Atg31 is required for autophagosome formation in Saccharomyces cerevisiae

Biochemical and Biophysical Research Communications 356 (2007) 405–410 www.elsevier.com/locate/ybbrc

Cis1/Atg31 is required for autophagosome formation in Saccharomyces cerevisiae q Yukiko Kabeya a, Tomoko Kawamata a, Kuninori Suzuki a

a,b

, Yoshinori Ohsumi

a,b,*

Division of Molecular Cell Biology, National Institute for Basic Biology, Myodaiji 38, Okazaki 444-8585, Japan b School of Life Science, The Graduate University for Advance Studies, Okazaki 444-8585, Japan Received 21 February 2007 Available online 7 March 2007

Abstract Autophagy is the bulk degradation of cytosolic materials in lysosomes/vacuoles of eukaryotic cells. In the yeast Saccharomyces cerevisiae, 17 Atg proteins are known to be involved in autophagosome formation. Genome wide analyses have shown that Atg17 interacts with numerous proteins. Further studies on these interacting proteins may provide further insights into membrane dynamics during autophagy. Here, we identify Cis1/Atg31 as a protein that exhibits similar phenotypes to Atg17. ATG31 null cells were defective in autophagy and lost viability under starvation conditions. Localization of Atg31 to pre-autophagosomal structures (PAS) was dependent on Atg17. Coimmunoprecipitation experiments indicated that Atg31 interacts with Atg17. Together, Atg31 is a novel protein that, in concert with Atg17, is required for proper autophagosome formation. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Autophagy; Starvation; ATG; Autophagosome; PAS; Rapamycin

Autophagy is an intracellular degradation system that responds to nutrient starvation. The process is highly conserved from yeast to higher eukaryotes. Upon induction, cytoplasmic components, including organelles, are enclosed in double membrane structures, termed autophagosomes, and delivered to the lysosome/vacuole to be degraded. From recent studies in yeast, it is thought that a structure near the vacuole, called the pre-autophagosomal structure (PAS) [1,2], plays a pivotal role in autophagosome formation.

Abbreviations: ATG genes, autophagy-related genes; Cvt, cytoplasm to vacuole targeting; GFP, green fluorescent protein; PAS, pre-autophagosomal structure. q This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. * Corresponding author. Address: Division of Molecular Cell Biology, National Institute for Basic Biology, Myodaiji 38, Okazaki 444-8585, Japan. Fax: +81 564 55 7516. E-mail address: [email protected] (Y. Ohsumi). 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.02.150

About 30 autophagy-related (ATG) genes have been identified in yeast and are required for the degradative autophagic pathway, the biosynthetic cytoplasm to vacuole (Cvt) pathway, or both. Both pathways are accompanied by topologically similar membrane phenomena. The Cvt pathway is a selective process that transports at least two vacuolar resident hydrolases, aminopeptidase I (Ape1) [3] and a-mannosidase [4], via small double-membrane vesicles, termed Cvt vesicles [5,6]. There is a significant difference in the sizes of the autophagosome (300–900 nm in diameter) and the Cvt vesicle (140–160 nm) [6]. Studies in yeast have shown that 17 Atg proteins, Atg1–10, 12–14, 16–18, and Atg29, are involved in autophagosome (AP) formation. Hereafter, these proteins will be referred to as AP-Atg proteins. Among these proteins, Atg17 and Atg29 are dispensable for Cvt vesicle formation, although most AP-Atg proteins are required for formation of Cvt vesicles. Atg17 was initially identified by two-hybrid screen using Atg1 as bait [7]. Atg13 and Atg17 are involved in activation of the kinase, Atg1 [7]. Previous studies have demonstrated

