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1 Department of Cell Biology, Division of Molecular Cell Biology, National Institute for Basic Biology, Nishigonaka 38, Myodaiji-cho, Okazaki, 444-8585 Japan
2 Department of Basic Biology, The Graduate University for Advanced Studies, Nishigonaka 38, Myodaiji-cho, Okazaki, 444-8585 Japan
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Abstract |
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Introduction |
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During autophagy, a double-membraned structure called the autophagosome emerges and non-selectively sequesters a portion of cytoplasm. The autophagosome fuses to a lytic compartment, the lysosome/vacuole, and is degraded within a couple of minutes (Suzuki et al. 2002). Sixteen ATG (autophagy-related) genes essential for autophagosome formation have been isolated from the yeast Saccharomyces cerevisiae (Tsukada & Ohsumi 1993). In addition to the autophagic pathway, the ATG genes are involved in a biosynthetic pathway called the cytoplasm-to-vacuole (Cvt) pathway, which selectively delivers aminopeptidase I and 
-mannosidase to the vacuole under growth conditions (Scott et al. 1997; Hutchins & Klionsky 2001). ATG17 is a specific factor for the autophagic pathway (Kamada et al. 2000), whereas several ATG genes, such as ATG11 and ATG19, contribute only to the Cvt pathway (Kim et al. 2001; Scott et al. 2001). Autophagosomes and Cvt vesicles are distinguishable based on their contents and size, which average 500 and 150 nm in diameter, respectively (Baba et al. 1997).
Recent morphological analyses of the budding yeast revealed that a number of Atg proteins accumulate to a perivacuolar structure termed the pre-autophagosomal structure (PAS) (Suzuki et al. 2001; Kim et al. 2002). Upon starvation, a cup-shaped membrane sac, called the isolation membrane, is produced from the PAS. The isolation membrane extends and finally matures into an autophagosome (Mizushima et al. 2001). As the PAS is an essential structure for autophagosome formation, we have focused our work on determining how the PAS is organized, in order to elucidate the complex protein and membrane dynamics that govern autophagosome formation.
In this report, we have studied the 16 ATG genes that are absolutely required for autophagosome formation (ATG1-ATG18 except ATG11 and ATG15) and have quantified their PAS localization by fluorescence microscopy. This comprehensive analysis allowed us to construct a hierarchy diagram of Atg proteins required for PAS organization, and suggested that Atg17p acts as a scaffold protein in PAS organization.
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Results |
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Our systematic analysis aims to elucidate the mechanisms of PAS organization. First, we examined whether each Atg protein localizes to the PAS. Second, we investigated whether PAS localization of each Atg protein is perturbed by disruption of another ATG gene using quantitative fluorescence microscopy.
We constructed 16 yeast strains expressing GFP-fused Atg proteins from their native loci (each of Atg1 to Atg18 except Atg11 and Atg15), thereby avoiding mislocalization owing to over-expression. We confirmed the autophagic activity of these strains and excluded the Atg3-GFP strain from further analysis since it did not show any autophagic activity (data not shown).
We examined the 15 different Atg-GFP strains using fluorescence microscopy and found that 11 out of the 15 Atg proteins exhibited perivacuolar punctuate structures that co-localized with Ape1p, showing that these Atg proteins were indeed localized to the PAS (Fig. 1 and Supplementary Table S1). The GFP signals corresponding to Atg4p-GFP, Atg7p-GFP, Atg10p-GFP and GFP-Atg12p were too weak to quantify (data not shown). To determine which specific Atg proteins were required for the PAS localization of the various Atg proteins, all of the ATG genes were individually disrupted in each Atg-GFP strain, for a total of 165 (11 x 15) disruptants. These cells were treated with rapamycin for 5 h to induce autophagy and analyzed by fluorescence microscopy. The PAS targeting index (PTI) was introduced to quantify the level of Atg protein recruitment to the PAS (see Experimental procedures). The PTI for each Atg protein in a wild-type strain was set at 1.00. A lower PTI indicates defects in PAS targeting of the Atg protein, as indicated by a lower frequency of dots, a lower intensity of dots, or both.
