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© 2016. Published by The Company of Biologists Ltd.

Atg20 and Atg24 family proteins promote organelle autophagy in
fission yeast

Dan Zhaoa,b, Xiao-Man Liub, Zhong-Qiu Yub, Ling-Ling Sunb, Xingchuang Xiongc, Meng-
Qiu Dongb, Li-Lin Dub

a
PTN Graduate Program, School of Life Sciences, Peking University, Beijing 100871, China;
b
National Institute of Biological Sciences, Beijing 102206, China; cNational Institute of
Metrology, Beijing 100013, China

Correspondence to: Li-Lin Du; Email: dulilin@nibs.ac.cn

Keywords: autophagy, autophagosome, Atg20, Atg24, PX-BAR, fission yeast,
Schizosaccharomyces pombe

Abbreviations: Atg, autophagy-related; PAS, pre-autophagosomal structure/phagophore
assembly site; Cvt pathway, cytoplasm to vacuole targeting pathway

Journal of Cell Science • Advance article

JCS Advance Online Article. Posted on 13 October 2016

Summary statement
We found that fission yeast Atg20 and Atg24 family proteins promote starvation-induced
autophagy of ER and mitochondria. We propose that their function is to ensure that
autophagosomes are of sufficient sizes.

Journal of Cell Science • Advance article

Abstract
Autophagy cargos include not only soluble cytosolic materials but also bulky organelles such
as ER and mitochondria. In budding yeast, two proteins that contain the PX domain and the
BAR domain, Atg20/Snx42 and Atg24/Snx4, are required for organelle autophagy and
contribute to general autophagy in a way that can be compensated. It remains unclear why
these proteins are important for organelle autophagy. Here, we show that in a distantly related
fungal organism the fission yeast Schizosaccharomyces pombe, autophagy of ER and
mitochondria is induced by nitrogen starvation and is promoted by three Atg20 and Atg24
family proteins, Atg20, Atg24, and Atg24b/SPBC1711.11. These proteins localize at the pre-
autophagosomal structure/phagophore assembly site (PAS) during starvation. Atg24 forms a
homo-oligomer, and acts redundantly with Atg20 and Atg24b, which form a hetero-oligomer.
The organelle autophagy defect caused by the loss of these proteins is associated with a
reduction of autophagosome size and a decrease of Atg8 accumulation at PAS. These results
provide new insights into the autophagic function of Atg20 and Atg24 family proteins.

Journal of Cell Science • Advance article

Introduction

Macroautophagy (hereafter autophagy) is an evolutionarily conserved process by which
intracellular components are transported into lysosomes or vacuoles using a double-
membraned vesicle called autophagosome. Unicellular eukaryotes such as yeasts depend
heavily on autophagy for their survival when facing starvation. In recent years, diverse roles
of autophagy have been uncovered in various aspects of human health, such as cancer,
infectious disease, and aging (Jiang and Mizushima, 2014).
The molecular machinery driving autophagy has been best characterized in the
budding yeast Saccharomyces cerevisiae (Xie and Klionsky, 2007; Inoue and Klionsky, 2010;
Mizushima et al., 2011; Feng et al., 2014; Ohsumi, 2014). Extensive studies in this organism
have identified dozens of autophagy-related (Atg) proteins. Some of the Atg proteins are
required for the autophagy of all types of cargos. They are called core Atg proteins and are
involved in the biogenesis of autophagosomes. In contrast, other Atg proteins are only
required for the autophagy of special cargos but not general cytosolic proteins. Many of these
latter factors contribute to the autophagy of a single type of cargo, for example by conferring
cargo selectivity in a selective autophagy pathway. However, there are also Atg proteins that
are required for the autophagy of several different types of special cargos but are dispensable
for general autophagy. These include Atg11, a scaffold protein bound by many cargo-
selectivity factors, and Atg20/Snx42 and Atg24/Snx4, two PX-BAR proteins whose exact
functions remain mysterious (Inoue and Klionsky, 2010).
S. cerevisiae ATG20/SNX42 (originally called CVT20) and ATG24/SNX4 (originally
called CVT13) genes were first implicated in autophagy when their mutants were isolated in
screens for mutants defective in the cytoplasm-to-vacuole targeting (Cvt) pathway, a selective
autophagy pathway constitutively transporting the cytosolic hydrolase Ape1 into the vacuole Journal of Cell Science • Advance article

(Nice et al., 2002). In the same study, it was shown that these two genes are also required for
the autophagy of peroxisomes (pexophagy). Subsequently, it was shown that they are also
required for the efficient autophagy of mitochondria (mitophagy) (Kanki and Klionsky, 2008;
Kanki et al., 2009b; Okamoto et al., 2009). Atg20/Snx42 and Atg24/Snx4 localize to the pre-
autophagosomal structure/phagophore assembly site (PAS) in a manner dependent on their
binding to phosphatidylinositol 3-phosphate through the PX domain (Nice et al., 2002).

The cellular functions of S. cerevisiae Atg20/Snx42 and Atg24/Snx4 are not limited
to selective autophagy. Combining atg20∆ or atg24∆ with any one of several Golgi-
endosomal trafficking mutants resulted in synergistic defects in starvation-induced bulk
autophagy, suggesting that Atg20/Snx42 and Atg24/Snx4 contribute to general autophagy in
a way that can be compensated (Ohashi and Munro, 2010; Shirahama-Noda et al., 2013). In
addition, Atg20/Snx42, Atg24/Snx4, and a paralog of Atg20/Snx42 called Snx41, are
required for the endosomal sorting of the SNARE protein Snc1 (Hettema et al., 2003). It is
unclear whether Snx41 participates in autophagy. The relationship between the autophagic
function and the endosomal sorting function of Atg20/Snx42 and Atg24/Snx4 has not been
thoroughly investigated.
Few studies have examined the autophagic roles of Atg20- and Atg24-related proteins
in species other than S. cerevisiae. In the methylotrophic yeast Pichia pastoris, the ortholog
of Atg24/Snx4 is required for pexophagy but the roles of the Atg20/Snx42- and Snx41-
related proteins are unknown (Ano et al., 2005). In the rice-blast fungus Magnaporthe oryzae,
the ortholog of Atg24/Snx4 is required for mitophagy (He et al., 2013), whereas an
Atg20/Snx42- and Snx41-related protein is required for pexophagy (Deng et al., 2012; Deng
et al., 2013). Mammalian homologs of Atg20/Snx42 and Atg24/Snx4 exist (van Weering et
al., 2010), but it is unknown whether they are involved in autophagy.
The fission yeast Schizosaccharomyces pombe is an important unicellular eukaryotic
model organism and is evolutionarily distant from the budding yeast S. cerevisiae (Hoffman
et al., 2015). We have previously comprehensively identified and characterized the fission
yeast genes required for starvation-induced autophagy, and unveiled interesting differences in
autophagic mechanisms between the two model yeasts (Sun et al., 2013). For example, unlike
its budding yeast counterpart, fission yeast Atg11 is required for general autophagy. Thus, Journal of Cell Science • Advance article
Atg11 dependence cannot be used as a criterion to judge whether a cargo is selectively
targeted by autophagy in fission yeast. No systematic analysis on the autophagy of special
cargos such as organelles has been performed in fission yeast.
In this study, we firstly demonstrated that two types of organelles, ER and
mitochondria, are subject to autophagy during nitrogen starvation in fission yeast. To
understand whether organelle autophagy has requirement different from general autophagy,
we studied the four fission yeast homologs of S. cerevisiae Atg20/Snx42 and Atg24/Snx4,
none of which has been experimentally characterized before. Out of the four proteins, we
found that at least three of them, Atg20, Atg24, and Atg24b, play redundant roles in organelle

Journal of Cell Science • Advance article .autophagy. and carried out in-depth analysis of the phenotypes of their mutants.and Atg24/Snx4-related proteins. Our study shed new light on the autophagic function of the Atg20/Snx42. We examined their subcellular localization patterns and the protein-protein interactions they engage in.

after entering the vacuole. 2011. we analyzed the localization patterns of ER-GFP and mito-mCherry. and S1A). 2007. and pronounced colocalization was observed between Ost4-CFP and Cpy1-mCherry and between Sdh2-mCherry and Cpy1- Venus (Figure 1A. and monitored the subcellular localizations of two organelle markers. Under nutrient-rich conditions (+N). are attacked by vacuolar proteases and the free forms of fluorescent proteins accumulate due to resistance to proteases. which is a trigger of general autophagy in S. 2010). Results Organelle autophagy is induced by nitrogen starvation in S. 2009. 2003). B. B. we applied nitrogen starvation treatment. Thus. the only report of organelle autophagy in S. To examine whether organelle autophagy occurs in wild-type cells... to corroborate the imaging data with a biochemical assay. 2010. 2013). pombe (Kohda et al. whereas clear colocalization between ER-GFP and Cpy1-mCherry and between mito-mCherry and Cpy1-Venus was observed after Journal of Cell Science • Advance article nitrogen starvation (Figure 1C. Sun et al.. D.. pombe is the observation that mitochondrial autophagy is required for the survival of a proteasome mutant in quiescent state (Takeda et al. In atg5Δ cells.. B.. which are fluorescent proteins targeted to ER lumen and mitochondrial matrix by a signal peptide and an ER retrieval sequence (Zhang et al. We used Cpy1 as a vacuole lumen marker (Tabuchi et al. and a mitochondrial targeting sequence (Yaffe et al. respectively. Under nutrient-rich conditions. both ER-GFP and mito- mCherry failed to enter vacuoles upon starvation treatment (Figure 1C.. Under . 1997). and S1A). and S1A). and S1A). 1987). Zhang et al. 2012). indicating that autophagy is required for the relocalization of ER and mitochondrial proteins into the vacuole lumen. Fluorescent protein-fused proteins. Thus. an ER integral membrane protein Ost4 and a mitochondrial matrix protein Sdh2.. and S1A). pombe To our knowledge. we examined the vacuolar processing of Ost4-CFP and Sdh2-mCherry using immunoblotting analysis. Atg5-dependent starvation-induced vacuole localization occurs to multiple ER and mitochondrial markers and most likely reflects the autophagy of these two organelles. Starvation-induced vacuole localization of Ost4-CFP and Sdh2-mCherry did not occur in atg5Δ cells (Figure 1A. Young and Fantes. neither marker showed overlap with Cpy1. D.. neither Ost4 nor Sdh2 showed obvious overlap with Cpy1 (Figure 1A. cells became shortened due to cell division without cell growth (Yanagida et al. After shifting cells to a nitrogen-free medium (-N) for 12 h. Mukaiyama et al. To determine whether other ER and mitochondrial proteins also undergo starvation- induced relocalization to the vacuole.

and one of the new bands ran at a position expected for the free CFP (Figure 1E). We chose to use the terms of ER autophagy and mitochondrial autophagy.. Taken together.nutrient-rich conditions. no free CFP or mCherry bands were generated after starvation (Figure 1E. the processing of Sdh2-mCherry also occurred after starvation (Figure 1F). Similarly. 2015. Sica et al. Thus. whereas two Atg1-associated proteins. 2013). whose sizes correspond to those of the full-length fusion proteins (Figure 1E. Okamoto.. starvation-induced organelle autophagy requires all of these five Atg proteins in fission yeast. pombe. 2014. and it is not yet clear whether the starvation-induced organelle autophagy processes we observed here are selective or nonselective. our results indicate that two types of organelles. and Atg101 (Figure S1B and S1C). 2010). in atg5Δ cells.. Khaminets et al.. when probed with antibodies against the fluorescent proteins. 2016). not ER-phagy or mitophagy. Atg13. Journal of Cell Science • Advance article . 2014. We found that starvation-induced vacuole localization of Ost4-CFP and Sdh2-mCherry was abolished in fission yeast mutants lacking either Atg1 or any one of four potential Atg1- associated proteins. are delivered into the vacuole by autophagy upon starvation treatment in S. because the latter terms usually refer to selective autophagy pathways (Jin et al. As expected. similar to the situation of starvation-induced bulk autophagy (Sun et al. after 6 h starvation. F). to describe these processes. are required for only selective autophagy or only nonselective autophagy. 2013. including Atg11. ER and mitochondria. For the Ost4-CFP expressing cells. Ost4- CFP and Sdh2-mCherry appeared as single bands. F).. Atg11 and Atg17. respectively (Inoue and Klionsky. Atg17. lower molecular weight CFP-antibody-reactive bands emerged. Atg1 kinase is required for both selective and nonselective autophagy. Rogov et al. In budding yeast.

