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Atg45 is an autophagy receptor for glycogen, a non-preferred cargo of bulk autophagy

in yeast

Takahiro Isoda, Eigo Takeda, Sachiko Hosokawa, Shukun Hotta-Ren, Yoshinori

Ohsumi

PII:

S2589-0042(24)01032-0

DOI:

https://doi.org/10.1016/j.isci.2024.109810

Reference:

ISCI 109810

To appear in:

ISCIENCE

Received Date: 31 May 2023
Revised Date: 3 July 2023
Accepted Date: 22 April 2024

Please cite this article as: Isoda, T., Takeda, E., Hosokawa, S., Hotta-Ren, S., Ohsumi, Y., Atg45 is an

autophagy receptor for glycogen, a non-preferred cargo of bulk autophagy in yeast,

ISCIENCE

(2024),

doi:

https://doi.org/10.1016/j.isci.2024.109810

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Atg45 is an autophagy receptor for glycogen, a non-preferred cargo of bulk autophagy in yeast

Takahiro Isoda

1,2,3

, Eigo Takeda

, Sachiko Hosokawa

, Shukun Hotta-Ren

, Yoshinori

Ohsumi

1,4*

Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology,

Yokohama, 226-8503, Japan.

School and Graduate School of Bioscience and Biotechnology, Tokyo Institute of

Technology, Yokohama, 226-8503, Japan.

Frontier Research Center, POLA Chemical Industries, Inc., Yokohama, 244-0812, Japan.

Lead contact

* Correspondence: Yoshinori Ohsumi, yohsumi@iri.titech.ac.jp.

Summary

The mechanisms governing autophagy of proteins and organelles have been well studied, but

how other cytoplasmic components such as RNA and polysaccharides are degraded remains

largely unknown. In this study, we examine autophagy of glycogen, a storage form of glucose.

We find that cells accumulate glycogen in the cytoplasm during nitrogen starvation, and that

this carbohydrate is rarely observed within autophagosomes and autophagic bodies. However,

sequestration of glycogen by autophagy is observed following prolonged nitrogen starvation.

We identify a yet-uncharacterized open reading frame, Yil024c (herein Atg45), as encoding

a cytosolic receptor protein that mediates autophagy of glycogen (glycophagy). Furthermore,

we show that during sporulation, Atg45 is highly expressed, and is associated with an increase

in glycophagy. Our results suggest that cells regulate glycophagic activity by controlling the

expression level of Atg45.

Introduction

The vacuole (lysosome in mammals) is an organelle responsible for degradation of cellular

components, as well as the storage of ions and molecules such as amino acids.

Macroautophagy (hereafter autophagy) is a major degradation system that delivers

cytoplasmic constituents to the vacuole. In autophagy, various cargoes are engulfed by an

expanding membrane sac called the isolation membrane, which eventually becomes a double-

membrane vesicle called an autophagosome. The outer membrane of the autophagosome

fuses with the vacuole, which results in the release of the inner membrane-bound structure,

the autophagic body, into the vacuolar lumen, allowing degradation by the vacuolar lipase

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Atg15 and a range of proteases

–

. Degradation products arising from autophagy, such as

amino acids, are subsequently transported back to the cytoplasm for reuse

–

Autophagosomes are generally thought to sequester cytoplasmic components in a random

manner (termed bulk autophagy). In some cases, autophagosomes selectively enclose specific

cargoes such as protein aggregates or organelles in a regulated process called selective

autophagy

. In selective autophagy, a specific cargo is engulfed by isolation membranes via

cargo specific receptors

. Receptors generally bind to Atg8-family proteins on the isolation

membrane, and the scaffold protein Atg11 (in yeast) or FIP200 (in mammals)

–

. These

scaffold proteins recruit autophagic machinery proteins, allowing the formation of

autophagosomal membranes that surround the cargo

–

Although much is known about autophagy of proteins and organelles, the mechanisms of

autophagy for other cellular components such as RNAs and polysaccharides are still largely

unclear. Glycogen, which is a highly branched polysaccharide, is reported to be delivered to

lysosomes by autophagy (glycophagy) in mammalian and insect cells

–

. When the lysosomal

acid



-glucosidase, which hydrolyzes glycogen to glucose, is defective in mammalian cells,

glycogen accumulates in the lysosome, causing Pompe disease

. Thus, control of the delivery

of glycogen to the lysosome is physiologically important. Although the mechanism of

glycophagy is not fully understood, a putative trans-membrane protein Stbd1 is reported to

be a glycophagy receptor in mammals

15,16

. This protein contains a glycogen binding domain

called CBM20, as well as an Atg8 family-interacting motif (AIM, also called the LC3-

interacting region (LIR))

15,16

In the budding yeast

Saccharomyces cerevisiae

, which is an excellent model organism for

studies of autophagy, glycogen is synthesized in the cytosol by glycogenins (Glg1, Glg2),

glycogen synthases (Gsy1, Gsy2), and glycogen branching enzyme (Glc3) in a concerted

manner

. Synthesis is induced under stress conditions, such as nitrogen starvation

Accumulated glycogen is subsequently broken down into glucose-1-phosphate via

glycogenolysis by glycogen phosphorylases (Gph1) and glycogen debranching enzyme

(Gdb1) in the cytosol according to cellular needs

. Glycogen is also degraded by the

glucoamylase Sga1 which localizes in the vacuole or to prospore membranes during

sporulation

21,22

. It has been suggested that autophagy may be involved in the delivery of

glycogen to vacuoles

23,24

, but the molecular details of this process remain uncharacterized

–

We recently conducted a comprehensive proteomic analysis of autophagic bodies and

reported that glycogen metabolism-related enzymes are excluded from autophagic bodies

