Induction of autophagy by phosphate starvation in an Atg11-dependent manner in Saccharomyces cerevisiae

Biochemical and Biophysical Research Communications xxx (2016) 1e6

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Induction of autophagy by phosphate starvation in an Atg11-dependent manner in Saccharomyces cerevisiae Hiroto Yokota, Katsuya Gomi, Takahiro Shintani* Department of Bioindustrial Informatics and Genomics, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 December 2016 Accepted 17 December 2016 Available online xxx

Upon nutrient starvation, eukaryotic cells exploit autophagy to reconstruct cellular components. Although autophagy is induced by depletion of various nutrients such as nitrogen, carbon, amino acids, and sulfur in yeast, it was previously ambiguous whether phosphate depletion could trigger the induction of autophagy. Here, we showed that phosphate depletion induced autophagy in Saccharomyces cerevisiae, albeit to a lesser extent than nitrogen starvation. It is known that rapid inactivation of the target of rapamycin complex 1 (TORC1) signaling pathway contributes to Atg13 dephosphorylation, which is one of the cues for autophagy induction. We found that phosphate starvation caused Atg13 dephosphorylation with slower kinetics than nitrogen starvation, suggesting that poor autophagic activity during phosphate starvation was associated with slower inactivation of the TORC1 pathway. Phosphate starvation-induced autophagy requires Atg11, an adaptor protein for selective autophagy, but not its cargo recognition domain. These results suggested that Atg11 plays important roles in low-level nonselective autophagy. © 2016 Elsevier Inc. All rights reserved.

Keywords: Yeast Autophagy Phosphate metabolism Target of rapamycin complex 1

1. Introduction Autophagy is a catabolic process by which cytoplasmic substrates are delivered to the lysosomes/vacuoles to be degraded and recycled. In response to environmental cues, the phagophore selectively or nonselectively enwraps target substrates to form double-membrane vesicles, called autophagosomes, which are then fused with lysosomes/vacuoles. Physiological roles of autophagy are categorized into three groups: cell survival during nutrient deprivation, removal of potentially cytotoxic compounds, and lysosome (vacuole) biogenesis. A number of studies using yeast Saccharomyces cerevisiae have identified 17 core autophagy-related (Atg) proteins required for autophagosome biogenesis, most of which are widely conserved in eukaryotic cells [1]. In order to initiate nutrient starvation-induced autophagy, the Atg17-Atg29Atg31 complex is required in addition to core machinery [2]. Upon nitrogen starvation, Atg13 is dephosphorylated by inactivation of target of rapamycin complex 1 (TORC1), which facilitates the

Abbreviations: TORC1, target of rapamycin complex 1; PSiA, phosphate starvation-induced autophagy; NSiA, nitrogen starvation-induced autophagy; Atg, autophagy-related; Cvt, cytoplasm to vacuole targeting. * Corresponding author. E-mail address: [email protected] (T. Shintani).

assembly of the supramolecular Atg1 complex composed of Atg1, Atg13, Atg17, Atg29, and Atg31; this activates Atg1 kinase activity and Atg9 vesicles are accumulated on this complex through Atg13Atg9 interaction [2e4]. In contrast, cells have evolved to acquire receptor proteins that recognize specific substrates for selective sequestration into autophagosomes. In some cases such as xenophagy (autophagy of invaded microorganisms), which occurs in animal cells and the cytoplasm to vacuole targeting (Cvt) pathway (autophagic transport of vacuolar enzymes) in yeast, cargos themselves act as signals and scaffolds for induction of autophagosome formation [5,6]. In the Cvt pathway, the vacuolar hydrolase aminopeptidase I (Ape1) forms the cytoplasmic aggregate, which is then recognized by the receptor Atg19 [7,8]. Atg19 further associates with the adaptor protein Atg11 to recruit Atg1 proteins and Atg9 vesicles on the surface of the cargo-receptor complex [7]. Recently, it was reported that the cargo-receptor-adaptor complex activates Atg1 kinase [9]. Upon depletion of macronutrients such as glucose, nitrogen, or phosphate, yeast cells arrest their growth at G1 phase and induce autophagy for survival [10e12]. Although starvation of these nutrients is known to stimulate autophagy [10], little attention has been paid to its magnitude. Furthermore, it is unknown whether the magnitude of autophagy affects its mechanism. In this study, we focused on the phosphate depleted condition because our

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Please cite this article in press as: H. Yokota, et al., Induction of autophagy by phosphate starvation in an Atg11-dependent manner in Saccharomyces cerevisiae, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.12.112

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knowledge of phosphate starvation-induced autophagy (PSiA) is very limited despite that fact that phosphate is an essential macronutrient for synthesis of various cellular constituents including nucleic acids, phospholipids, and high energy compounds such as ATP [11]. Here, we found that a depletion of phosphate elicited bulk autophagy to a lesser extent than nitrogen starvation. Additionally, PSiA was found to be under the control of TORC1 signaling pathway, but required Atg11 in addition to Atg17.