2007). Suzuki and Ohsumi. C). probably have arisen through independent gene duplication events occurring after the divergence of the ancestors of S..08 (named Atg20 in PomBase) (McDowall et al. SPBC1711. cerevisiae Atg20/Snx42 and Snx41. In a phylogenetic tree we constructed using PX-BAR proteins of representative Ascomycota fungal species. 2010. the two S. extensive colocalization was . CFP-Atg8. Journal of Cell Science • Advance article To assess which fission yeast Atg20 and Atg24 family proteins are involved in autophagy. Vps17. 2013. and Mug186 localize to PAS Budding yeast Atg20/Snx42 and Atg24/Snx4. van Weering et al. a PX domain and a BAR domain (Teasdale and Collins. which are required for organelle autophagy but not general autophagy. SPCC16A11. Starvation treatment induced Atg24b to form a few cytoplasmic puncta per cell (Figure 2D). There are at least one Atg20 family protein and one Atg24 family protein in each of the fungal species surveyed.12 (named Atg24 in PomBase). cerevisiae Atg20/Snx42. Within the Atg24 family. 2013). Budding yeast Atg20/Snx42 and Atg24/Snx4 are PAS-localizing proteins (Nice et al.11 (unnamed in PomBase. Our homology searches showed that there are seven PX-BAR proteins in S. SPAC6F6. Under nutrient-rich conditions. 2015). S. Atg20 formed a few cytoplasmic puncta per cell (Figure 2A). cerevisiae. We will refer to these two branches as Atg20 family and Atg24 family. and the two S. and Mvp1 proteins. where many Atg proteins also accumulate (Sun et al. hereafter referred to as Atg24b). CFP-Atg8 is diffusely distributed in the cytoplasm under nutrient-rich conditions. pombe Atg24 is a closer homolog of S. Autophagy proteins in both budding yeast and fission yeast usually localize to a subcellular site called pre-autophagosomal structure or phagophore assembly site (PAS) when autophagy occurs (Sun et al. 2002). are PX-BAR proteins each harboring two membrane- binding domains. Atg24. Xie and Klionsky. cerevisiae Atg24/Snx4 in another branch (Figure S2B). and upon starvation treatment forms bright puncta.. and examined whether they colocalize with a PAS marker. respectively. Four of these seven proteins. Atg20 and Mug186 fall into the same branch with S. cerevisiae proteins Atg20/Snx42 and Snx41. pombe proteins Atg20 and Mug186. pombe Atg24b.. whereas Atg24 and Atg24b cluster together with S. and Atg24b was diffusely distributed in the cytoplasm and the nucleus (Figure 2D). are more closely related to S. Atg24/Snx4. Atg24 and Mug186 formed dozens of cytoplasmic puncta per cell (Figure 2B. and Snx41. we tagged their endogenous genes with a C-terminal YFP tag. pombe (Figure S2A). and SPBC14F5. cerevisiae Atg24/Snx4 than is S. Atg24b. 2010). After 2 h starvation. These two branches are more closely related to each other than to the other three branches formed by Vps5. 2012.Atg20. pombe and S.11c (named Mug186 in PomBase).. Within the Atg20 family..

Snx41. still formed starvation-induced puncta (Figure S3B). and Atg24b (Figure 2E. only a minority of CFP-Atg8 puncta overlapped with Mug186 puncta (Figure 2G. Atg20 no longer formed cytoplasmic puncta (Figure 2E). Atg24. which is the autophagy-specific subunit of the phosphatidylinositol 3-kinase complex I that generates phosphatidylinositol 3-phosphate at PAS (Kihara et al. 2003). We next examined whether deleting the genes encoding these four proteins causes autophagy defect. indicating that Atg20 and Atg24b accumulate on the PAS. we constructed a quadruple deletion mutant that lacks all four proteins. Atg20. Thus. and Mug186. especially for Atg24 and Mug186. pombe Because of their sequence similarities. Atg24. Journal of Cell Science • Advance article Atg20. In budding yeast. We hypothesized that. most of the cytoplasmic puncta formed by Atg24/Snx4. PAS localization of Atg20 and Atg24 family proteins should require Atg14. G). 2001). Atg24b. We first analyzed whether the quadruple mutant (QM) has a defect in general autophagy by monitoring the processing of Tdh1-YFP (Figure 3A). After starvation treatment.. Thus.. Indeed. Atg24. which generates phosphatidylinositol 3-phosphate at the endosome (Kihara et al. Atg24.. pombe Atg20 and Atg24 family proteins localize to PAS. D. we may eliminate the endosomal accumulation of Atg20 and Atg24 family proteins and better visualize their PAS localization. puncta formation by these four proteins was completely abolished in atg14Δ vps38Δ double mutant cells (Figure S3A). F. In contrast. Vps38 is a complex-specific subunit of the phosphatidylinositol 3-kinase complex II. implying that they play direct roles in autophagy. Tdh1 is the major form of fission yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Morigasaki et al. and between CFP-Atg8 puncta and Atg24b puncta (Figure 2A. 2013). The large numbers of Atg24 and Mug186 puncta per cell precluded us from reliably determining the extent of their colocalization whith CFP-Atg8 puncta. Indeed. Atg17. H. in which an Atg14-independent PAS marker. and Atg20/Snx42 are abolished in vps38∆ mutant (Hettema et al. I). 2008). I).observed between CFP-Atg8 puncta and Atg20 puncta.. I). 2001). and Atg24b have especially pronounced association with the PAS. by using a vps38Δ fission yeast mutant background. and the numbers of puncta formed by Atg24 and Mug186 dramatically decreased (Figure 2F. we concluded that all four S.. in vps38Δ cells grown under nutrient-rich conditions. Free YFP was generated after starvation . and has been used as a representative marker for nonselective bulk autophagy (Sun et al. most of the CFP-Atg8 puncta were found to colocalize with the puncta formed by Atg20. and Atg24b act redundantly to promote ER autophagy in S. we hypothesized that functional redundancy may exist between Atg20.

Among the double and triple mutants lacking Atg24. Vacuole localization of ER-GFP was defective in three of the four triple mutants (Figure 3D). we examined the triple mutants lacking three of the proteins. We then examined double mutants lacking two of the four proteins. suggesting that the ER autophagy defect of the quadruple mutant is due to the loss of more than one protein. Next. Atg24b. Wild-type-level Tdh1-YFP processing occurred in the quadruple mutant. Atg24. indicating that general autophagy is normal in the absence of Atg20. . Similarly. cannot even partially fulfill the autophagic function carried out by Atg24 or the combination of Atg20 and Atg24b. in the absence of Atg24. The exception was atg20Δ atg24bΔ mug186Δ. indicating that either Atg24 alone or the combination of Atg20 and Atg24b can fully support ER autophagy. Thus. but not in the other four double mutants (Figure 3D). atg20Δ atg24bΔ mug186Δ triple mutant and atg24Δ mug186Δ double mutant behaved like wild type. we analyzed the processing of Ost4-CFP (Figure Journal of Cell Science • Advance article 3E).treatment in wild type but not atg5Δ mutant. Starvation-induced processing of Ost4-CFP was diminished in atg20Δ atg24Δ and atg24Δ atg24bΔ double mutants. and Atg24b play crucial but redundant functions in ER autophagy. The severity of the defect of these two double mutants was similar to that of the quadruple mutant (QM). Thus. Atg24. To further characterize which ones of these four proteins are involved in this process. we investigated whether these four proteins participate in organelle autophagy. and examined the localization of the other ER marker ER-GFP upon starvation treatment (Figure 3D). Atg24. and found that the vacuole localization of ER-GFP was strongly diminished in atg20Δ atg24Δ and atg24Δ atg24bΔ. the processing of Ost4-CFP was reduced in the quadruple mutant (Figure 3C). we generated single mutants that lack either of Atg20. either the combination of Mug186 and Atg24b. We first examined the behavior of the ER marker Ost4-CFP by imaging analysis and found that starvation-induced entry of Ost4-CFP into vacuoles was dramatically impeded in the quadruple mutant cells (Figure 3B). only atg24Δ mug186Δ double mutant behaved like wild type. both Atg20 and Atg24b but not Mug186 become important. the autophagic delivery of ER into the vacuole is defective in the absence of these four proteins. and Mug186. Consistent with the imaging results obtained with the ER-GFP marker. suggesting that Atg20. suggesting that Atg24 can fully support ER autophagy in the absence of the other three proteins. suggesting that the remaining proteins in these two double mutants. or Mug186. To further confirm these results. We observed wild-type-like colocalization between ER-GFP and Cpy1 after 12 h starvation in all four single mutants. or the combination of Mug186 and Atg20. Finally. Atg24b.