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Atg8p is a protein that was first identified as an autophagosome marker (Kirisako et al. 1999). Our previous works showed that an ubiquitin-like conjugation system attaches Atg8p to phosphatidylethanolamine (PE) (Ichimura et al. 2000). The analysis presented herein showed that no Atg proteins were mislocalized in the absence of Atg8p (Fig. 2A), suggesting that ATG8 is located downstream of the other ATG genes in terms of PAS organization. In contrast, we identified ten ATG genes responsible for PAS targeting of Atg8p (Fig. 2A and for details, see Supplementary Fig. S1). In the absence of a functional Atg8 system, Atg8p-PE could not be produced (Suzuki et al. 2001), resulting in a dramatically lower PTI for Atg8. A similar pattern of Atg8 modification and targeting was observed in strains lacking a functional Atg12 system (Suzuki et al. 2001). We concluded that Atg8p-PE formation is requisite for Atg8p targeting to the PAS.
Atg12p forms a conjugate with Atg5p (Mizushima et al. 1998). Subsequently, the conjugate associates with Atg16p to form the Atg12p-Atg5p·Atg16p complex (Kuma et al. 2002). Atg5p is also capable of associating with Atg16p regardless of its conjugation to Atg12p (Mizushima et al. 1999). The Atg12p-Atg5p·Atg16p complex finally reaches the PAS and is involved in autophagosome formation. Both Atg5p-GFP and Atg16p-GFP localize to the PAS normally in mutants of the Atg8 system (Fig. 2A and for details, see Supplementary Fig. S2), indicating that the Atg12 system is located upstream of the Atg8 system in the context of PAS organization. Atg16p-GFP localization to the PAS was dependent upon Atg5p; the converse was also true: PAS localization of Atg5p-GFP required Atg16p (Fig. 2A and for details, see Supplementary Fig. S2). This result indicates that, association of Atg5p with Atg16p, but not conjugation of Atg5p with Atg12p, is absolutely required for formation of the Atg12p-Atg5p·Atg16p complex and its targeting to the PAS.
Atg14p is a member of the PtdIns(3)-kinase complex I, together with Vps30p/Atg6p (Kihara et al. 2001). Both Atg14p and Vps30p are required for the recruitment of Atg8p-PE and the Atg12p-Atg5p·Atg16p complex to the PAS (Fig. 2A). Our group recently reported that Atg14p determines the localization of the PtdIns(3)-kinase complex in the autophagic pathway (Obara et al. 2006). The PAS localization of Atg14p-GFP was abolished in 
vps30, atg9, atg13 and atg17 cells (Fig. 2A and for details, see Supplementary Fig. S3).
As shown in Fig. 2A, Atg2p and Atg18p showed similar patterns. Moreover, Atg2p and Atg18p localize to the PAS interdependently (Fig. 2A and for details, see Supplementary Fig. S4). This indicates that these proteins are functionally related. As genome-wide analyses suggested that Atg2p and Atg18p form a complex (Gavin et al. 2002; Ho et al. 2002; Krogan et al. 2006), we examined a physical interaction between Atg2p and Atg18p; such interaction was detectable by immunoprecipitation analysis at native expression levels (Fig. 3A). Hereafter, we will refer to these two proteins as the Atg2p·Atg18p complex. The localization of this complex was abolished in the absence of the PtdIns(3)-kinase complex I (Vps30p and Atg14p), Atg1p protein kinase and its regulators (Atg1p, Atg13p and Atg17p), and Atg9p (Fig. 2A and for details, see Supplementary Fig. S4). The localization of other Atg proteins was not affected in the absence of Atg2p or Atg18p (Fig. 2A). This result indicates that the Atg2p·Atg18p complex is not involved in the recruitment of other Atg proteins to the PAS.
