability under conditions of prolonged nitrogen-starvation, whereas the WT cells survived. Furthermore, these mutants were incapable of undergoing sexual differentiation upon nitrogen deprivation. Thus, as is the case for other eukaryotic organisms (Levine & Klionsky 2004), fission yeast autophagy plays a major role in the adaptation processes that result from environmental changes.
As a major player of cytosolic protein degradation, two functions of autophagy have been proposed. One is to simply remove deadwood proteins and the other is to recycle such surplus proteins. These two functions may not be mutually exclusive, however, and in many cases both of them may in fact be operating in the cell. It is thus possible that in some cases one of these aspects of autophagy may be more emphasized than the other in certain cellular contexts. For example, the former function has been highlighted in reports that demonstrated autophagy as a major mechanism to dissolve aggregated huntingtin, which triggers Huntington's disease (Iwata et al. 2005; Yamamoto et al. 2006). The latter "recycling" role of autophagy was underscored in studies of amino-acids supply systems that assist protein synthesis (Kuma et al. 2004; Onodera & Ohsumi 2005). In this report, we observe that fission yeast autophagy-deficient mutants progressed through the sexual differentiation process once a nitrogen source was provided exogenously. Thus, we propose that fission yeast autophagy supports adaptation by creating a nitrogen source via the recycling of proteins rather than by removing obstacles. We note, however, that spores produced by autophagy-defective mutants in the presence of a nitrogen supplement were somewhat aberrant in their morphology, although their viability was comparable of the WT cells. Therefore, autophagy might play some role during the formation of a forespore membrane by eliminating unwanted proteins. Alternatively, the formation of spores might require full supply of nitrogen which could be achieved only in the presence of autophagy.
It was of considerable further interest also that the ectopic induction of meiosis did not induce autophagic protein degradation activity, and the deletion of the ste11 gene did not perturb the full induction of autophagy upon nitrogen deprivation in fission yeast. This indicates that sexual differentiation is not involved in the regulation of autophagy in this organism. Thus, we propose that nitrogen starvation, an environmental signal, will trigger sexual differentiation and autophagy in parallel. The autophagic response thus plays an essential role in driving sexual differentiation when an environmental nitrogen source is lacking (Fig. 7C).
Regulation of autophagy in fission yeast
In budding yeast, the availability of a carbon source is a critical determinant of autophagy (Takeshige et al. 1992). Transferring budding yeast cells from a fermentable glucose medium to a non-fermentable glycerol medium induces autophagy. The addition of cAMP, the concentration of which increases momentarily when cells are exposed to a high concentration of glucose (Thevelein 1994), inhibits ongoing autophagic protein degradation (Noda & Ohsumi 1998). In this study in fission yeast, we observed no obvious induction of autophagy by carbon starvation. Furthermore, the addition of cAMP did not interfere with the induction of autophagy in this organism. These results, together with the previous findings of Nakashima et al. (2006), states that PKA is unlikely to be involved in autophagy regulation, and suggest that the levels of available environmental carbon are not critical for the regulation of autophagy in fission yeast.
In both fission and budding yeast, the supply of an exogenous nitrogen source and autophagic protein degradation are both likely to generate amino acids for subsequent protein production. The possibility that this synergy functions to facilitate effective protein synthesis, however, is unlikely as an exogenously supplied nitrogen source inhibits the ongoing autophagy. At the molecular level, Kamada et al. (2000) have shown that upon a shift to rich medium, Atg13 is quickly phosphorylated, which is proposed to shut off its function. We also observed rapid phosphorylation of SpAtg13 under these conditions. Taken together, these data indicate that in both yeast species, a rich nutrition supply may negatively regulate autophagy through the phosphorylation of Atg13, which may result in no activation of Atg1 kinase.
While our present study demonstrates that fission yeast autophagy is regulated by the amount of environmental nitrogen available, the exact properties of nitrogen in this context and the molecular mechanisms that detect it are currently enigmatic. One possibility is that the levels of extracellular nitrogen might be a regulator of autophagy. Some transporter(s) which recognize and uptake exogenous nitrogen might also play a critical role. Another possibility is that the intracellular amino acid pool might act directly as an "on/off" switch for autophagy. The possible involvement of the TOR kinase pathway in this regulation has also been explored extensively and we and others have observed that one of the two fission yeast TOR genes, Tor1, is dispensable for the activation of autophagy (Nakashima et al. 2006; our unpublished result), whereas the other, Tor2, might play a substantial role (Uritani et al. personal communication; Kohda and Yamamoto unpublished result). The further identification and elucidation of these regulatory mechanisms will be a challenging future task.