Atg24-Mug186 interaction was also supported by reciprocal coimmunoprecipitation (Figure 5C. Atg24 and Atg24b act redundantly to promote mitochondrial autophagy in S. are sufficient for ER autophagy. the processing of Sdh2-mCherry was defective in atg20Δ atg24Δ and atg24Δ atg24bΔ double mutants. Atg24 and Atg24b may function in mitochondrial autophagy in a redundant manner. Nice et al. Atg24. We first performed affinity purification coupled with mass spectrometry (AP-MS) analysis and observed co-purification between Atg20 and Atg24. we examined a number of selected double and triple mutants. In conclusion. Atg20. mitochondrial autophagy is promoted redundantly by Atg24. robust coimmunoprecipitation was observed no matter which protein was used as bait (Figure 5B. but not in atg24Δ mug186Δ double mutant and atg20Δ atg24bΔ mug186Δ triple mutant (Figure 4B). Consistent with the findings for ER autophagy. including the interaction between Atg24/Snx4 and Atg20/Snx42. Atg20 and Atg24 family proteins were found to engage in heterotypic and homotypic interactions.. These interactions were confirmed by coimmunoprecipitation experiments (Figure 5B-E).. Therefore. 2003. the mitochondrial autophagy-promoting function can be fulfilled equally well by the combined actions of Atg20 and Atg24b. Either Atg24 alone. we examined the physical interactions between the Atg20 and Atg24 family fission yeast proteins. One explanation is that these two proteins may act together in a hetero-oligomer. Fission yeast Atg20 and Atg24 family proteins form multiple oligomers In S. we found that. 2002). among the four Atg20 and Atg24 family fission yeast proteins. these data demonstrate that like ER autophagy. Thus. at least three. and between Atg24 and Mug186 (Figure 5A). cerevisiae. and Atg24b. D). For the Atg20-Atg24 pair. but not in atg24Δ mug186Δ double mutant and atg20Δ atg24bΔ mug186Δ triple mutant (Figure 4A). Atg20. contribute to ER autophagy in a redundant manner. For the Atg20-Atg24b interaction. strong . the interaction between Atg24/Snx4 and Snx41. or Atg20 and Atg24b together. the vacuolar entry of the mitochondrial marker mito-mCherry was impeded in atg20Δ atg24Δ and atg24Δ atg24bΔ double mutants. and Atg24b. similar to the way they act in ER autophagy. between Atg20 and Atg24b. but the presence Journal of Cell Science • Advance article of both was sufficient. Together. pombe We hypothesized that Atg20. Atg20. Our analysis of double and triple mutants indicates that Atg20 alone or Atg24b alone was not sufficient for organelle autophagy. Similarly. In its absence. Atg24 appears to be sufficient by itself. and the self-interaction of Atg24/Snx4 (Hettema et al. E).

respectively. we speculate that the functional forms of these proteins may be oligomers (including dimers). Similarly. Atg20 can interact with either Atg24 or Atg24b. Atg13. Under nutrient-rich Journal of Cell Science • Advance article conditions. Atg20 no longer formed puncta in atg24Δ cells (Figure S4C). we examined whether their subcellular localizations were affected by each other. and either the homo-oligomer of Atg24 or the hetero- oligomer of Atg20 and Atg24b is sufficient for promoting organelle autophagy (Figure 5F). Atg24 and Atg24b. Atg24 can engage in not only hetero- interactions with Atg20 and Mug186. Atg24. and Atg24b interact with Atg17 and several other Atg proteins that may act together with Atg17. no interaction was found between Atg20 and Mug186. No other Atg proteins were found in our AP-MS analysis. but also a homo-interaction with itself. puncta formation by Atg24b was completely abolished in atg20Δ cells (Figure S4A). Atg8-colocalizing Atg20 puncta were severely diminished (Figure S4C). Deleting mug186 strongly reduced the numbers of puncta formed by Atg24. Thus. Because budding yeast Atg20/Snx42 and Atg24/Snx4 interact with Atg17 (Nice et al. we used the yeast two- hybrid (Y2H) assay to examine whether fission yeast Atg20. The results were all negative (Figure S3C). 2002). the PAS localization of Atg20 redundantly depends on its two interaction partners. in atg24Δ atg24bΔ double mutant. C). Even though both Atg20 and Mug186 interact with Atg24. and Atg101. In addition to the hetero-interactions.interaction was only detected when Atg20 was used as bait (Figure 5B. no interaction was found between Atg24 and Atg24b. Given the results of the mutant phenotype analysis. consistent with the fact that Atg24 but not Atg24b forms puncta under such conditions. Supporting the importance of these interactions. and puncta formation by Mug186 was completely abolished in atg24Δ cells (Figure S4B). despite both of them being able to interact with Atg20. The only interactions we have observed for Atg24b and Mug186 are their hetero-interactions with Atg20 and Atg24. and actually rendered the observation of PAS localization of Atg24 . In contrast. suggesting that Atg24 can engage only one interaction partner at a time. but did not affect the PAS localization of Atg24. Atg11. the coimmunoprecipitation analysis also revealed that Atg24 can engage in a homotypic interaction (Figure 5C).. The PAS localization of Atg20 in starved cells remained largely normal in atg24Δ or atg24bΔ single mutants (Figure S4C). Fission yeast Atg20 and Atg24 family proteins influence the subcellular localizations of their interaction partners To further understand the functional relevance of the hetero-interactions between Atg20 family and Atg24 family proteins. perhaps due to the C-terminal YFP tag on Atg24 interfering with this interaction. including Atg1.

2006. 2004). the Atg24-Mug186 interaction. we also observed cytoplasmic puncta of Syb1. 2003. 2005... because Atg24 by itself can support organelle autophagy.. Kita et al. these localization data suggest that. In contrast. However. Deleting atg20 either in wild-type or mug186Δ background did not obviously alter the localization pattern of Atg24 (Figure S4D). Together. and the Atg20-Atg24b interaction. testing these possibilities will have to await the creation of separation-of-function atg24 mutations that abolish the homotypic but not heterotypic interactions. all contribute to the normal localization patterns of the Atg20 and Atg24 family proteins. consistent with the model that Atg24 homo-oligomers can function in the absence of both Atg20 and Mug186.easier than in the wild-type cells (Figure S4D). 2003). namely. similar to that of its budding yeast homolog (Hettema et al.. 2004). some of which may Journal of Cell Science • Advance article represent endosomes where Syb1 traffics through during its recycling (Gachet and Hyams. as previously reported (Edamatsu and Toyoshima.. The loss of Atg24 alone causes a defect in the endosomal sorting of the SNARE protein Syb1 Budding yeast Atg20/Snx42 and Atg24/Snx4 have been implicated in not only autophagy. He et al. It remains possible that the Atg24-Atg20 hetero- oligomer and/or the Atg24-Mug186 hetero-oligomer also function in autophagy. In addition. in particular cell tips and septa. the Atg24-Atg20 interaction. It is not entirely clear whether the autophagic function of these proteins is independent of their endosomal sorting function. 2003). The fact that atg24Δ cells showed no defect in ER autophagy (Figure 3) indicates that impeding endosomal sorting does not cause defects in organelle autophagy. The results of these analyses are summarized in Figure S4E. In wild-type cells. Wild-type-like Syb1 localization patterns were observed in single mutant cells lacking Atg20. Furthermore. Kita et al. the two . but also the sorting of a SNARE protein Snc1 at the endosome (Hettema et al. the three hetero-interactions observed by our biochemical analysis. Syb1 (Figure 6). These sites are presumably plasma membrane regions where polarized exocytosis occurs. suggesting that Atg24 is important for the proper plasma membrane localization of Syb1. probably due to an endosomal sorting function of Atg24. or Mug186. including their localization at PAS. GFP-Syb1 still localized to cytoplasmic puncta but was seldom found concentrated at the cell cortex (Figure 6). in atg24Δ single mutant cells. Atg24b. We examined the localization of fission yeast Snc1 homolog. GFP-Syb1 concentrated at the cell cortex. The autophagy phenotype data shown in Figure 3 and 4 suggest that the Atg20-Atg24b hetero- oligomer functions in organelle autophagy.

the deletion of atg24. Significant size difference was observed between autophagosomes accumulated in fsc1Δ cells and those accumulated in fsc1Δ atg20Δ atg24Δ atg24bΔ cells. B) (Sun et al. these data suggest that the organelle autophagy defect caused by the . we performed transmission electron microscopy (TEM) (Figure 7C. To visualize the accumulated autophagomes. D). which hinder organelle autophagy. The average diameter of autophagosomes in fsc1Δ was 462 nm. or the double deletion of both atg20 and atg24b. caused a marked reduction of the autophagosome size. whereas the average diameter of autophagosomes in fsc1Δ atg20Δ atg24Δ atg24bΔ cells was 300 nm. which do not cause organelle autophagy defect. 2013). Organelle autophagy defect caused by the loss of Atg20 and Atg24 family proteins is associated with reduced autophagosome size For organelle autophagy to occur efficiently.double mutants defective in organelle autophagy. Taken together. Introducing into the fsc1Δ background the deletion of both atg24 and atg24b. autophagosomes need to accommodate the large sizes of the cargos. we employed a fluorescence loss in photobleaching (FLIP) assay (Figure 7A. or the deletion of both atg20 and atg24. repetitive photobleaching at a site near one cell tip diminishes the fluorescence signal of diffusible cytoplasmic Tdh1-YFP but leaves the fluorescence signal of autophagosome- enclosed Tdh1-YFP intact. in order to accurately measure autophagosome size. in contrast. endosomal trafficking is altered so that the double mutants suffer a more severe endosomal transport defect than the atg24Δ single mutant. 2013). However. We hypothesized that Atg20 and Atg24 family proteins may promote organelle autophagy by ensuring autophagosomes to be sufficiently large.. Because fluorescence microscopy has limited spatial resolution. 2013).. Thus. did not exhibit more severe Syb1 localization defect than atg24Δ single mutant (Figure 6). In this assay. we examined the sizes of autophagosomes in mutants lacking Atg20 and Atg24 family proteins.. We used the deletion of the fsc1 gene to block autophagome-vacuole fusion so that autophagosomes would accumulate after starvation treatment (Sun et al. there appears to be a correlation between the severity of the organelle autophagy defect and the extent of autophagosome size reduction. we cannot rule out the possibility that under starvation conditions. Thus. Journal of Cell Science • Advance article did not alter or only moderately reduced the sizes of autophagosomes. To investigate this possibility. atg20Δ atg24Δ and atg24Δ atg24bΔ. the autophagy defects of these double mutants do not appear to result from their endosomal sorting defect. It has been proposed that endosomal trafficking is rerouted in response to starvation in budding yeast (Shirahama-Noda et al.

Thus. Thus. Jin et al.. mutants that form smaller autophagosomes accumulated more autophagosomes than the wild type (Figure 8B). D). 2007. In budding yeast. 2014. Xie et al. we noticed that the intensities of CFP-Atg8 puncta appeared to differ somewhat between wild type and certain mutants lacking Atg20 and Atg24 family proteins. 2008). autophagosome number is controlled by Atg9 (Jin and Klionsky. and found that. We hypothesized that autophagosome size reduction may be compensated by increased autophagosome numbers. we quantitated the number of autophagosomes using the FLIP analysis data and found that. 2014). Nakatogawa et al. After starvation treatment. 2014. we examined Atg9 localization (Figure 8C. . indeed. approximately 30% CFP-Atg8 puncta overlapped with Atg9-YFP puncta in wild- type cells.. We quantitated the intensities of CFP-Atg8 puncta in starved cells.. indicating that enhanced Atg9 accumulation at PAS may underlie the compensatory increase of autophagosome number in mutants defective in organelle autophagy but not general autophagy. the CFP-Atg8 puncta in mutants that form smaller autophagosomes were not as bright as the puncta in wild type or mutants that form relatively normal-sized autophagosomes (Figure 8A). our Tdh1-YFP processing data showed that general autophagy remains normal in these mutants. In budding yeast.loss of Atg20 and Atg24 family proteins may be due to a failure to form autophagosomes of sufficiently large sizes. Organelle autophagy defect caused by the loss of Atg20 and Atg24 family proteins is associated with reduced Atg8 accumulation at PAS and enhanced Atg9 accumulation at PAS When observing CFP-Atg8 puncta. Atg8 promotes phagophore expansion and controls the sizes of autophagosomes (Jin and Klionsky. Thus. Even though the mutants defective in organelle autophagy form smaller autophagosomes. Significantly higher levels of overlap between CFP-Atg8 puncta and Atg9-YFP Journal of Cell Science • Advance article puncta were observed for mutants that form smaller but more numerous autophagosomes. we hypothesized that the reduced autophagosome size may be due to a reduced level of Atg8 at PAS. indeed.