L kit (Nacalai Tesque, Japan) and image analyzer ImageQuant LAS4000 (GE Healthcare, Piscataway, NJ). For the CFP* processing assay, protein amounts were quantified using ImageQuant TL software (GE Healthcare, Piscataway, NJ), and free CFP was normalized by Pgk1. To calculate relative amounts of free CFP, samples from parent strains incubated in SDN for 4 h were analyzed in every experiment and used as controls. Relative amounts of free CFP were calculated by setting values of parent strains in SDN for 4 h to 100%.

2. Materials and Methods 2.3. Miscellaneous methods 2.1. Yeast strains, plasmids, and growth conditions The yeast S. cerevisiae strains and plasmids used in this study are listed in Table S1. The prototrophic strains were generated from BY4742 (MATa leu2D0 his3D1 lys2D0 ura3D0) by replacement of the leu2D0, his3D1, and lys2D0 alleles with PCR-generated DNA fragments containing LEU2, HIS3, or LYS2 genes, respectively. The remaining ura3D0 allele was used for selection of strains transformed with plasmids expressing reporter proteins. Gene disruptions were performed by the method described by Janke et al. [13] using disruption cassettes containing drug-resistant genes such as kanMX, hphNT1, or natNT2. Oligonucleotides used for plasmid construction are listed in Table S2. To generate pCu416-CFP, the DNA fragment encoding yEmCFP was PCR-amplified using oligonucleotides oTAKA195 and oTAKA197 from pKT212 [14] as template DNA, followed by digestion with BamHI and SpeI and ligation to pCu416 [15]. The CFP derivative CFP* (see Results) was expressed from pCu416-CFP. p3FLAG-ATG11 was constructed by homologous recombination in yeast [16]. The ATG11 promoter region with a 3FLAG-coding sequence and the ORF of ATG11 with a 3FLAG tag were PCRamplified with the primer combination of oTAKA349/oTAKA352 and oTAKA351/oTAKA350, respectively. BY4742 was transformed with these DNA fragments and pCu415 [15] digested with SacI and XhoI. The plasmid was rescued from Leuþ yeast clones. To generate p3FLAG-ATG11(1e859), the DNA fragment of 3FLAG ATG11(1e859) was amplified with oTAKA351 and oTAKA356, and then used for transformation of yeast with the ATG11 promoter fragment and SacI-XhoI digested pCu415. Yeast cells were grown at 30  C in synthetic dextrose (SD) medium (0.17% yeast nitrogen base without amino acids and ammonium sulfate [Formedium, Hunstanton, UK], 0.5% ammonium sulfate, 2% glucose). For nitrogen starvation, glucose starvation, and phosphate starvation, SDeN medium (0.17% yeast nitrogen base without amino acids and ammonium sulfate and 2% glucose), SDeC (0.17% yeast nitrogen base without amino acids and ammonium sulfate, and 0.5% ammonium sulfate), and SDeP medium (0.57% yeast nitrogen base without amino acids and phosphate [Formedium, Hunstanton, UK], 7.4 mM potassium chloride and 2% glucose) were used, respectively. To induce autophagy, cells grown in SD medium to an early log phase were washed with sterile water twice, resuspended in starvation medium at OD600 of 1.0, and incubated at 30  C for the appropriate periods. 2.2. Immunoblotting Whole cell extracts were prepared as described by Huang et al. [17]. For the CFP* processing assay, 1 ml of culture was harvested at the time points indicated and used for preparation of cell lysates. The samples equivalent to 0.1 OD600 were subjected to immunoblot analysis probed with anti-GFP (mFX75, Wako Chemical, Japan), anti-HA (F7, Santa Cruz Biotechnology, TX), anti-PGK (22C5D8, Abcam, UK), and anti-Ape1 (yH-16, Santa Cruz Biotechnology, TX) antibodies. Signal detection was performed using Chemi-Lumi One