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that Atg17 forms a complex with Atg1 and Atg13 under starvation conditions, and this physical interaction is required for proper autophagosome formation [8]. A systematic fluorescence microscopy analysis showed that Atg17 is essential for organization of the PAS [9]. A genome wide two-hybrid assay and a global mass spectrometry analysis in S. cerevisiae suggested that Atg17 interacts with numerous proteins [10,11]. These proteins are additional candidates to function together with Atg17 in autophagy, and we have now tested whether they are required for autophagy. Intriguingly, one disruptant, ydr022cD/cis1D, showed a defect in autophagosome formation. CIS1 was originally identified as a suppressor of CIK1, a kinesin-associated protein that is localized to the spindle pole bodies and is required for both karyogamy and mitotic spindle organization [12–14]. A previous study showed that overexpression of Cis1 rescued the temperature-sensitive growth defect of cik1D cells [12]. However, in our hands, the function of CIS1 as a suppressor of CIK1 was quite weak. Rather, cis1D cells showed a distinctive defect in autophagy, similar to atg17D cells. Here, we demonstrate that the Cis1 protein functions mainly in autophagosome formation and propose to rename Cis1 as Atg31. Materials and methods Yeast strains, media, and plasmids. The strains utilized in this study are listed in Table 1. Media for growth of yeast cells, starvation, and induction of autophagy have been described previously [7,15]. YKY6, YKY61, and YKY62 were generated using the one-step gene replacement method described previously [16]. The region containing the disruption marker and the flanking sequences was amplified by PCR using genomic DNA prepared from the BY4741 cis1D::kanMX strain [17]. The resulting cassette was transformed into the YKY6 strain to generate YKY79. The atg17D strain was constructed using a previously described method [7]. To obtain pATG31, a 1.6 kb fragment containing the entire ATG31 gene was cloned from yeast genomic DNA into pRS316 [18]. Antibodies. Antisera against Ape1 [19], Atg8 [20], Atg12 [21], Atg13 [7], and PGK [22] were described previously. Mouse monoclonal anti-Myc epitope antibody 9E10 was purchased from BabCo.

Table 1 Yeast strains used in this study Strain

Genotype

Source

BY4741 atg1D atg7D atg13D atg17D atg31D/cis1D YKY6 YKY61 YKY62 YKY77 YKY79 SEY6210 STY1133 YKY65

MATa his3D leu2D met15D ura3D BY4741 atg1D::kanMX BY4741 atg7D::kanMX BY4741 atg13D::kanMX BY4741 atg17D::kanMX BY4741 cis1D::kanMX BY4741 ATG17::ATG17-GFP-HIS3 BY4741 ATG31::ATG31-13Myc-kanMX BY4741 ATG31::ATG31-GFP-kanMX YKY62 atg17D::HIS3 YKY6 atg31D::kanMX MATa leu2 his3 trp1 lys2 suc2 ura3 SEY6210 APE1::mRFP-APE1-LEU2 MATa/a his3/his3 leu2/leu2 LYS2/lys2 met15/MET15 SUC2/suc2 ura3/ura3 ATG31-GFP-kanMX/ATG31 APE1/mRFP-APE1-LEU2

ResGen ResGen ResGen ResGen ResGen ResGen This study This study This study This study This study Ref. [31] Ref. [29] This study

Other methods. Fluorescence microscopy [1], alkaline phosphatase (ALP) assays [23,24], cell viability assays [25], protein extraction [8], immunoblot analysis [8,26], and immunoprecipitations [8] were conducted as described previously.