.. Kanki et al. 2009a. It is commonly believed that starvation-triggered ... S. 2008. also functions in endosomal sorting.. cerevisiae Atg19 (Kraft et al. pombe during nitrogen starvation. we established that ER autophagy and mitochondrial autophagy occur in the fission yeast S.. Atg24. At least eight autophagy receptors have been identified in fungal species: S. and imaging analysis showed that the heterotypic interactions influence the subcellular localization of these proteins. Okamoto et al... 2010). which is a yet uncharacterized protein. 2015). 2015. 2015). We found that Atg20 and Atg24 family proteins are important for organelle autophagy but are dispensable for general autophagy. Scott et al. cerevisiae Atg39 for nucleophagy (Mochida et al. 2001.. S. 2016. Interestingly. pastoris Atg36 for pexophagy (Farré et al. Liu et al. cerevisiae Atg40 for ER-phagy (Mochida et al. S. and S. However. A defining feature of selective autophagy pathways is the requirement for cargo specificity- determining factors called autophagy receptors (Jin et al. Besides the molecular hallmark of receptor proteins.. Suzuki et al..02c). it remains unclear whether conventional autophagy receptors exist in S. but this role appears to be separate from its role in autophagy. 2015). 2009a. cerevisiae Cue5 for aggrephagy (Lu et al. 2015).. S. 2001. 2013. Thus. In budding yeast. pombe Journal of Cell Science • Advance article homolog (SPBC16E9. ER and mitochondria are cargos of selective autophagy pathways (Kanki and Klionsky. 2009).. Okamoto et al. Mochida et al. especially to the Atg8 protein. 2010. cerevisiae Atg19 and Atg34 for the Cvt pathway (Leber et al.. pombe Nbr1 is a homolog of mammalian autophagy receptor NBR1 and is remotely related to S.. these proteins act in a redundant fashion. Finally. 2015). Nbr1 mediates an unconventional selective autophagy pathway that does not rely on the core autophagy machinery (Liu et al. S.. 2009). One of these proteins. Rogov et al.. Among these receptors.. Our biochemical analysis revealed one homotypic and three heterotypic interactions among the four Atg20 and Atg24 family proteins. we showed that the underlying cause of the organelle autophagy defect may be a failure to maintain proper autophagosome size. including their localization at the PAS..Discussion In this study. 2014). Khaminets et al. Motley et al. 2014). only Cue5 has an obvious S. cerevisiae Atg32 for mitophagy (Kanki et al. Autophagy receptors link selective cargos to the core autophagy machinery. 2008. and thus only two combinations of double mutants but none of the single mutants exhibit defects in organelle autophagy. pombe.. cerevisiae Atg30 and P. another criterion that has been applied to distinguish selective and nonselective autophagy pathways is the autophagy- triggering stimuli (Sica et al. 2012).

these hetero-interactions all happen between an Atg20 family protein and an Atg24 family protein. However. 2010). However. the identification of pathway-specific receptors will be needed to draw definitive conclusions. Fission yeast Atg24 is capable of acting alone in the absence of any Atg20 family protein. In their well-established roles in the Cvt pathway. Mochida et al. suggesting that these interactions are either not conserved or difficult to observe under our experimental conditions. 2010. 2008). but not Snx41-Atg20/Snx42 interaction. . and for the starvation-induced autophagy of fatty acid synthase (Shpilka et al.... as Atg24/Snx4 but not Atg20/Snx42 is important for the piecemeal microautophagy of the nucleus (PMN) (Krick et al. 2015). 2002). pexophagy. 2005). This is reminiscent of the situation in the budding yeast. we uncovered three pair-wise hetero-interactions including Atg20-Atg24. Fission yeast Atg20 and Atg24b behave similarly.autophagy tends to be nonselective and can randomly engulf any cellular components to meet the heightened demand of cellular material recycling.. COG complex subunits (Yen et al. and the interaction between Atg20/Snx42 and Atg11 (Yorimitsu and Klionsky. interacting with each other and being equally indispensible for organelle Journal of Cell Science • Advance article autophagy when Atg24 is absent. It is possible that the starvation-induced organelle autophagy processes we observed in fission yeast are also selective. an Atg20 family protein and an Atg24 family protein acting together in a heterotypic complex appears to be a conserved mode of action for these two families of PX-BAR proteins. Thus. 2002).. 2015). 2003. Budding yeast Atg24/Snx4 may also adopt such a mode of action under certain situations.. including their interactions with Atg17 (Nice et al. Nice et al. budding yeast Atg20/Snx42 and Atg24/Snx4 are equally important. Atg20-Atg24b. We found that Atg24 can self interact. this is not the only way these proteins function. and mitophagy. presumably because they are only functional when forming a hetero complex together. Thus... However.. like its budding yeast ortholog (Nice et al. the autophagy of the hydrolase Ams1 and the autophagy of ER under the starvation conditions are receptor-dependent processes (Suzuki et al. However. 2002). Budding yeast Atg20/Snx42 and Atg24/Snx4 also engage in other protein-protein interactions. in S. Interestingly. certain cargos appear to be preferentially targeted by selective autophagy even during starvation. have been detected (Hettema et al. our AP-MS analysis and pair-wise Y2H analysis failed to detect similar interactions in fission yeast. cerevisiae. where Atg20/Snx42-Atg24/Snx4 interaction and Snx41- Atg24/Snx4 interaction. In addition. and Atg24-Mug186 interactions.

We speculate that Atg20 and Atg24 family proteins may contribute to autophagosome size regulation by modulating the membrane curvature of certain regions of the expanding phagophore. Previously. through their association with the autophagic membrane. cerevisiae and P.. 2005. Kanki and Klionsky. are important for organelle autophagy (Nice et al. 2002. a more basal subphylum of Ascomycota (Stajich et al. 2008.. 2014). our findings here provide further evidence that the involvement of Atg20 and Atg24 family proteins in organelle autophagy is a conserved mechanism. PX-BAR proteins have intrinsic capabilities of curvature sensing. 2013. We found in this study that organelle autophagy defect and a reduction of autophagosome size are two correlated phenotypes of mutants lacking Atg20 and Atg24 family proteins.. we propose that a failure to maintain proper autophagosome size may be the underlying cause of the organelle autophagy defect. pastoris. Our observation here that the reduction of autophagosome size caused by the loss of Atg20 and Atg24 family proteins is compensated by an increase of autophagosome numbers suggests that fission yeast Atg20 and Atg24 family proteins also contribute to general autophagy in a way that can be compensated. Mao et al. S. Thus.. Thus.. at least for the soluble cargos. and SNX30 also play similar roles in autophagy (van Weering et al. two Ascomycota species belonging to the Saccharomycotina subphylum. 2010. 2016). Rao et al.. 2012). curvature inducing. it has been shown that Atg20 and Atg24 family proteins in S.. pombe belongs to Journal of Cell Science • Advance article Taphrinomycotina. efficient organelle autophagy requires the participation of organelle fission machinery. It has been shown that budding yeast Atg20/Snx42 and Atg24/Snx4 contribute to general autophagy but their roles can be masked by compensatory mechanisms (Ohashi and Munro. 2010). and membrane remodeling (van Weering and Cullen. suggesting that it is important to match the size of the organelle cargo with the size of the autophagosome (Mao et al. .. 2012). and those in Magnaporthe oryzae. 2016. 2009). Ano et al.. Deng et al. they may alter the local concentrations of certain Atg proteins such as Atg8. It will be worthwhile to investigate whether their mammalian homologs SNX4. van Weering et al. In budding yeast. Further studies using in vitro reconstitution systems may provide detailed insights into how these proteins influence autophagosome formation (Brier et al. an Ascomycota species belonging to the Pezizomycotina subphylum. 2014). SNX7.. He et al. Alternatively. 2013..

Deletion strains were constructed by PCR amplifying the deletion cassettes in the Bioneer deletion strains and transforming the PCR product into strains in our lab strain collection. . The resulting plasmid was linearized by NotI digestion and integrated at the his3 locus. For the construction of the strain expressing GFP-Syb1. the coding sequence of Syb1 was amplified by PCR from genomic DNA and inserted into a modified pDUAL vector.. Genetic methods for strain construction and composition of media are as described (Forsburg and Rhind. 2012). Strains expressing CFP-Atg8 and Tdh1-YFH were as reported before (Sun et al. Protein bands were quantitated using the “Analyze tool” in the Fiji distribution of ImageJ software (Schindelin et al. which contains the P41nmt1 promoter and the sequence encoding GFP (Wei et al. 2009).Materials and Methods Fission yeast strains and media Fission yeast strains used in this study are listed in Table S1. and the resulting plasmid was linearized by NotI digestion and integrated at the leu1 locus. The strain expressing Sdh2-mCherry under the native promoter was constructed by PCR-based tagging. cerevisiae Cox4 and mCherry into the integrating vector pHIS3K-Ptub1 (Matsuyama et al. 2013). 2013).. The resulting plasmid was linearized by NotI digestion and integrated at the leu1 locus. 2010).. An integrating plasmid expressing Ost4-CFP from the Pnmt1 promoter was constructed by performing a Gateway LR reaction between the ORFeome library entry clone of the ost4 gene and the pDUAL-CFH1c destination plasmid (Matsuyama et al.... 2014). Samples were separated on 10% SDS-PAGE and then immunoblotted with an antibody that can recognize GFP (also YFP and CFP) or mCherry tag. 2006). Strains expressing ER-GFP (called GFP-AHDL in previous publications) were provided by Snezhana Oliferenko (Zhang et al. 2008).. Processing assay of fluorescent protein-fused proteins Journal of Cell Science • Advance article Cell lysates were prepared from about 5 OD600 units of yeast cells using a TCA lysis method (Ulrich and Davies. Strains expressing PX-BAR proteins fused with the YFP-FLAG-His6 (YFH) tag from their native promoters were constructed by an overlap-extension PCR approach (Yu et al. An integrating plasmid expressing mito-mCherry was constructed by inserting sequences encoding the mitochondrial targeting sequence of S. 2006).

and dissolved in 8 M urea.4-NA objective. Images were acquired with a 100×. then reduced with 5 mM TCEP for 20 min. Proteins bound to beads were eluted by boiling in SDS-PAGE loading buffer.5. Affinity purification coupled with mass spectrometry (AP-MS) analysis
 About 1000 OD600 units of cells were harvested and lysate was prepared using the bead- beating lysis method. and analyzed with the SoftWoRx software. The cell lysate was cleared by centrifugation at 13000 rpm for 30 min. Peptides were loaded on a pre-column (100 μM ID.05% NP-40. Formic acid was added to the final concentration of 5 % to stop digestion reaction. 2001). 150 mM NaCl. 1 mM PMSF. the agarose beads were washed 3 times with lysis buffer. pH 8. Punctum intensity was determined by measuring the fluorescence intensities of two concentric square regions (7 × 7 and 9 × 9 pixels. After incubation. and alkylated with 10 mM iodoacetamide for 15 min in the dark. Cells were lysed by beating with 0. 0. the LC-MS/MS analysis was performed on an Easy-nLC II HPLC (Thermo Fisher Scientific) coupled to a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific). 1× Roche protease inhibitor cocktail) using a FastPrep-24 instrument.5. Immunoprecipitation
 Approximately 100 OD600 units of log-phase cells were harvested and washed once with water.Fluorescence microscopy Except for the FLIP assay. After incubation. the agarose beads were washed 2 times with lysis buffer and another time with
 lysis buffer without NP-40.5-mm-diameter glass beads in the lysis buffer (50 mM HEPES. pH 8. . 10% glycerol. Protein precipitates were washed three times using ice-cold acetone. live cell imaging was performed using a DeltaVision PersonalDV system (Applied Precision) equipped with a mCherry/GFP/CFP filter set (Chroma 89006 set) and a Photometrics CoolSNAP HQ2 camera. respectively) centered around the punctum and calculating background subtraction using the method described in Hoffman et al (Hoffman et al. 100 mM Tris. After digestion. 100 mM Tris. 1 mM EDTA. The supernatant was incubated with GFP-Trap agarose beads for 3 h. 1 mM DTT. pH 7. The supernatant was incubated with GFP-Trap agarose beads (Chromotek). Eluted proteins were precipitated with 20% TCA for 1 h.. 4 cm long. 1 mM CaCl2. Then the samples were diluted by a factor of 4 and digested overnight at 37° with trypsin (dissolved in 2 M urea.0). 100 mM Tris. Bead-bound proteins were eluted by incubation at 65° with Journal of Cell Science • Advance article elution buffer (1% SDS. pH 8. 1.5).