The activity of repressible acid phosphatase (rAPase) was measured as previously described [18,19]. The b-galactosidase assay was performed by the method described by Rose and Botstein [20]. For ultrastructural observation of autophagic bodies, pho91D pep4Dprb1Dvps4D cells (YHY32) were incubated in SDN for 2 h or in SDP for 6 h and used for transmission electron microscopy, which was performed by Tokai-EMA Inc. based on rapid freezing and freeze-fixation methods as described by Okamoto et al. (2009) [21]. 3. Results 3.1. Autophagy was induced under a phosphate starvation condition In order to compare magnitudes of autophagy induced by depletion of glucose, nitrogen, or phosphate in yeast, we used a prototrophic strain as a wild-type strain so that we could exclude unexpected effects of supplemental amino acids and nucleic acids on autophagy. To determine the autophagic activity, we utilized a newly developed substrate called CFP*, which was a cyan fluorescent protein (CFP) fused with a 22-amino-acid peptide derived from a multiple cloning site of pRS416. Yeast cells expressing CFP* under the control of the CUP1 promoter were grown in normal SD medium or SD medium depleted of nutrients. First, we confirmed that the expression levels of CFP* were comparable among the conditions tested (Fig. 1A) and that CFP* was uniformly localized in the cytoplasm (Fig. S1). In wild-type cells, CFP* was converted to free CFP, which is relatively resistant to the vacuolar proteinase [22], under nitrogen starvation, while not in the atg1D cells (Fig. S1). The CFP* processing normally occurred in the cells lacking Atg11, an adaptor protein for selective autophagy, in this condition (Fig. S1). These results indicate that CFP* is usable as a reporter for bulk autophagy. We next evaluated the magnitude of autophagy stimulated by depletion of nitrogen, glucose, or phosphate. Cells expressing CFP* were grown to a mid-log phase in minimal medium (SD medium) and were then shifted to SD medium depleted with nitrogen (SDeN), glucose (SDeC), or phosphate (SDeP). The processing of CFP* occurred quickly in SDeN, and to a lesser extent in SDeC (Fig. 1A and C), which was consistent with the previous report by Takeshige et al. (1992) [10]. In contrast, the generation of free CFP was greatly reduced but reliably detectable under conditions of phosphate starvation (Fig. 1A and C). When the cells were shifted to SD medium, CFP* processing was not detected (Fig. 1C). These results indicated that bulk autophagy was only modestly induced by phosphate starvation. Because yeast cells can accumulate large amounts of phosphate as polyphosphate in the vacuole when grown in media containing high concentrations of phosphate [23], we assumed that this reservoir might buffer PSiA activity. Accordingly, the PHO91 gene was deleted, which encodes a vacuolar phosphate transporter implicated in export of orthophosphate from the vacuolar lumen to the cytoplasm [19], and thus its deletion was expected to reduce the available phosphate in the cytoplasm.

Please cite this article in press as: H. Yokota, et al., Induction of autophagy by phosphate starvation in an Atg11-dependent manner in Saccharomyces cerevisiae, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.12.112

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Fig. 1. Comparison of magnitude of bulk autophagy induced by depletion of nitrogen, carbon, and phosphate. (A) Wild-type strain (YHY36) harboring pCu416-CFP was grown in SD medium to an early log phase, and then transferred to SDN, SDC, or SDP medium. Total cell lysates were prepared at the indicated time points, analyzed by immunoblotting probed with anti-GFP and anti-Pgk1 antibodies. (B) The pho91D strain (YHY38) harboring pCu416-CFP grown in SD was incubated in SDN (left panel) and SDP (right panel) and subjected to CFP* processing assay. (C) Quantification of processed CFP. The intensities of free CFP signals from immunoblot analysis of Fig. 1A and B were normalized with Pgk1 signals at each time point. The amount of CFP in wild-type cells starved with nitrogen for 4 h was set to 100%. Solid and dashed lines indicate wild-type strain and pho91D strain, respectively. Circle: SDN; diamond: SDC; triangle: SDP; square: SD. Error bars represent standard deviation of three independent experiments. (D) Electron microscopy of autophagic bodies. The pho91Dpep4Dprb1Dvps4D cells incubated in SDP for 6 h were subjected to transmission electron microscopy. Arrowheads indicate examples of autophagic bodies. (E) Cross-sectional area of autophagic bodies was quantified using Image-J software (https://imagej.nih.gov/ij/) and presented as scatter dot plot with mean (closed diamonds) and standard deviation (error bars).