Results cis1D/atg31D is specifically defective in autophagy Currently, it is thought that Atg17 is the most important protein for organization of the pre-autophagosomal structure (PAS); Atg17 has a role in localization of other APAtg proteins to the PAS [9]. We examined whether any of the large number of proteins that interact with Atg17 [10,11] are involved in autophagy. Interestingly, a disruptant, ydr022cD/cis1D, showed a defect in autophagosome formation. Manning et al. originally identified the CIS1 gene as a suppressor of CIK1 (chromosome instability and karyogamy 1) [12]. In that study, a multi copy plasmid harboring CIS1 allowed cik1D cells to grow on solid medium at the restrictive temperature. To substantiate the suppressive activity of Cis1, we overexpressed Cis1 in cik1D mutant cells. We observed minimal suppressive activity of Cis1 (data not shown), suggesting that Cis1 is mainly involved in autophagy. Wild-type cells accumulated autophagic bodies in the vacuole under starvation conditions in the presence of phenylmethanelusulfonylfluoride (PMSF), while cis1D cells did not (Fig. 1A). A defect of cis1D mutant cells was also confirmed by examining autophagic activity using the ALP assay (Fig. 1B). A truncated form of Pho8 (Pho8D60) expressed in the cytosol is delivered to the vacuole via autophagy and processed to an active form [23,24]. ALP activity did not increase when the cis1D mutant cells were cultured in starvation medium SD(-N) for 6 h, providing further evidence that Cis1 is essential for autophagy. A low copy plasmid harboring CIS1 rescued the autophagy defect in the cis1 mutant strain (Fig. 1A and B). The cis1D cells also showed a loss of viability phenotype under nitrogen starvation (Fig. 1C). Together, these results show that Cis1 plays a critical role in autophagosome formation. Hereafter, we refer to Cis1 as Atg31. The phosphorylation state of Atg13 is extremely sensitive to nutrient conditions in a TOR-dependent manner [7]. We examined the phosphorylation of Atg13 by immunoblot in the presence and the absence of rapamycin (Fig. 2A). Both in wild-type and atg31D cells, Atg13 was mostly hyperphosphorylated in growth medium, and was efficiently dephosphorylated after rapamycin treatment. These results suggest that Atg31 is not involved in dephosphorylation of Atg13. Autophagy requires two ubiquitin-like conjugation systems, in which Atg8 and Atg12 are covalently bound to phosphatidylethanolamine (PE) and Atg5, respectively [27,28]. We assessed the amounts of PE-conjugated Atg8 and Atg5-conjugated Atg12 in the atg31D mutant by SDS–PAGE in the presence of 6 M urea. As shown in

Y. Kabeya et al. / Biochemical and Biophysical Research Communications 356 (2007) 405–410

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Strain Fig. 1. CIS1/ATG31 is essential for autophagy. (A) The Cis1 mutant is defective in accumulation of autophagic bodies. Wild-type (BY4741; a) and cis1D (BY4741 background) strains transformed with either vector alone (b) or a low copy CIS1 plasmid (c) were incubated for 6 h under nitrogen and carbon starvation conditions in the presence of 1 mM phenylmethanelusulfonylfluoride. DIC (differential interference contrast) images are shown. Bar = 5 lm. (B) Autophagic activity in cis1D cells. Wild-type cells (BY4741) or the indicated mutant strains (in the BY4741 background) expressing Pho8D60 were shifted from YEPD medium (white bars) to SD(-N) medium and incubated for 6 h (black bars). Autophagic activity was measured by alkaline phosphatase (ALP) assay as described in Materials and methods. Error bars indicate the SD of three independent experiments. (C) The cis1D strain is sensitive to nitrogen starvation. Wild-type (BY4741, closed circle), atg16D (open square), and cis1D (open circle) cells were cultured in nitrogen starvation medium. Aliquots were removed at the indicated times and spread onto YEPD plates. The number of colonies was determined after 2–3 days.

Fig. 2B, the atg31D mutant possessed normal levels of Atg8-PE and Atg12–Atg5 conjugates, indicating that the Atg31 protein is not involved in these two ubiquitin-like reactions. Although the atg31D mutant is defective in autophagosome formation, we found that prApe1 import occurred under both growing and starvation conditions in atg31D cells (Fig. 2C). These results show that the phenotype of atg31D cells is very similar to those of the atg17D and atg29D mutants, suggesting that Atg31 is the third APAtg protein that is not required for the Cvt pathway. Atg31 is localized at the pre-autophagosomal structure (PAS) Next, we examined the localization of Atg31-GFP by fluorescence microscopy. ATG31-GFP was chromosomally integrated, and the Atg31-GFP strain was normal in autophagy judging by ALP assay (data not shown). The cells were grown in SD + CA medium and treated with rapamycin for 3 h. Atg31-GFP was visible as a punctate dot near the vacuole and diffused in the cytosol prior to rapamycin treatment (Fig. 3A). After treatment with rapamycin for 3 h, brighter dots were observed. It is known that most AP-Atg proteins are localized at the PAS, very prox-