Electron microscopy Approximately 100 OD600 units of cells were starved for 3 h and harvested. The sizes of autophasosomes were determined using a method previously used for measuring autophagic body size (Xie et al.. Journal of Cell Science • Advance article . The samples were fixed with 2% glutaraldehyde and 4% KMnO4. Quantitative analysis of autophagosome size was performed with the Volocity software. 2008).000) were isolated for CID MS2 (normalized collision energy 35) with a dynamic exclusion time of 60 s. packed with Luna C18 3 μm 100 Å resin from Phenomenex) using an acetonitrile gradient from 0–28% in 100 min at a flow rate of 200 nl/min. using a 100× objective. The search results were filtered with DTASelect. Precursors with less than 2+ or unassigned charge states were excluded. The MS/MS spectra were searched with Prolucid against an S. and embedded in Spurr’s resin. then washed once with 10 ml of water. FLIP assay The photobleaching of the Tdh1-YFP signal and image acquisition were carried out with a PerkinElmer Ultraview VoX spinning disk system. then dehydrated with graded ethanol.packed with C12 10 μm 120 Å resin from YMC) and separated on an analytical column (75 μM ID. Thin sections were examined using an FEI Tecnai G2 Spirit electron microscope equipped with a Gatan 895 4k x 4k CCD camera. pombe protein database. The top 8 most intense precursor ions from each full scan (resolution 60. 10 cm long. MS/MS fragment ions were detected by linear ion trap in a normal scan mode.

L.. Journal of Cell Science • Advance article .D. X. Jia-Min Zhang for help constructing the Sdh2-mCherry strains.. 2014CB849901 and 2014CB849801).-M. and M.D..-Q. D. and L.-L. Wan-Zhong He for help with electron microscopy.-L. S. This work was supported by the National Basic Research Program of China (973 Program. and L. Z. Author contributions D. Meng Ouyang for constructing the mito-mCherry plasmid. conceived the study and participated in the writing of the manuscript.Acknowledgments We thank Snezhana Oliferenko for providing the ER-GFP (GFP-AHDL) strains. X. performed some of the experiments. Z. carried out most of the experiments and prepared the manuscript.-Q. Competing interests No competing interests declared.-Q. and by grants from the Chinese Ministry of Science and Technology and the Beijing municipal government to M.-L. D. Y. L. contributed to the mass spectrometry analysis. X.

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Figures Journal of Cell Science • Advance article .

White rectangles enclose cells shown at higher magnification at right. For the lanes where the free form of fluorescent protein appeared. (B) A mitochondrial marker Sdh2-mCherry localizes to vacuole lumen upon starvation treatment in an Atg5-dependent manner. (D) A mitochondrial marker mito-mCherry localizes to vacuole lumen upon starvation treatment in an Atg5-dependent manner. (E) and (F) Starvation induces the processing of Ost4-CFP (E) and Sdh2-mCherry (F) in Journal of Cell Science • Advance article an Atg5-dependent manner. (A) An ER marker Ost4-CFP localizes to vacuole lumen upon starvation treatment in an Atg5-dependent manner. Wild-type (WT) and atg5Δ cells expressing GFP fused with the signal peptide and ER-retention signal of Bip1 from the bip1 promoter were examined before and after 12 h starvation. Wild-type (WT) and atg5Δ cells expressing mCherry fused with the first 33 amino acids of S. Coomassie Brilliant Blue R-350 (CBB) staining of PVDF membrane after immunodetection was used to control for protein loading and blotting efficiency (Welinder and Ekblad. Cpy1- Venus serves as a vacuole lumen marker. . Wild-type (WT) and atg5Δ cells expressing endogenously mCherry-tagged Sdh2 were collected before (+N) and after shifting to nitrogen-free medium (-N) for 12 h.Figure 1. and examined by fluorescence microscopy. Bars. the ratio of free form of fluorescent protein vs. 2011). 3 µm. cerevisiae Cox4 from the tub1 promoter were examined before and after 12 h starvation. Cpy1-mCherry serves as a vacuole lumen marker. and examined by fluorescence microscopy. Wild-type (WT) and atg5Δ cells expressing CFP-tagged Ost4 from the Pnmt1 promoter were collected before (+N) and after shifting to nitrogen-free medium (-N) for 12 h. (C) An ER marker ER-GFP localizes to vacuole lumen upon starvation treatment in an Atg5-dependent manner. Total lysates of wild-type (WT) and atg5Δ cells were analyzed by immunoblotting using an antibody that can recognize CFP (E) or mCherry (F). the full-length tagged protein (free/FL ratio) was quantitated using the Fiji software. Nitrogen starvation induces the autophagy of ER and mitochondria in fission yeast. Insets enclose cells shown at higher magnification at right.

Journal of Cell Science • Advance article .

Atg24b. CFP-Atg8 serves as a marker for starvation- induced PAS. and Atg24b- YFP (D) before and 2 h after starvation in wild-type background. ND. not determined. (A-D) Localization of Atg20-YFP (A). (E-H) Localization of Atg20-YFP (E). Arrowheads point to representative puncta where colocalization with CFP- Atg8 occurs. and Mug186 form puncta at the PAS during starvation. Atg24-YFP (B). Bars. 100 Atg8 puncta were examined each time). Journal of Cell Science • Advance article . YFP-tagged proteins were expressed from endogenous promoters. Mug186-YFP (G).Figure 2. Atg20. Atg24-YFP (F). and Atg24b- YFP (H) before and 2 h after starvation in vps38Δ background. Mug186-YFP (C). Atg24. 3 µm. (I) Quantitation of the percentages of CFP-Atg8 puncta overlapping with puncta formed by Atg20 and Atg24 family proteins in starved cells (mean ± SEM.

Journal of Cell Science • Advance article .

Figure 3. (A) Starvation-induced processing of Tdh1-YFP was monitored in the wild type (WT). atg5Δ mutant. and the total lysates were analyzed by immunoblotting using an antibody that can recognize YFP. and quadruple mutants lacking Atg20 and Atg24 family proteins. triple. (E) Starvation-induced processing of Ost4-CFP was monitored in selected double and triple mutants. (B) Starvation-induced vacuole localization of Ost4-CFP was diminished in the quadruple mutant. Bars. Journal of Cell Science • Advance article . (C) Starvation-induced processing of Ost4-CFP was reduced in the quadruple mutant. Atg20. double. and quadruple mutant (QM) lacking all four Atg20 and Atg24 family proteins. Atg24 and Atg24b are important for ER autophagy but not general autophagy. Cells expressing YFP-tagged Tdh1 from the Pnmt1 promoter were collected before and after starvation. Cell lysates were analyzed by immunoblotting with an antibody that can recognize CFP. 3 µm. (D) Starvation-induced vacuole localization of ER-GFP was monitored in all combinations of single.

(A) Starvation-induced vacuole localization of mito-mCherry was monitored in selected double and triple mutants.Figure 4. (B) Starvation-induced processing of Sdh2-mCherry was monitored in selected double and triple mutants. Mitochondrial autophagy is promoted redundantly by Atg20. Bar. and Atg24b. 3 µm. Atg24. Journal of Cell Science • Advance article .

Journal of Cell Science • Advance article Figure 5. (A) Affinity purification coupled with mass spectrometry analysis revealed interactions between Atg20 and Atg24 family proteins. Atg24. Endogenously YFP-tagged Atg20. . Atg20 and Atg24 family proteins form multiple oligomers.

Atg24-mCherry (C). SE. Journal of Cell Science • Advance article . (F) A summary of the interactions found between Atg20 and Atg24 family proteins. and bead-bound proteins were identified using mass spectrometry. mCh. and Mug186 were purified using GFP-Trap agarose beads. YFP-tagged proteins expressed from the endogenous promoter were immunoprecipitated using GFP-Trap agarose beads. LE. short exposure. Atg24b- mCherry (D). (B-E) Coimmunoprecipitation analysis to detect possible interactions between YFP-taged Atg20 and Atg24 family proteins and Atg20-mCherry (B). long exposure. Arrowed lines represent protein-protein interactions. and the coimmunoprecipitation of mCherry-tagged proteins expressed from the P41nmt1 promoter was analyzed by immunoblotting. mCherry.Atg24b. Dashed ovals indicate the two interactions (Atg20-Atg24b hetero-interaction and Atg24 homo-interaction) that likely contribute to the organelle autophagic function. and Mug186-mCherry (E).

Bar. (B) Quantitation of the fractions of cells with Syb1-GFP signal at the cell tip or septum (mean ± SEM. atg20Δ atg24Δ. 100 cells were examined each time). Cells expressing GFP-tagged Syb1 from the P41nmt1 promoter were grown to mid-log phase and examined by fluorescence microscopy.Figure 6. 3 µm. . atg24Δ single mutant is defective in the endosomal sorting of Syb1 Journal of Cell Science • Advance article (A) Polarized Syb1 localization was diminished in atg24Δ. and atg24Δ atg24bΔ.

and rendered visible by fluorescence loss in photobleaching (FLIP) that abolished diffusible cytoplasmic fluorescent signal of Tdh1- YFP. Mutants defective in organelle autophagy have smaller autophagosomes (A) Autophagosomes were accumulated using a fsc1Δ mutations that blocks autophagosome-vacuole fusion. Journal of Cell Science • Advance article Figure 7. a nuclear pool of Tdh1-YFP . Besides Tdh1-YFP trapped inside autophagosomes.

vacuole. Cells expressing YFP-tagged Tdh1 were collected after 3 h starvation. and FLIP assay was performed. Two representative cells were shown for each strain. Horizontal bars represent mean ± SD (n=30). M. Journal of Cell Science • Advance article .. nucleus. P values were calculated using Welch’s t-test. Horizontal bars represent mean ± SD (n=45).also remained visible post FLIP. (D) Quantitation of sizes of autophagosomes observed in the EM analysis. mitochondrion. V. (B) Quantitation of sizes of autophagosomes observed in the FLIP analysis. N. 2013). White squares enclose regions shown at higher magnification below. AP. P values were calculated using Welch’s t-test. Yellow dots mark the sites of photobleaching. as shown before (Sun et al. autophagosome. (C) Representative EM images of fsc1Δ and fsc1Δ atg20Δ atg24Δ atg24bΔ cells.

Journal of Cell Science • Advance article .