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Experimental procedures |
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The Schizosaccharomyces pombe strains used in this study are listed in Supplemental Table S1. Yeast extract medium (YE) (Moreno 1990), minimal medium (MM), which contains 2% glucose as a carbon source and 93.5 mM NH4Cl as a nitrogen source, its nitrogen-free version MM-N (Watanabe et al. 1988), and its low glucose version MM-C (3% glycerol + 0.1% glucose) (Hoffman & Winston 1990) were employed as growth media. General genetic methods for analyzing S. pombe have been described previously (Gutz et al. 1974). A temperature-sensitive h–/h– pat1-114/pat1-114 diploid strain was used to induce meiosis regardless of the nutritional conditions (Iino 1985).
Gene disruption
Using BLAST homology search algorithms, the fission yeast genes SPCC63.08c, SPBP8B7.24c, SPAC4F10.07c, and isp6 (Sato et al. 1994) were identified as putative orthologs of the budding yeast ATG1, ATG8, ATG13, and PRB1 genes, respectively. We designated the former three genes as atg1, atg8, and atg13. Gene disruption was performed using the direct chromosomal integration method as described previously (Bahler et al. 1998). The entire atg1, atg8, and atg13 ORFs were then substituted by ura4+ or hphr (Sato et al. 2005), the isp6 and ste11 ORFs were substituted by kanr and the tor1 ORF was substituted by hphr.
Construction of mutant strains
For construction of atg1.NPk allele or atg1.K43ANPk allele, we placed a pk DNA fragment at the 5' of the atg1 ORF or atg1K43A ORF that had been cloned in a vector. The K43A mutation was introduced into the atg1 gene according to a standard method (Kunkel, 1985). The 5' UTR of atg1 (atg1 5'UTR) was obtained by PCR amplification employing genomic DNA as the template and was inserted to 5' of the pk-atg1/pk-atg1K43A ORF to yield a plasmid containing "atg1 5'UTR-pk-atg1 ORF" or "atg1 5'UTR-pk-atg1K43A ORF." The "atg1 5'UTR-pk-atg1 ORF" or "atg1 5'UTR-pk-atg1K43A ORF" insert was subsequently excised, and transformed into the atg1Nura4+ strain, where 5' half of the atg1 ORF was substituted with ura4+. Correct replacement of ura4+ by "atg1 5'UTR-pk-atg1 ORF" or "atg1 5'UTR-pk-atg1K43A ORF" resulted in resistance to 0.1% 5-fluoroorotic acid (5-FOA) and transformants were selected on YE plates containing 0.1% 5-FOA (Wako). For the construction of the atg8.NGFP allele, we placed the ORF and 3'UTR of atg8 (atg8 ORF-3'UTR) at the 3' of an EGFP DNA fragment and inserted the 5'UTR of atg8 (atg8 5'UTR) 5' of EGFP DNA. The resultant construct ("atg8 5'UTR-gfp-atg8 ORF-3'UTR") was transformed into the atg8ura4+ strain and transformants were again selected on YE plates containing 0.1% 5-FOA.
Measurement of protein degradation
Measurements of protein degradation were carried out as previously described with some modifications (Takeshige et al. 1992). Briefly, cells were cultured in 2 mL of MM medium containing 0.011 Mbq/mL of [14C]-leucine (Amersham) for 12–14 h at 30 °C, and then chased for one generation (2.5 h) by adding 4 mL of liquid YE. They were then washed twice with MM-N, and incubated in 1.8 mL of MM-N with or without the addition of 1 mM PMSF (3.6 µL of a 500 mM stock solution in methanol). Aliquots of these cultures (270 µL) were sampled every 2 h, mixed with 30 µL of 100% TCA and kept at 4 °C overnight. The cell suspensions were then centrifuged at 2500x g for 5 min and 200 µL of the supernatant was aliquoted to measure radioactivity in the presence of 800 µL ACS II aqueous counting scintillant (Amersham Bioscience) with a liquid scintillation counter. The radioactivity released into the supernatant was calculated as a percentage of the initial total cellular radioactivity. In Fig. 3A, we used MM-C in the place of MM-N. To examine the effects of the loss of pat1 function, [14C]-labeled cells grown at 25 °C were chased and washed with MM+N. They were then suspended in MM+N and shifted to 34 °C, followed by the degradation assay.
Light and fluorescence microscopy
Cells were observed under a microscope (Axioplan2, Zeiss) with a Plan APOCHROMAT objective lens (63x/1.40, Zeiss). A cooled CCD camera (CoolSNAP HQ, Photometrics) and the SlideBook software (Intelligent Imaging Innovations) were used to capture images.
Construction of antibodies against Atg13p
Rabbit polyclonal SpAtg13 antibodies were raised against a glutathione-S-transferase (GST)-SpAtg13 fusion protein in a similar manner to the method described by Kamada et al. (2000). A 1.2 kb PCR fragment of the C-terminus of atg13 encoding 398 amino acids of the SpAtg13 protein was cloned into the pGEX-KG vector, and the resulting fusion protein was expressed in the E. coli strain BL21. Glutathione-Sepharose 4B (Amersham Biosciences) was then employed to purify the expressed fusion protein, which was subsequently used to immunize rabbits and raise the antiserum (Scrum Inc.). Antibodies were purified from the resultant antiserum using His-tagged SpAtg13 in a conventional method (Harlow & Lane 1988).