imal to the vacuole [1]. We next examined the distribution of Atg31 and Ape1, a PAS marker; these two proteins were shown to colocalize (Fig. 3B). Thus, the granular staining of Atg31 presented in the literature presumably represented the PAS; we were not able to see Atg31 localization at the spindle pole bodies. The localization of AP-Atg proteins is intimately interconnected [9]. In particular, Atg17 is required for the localization of other AP-Atg proteins to the PAS [9]. We examined the localization of Atg31-GFP in the atg17D strain by fluorescence microscopy after treatment with rapamycin for 3 h. As shown in Fig. 3C, in the atg17D strain, Atg31-GFP punctate staining disappeared. In contrast, in atg31D cells, Atg17-GFP staining primarily showed a PAS pattern, as in wild-type cells (Fig. 3C). Therefore, the PAS localization of Atg31-GFP requires Atg17. Atg31 physically associates with Atg17 The analysis of the GFP localization pattern along with the previous two-hybrid and mass spectrometry analyses suggested that Atg31 and Atg17 form a protein complex. To confirm that Atg31 indeed interacts with Atg17, coimmunoprecipitation experiments were performed. For the biochemical analysis, we constructed cells that had chro-

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Fig. 2. Phenotypes of atg31D cells are similar to those of atg17D cells. (A) Rapamycin-dependent dephosphorylation of Atg13 is normal in atg31D cells. Wild-type (BY4741) or the indicated mutant (BY4741 background) strains overexpressing Atg13 were grown in YEPD and then treated with rapamycin for 1 h. Samples were collected and Atg13 was detected by immunoblotting using an anti-Atg13 antibody. Equal amounts of cell lysates were loaded onto the gel. PGK served as a loading control. (B) Atg31 is not involved in two ubiquitin-like conjugation reactions. Wild-type (BY4741) or the indicated mutant (BY4741 background) cells were grown in YEPD or treated with rapamycin for 3 h. Total cell lysates were separated by SDS–PAGE containing 6 M urea and subjected to immunoblotting using anti-Atg8 (top) and anti-Atg12 (bottom) antibodies. (C) Atg31 is dispensable for the Cvt pathway. Wild-type (BY4741) or the indicated mutant (BY4741 background) strains were grown in YEPD medium (lanes: 1, 3, 5, 7, and 9), washed, and incubated in SD(-N) medium for 6 h (lanes: 2, 4, 6, 8, and 10). Cell lysates were prepared and analyzed by immunoblotting using an anti-Ape1 antibody.

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Fig. 3. Localization of Atg31-GFP. (A) Atg31-GFP displayed punctate perivacuolar dots and diffuse cytosolic staining. Wild-type cells expressing Atg31-GFP (YKY62) were grown in SD + CA medium (0 h) and treated with 0.2 lg/ml rapamycin for 3 h prior to fluorescence microscopy analysis. DIC, differential interference contrast. Bar = 3 lm. (B) Atg31 colocalizes with Ape1. Wild-type cells (YKY65) expressing combinations of Atg31-GFP and mRFP-Ape1 were incubated with rapamycin for 3 h. Fluorescence and DIC images are shown. Bar = 2 lm. (C) Atg31 localization to the PAS depends on Atg17. Wild-type (YKY62) and atg17D (YKY77) cells expressing Atg31-GFP were prepared and examined as indicated. Wild-type (YKY6) and atg31D (YKY79) strains expressing Atg17-GFP were examined as indicated. Bar = 5 lm.