Horizontal bars represent mean ± SD (n=20). Cells expressing endogenously CFP-tagged Atg8 were examined after 2 h starvation. 100 Atg8 puncta were examined each time).Figure 8. Horizontal bars represent mean ± SD (n=30). P values were calculated using Welch’s t-test. 3 µm. Bar. Cells expressing endogenously YFP-tagged Atg9 were examined after 2 h starvation. (B) Quantitation of the numbers of autophagosomes per cell. The values are log10 transformed average pixel intensities in 7 × 7 pixel regions surrounding the puncta after background subtraction. Mutants defective in organelle autophagy have reduced Atg8 accumulation and enhanced Atg9 accumulation at the PAS (A) Quantitation of the intensity of starvation-induced CFP-Atg8 puncta. Images of the FLIP assay shown in Figure 8A were analyzed. P values were calculated using Welch’s t-test. Journal of Cell Science • Advance article . (C) Localization patterns of Atg9-YFP. (D) Quantitation of the percentages of CFP-Atg8 puncta overlapping with Atg9-YFP puncta in the analysis shown in (C) (mean ± SEM.

8 PCC (colocalization with Cpy1) 0.1242/jcs. 129: doi:10.194373: Supplementary information A 1.J.4 0. .6 0.0 0.2 0 FP FP ry ry FP FP ry ry FP FP ry ry FP FP ry ry er er er er er er er er -C -G -C -G -C -G -C -G Ch Ch Ch Ch Ch Ch Ch Ch t4 t4 t4 t4 ER ER ER ER -m -m -m -m -m -m -m -m Os Os Os Os h2 ito h2 ito h2 ito h2 ito m m m m Sd Sd Sd Sd WT WT +N -N 12 h B WT +N Ost4-CFP -N 12 h Journal of Cell Science • Supplementary information C WT +N Sdh2-mCherry -N 12 h Figure S1. Colocalization of ER and mitochondrial markers with the vacuole marker Cpy1 upon starvation treatment and the organelle autophagy phenotype of five atg mutants. Cell Sci.

and atg101∆.1242/jcs. B. 129: doi:10. atg13∆. and D were analyzed using the Coloc 2 plugin of the Fiji distribution of ImageJ software (http://imagej. Values shown represent mean ± SD (10 cells).net/Coloc_2). Pearson’s R value (no threshold) reported in the ImageJ Log window was recorded for each cell. After running the plugin.J. Cell Sci. Colocalization was quantitated using Pearson's correlation coefficient (PCC). (C) Mitochondrial autophagy is defective in atg1∆.1 Imaging data from the experiments shown in Figure 1A.2 Individual cells were selected as regions of interest (ROIs) using the freehand selection tool before running the plugin. atg11∆. atg17∆. atg17∆.194373: Supplementary information (A) Colocalization of ER and mitochondrial markers with the vacuole marker Cpy1 upon starvation treatment. (B) ER autophagy is defective in atg1∆. atg11∆. and atg101∆. Journal of Cell Science • Supplementary information . C. atg13∆.

194373: Supplementary information A 27 145 275 524 Atg20 PX BAR 1 534 75 193 241 578 Mug186 PX BAR 1 586 19 145 176 400 Atg24 PX BAR 1 401 4 129 161 390 Atg24b PX BAR 1 390 204 324 366 574 Vps5 PX BAR 1 576 101 220 243 451 Vps17 PX BAR 1 549 283 406 432 656 Mvp1 PX BAR 1 664 B 15 gi_254570152__Komagataella_pastoris gi_6320090__Saccharomyces_cerevisiae (Atg20) 34 gi_6320633__Saccharomyces_cerevisiae (Snx41) 66 gi_254572555__Komagataella_pastoris 52 gi_389638938__Magnaporthe_oryzae 34 100 Atg20 gi_67540362__Aspergillus_nidulans 100 93 family gi_50550139__Yarrowia_lipolytica gi_19113529__Schizosaccharomyces_pombe (Mug186) 99 gi_745750417__Schizosaccharomyces_japonicus gi_19075495__Schizosaccharomyces_pombe (Atg20) 66 100 gi_213409135__Schizosaccharomyces_japonicus gi_19112676___Schizosaccharomyces_pombe (Atg24b) 100 gi_213403232__Schizosaccharomyces_japonicus gi_19114817__Schizosaccharomyces_pombe (Atg24) 100 gi_213403720__Schizosaccharomyces_japonicus Atg24 gi_210075929__Yarrowia_lipolytica 96 family gi_389637225__Magnaporthe_oryzae 88 73 100 gi_67526253__Aspergillus_nidulans 93 gi_254568362__Komagataella_pastoris 43 gi_6322424__Saccharomyces_cerevisiae (Snx4/Atg24) gi_19075429__Schizosaccharomyces_pombe (Vps5) Journal of Cell Science • Supplementary information 96 gi_213409243__Schizosaccharomyces_japonicus gi_389623607__Magnaporthe_oryzae Vps5 100 gi_67526273__Aspergillus_nidulans 100 56 family gi_50545211__Yarrowia_lipolytica 55 gi_254572243__Komagataella_pastoris 52 gi_46562125__Saccharomyces_cerevisiae (Vps5) 76 gi_19113959__Schizosaccharomyces_pombe (Vps17) 56 gi_745750485__Schizosaccharomyces_japonicus 47 gi_3896334617__Magnaporthe_oryzae 66 Vps17 41 100 gi_67523537__Aspergillus_nidulans 100 family gi_398365299__Saccharomyces_cerevisiae (Vps17) 60 gi_254569234__Komagataella_pastoris gi_50551269__Yarrowia_lipolytica gi_389638532__Magnaporthe_oryzae 99 gi_67900524__Aspergillus_nidulans 73 83 gi_50543118__Yarrowia_lipolytica Mvp1 100 family gi_6323646__Saccharomyces_cerevisiae (Mvp1) gi_19115109__Schizosaccharomyces_pombe (Mvp1) Figure S2. . 129: doi:10.J. Cell Sci. Domain organization diagrams of fission yeast PX-BAR proteins and a phylogenetic tree of fungal PX-BAR proteins.1242/jcs.

whose structure has been solved (PDB entry 2RAJ).3 PX and BAR domains depicted are the regions homologous to SNX9(252–370) and SNX9(392-589). Snx12 proteins. Yarrowia lipolytica (taxid:4952). Domain prediction was based on a multiple sequence alignment including human SNX9.ncbi.0001 were collated and duplicate sequences were removed.J. We performed BLASTP searches using the NCBI BLAST server (http://blast. 129: doi:10.194373: Supplementary information (A) Domain organization of fission yeast PX-BAR proteins.4 Default MAFFT settings were used except that the E-INS-i iterative refinement method was selected as the alignment strategy. Vps5. Bootstrap resampling = 100. Cell Sci.cbrc. and a truncated version of Komagataella pastoris Mvp1 (GI:254570755). Magnaporthe oryzae (taxid:318829). (B) Phylogenetic tree of fungal PX-BAR proteins. BLASTP hits with E-value < 0. Aspergillus nidulans (taxid:162425). Mug186. Atg24b. pombe Atg20. From these sequences a multiple sequence alignment was generated using the MAFFT online server (http://mafft.jp/alignment/server/). a Neighbor-Joining (NJ) tree was constructed using the MAFFT online server with the parameters of Model = JTT. Using the 235 gap-free sites in the resulting alignment. Vps17. Mvp1. Komagataella pastoris (taxid:4922). and Vps17 were used as queries. and Mvp1 proteins as outgroup.6 The tree was rooted using Vps5.5 The NJ tree was visualized using Archaeopteryx.1242/jcs. After filtering out Snx3/Grd19 proteins.nlm. respectively. Atg24. and Schizosaccharomyces japonicus (taxid:4897). Schizosaccharomyces pombe (taxid:4896). . Alpha = 2.06 (estimated). The searches were carried out using the RefSeq database and we limited the organisms to seven representative fungal species including Saccharomyces cerevisiae S288c (taxid:559292). Journal of Cell Science • Supplementary information a total of 39 sequences were retained.nih. S.gov).

.J. Atg11. Atg24. Atg24b. and Mug186 did not form puncta in atg14∆ vps38∆. 129: doi:10.1242/jcs. Atg24. Localization patterns in atg14∆ vps38∆ double mutant and the Y2H assay examining the interactions between Atg20 and Atg24 family proteins and Atg1- associated proteins. Atg13. Atg17. and Atg24b do not interact with Atg1. (B) The PAS marker Atg17 still formed puncta in atg14∆ vps38∆. (C) Atg20. Journal of Cell Science • Supplementary information (A) Atg20.194373: Supplementary information A C Bait Atg24b Atg24b Atg20 Atg24 Atg20 Atg24 Crb2 Crb2 YFP Merge DIC Atg1 Atg20-YFP Atg11 Atg17 Atg24-YFP Prey Atg101 Atg24 Atg20 Crb2 Atg24b-YFP B Atg17-YFP DIC +N Figure S3. Cell Sci. or Atg101 in the Y2H assay.

more obvious mug186 WT-like WT-like localization at PAS atg24 atg24b Puncta severely diminished ND atg20 mug186 Similar to mug186 ND .194373: Supplementary information A C WT WT atg24b Atg24b-YFP CFP-Atg8 Atg20-YFP CFP-Atg8 Atg20-YFP CFP-Atg8 +N +N +N -N 2 h -N 2 h -N 2 h atg20 atg24 atg24 atg24b Atg24b-YFP CFP-Atg8 Atg20-YFP CFP-Atg8 Atg20-YFP CFP-Atg8 +N +N +N -N 2 h -N 2 h -N 2 h B D WT WT mug186 Mug186-YFP CFP-Atg8 Atg24-YFP CFP-Atg8 Atg24-YFP CFP-Atg8 +N +N +N -N 2 h -N 2 h -N 2 h atg24 atg20 atg20 mug186 Mug186-YFP CFP-Atg8 Atg24-YFP CFP-Atg8 Atg24-YFP CFP-Atg8 +N +N +N Journal of Cell Science • Supplementary information -N 2 h -N 2 h -N 2 h E Atg20-YFP Atg24-YFP Atg24b-YFP Mug186-YFP atg20 WT-like No longer forming puncta WT-like No longer forming puncta before starvation.J. 129: doi:10. Cell Sci. more atg24 WT-like No longer forming puncta obvious localization at PAS after starvation atg24b WT-like WT-like WT-like Fewer puncta.1242/jcs.

3 µm. (E) A summary of the effects of the deletion mutations on the localization patterns of Atg20 and Atg24 family proteins.194373: Supplementary information Figure S4. Journal of Cell Science • Supplementary information . 129: doi:10. (B) Localization of Mug186-YFP in wild-type and atg24Δ backgrounds. atg24bΔ. (C) Localization of Atg20-YFP in wild-type. atg20Δ. and atg24Δ atg24bΔ backgrounds. ND. mug186Δ. and atg20Δ mug186Δ backgrounds. Bars. Cell Sci. not determined. Atg24. and Mug186 are influenced by their interaction partners (A) Localization of Atg24b-YFP in wild-type and atg20Δ backgrounds.J. CFP-Atg8 serves as a marker for starvation-induced PAS. (D) Localization of Atg24-YFP in wild-type. Atg24b. The localization patterns of Atg20.1242/jcs. atg24Δ.