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Finally, we analyzed Atg9p, Atg13p and Atg17p, which affect the PAS localization of many Atg proteins. The PTIs of Atg9p and Atg13p were lower in the absence of Atg17p, but not vice versa (Fig. 2A). Moreover, the PTI of Atg17p was not low in any atg mutants (Fig. 2A), indicating that Atg17p acts upstream of all the other ATG genes in PAS organization.
Atg11p recruits Atg proteins for the Cvt pathway under autophagy-inducing conditions
Localization of most Atg proteins was impaired in the absence of Atg17p (Fig. 2). This fact led us to hypothesize that Atg17p is a scaffold protein for PAS organization. If this is true, no Atg proteins should localize to the PAS in the absence of Atg17p. However, the PTI of Atg8p in atg17 cells was at the similar level as in wild-type cells (Fig. 2A). The atg17 mutant, which is specifically defective in autophagy, retains the ability to transport Ape1p to the vacuole by way of the Cvt pathway under growth conditions (Kabeya et al. 2005). To estimate the contribution of the Cvt pathway to the recruitment of GFP-Atg8p under autophagy-inducing conditions, we disrupted each of the ATG11, ATG19 and APE1 genes in atg17 cells. In atg17atg19 and atg17ape1 cells, GFP-Atg8p dots were still detectable; however, they disappeared in atg11atg17 cells (Fig. 4A). Moreover, transport of GFP-Atg8p and Ape1p to the vacuole was completely blocked in these mutants (Fig. 4B,C). We concluded that GFP-Atg8p transport to the vacuole in the absence of Atg17p depends on the Cvt pathway and that this pathway is blocked in the absence of Atg11p. Presumably, Atg11p is a scaffold protein for the Cvt pathway. This assumption is consistent with the finding that Atg11p facilitates the recruitment of Atg1p, Atg2p, Atg8p and Atg20p to a punctate structure under growth conditions (Shintani & Klionsky 2004b). In combination with this prior study, our data suggest that Atg17p plays a role in the recruitment of Atg proteins for autophagosome formation and that Atg11p plays a similar role for Cvt vesicle formation, and that the autophagic pathway and the Cvt pathway can run independently under autophagy-induced conditions.
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atg17 disruptants, PTIs of the other Atg proteins were in general lower, but always maintained certain basal levels (Fig. 2A). It is likely that the Cvt pathway contributes to the recruitment of these Atg proteins. To examine the effect of Atg11p disruption on the recruitment of Atg13p and Atg9p, which are other upstream factors (Fig. 2B), we analyzed their PTIs. As expected, both of their PTIs were drastically lower in the atg11atg17 strain (Fig. 4D,E). These data strongly support the possibility that Atg17p and Atg11p work as scaffold proteins that recruit Atg proteins. Atg17p is a scaffold protein that is responsible for PAS organization
The PAS localization of Atg proteins is completely absent in the atg17atg11 mutant (Fig. 4). If Atg17p actually functions as a scaffolding protein in PAS organization, Atg proteins must be recruited to the site where Atg17p localizes. We constructed Atg17p fused with a plasma membrane-targeting sequence of Gpa2p (Harashima & Heitman 2005) (PM-Atg17p), then examined whether other Atg proteins were targeted to the plasma membrane in atg17atg11 cells expressing PM-Atg17p.
Atg9p, Atg13p and Atg8p exhibited a cytoplasmic expression pattern in atg11atg17 cells carrying an empty vector (Fig. 5, Vector). C-terminally mRFP1-fused PM-Atg17p (PM-Atg17p-RFP) thoroughly localized to the plasma membrane (Fig. 5, left column). Intriguingly, other Atg proteins were also recruited to the plasma membrane and essentially co-localized with Atg17p (Fig. 5), showing that Atg17p indeed acts as a scaffold to recruit Atg proteins. Also, we found slight differences in the localization of Atg proteins: whereas Atg13p was closely co-localized with PM-Atg17p (Fig. 5A), Atg9p was recruited to the plasma membrane, as visualized by the presence of a small number of punctate fluorescence (Fig. 5B), and Atg8p formed a single dot just inside the plasma membrane (Fig. 5C).