Western blotting and immunoprecipitation
For the Western blotting of Pk-SpAtg1, GFP-SpAtg8 and SpAtg13, harvested cells were boiled for 5 min, and then disrupted with glass beads in HB buffer [150 mM KCl, 25 mM MOPS, 5 mM EGTA, 15 mM MgCl2, 50 mM ß-glycerophosphate, 0.2% NP-40, 10% Glycerol, 0.1 mM Na2VO3, 15 mM p-nitrophenyl phosphate (p-NPP), 1 mM PMSF, 1 mM dithiothreitol (DTT); one tablet of Complete (Roche) was added to 10 mL of HB buffer]. Equal amounts of 2x sample buffer [100 mM Tris–HCl (pH 6.8), 4% SDS, 0.2% BPB, 20% Glycerol, 200 mM DTT] were then added to the cell extracts and samples were boiled prior to analysis by SDS-PAGE. We used 6% separating acrylamide gels to detect Pk-SpAtg1 or SpAtg13, and a 10% gel for the detection of GFP-SpAtg8. The mouse monoclonal anti-Pk MCA1360 (Serotec), Rabbit polyclonal anti-GFP (Molecular Probes) and Rabbit polyclonal anti-SpAtg13 were used as the primary antibodies. Sheep anti-mouse IgG conjugated with horseradish peroxidase (Amersham Pharmacia) and donkey anti-rabbit IgG conjugated with horseradish peroxidase (Amersham Bioscience) were used as the secondary antibodies. Enhanced chemiluminescence (Amersham Pharmacia) was used for immunodetection.
For phosphatase treatment of SpAtg13 or Pk-SpAtg1, cells were disrupted in HB buffer. The supernatant was then immunoprecipitated with either anti-SpAtg13 antibodies loaded onto Protein A Dynabeads (Dynal Biotech) or anti-Pk MCA1360 loaded onto Protein G Dynabeads (Dynal Biotech). The beads were washed twice with HB buffer and suspended in 
-PPase reaction buffer [50 mM Tris-HCl, 100 mM NaCl, 0.1 mM EGTA, 2 mM dithiothreitol (DTT), 0.01% Brij 35, 2 mM MnCl2]. Samples were then incubated with 400 U of -PPase (New England Biolabs) at 30 °C for 20 min, either with or without the inhibitor mixture [10 mM EGTA, 0.1 mM Na3VO4, 20 mM ß-glycerophosphate, 15 mM p-NPP].
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Assay of the kinase activity of SpAtg1 |
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-32P]ATP (Amersham Pharmacia) and 10 µg myelin basic protein (Wako). After incubation for 20 min at 30 °C, the reaction was terminated by the addition of 60 µl 2x sample buffer. The reaction mix was boiled and subjected to SDS-PAGE on 14% acrylamide gels. Radioactivity of dried gels was quantitated using FLA-2000 (FUJIFILM). To estimate the amount of immunoprecipitated SpAtg1 protein, the beads remaining in 54 µL kinase reaction buffer were boiled after the addition of 54 µL 2x sample buffer, and the supernatants were subjected to SDS-PAGE on 10% acrylamide gels. Northern blotting
Total RNA was prepared from S. pombe cells and Northern blotting was performed as previously described (Watanabe et al. 1988). The DNA probes used were PCR-amplified fragments that covered the entire open reading frames of mei2 and fbp1.
Electron microscopy
Cultured cells were harvested by centrifugation, pipetted into aluminum specimen carriers (BAL-TEC, Liechtenstein) and frozen by high pressure freezing (Moor 1987) using HPM-010 (BAL-TEC, Liechtenstein) as described previously (Konomi et al. 2003). The samples were then freeze-substituted in 2.0% osmium tetroxide in absolute acetone at –80 °C for 2 days, and then warmed to –20 °C for 2 h, to 4 °C for 2 h and then at room temperature for 1 h. After washing with absolute acetone, the specimens were passed through QY-2 (methyl glycidyl ether; Nisshin EM, Japan) and embedded in Quetol 812 resin followed by polymerization at 60°C for 48 h. The ultra-thin sections were stained in 4% uranyl acetate and 0.4% lead citrate and viewed with a TEM H-800 (Hitachi, Japan) at 125 kV.
Flow cytometric analysis
We prepared samples for flow cytometry essentially as described previously (Imai & Yamamoto 1994). Cells were analyzed with a flow cytometer FACScan (Beckton-Dickinson, San Jose, CA, USA).
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Acknowledgements |
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Footnotes |
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aPresent address: Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.
* Correspondence: E-mail: yamamoto{at}biochem.s.u-tokyo.ac.jp |
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Received: 25 March 2006
Accepted: 30 October 2006
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