Preibisch S. EMBO J 2007.J. Nat Methods 2012. Katoh K. Arganda-Carreras I. 30:772–80. 5. Nucleic Acids Res 2013. 9:676–82. Standley DM. Zmasek CM. Kuraku S. Cell Sci. Rueden C. Saalfeld S. Han MV. Schmid B. Journal of Cell Science • Supplementary information . Am J Physiol Cell Physiol 2011. The PX-BAR membrane-remodeling unit of sorting nexin 9. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Frise E.1242/jcs. aLeaves facilitates on-demand exploration of metazoan gene family trees on MAFFT sequence alignment server with enhanced interactivity. McDonald JH. A practical guide to evaluating colocalization in biological microscopy. 3. Kaynig V. Rasmuson E. Schindelin J. Katoh K. phyloXML: XML for evolutionary biology and comparative genomics.194373: Supplementary information Supplementary references 1. Pylypenko O. 41:W22-28. BMC Bioinformatics 2009. Fiji: an open-source platform for biological-image analysis. Carlsson SR. 2. Zmasek CM. 10:356. Pietzsch T. 6. Rak A. Mol Biol Evol 2013. 4. 300:C723- 742. Longair M. Kamocka MM. 26:4788–800. Lundmark R. 129: doi:10. et al. Dunn KW. Nishimura O.

Cell Sci. D1 3B. Figure 1B. CFP-atg8::leu1+ his3-D1 4B. leu1-32::Pnmt1-ost4-CFH(leu1+) cpy1-mCherry::natMX his3. 1F. Figure 1C. vps38Δ::kanMX atg20-YFH::leu1+ CFP-Atg8::leu1+ his3-D1 Figure 2E.194373: Supplementary information Table S1.1242/jcs. atg5Δ::kanMX leu1-32:: Pnmt1-tdh1-YFP(leu1+) cpy1. vps38Δ::kanMX atg24b-YFH::leu1+ CFP-atg8::leu1+ his3-D1 Figure 2H. S1A DY29568 h? atg5Δ::hphMX sdh2-mCherry::kanMX cpy1-venus::natMX Figure 1B. S1A his3-D1 ade6 DY29565 h? atg5Δ::kanMX Pbip1-GFP-AHDL::ura4+ cpy1. Pbip1-GFP-AHDL::ura4+ leu1-32 Figure 3D DY29537 h? atg20Δ::kanMX Pbip1-GFP-AHDL::ura4+ leu1-32 his3-D1(or Figure 3D his3+) DY29538 h? atg24Δ::natMX Pbip1-GFP-AHDL::ura4+ leu1-32 his3-D1(or Figure 3D his3+) DY29539 h? atg24bΔ::hphMX Pbip1-GFP-AHDL::ura4+ leu1-32 his-3. 1F.J. S1A leu1-32 DY29567 h? atg5Δ::kanMX Ptub1-cox4(1-33)-mCherry::kanMX cpy1. leu1-32::Pnmt1-tdh1-YFH(leu1+) cpy1-mCherry::natMX his3. 4A. vps38Δ::kanMX mug186-YFH::leu1+ CFP-atg8::leu1+ his3. Pbip1-GFP-AHDL::ura4+ leu1-32 Figure 3D DY29543 h? atg5Δ::kanMX. S4A DY29588 h. vps38Δ::kanMX atg24-YFH::leu1+ CFP-Atg8::leu1+ his3-D1 Figure 2F. Figure 3D D1(or his3+) DY29542 h? atg20Δ::kanMX atg24bΔ::hphMX Pbip1-GFP-AHDL::ura4+ Figure 3D leu1-32 his-3-D1(or his3+) DY29540 h? atg20Δ::kanMX atg24Δ::natMX Pbip1-GFP-AHDL::ura4+ Figure 3D leu1-32 his-3-D1(or his3+) . 2I D1 leu1-32 DY29536 h. Figure 2G. 2I leu1-32 DY29589 h. 1E. S4D DY29487 h+ mug186-YFH::leu1+ CFP-atg8::leu1+ his3-D1 leu1-32 Figure 2C. venus::natMX leu1-32 S1A DY29586 h+ atg20-YFH::leu1+ CFP-atg8::leu1+ his3-D1 leu1-32 Figure 2A. Figure 3D D1(or his3+) DY29555 h? mug186Δ::kanMX Pbip1-GFP-AHDL::ura4+ leu1-32 his-3. 2I leu1-32 DY29535 h. Strains used in this study Strain Genotype Use DY29585 h. S1A DY29564 h? Pbip1-GFP-AHDL::ura4+ cpy1-mCherry::natMX leu1-32 Figure 1C. 1E. 2I leu1-32 DY29479 h. Figure 3A mCherry::natMX his3-D1 DY29590 h? atg20Δ::natMX atg24Δ::hghMX mug186Δ::kanMX Figure 3B. 3E. 2I. Figure 1A. 2I. Figure 1D. Figure 3A D1 Journal of Cell Science • Supplementary information DY29480 h. S1A DY29485 h? atg5Δ::kanMX leu1-32::Pnmt1-ost4-CFH(leu1+) cpy1. 129: doi:10. S1A mCherry::natMX leu1-32 his3-D1 ade6 DY29566 h? his3::Ptub1-cox4(1-33)-mCherry::kanMX cpy1-venus::natMX Figure 1D. Figure 1A. mCherry::natMX his3-D1 3C. 3C atg24bΔ::natMX leu1-32::Pnmt1-ost4-CFH(leu1+) cpy1- mCherry::kanMX his3-D1 DY29570 h. S4C DY29587 h+ atg24-YFH::leu1+ CFP-atg8::leu1+ his3-D1 leu1-32 Figure 2B. his3-D1 4B. S1A DY29569 h? sdh2-mCherry::kanMX cpy1-venus::natMX CFP-atg8::leu1+ . 3C. S4B DY29486 h+ atg24b-YFH::leu1+ CFP-atg8::leu1+ his3-D1 leu1-32 Figure 2D.

Figure 3D GFP-AHDL::ura4+ leu1-32 his-3-D1(or his3+) DY29562 h? atg20Δ::natMX atg24bΔ::natMX atg24Δ::hphMX Figure 3D mug186Δ::kanMX Pbip1-GFP-AHDL::ura4+ leu1-32 his-3-D1(or his3+) DY29563 h? atg20Δ::natMX atg24bΔ::kanMX mug186Δ::hphMX leu1. Figure 3D GFP-AHDL::ura4+ leu1-32 his-3-D1(or his3+) DY29559 h? atg20Δ::natMX atg24bΔ::natMX mug186Δ::kanMX Pbip1.mug186-YFH::hphMX leu1-32 his3-D1 Figure 5A DY29508 h+ leu1-32::P41nmt1-atg20-mCherry(leu1+) his3-D1 Figure 5B DY29512 h+ leu1-32::P41nmt1-atg20-mCherry(leu1+) atg20-YFH::hphMX Figure 5B his3-D1 DY29516 h+ leu1-32::P41nmt1-atg20-mCherry(leu1+) atg24-YFH::hphMX Figure 5B . Figure 3E 32::Pnmt1-ost4-CFH(leu1+) his3-D1 DY29528 h? atg20Δ::natMX atg24Δ::hghMX mug186Δ::kanMX Figure 3E atg24bΔ::natMX leu1-32::Pnmt1-ost4-CFH(leu1+) his3-D1 DY29531 h? atg24Δ::hphMX atg24bΔ::kanMX leu1-32::Pnmt1-ost4. atg20-YFH::leu1+ leu1-32 his3-D1 Figure 5A DY29530 h. Figure 4B mCherry::kanMX leu1-32 his3-D1 DY33714 h? atg24Δ::natMX mug186Δ::kanMX sdh2-mCherry::kanMX Figure 4B leu1-32 his3-D1 DY29529 h. Figure 4A mCherry::ura4+ leu1-32 DY29575 h? atg20Δ::natMX atg24bΔ::hphMX mug186Δ::kanMX Figure 4A his3::Ptub1-cox4(1-33)-mCherry::ura4+ leu1-32 DY31748 h? atg24Δ::hphMX atg20Δ::kanMX sdh2-mCherry::kanMX leu1. atg24-YFH::leu1+ leu1-32 his3-D1 Figure 5A DY29503 h.1242/jcs.J. Figure 3D GFP-AHDL::ura4+ leu1-32 his-3-D1(or his3+) DY29560 h? atg20Δ::natMX atg24Δ::hphMX mug186Δ::kanMX Pbip1. S1B DY29545 h.194373: Supplementary information DY29541 h? atg24Δ::natMX atg24bΔ::hphMX Pbip1-GFP-AHDL::ura4+ Figure 3D leu1-32 his-3-D1(or his3+) DY29556 h? atg24Δ::hphMX mug186Δ::kanMX Pbip1-GFP-AHDL::ura4+ Figure 3D leu1-32 his-3-D1(or his3+) DY29557 h? atg24bΔ::natMX mug186Δ::kanMX Pbip1-GFP-AHDL::ura4+ Figure 3D leu1-32 his-3-D1(or his3+) DY29558 h? atg20Δ::natMX mug186Δ::kanMX Pbip1-GFP-AHDL::ura4+ Figure 3D leu1-32 his-3-D1(or his3+) DY29544 h? atg20Δ::kanMX atg24bΔ::hphMX atg24Δ::natMX Pbip1. Figure 4B 32 his3-D1 DY31749 h? atg24Δ::natMX atg24bΔ::hphMX sdh2-mCherry::kanMX Figure 4B leu1-32 his3-D1 DY31750 h? atg20Δ::natMX atg24bΔ::hphMX mug186Δ::kanMX sdh2. Cell Sci. Figure 3E CFH(leu1+) his3-D1 DY29591 h? atg24Δ::hphMX mug186Δ::kanMX leu1-32::Pnmt1-ost4. Figure 3D GFP-AHDL::ura4+ leu1-32 his-3-D1(or his3+) DY29561 h? atg24bΔ::natMX atg24Δ::hphMX mug186Δ::kanMX Pbip1.atg24b-YFH::hphMX leu1-32 his3-D1 Figure 5A DY29504 h. Figure 4A mCherry::ura4+ leu1-32 Journal of Cell Science • Supplementary information DY29574 h? atg24Δ::natMX atg24bΔ::hphMX his3::Ptub1-cox4(1-33). Figure 4A mCherry::ura4+ leu1-32 DY29572 h? atg24Δ::natMX mug186Δ::kanMX his3::Ptub1-cox4(1-33). his3::Ptub1-cox4(1-33)-mCherry::kanMX leu1-32 Figure 4A DY29571 h? atg20Δ::kanMX atg24Δ::natMX his3::Ptub1-cox4(1-33). Figure 3E CFH(leu1+) his3-D1 DY29532 h? atg24Δ::hphMX atg20Δ::kanMX leu1-32::Pnmt1-ost4. Figure 3E CFH(leu1+) his3-D1 DY29581 h? leu1-32::Pnmt1-ost4-CFH(leu1+) ura4-D18 his3-D1 Figure 3E. 129: doi:10.