The pho91D cells grew slower than wild-type cells in SDeP medium, although there was no difference in the growth rate in SD medium, suggesting that stored phosphate was used to maintain cell growth during phosphate starvation (Fig. S2A). It is known that the expression of PHO5, encoding rAPase, is negatively correlated with the concentration of intracellular orthophosphate and thus rAPase activity can be an indicator for phosphate availability [24]. In the pho91D strain, rAPase activity was hyperactivated in response to phosphate starvation (Fig. S2B), suggesting that a concentration of intracellular phosphate (most probably cytoplasm phosphate) was more decreased in this strain. Notably, we found that the PHO91 deletion promoted PSiA, but not nitrogen starvationinduced autophagy (NSiA) (Fig. 1). We also confirmed that the level of PSiA varied dependently of the phosphate concentration of the medium (Fig. S3). Taken together, these results suggested that yeast cells sense the availability of storage and environmental phosphates to induce autophagy. We verified that autophagic bodies were accumulated in the vacuoles in the strain deficient of vacuolar proteinases in response to phosphate starvation by electron microscopy analysis (Fig. 1D). It should be noted that the vps4D background strain was used for electron microscopy analysis to prevent accumulating vesicles derived from a multivesicular body pathway in the vacuole [25]. We found that the size of the autophagic bodies was significantly smaller under phosphate starvation conditions than that under nitrogen starvation conditions (Fig. 1E, Fig. S4).

whether PSiA could be under the control of the PHO pathway. At a low phosphate concentration, Pho81 inhibits Pho85 kinase to prevent the transcriptional activator Pho4 from phosphorylation, resulting in its nuclear localization and transcriptional activation of

3.2. PSiA is regulated by TORC1, but not by the PHO pathway Intracellular phosphate levels are maintained by the phosphateresponsive signaling (PHO) pathway that modulates expressions of phosphate-responsive genes such as PHO84 (coding a high affinity phosphate transporter) and PHO5 to scavenge a variety of phosphate sources from the environment [26]. We therefore examined

Fig. 2. Autophagic activity in strains deleted for genes involved in PHO and TORC1 signaling pathways. CFP* processing assay was performed in pho91D background strains as described in Fig. 1. Error bars represent standard deviation of 3 independent experiments.

Please cite this article in press as: H. Yokota, et al., Induction of autophagy by phosphate starvation in an Atg11-dependent manner in Saccharomyces cerevisiae, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.12.112

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Fig. 3. Dephosphorylation of Atg13 in response to a nitrogen and phosphate starvation. Wild-type (A) and pho91D (B) cells expressing Atg13-6HA were incubated in SDN and SDP and total cell lysates were subjected to immunoblot analysis probed with anti-HA antibody.

PHO genes [26]. We first investigated whether this Pho4-dependent transcription could be involved in PSiA. Accordingly, we deleted

PHO4 gene in the pho91D strain and performed the CFP* processing assay. As shown in Fig. 2, autophagy activity was not affected by the deletion of PHO4, although its deletion caused loss of PPHO84-lacZ activation (Fig. S5), indicating that PSiA was not downstream of Pho4-dependent transcription. Similarly, deletion of the PHO81 or PHO85 gene did not affect PSiA (Fig. 2). We therefore concluded that PSiA was not under the control of the PHO pathway. To gain further insight into the signal transduction pathway for PSiA, we next examined the involvement of TORC1 in PSiA. The TORC1 signaling pathway senses nutrients and negatively regulates autophagy [27]. Recently, it has been reported that Npr2 and Npr3 are negative regulators of TORC1 and disruptions of these proteins impair NSiA [28,29]. We therefore measured PSiA in npr2Dpho91D and npr3Dpho91D strains and found that both PSiA and NSiA were significantly attenuated in these mutants (Fig. 2), indicating that PSiA was also regulated by the TORC1 pathway. When the pho91D strain was incubated in SDP medium containing rapamycin, a TORC1 inhibitor, autophagic activity was elevated to a similar extent as in SD containing rapamycin (Fig. S6). These results suggested that lower autophagic activity under phosphate starvation might be attributed to weak inhibition of the TORC1 pathway rather than lack of phosphate-containing compounds required for autophagic progression. In order to verify this hypothesis, we examined TORC1-dependent phosphorylation of Atg13, one of the components of the Atg1 complex. Upon nitrogen starvation, TORC1 becomes inactivated to dephosphorylate Atg13, thereby