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atg17atg11 mutant, as determined by the failure of GFP-Atg8p delivery to the vacuole (Fig. 5C), suggesting that these plasma membrane-targeted Atg proteins are unable to generate autophagosomes. Presumably, PAS organization at the proper site (next to the vacuole) is required for normal autophagy. Again, this experiment clearly showed that Atg17p is a determinant of the site of PAS organization during autophagy. |
Discussion |
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From this analysis, we have developed a hierarchy diagram of Atg proteins in PAS organization (Fig. 2B). Atg1p protein kinase and its regulators (blue), Atg9p (white), and the PtdIns(3)-kinase complex I (green) act in initial Atg protein recruitment to the PAS. Atg8p-PE (red), the Atg16p-Atg5p·Atg12p complex (yellow) and the Atg2p·Atg18p complex (violet) are recruited to the PAS with the help of the Atg proteins belonging to the above group, and work at the PAS to generate autophagosomes. The two ubiquitin-like systems confer autophagic activity to Atg8p and Atg12p by modifying them. This diagram suggests that Atg17p is the most upstream component in PAS organization (Fig. 2B).
Under autophagy-inducing conditions, the PAS localization of most Atg proteins was lost in the absence of Atg17p (Fig. 2A). The remaining PAS localization was completely abrogated by additional disruption of the ATG11 gene (Fig. 4). This data suggest that the Cvt pathway is able to operate in the absence of ATG17 and that Atg11p facilitates the recruitment of Atg proteins to the Cvt pathway under autophagy-inducing conditions. Atg17p and Atg11p were initially identified as proteins that associate with Atg1p (Kamada et al. 2000; Kim et al. 2001), but their actual roles in the autophagic and Cvt pathways were initially unclear. In this study, we propose that Atg17p and Atg11p play roles in the recruitment of Atg proteins: Atg17p functions in autophagosome formation, whereas Atg11p functions in the Cvt vesicle formation.
In the absence of Atg17p and Atg11p, the assembly of Atg proteins in the PAS was abolished (Fig. 4). To confirm the activity of Atg17p as a scaffold protein, we forced Atg17p to localize to the plasma membrane in the atg17atg11 mutant and then examined whether Atg proteins were recruited to the plasma membrane. This experiment clearly demonstrated the scaffold activity of Atg17p (Fig. 5). Autophagic activity in this strain was estimated by staining of the vacuolar lumen by GFP-Atg8p; cells containing plasma membrane-targeted Atg17p did not recover autophagic activity (Fig. 5C), whereas atg11 cells did (Fig. 4A). This suggests that the perivacuolar localization of the PAS is crucial for autophagy. The vacuolar membrane and/or proteins on the vacuole might have some roles in autophagosome formation from the PAS. Atg17p has several coiled-coil motifs (Cheong et al. 2005) and interacts with many proteins (Gavin et al. 2002; Ho et al. 2002; Krogan et al. 2006), suggesting that this protein plays a role not only in Atg protein recruitment but also in connecting the autophagic machinery with other membrane-trafficking pathways. Further analysis is required to determine more precisely the mechanisms by which Atg17p is localized to the PAS.
Under nutrient-rich conditions, Atg11p is responsible for recruitment of Atg proteins (Shintani & Klionsky 2004b), implying the Atg17p is not functional. It is likely that starvation signals confer the ability to recruit Atg components upon Atg17p. Recently, we have identified ATG29 as a new gene required for autophagy (Kawamata et al. 2005). Similarly to Atg17p, Atg29p localizes to the PAS and is not required for the Cvt pathway (Kawamata et al. 2005). Atg29p and Atg17p may act closely together during autophagy. We hope that further analysis of Atg29p will reveal the role of Atg17p in the recruitment of Atg proteins during autophagy.