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mosomally integrated C-terminally Myc tagged ATG31. Cells expressing Atg31-Myc showed normal autophagic activity, which was comparable to the level in wild-type cells (data not shown). Cells were grown in YEPD, converted to spheroplasts, and treated with rapamycin for 1 h. Total lysates were subjected to immunoprecipitation using anti-Myc antibody. Immunoblotting showed that Atg17 was present in anti-Myc immunoprecipitates prepared from cells expressing Atg31-Myc (Fig. 4), confirming that Atg31 and Atg17 interact. The amount of Atg17 bound to Atg31-Myc did not change with rapamycin treatment, suggesting that Atg31 constitutively forms a complex with Atg17.

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Fig. 4. Atg31 physically associates with Atg17. Wild-type cells expressing Atg31-Myc (YKY61) were converted to spheroplasts and treated with rapamycin for 1 h. Total lysates were immunoprecipitated with an antiMyc antibody and immunoblotted with an anti-Myc and Atg17 antibodies. A control was performed with wild-type cells (BY4741; ‘untagged’). For the experiment, approximately 0.5% of the total lysate or 20% of the total immunoprecipitated sample was loaded per lane.

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Acknowledgments

This study demonstrates that the yeast Atg31/Cis1 protein plays a role in autophagy. Atg31 functions with Atg17 at the PAS in order to form normal autophagosomes under starvation conditions. A recent study showed that Atg17 acts as a scaffolding protein to organize AP-Atg proteins to the PAS [9]. Twohybrid assay [10], mass spectrometry analysis [11], and coimmunoprecipitation demonstrate that Atg31 forms a complex with Atg17 and suggest that Atg31 functions coordinately with Atg17 at the PAS. Expectedly, defects in the atg31D mutant are very similar to those of atg17D; for example, in atg31D cells, autophagic bodies are not accumulated in the vacuole, but prApe1 is transported normally into the vacuole. Furthermore, the atg31D mutant also displays phenotypes common to mutant cells lacking other AP-ATG genes, including loss of viability under starvation conditions and defective sporulation [30]. Atg31 is not required for the dephosphorylation of Atg13, Atg8PE formation, or Atg12–Atg5 conjugation. These observations indicate that Atg31 belongs to the same functional unit as Atg17. It was shown previously that overexpressed Cis1/Atg31 restores the growth of cik1D cells at the restrictive temperature [12]. However, the role of Cis1/Atg31 in karyogamy remains to be elucidated. Cik1 was reported to localize to cytoplasmic microtubules and spindle pole bodies in a Kar3-dependent manner [13,14]. Cis1/Atg31 was predicted to share this localization. However, Atg31-GFP did not colocalize with Spc42-RFP, a marker protein for spindle pole bodies (our unpublished results). Instead, we showed that Atg31 locates to the PAS. We have also shown that Cik1 and Kar3 are dispensable for autophagy (our unpublished results). In addition, any other AP-Atg proteins are not required for karyogamy. These data support a role for Atg31 in autophagy rather than karyogamy and mitosis. Loss of Atg17 abolishes the PAS localization of most AP-Atg proteins [9]. Atg31 is primarily located to the PAS and requires Atg17 for correct localization. Similarly, several AP-Atg proteins failed to target to the PAS in atg31D mutant cells (our unpublished results), suggesting that Atg31 and Atg17 coordinately play a role in the recruitment of AP-Atg proteins to the PAS as a scaffolding protein complex. In addition to Atg31, Atg17 interacts with Atg29 [29]. Because Atg29 is required for the formation of autophagosomes, but not Cvt vesicles [29], we speculate that Atg31 and Atg29 cooperate to orchestrate PAS organization via interaction with Atg17, or that Atg31 forms a complex with Atg29 through Atg17. Considering that Atg17 is the most upstream component of PAS organization in our hierarchy map [9], it is an attractive idea that Atg31 functions in PAS organization as well as Atg17. Further analysis of these proteins is needed to provide additional information about the mechanism of PAS organization and autophagosome formation.

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