Fig. 4. Atg11 is crucial for nonselective autophagy during phosphate starvation. (A) CFP* processing assay was performed in pho91D (closed circle), pho91Datg11D (open square), and pho91Datg17D (open triangle) strains as in Fig. 1 (B) pho91D cells harboring empty vector (pRS415) and pho91Datg11D cells harboring empty vector, p3FLAG-ATG11, and p3FLAG-ATG11(1e859) respectively, were grown in SD medium. Total cell lysates were subjected to immunoblot analysis with anti-FLAG, anti-Ape1, and anti-Pgk1 antibodies. (C) CFP* processing assay was performed for the strains used in Fig. 4B. Cells were incubated in SDN and SDP. Total cell lysates were subjected to immunoblot analysis probed with anti-GFP and anti-Pgk1 antibodies, and relative amounts of free CFP were calculated as described in Materials and Methods. Error bars represent standard deviation of 3 independent experiments. *p < 0.05 and ***p < 0.001.

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accelerating the assembly of this complex [2,3]. To examine a phosphorylation state of Atg13 during phosphate starvation, we expressed Atg13-6HA in wild-type and pho91D strains. In both strains, Atg13-6HA underwent rapid dephosphorylation (within 10 min), which was observed as faster migration on SDS-PAGE as previously reported [3], during nitrogen starvation (Fig. 3A and B). During phosphate starvation, change in migration of the Atg136HA band was only limited in the wild type strain, suggesting that the inactivation of TORC1-dependent phosphorylation of Atg13 occurred moderately under phosphate starvation (Fig. 3A). Importantly, in pho91D cells the band shift of Atg13-6HA was more evident after 30 min of incubation in SDP, albeit nitrogen starvation did not affect the kinetics of Atg13 dephosphorylation (Fig. 3B). This result implies that the availability of intracellular phosphate is tightly associated with the phosphorylation state of Atg13. 3.3. Atg11, an adaptor protein for selective autophagy, is essential for PSiA It is known that Atg17 and Atg11 are factors specifically required for initiation of starvation-induced bulk autophagy and selective autophagy, respectively [3]. Because PSiA is nonselective autophagy, we assumed that Atg17, but not Atg11, might be necessary for PSiA. To test this idea, we measured autophagy in atg17D and atg11D strains (pho91D background strains). As expected, both PSiA and NSiA were completely impaired in atg17D (Fig. 4A). Intriguingly, PSiA but not NSiA was almost completely inhibited in atg11D (Fig. 4A), suggesting that Atg11 appeared to have some essential roles in bulk autophagy under phosphate starvation. This is surprising, because it has been widely accepted that Atg11 is an adaptor protein that bridges selective cargos and core autophagy machinery proteins and is thereby specifically required for selective autophagy [30]. To verify that Atg11 fulfills its role in bulk autophagy, we examined if an Atg11 mutant lacking its C-terminal cargo-binding domain (860e1178 amino acids) was enough to support PSiA. In the Cvt pathway, a propeptide of precursor Ape1 (prApe1) is removed by vacuolar proteinases after reached at the vacuolar lumen. As previously reported [31], prApe1 transport was defective in the strain expressing 3FLAG-Atg11(1e859), but active in the strain expressing wild-type 3FLAG-Atg11 (Fig. 4B). We next analyzed PSiA in these strains. PSiA was entirely blocked in the atg11Dpho91D strain with an empty plasmid, whereas NSiA occurred as normal in this strain (Fig. 4C). Notably, 3FLAGAtg11(1e859) could partially complement the defect in PSiA in the atg11Dpho91D strain (Fig. 4C), indicating that Atg11(1e859) was enough to achieve bulk autophagy in phosphate starvation conditions. 4. Discussion Here we demonstrated that a significantly low but reliable level of bulk autophagy was induced in S. cerevisiae during phosphate starvation. Although PSiA is regulated by the TORC1 signaling pathway similar to NSiA, the magnitude of PSiA is much lower than that of NSiA. This may be attributed to slower kinetics of TORC1 inactivation in phosphate-free medium [32]. Consistent with slow inactivation of TORC1, Atg13 is dephosphorylated slower during phosphate starvation than during nitrogen starvation (Fig. 3). This implies that the assembly of the supramolecular Atg1 complex as a scaffold for autophagosome formation (i.e., 1:1 interactions between Atg13 and Atg1 and multimeric interaction between Atg13 and Atg17) may be inefficient during phosphate starvation, which in turn causes lower activation of Atg1 kinase activity and inefficient recruitment of Atg9 vesicles to this complex through Atg13-