Our hierarchy map accounts for several phenomena previously reported in mammalian cells. This map suggests that the Atg16p·Atg5p-Atg12p complex and the PtdIns(3)-kinase complex are crucial for recruitment of Atg8p (Fig. 2B). This is the case in mammalian cells; atg5-deficient cells show no LC3 (mammalian Atg8p) dots (Mizushima et al. 2001), suggesting the necessity of the Atg16p·Atg5p-Atg12p complex. In addition, wortmannin and 3-methyladenine, inhibitors of PtdIns(3)-kinases, prevent Atg5p from accumulating to the dot structures (Mizushima et al. 2001), leading to the defect in recruitment of LC3 (Ogata et al. 2006). Presumably, the molecular mechanisms of LC3 recruitment involving the Atg16p·Atg5p-Atg12p complex and the PtdIns(3)-kinase complex are conserved between the yeast cells and mammals.
The most prominent difference between yeast and mammalian cells is the number of Atg8p dots emerging upon starvation. Yeast cells mostly have a single dot per cell, whereas mammalian cells form multiple dots. The PAS is defined as the structure that contains most Atg proteins and that produces autophagosomes (Suzuki et al. 2001). In this context, dot structures labeled with Atg5p, Atg12p and Atg16p could be defined as the PAS in mammalian cells (Mizushima et al. 2001, 2003). In this study, we found that Atg17p determines the site of PAS organization (Fig. 5). To answer the question whether mammalian cells have the PAS as a functional counterpart of that in the yeast, functional homologue(s) of Atg17p should be identified.
Our study has provided a framework for analyzing Atg proteins in the context of PAS organization. Moreover, the knowledge obtained from these analyses will be generally applicable to the study of de novo biogenesis of intracellular membranes.
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Experimental procedures |
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Standard methods were used for yeast manipulation (Adams et al. 1998). Cells were grown in either YEPD or SD + CA medium (0.17% yeast nitrogen base w/o amino acids and ammonium sulfate, 0.5% ammonium sulfate, 0.5% casamino acid and 2% glucose) with appropriate supplements. Autophagy was induced in growth media containing 400 ng/mL rapamycin (Sigma). The yeast strains used in this study are listed in Supplementary Table S2. ORY strains were generated using a PCR-based gene modification method (Longtine et al. 1998). A two-step gene replacement method (Adams et al. 1998) was used to generate the GFP-Atg8 and GFP-Atg12 strains. Cells were transformed with the pRS306 YEGFP-ATG8 plasmid digested with SnaBI or the pRS306 YEGFP-ATG12 plasmid digested with EcoRI. To disrupt each ATG gene, Schizosaccharomyces pombe HIS5 (spHIS5) fragments amplified from pDH5 were used. Construction of the strains was carried out by Oriental Yeast Co., LTD. (Japan).
An Ape1-RFP strain was generated using an mRFP1 sequence amplified from the pFA6a-mRFP1-kanMX plasmid. The GYS516 strain was obtained by tetrad dissection. The STY1530-1533 strains were generated by transforming ORY0900 cells with pPS128 digested with AflII or pPS129 digested with AvrII (plasmids were gifts from Dr Daniel J. Klionsky, University of Michigan, USA). Subsequently, the ATG11 and ATG17 genes were disrupted as described previously (Kamada et al. 2000; Kim et al. 2001; Kabeya et al. 2005). The ATG5 gene was disrupted as described (Mizushima et al. 1998).
The ATG17-RFP and PM-ATG17-RFP (plasma membrane-targeted ATG17-mRFP1) plasmids were generated by successive cloning of ATG17 and mRFP1 sequences into the BamHI site of pYEX-BX vector (Clontech). The plasma membrane-targeting sequence of Gpa2p (2-10: GLCASSEKN) (Harashima & Heitman 2005) was inserted just after the start codon of ATG17. Cells carrying these plasmids were cultured in SD + CA medium containing 250 µM CuSO4 to drive the Cu2+-inducible CUP1 promoter in pYEX-BX.