Figure 5E YFH::hphMX his3-D1 DY29526 h+ leu1-32::P41nmt1-mug186-mCherry(leu1+) atg24. 32::Pnmt1-tdh1-YFH(leu1+) cpy1-mCherry::kanMX his3-D1 8B DY29592 h? fsc1Δ::kanMX atg20Δ::kanMX atg24Δ::natMX leu1-32::tdh1.1242/jcs. YFH(leu1+) cpy1-mCherry::kanMX his3-D1 8B . 6B syb1(leu1+) his3-D1 DY29553 h? atg20Δ::natMX mug186Δ::kanMX leu1-32::P41nmt1-GFP. 7B. Figure 5D YFH::hphMX his3-D1 DY29511 h+ leu1-32::P41nmt1-mug186-mCherry(leu1+) his3-D1 Figure 5E DY29515 h+ leu1-32::P41nmt1-mug186-mCherry(leu1+) mug186.mug186. 7B. Figure 5E YFH::hphMX his3-D1 DY29546 h. 8B DY29577 h? fsc1Δ::kanMX atg24Δ::natMX atg24bΔ::hphMX leu1. leu1-32::P41nmt1-GFP-syb1(leu1+) his3-D1 Figure 6A.J. 7B. Figure 5E YFH::hphMX his3-D1 DY29527 h+ leu1-32::P41nmt1-mug186-mCherry(leu1+) atg24b. mCherry::kanMX 7C. 7B. Cell Sci. Figure 7A. 6B syb1(leu1+) his3-D1 DY29551 h? atg20Δ::kanMX atg24Δ::natMX leu1-32::P41nmt1-GFP. Figure 6A. Figure 5B YFH::hphMX his3-D1 DY29509 h+ leu1-32::P41nmt1-atg24-mCherry(leu1+) his3-D1 Figure 5C DY29513 h+ leu1-32::P41nmt1-atg24-mCherry(leu1+) atg24-YFH::hphMX Figure 5C his3-D1 DY29519 h+ leu1-32::P41nmt1-atg24-mCherry(leu1+) atg20-YFH::hphMX Figure 5C his3-D1 DY29520 h+ leu1-32::P41nmt1-atg24-mCherry(leu1+) atg24b-YFH::hphMX Figure 5C his3-D1 DY29521 h+ leu1-32::P41nmt1-atg24-mCherry(leu1+) mug186. 6B DY29548 h? atg24Δ::kanMX leu1-32::P41nmt1-GFP-syb1(leu1+) his3-D1 Figure 6A.194373: Supplementary information his3-D1 DY29517 h+ leu1-32::P41nmt1-atg20-mCherry(leu1+) atg24b-YFH::hphMX Figure 5B his3-D1 DY29518 h+ leu1-32::P41nmt1-atg20-mCherry(leu1+) mug186. 7D. 6B syb1(leu1+) his3-D1 DY29573 h. fsc1Δ::kanMX leu1-32::Pnmt1-tdh1-YFH(leu1+) cpy1. Figure 5D YFH::hphMX his3-D1 DY29522 h+ leu1-32::P41nmt1-atg24b-mCherry(leu1+) atg20-YFH::hphMX Figure 5D his3-D1 DY29523 h+ leu1-32::P41nmt1-atg24b-mCherry(leu1+) atg24-YFH::hphMX Figure 5D his3-D1 DY29524 h+ leu1-32::P41nmt1-atg24b-mCherry(leu1+) . YFH(leu1+) cpy1-mCherry::kanMX his3-D1 8B DY29593 h? fsc1Δ::kanMX atg24Δ::natMX leu1-32::Pnmt1-tdh1. Figure 7A. 6B syb1(leu1+) his3-D1 DY29552 h? atg24Δ::natMX atg24bΔ::hphMX leu1-32::P41nmt1-GFP. Figure 7A. Figure 6A. 6B DY29547 h? atg20Δ::kanMX leu1-32::P41nmt1-GFP-syb1(leu1+) his3-D1 Figure 6A. Figure 5C YFH::hphMX his3-D1 DY29510 h+ leu1-32::P41nmt1-atg24b-mCherry(leu1+) his3-D1 Figure 5D DY29514 h+ leu1-32::P41nmt1-atg24b-mCherry(leu1+) atg24b. 6B D1 DY29550 h? atg20Δ::natMX atg24bΔ::kanMX leu1-32::P41nmt1-GFP. 6B DY29554 h? mug186Δ::hphMX leu1-32::P41nmt1-GFP-syb1(leu1+) his3. Figure 6A. Figure 7A. 129: doi:10. Figure 6A. 6B Journal of Cell Science • Supplementary information DY29549 h? atg24bΔ::kanMX leu1-32::P41nmt1-GFP-syb1(leu1+) his3-D1 Figure 6A. Figure 5E YFH::hphMX his3-D1 DY29525 h+ leu1-32::P41nmt1-mug186-mCherry(leu1+) atg20. Figure 6A.

atg9-YFP::kanMX CFP-atg8::leu1+ leu1-32 Figure 8C. Figure S4E D1 leu1-32 DY29495 h? atg24Δ::kanMX atg24b-YFH::leu1+ CFP-atg8::leu1+ his3-D1 Figure S4E leu1-32 DY29502 h? mug186Δ::kanMX atg24b-YFH::leu1+ CFP-atg8::leu1+ his3. Figure S3A Atg8::leu1+ his3-D1 leu1-32 DY33717 h? atg14Δ:: hphMX vps38Δ::kanMX mug186-YFH::leu1+ CFP. S4E DY29501 h? mug186Δ::kanMX atg24-YFH::leu1+ CFP-atg8::leu1+ leu1. 32::Pnmt1-tdh1-YFH(leu1+) cpy1-mCherry::kanMX his3-D1 8B DY29578 h? fsc1Δ::kanMX atg20Δ::kanMX atg24Δ::natMX Figure 7C. Figure S3A . 8D leu1-32 DY29582 h? atg24Δ::natMX atg20Δ::kanMX atg9-YFP::kanMX CFP. 8D atg8::leu1+ his3-D1 leu1-32 DY29583 h? atg24Δ::natMX atg24bΔ::hphMX atg9-YFP::kanMX CFP. Figure S4D. S4E DY29497 h? atg24bΔ::kanMX atg20-YFH::leu1+ CFP-atg8::leu1+ his3-D1 Figure S4C. S4E leu1-32 DY29534 h? atg24Δ::natMX atg24bΔ::hphMX atg20-YFH::leu1+ CFP.194373: Supplementary information DY29594 h? fsc1Δ::kanMX atg20Δ::kanMX atg24bΔ::hphMX leu1. S4E atg8::leu1+ his3-D1 leu1-32 DY29490 h? atg20Δ::kanMX atg24-YFH::leu1+ CFP-atg8::leu1+ leu1-32 Figure S4D. Figure S4B. Figure 8C. Figure S3A Atg8::leu1+ his3-D1 leu1-32 DY33718 h? atg14Δ:: hphMX vps38Δ::kanMX atg24b-YFH::leu1+ CFP.YFH::leu1+ CFP-atg8::leu1+ his3. 8D DY29576 h? atg24Δ::hphMX atg9-YFP::kanMX CFP-atg8::leu1+ his3-D1 Figure 8C. S4E D1 leu1-32 DY29492 h? atg24Δ::kanMX atg20-YFH::leu1+ CFP-atg8::leu1+ leu1-32 Figure S4C.1242/jcs. 8D DY29491 h? atg20Δ::kanMX atg24b-YFH::leu1+ CFP-atg8::leu1+ his3-D1 Figure S4A. Figure S4E D1 leu1-32 DY29498 h? atg24bΔ::kanMX atg24-YFH::leu1+ CFP-atg8::leu1+ leu1-32 Figure S4E DY29494 h? atg20Δ::kanMX mug186-YFH::leu1+ CFP-atg8::leu1+ his3. Figure S4E D1 leu1-32 DY29499 h? atg24bΔ::kanMX mug186. Figure 8C. Figure 8C. Figure S4D. 8D atg8::leu1+ his3-D1 leu1-32 DY29580 h. Figure S4C. Figure S3A Atg8::leu1+ his3-D1 leu1-32 DY33716 h? atg14Δ:: hphMX vps38Δ::kanMX atg24-YFH::leu1+ CFP. Cell Sci. Figure S4E D1 leu1-32 DY33715 h? atg14Δ:: hphMX vps38Δ::kanMX atg20-YFH::leu1+ CFP.J. 129: doi:10. Figure 7A. S4E atg8::leu1+ his3-D1 leu1-32 DY29500 h? mug186Δ::kanMX atg20-YFH::leu1+ CFP-atg8::leu1+ his3. 7D atg24b::hphMX. S4E leu1-32 DY29496 h? atg24Δ::kanMX mug186-YFH::leu1+ CFP-atg8::leu1+ his3. S4E 32 Journal of Cell Science • Supplementary information DY29533 h? atg20Δ::natMX mug186Δ::kanMX atg24-YFH::leu1+ CFP. 8D atg8::leu1+ his3-D1 leu1-32 DY29584 h? atg20Δ::kanMX atg24bΔ::hphMX atg9-YFP::kanMX CFP. 7B. leu1-32::Pnmt1-tdh1-YFH(leu1+) cpy1- mCherry::kanMX his3-D1 DY29488 h+ CFP-atg8::leu1+ his3-D1 leu1-32 Figure 8A DY29489 h+ atg24Δ::kanMX CFP-atg8::leu1+ his3-D1 leu1-32 Figure 8A DY29505 h? atg20Δ::natMX atg24Δ::hphMX CFP-atg8::leu1+ his3-D1 Figure 8A leu1-32 DY29506 h? atg20Δ::natMX atg24bΔ::hphMX CFP-atg8::leu1+ his3-D1 Figure 8A leu1-32 DY29507 h? atg24Δ::natMX atg24bΔ::hphMX CFP-atg8::leu1+ his3-D1 Figure 8A leu1-32 DY29579 h. atg1Δ::kanMX atg9-YFP::kanMX CFP-atg8::leu1+ leu1-32 Figure 8C.

129: doi:10.1242/jcs.194373: Supplementary information Atg8::leu1+ his3-D1 leu1-32 DY33719 h? atg14Δ:: hphMX vps38Δ::kanMX atg17-YFH::leu1+ his3-D1 Figure S3B leu1-32 DY33720 h? atg1Δ::kanMX leu1-32::Pnmt1-ost4-CFH(leu1+) his3-D1 Figure S1B DY33721 h? atg11Δ::kanMX leu1-32::Pnmt1-ost4-CFH(leu1+) his3-D1 Figure S1B DY33722 h? atg101Δ::kanMX leu1-32::Pnmt1-ost4-CFH(leu1+) his3-D1 Figure S1B DY33723 h? atg13Δ::kanMX leu1-32::Pnmt1-ost4-CFH(leu1+) his3-D1 Figure S1B DY33724 h? atg17Δ::kanMX leu1-32::Pnmt1-ost4-CFH(leu1+) his3-D1 Figure S1B DY33725 h+ sdh2-mCherry::kanMX leu1-32 his3-D1 Figure S1C DY33726 h? atg1Δ::kanMX sdh2-mCherry::kanMX leu1-32 his3-D1 Figure S1C DY33727 h? atg11Δ::kanMX sdh2-mCherry::kanMX leu1-32 his3-D1 Figure S1C DY33728 h? atg101Δ::kanMX sdh2-mCherry::kanMX leu1-32 his3-D1 Figure S1C DY33729 h? atg13Δ::kanMX sdh2-mCherry::kanMX leu1-32 his3-D1 Figure S1C DY33730 h? atg17Δ::kanMX sdh2-mCherry::kanMX leu1-32 his3-D1 Figure S1C Journal of Cell Science • Supplementary information .J. Cell Sci.