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Atg9 interaction [2,4,33]. This inefficient assembly of the scaffold complex might result in smaller autophagosome formation in phosphate starvation conditions. Our analyses revealed that Atg11 was necessary for bulk autophagy in addition to Atg17 (Fig. 4). In the Cvt pathway, the cargoreceptor complex (Ape1-Atg19 complex) associates with Atg11, which further binds to Atg1 kinase to activate its kinase activity [9]. An Atg11-Atg9 interaction is also known to be important for the Cvt pathway, but not for NSiA [34]. These facts suggest that the Atg11Atg1 system may compensate for inefficient assembly of a supramolecular Atg1 complex during phosphate starvation. Because Atg11 is known to interact with Atg29 [35], Atg11 could be an additional component of the Atg1 complex to ensure the enrichment of Atg1 kinase and Atg9 vesicles. Both Atg11 and Atg17 are conserved in many fungi, but their requirements for bulk autophagy are not necessarily the same. For instance, a single deletion of ATG11 and ATG17 in Kluyveromyces marxianus causes a partial defect in NSiA, but double deletion results in a complete block of NSiA [36], indicating that Atg11 and the Atg17-Atg29-Atg31 complex are redundant in NSiA. On the other hand, Atg11 and Atg17 are both essential for NSiA in Schizosaccharomyces pombe [37], suggesting that these proteins act in the same pathway similar to PSiA in S. cerevisiae. As we have shown, the requirement of these molecules can vary among nutrient conditions as well. We conclude that fungi may use these two systems to optimize the Atg1 complex for adaption to fluctuating environments. Further analysis is needed to understand how Atg11 and the Atg17-Atg29-Atg31 complex coordinate to modulate the Atg1 complex. Conflict of interest The authors declare no conflict of interest. Acknowledgements This work was supported by the Institute for Fermentation, Osaka (IFO). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.12.112. References [1] M. Jin, D.J. Klionsky, The core molecular machinery of autophagosome formation, in: H.-G. Wang (Ed.), Autophagy and Cancer, Current Cancer Research, vol. 8, Springer ScienceþBusiness Media LLC, New York, 2013, pp. 25e45. [2] H. Yamamoto, Y. Fujioka, S.W. Suzuki, D. Noshiro, H. Suzuki, C. Kondo-Kakuta, Y. Kimura, H. Hirano, T. Ando, N.N. Noda, Y. Ohsumi, The intrinsically disordered protein Atg13 mediates supramolecular assembly of autophagy initiation complexes, Dev. Cell. 38 (2016) 86e99. [3] Y. Kamada, T. Funakoshi, T. Shintani, K. Nagano, M. Ohsumi, Y. Ohsumi, Tormediated induction of autophagy via an Apg1 protein kinase complex, J. Cell. Biol. 150 (2000) 1507e1513. [4] Y. Kamada, K. Yoshino, C. Kondo, T. Kawamata, N. Oshiro, K. Yonezawa, Y. Ohsumi, Tor directly controls the Atg1 kinase complex to regulate autophagy, Mol. Cell. Biol. 30 (2010) 1049e1058. [5] T. Shintani, D.J. Klionsky, Cargo proteins facilitate the formation of transport vesicles in the cytoplasm to vacuole targeting pathway, J. Biol. Chem. 279 (2004) 29889e29894. [6] S.T. Shibutani, T. Yoshimori, Autophagosome formation in response to intracellular bacterial invasion, Cell. Microbiol. 16 (2014) 1619e1626. [7] T. Shintani, W.-P. Huang, P.E. Stromhaug, D.J. Klionsky, Mechanism of cargo selection in the cytoplasm to vacuole targeting pathway, Dev. Cell. 3 (2002) 825e837. [8] A. Yamasaki, Y. Watanabe, W. Adachi, K. Suzuki, K. Matoba, H. Kirisako, H. Kumeta, H. Nakatogawa, Y. Ohsumi, F. Inagaki, N.N. Noda, Structural basis for receptor-mediated selective autophagy of aminopeptidase I aggregates,

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Please cite this article in press as: H. Yokota, et al., Induction of autophagy by phosphate starvation in an Atg11-dependent manner in Saccharomyces cerevisiae, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.12.112