Measurement of autophagic activity
A proform of aminopeptidase I (Ape1p) is synthesized in the cytoplasm and is subsequently transported to the vacuole via the autophagosome for maturation. Autophagic activity was estimated by measuring the maturation of Ape1p using immunoblot. The autophagic activity in the Atg17-GFP strain was confirmed by accumulation of autophagic bodies (Takeshige et al. 1992).
Quantification of co-localization between GFP-fused Atg proteins and Ape1p
To ascertain whether the punctate fluorescence arising from GFP-fused Atg proteins corresponded to the PAS, a strain of opposite mating type that chromosomally expressed Ape1p-mRFP1 was generated (GYS516). This strain was mated with each GFP-expressing strain, and the resulting diploids were examined by fluorescence microscopy. The extent of co-localization was estimated by counting dots in cells where both GFP and RFP dots were detectable.
Microscopy
Fluorescence microscopy was performed using a TIR-FM microscope system (Olympus) equipped with a 100x objective lens (Apo100xOHR, NA: 1.65) and a CoolSNAP HQ CCD camera (Nippon Roper). GFP was excited with a mercury lamp and a U-MGFPHQ filter set. For cells labeled with both GFP and RFP, a blue laser (Sapphire 488-20, Coherent) and a yellow laser (85-YCA-010, Melles Griot) were used. A U-MNIBA2, from which the excitation filter was removed, was used for GFP visualization, and an FF593-Di02 dichroic mirror and an FF593-Em02 excitation filter (Semrock) were used to analyze RFP. A UPlanSApo100xOil (NA: 1.40) objective lens was used for microscopic observation. Images were acquired using MetaMorph software (Molecular Devices).
Calculating PTI (pre-autophagosomal structure targeting index)
Cells were treated with rapamycin for 5 h before images were acquired. The PAS always localizes next to the vacuole and relatively immobile; therefore, by a few-second exposure, we can distinguish the PAS from other mobile structures. The images were processed with MetaMorph software (Version 6.2r6). First, the dot number and the average intensity of dots were counted as follows. (1) Dots were extracted with a "Tophat" operation. (2) Background signals were subtracted using a "Threshold Image" operation. (3) Regions of interest were determined by selecting a "Create Regions Around Objects" operation. (4) To extract the PAS-related dots, we removed dots exhibiting abnormal localization (apart from the vacuolar membrane) and abnormal shapes (tubular or circular). (5) Using a "Region Measurements" operation, the number of dots was displayed in the "Region Labels" column. Values in the "Integrated Intensity" (of each dot) column were used for further analysis. The average intensity of the dots was calculated using the logged data.
Next, the cell number was counted. At least 200 cells were used to calculate the PTI. The number of dots per cell was calculated by dividing the number of dots by the number of cells. The number of dots per cell obtained from each disruptant was divided by the number of dots per cell in wild-type cells. Similarly, the intensity of dots in each disruptant was divided by the intensity of dots in wild-type cells. Consequently, the relative dot number and the relative dot intensity were obtained. The PTI was calculated by multiplying the relative dot number and the relative dot intensity.
Immunoblot analysis
Immunoprecipitation analysis was performed as described (Siniossoglou et al. 2000) with a slight modification. Atg2p was immunoprecipitated with affinity-purified anti-Atg2p antibodies (Shintani et al. 2001). Atg18p was probed with an anti-Atg18p antiserum raised against bacterially expressed Atg18p (gift from Dr Fuyuhiko Inagaki, Hokkaido University, Japan). For GFP and Ape1p, detection was performed with anti-GFP antiserum (Molecular Probes) or anti-Ape1p antiserum (Suzuki et al. 2002).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: yohsumi{at}nibb.ac.jp
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References |
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Received: 24 September 2006
Accepted: 8 November 2006
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