In this study, we find that glycogen is a non-preferred cargo of bulk autophagy. We further

determine that glycogen metabolism-related enzymes escape from autophagy, most likely due

to their binding to glycogen. However, we find that glycogen becomes a target of autophagy

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Results

Glycogen and glycogen metabolism-related enzymes are non-preferred cargoes of conventional

nitrogen starvation-induced autophagy

In our comprehensive proteomic analysis of autophagic bodies, we found that some

glycogen metabolism-related enzymes (e.g. Gsy2, Glc3, Gph1) are non-preferred cargoes of

bulk autophagy (Takeda et al.*accepted in EMBO J). To validate this result, we assessed

autophagic degradation of Gsy2, Glc3, and Gph1 by the GFP-cleavage assay, whereby a

protein of interest is expressed in fusion with GFP. Upon delivery to the vacuole, vacuolar

protease-resistant GFP moieties (free GFP) are generated and can be detected using

antibodies, allowing the assessment of the degree of autophagic degradation of the GFP-

tagged protein by measuring the amounts of free GFP and intact GFP-tagged proteins. We

constructed GFP-tagged Gsy2, Glc3, and Gph1, as well as Pgk1 and Trp4 (cytosolic proteins),

expressing each of these from their native promoter in WT cells. Consistent with our

comprehensive proteomic analyses, western blotting with anti-GFP antibodies revealed that

Gsy2, Glc3, and Gph1 were only marginally degraded up to 8 h of nitrogen starvation (Figure

1A). Previous works have suggested that some glycogen metabolism-related enzymes bind to

glycogen

–

(Figure 1B). For example, glycogen synthetase tightly binds to glycogen, which

is observed as co-localization by microscopy

27,28,33

. Upon nitrogen starvation, we observed the

synthesis and accumulation of glycogen in the cytoplasm of wild-type (WT) cells, as

previously reported

; this was completely absent in

glg1



glg2



cells (Figure S1A). Thus, we

assumed that glycogen metabolism-related enzymes would escape from autophagy by binding

100

to glycogen. We investigated the localization of the glycogen synthetase Gsy2 using

101

fluorescence microscopy to observe cells subjected to nitrogen starvation. 2 h after shifting to

102

nitrogen starvation medium (SD-N medium), Pgk1 in the cytoplasm was uniformly

103

distributed, whereas Gsy2 was unevenly distributed (Figure S1B). In addition, Gsy2 co-

104

localized with other glycogen metabolism-related proteins such as Glc3 and Gph1, but not

105

with membrane-bound organelles such as the ER (Sec63), nucleus (Ndc1) or mitochondria

106

(Om45) (Figures S1C and S1D). In addition, in

glg1



glg2



cells, Gsy2 showed a

107

cytoplasmically diffused pattern under nitrogen starvation (Figure S1E). These observations

108

suggest that glycogen metabolism-related

enzymes bind to glycogen granules. Furthermore,

109

the majority of Gsy2 was recovered in the pellet fraction after centrifugation of cellular lysates

110

at 100,000

in the presence of endogenous glycogen (WT cell lysate) or exogenously added

111

glycogen (

glg1



glg2



cells lysate with added glycogen) (Figure 1C, see also Figure 5A),

112

confirming that Gsy2 binds glycogen in this condition.

113

Next, we assessed autophagic degradation of glycogen metabolism-related enzymes in

114

glg1



glg2



cells and found that autophagic degradation of Gsy2, Glc3, and Gph1 was largely

115

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enhanced, while Pgk1 and Trp4 remained constant (Figure 1D). As expected, a similar

116

increase was observed in an alternative glycogen-deficient strain,

gsy1



gsy2



(Figure S1F).

117

These results indicate that glycogen suppresses autophagic degradation of these enzymes. To

118

investigate whether glycogen is also a non-preferred cargo of autophagy under these

119

conditions, we examined the localization of glycogen by transmission electron microscopy

120

(TEM). To this end, we used

atg15



cells, which accumulate autophagic bodies in the vacuole

121

(Figures 1E and S1G)

1,3,6

. Periodic acid-thiocarbohydrazide-silver proteinate (PATAg) was

122

used to stain polysaccharides including glycogen

–

. Under nitrogen starvation,

atg15



cells

123

accumulated PATAg-stained structures that were not seen in

glg1



glg2



atg15



cells (Figure

124

1E). The size of these glycogen structures in the cytoplasm varied from about 40 nm to 200

125

nm (Figure 1E), suggesting the formation of higher-order glycogen assemblies such as



126

granules, as observed in the mammalian liver

. Autophagic bodies rarely contained glycogen,

127

though glycogen was widely distributed in the cytoplasm (Figure 1E), indicating that glycogen

128

is a non-preferred cargo of nitrogen-starvation induced autophagy. These results support the

129

conclusion that glycogen is a non-preferred cargo of nitrogen-starvation induced autophagy,

130

and that proteins bound to glycogen (such as Gsy2) thereby escape autophagic degradation

131

upon nitrogen starvation,

132

133

Glycophagy occurs after prolonged nitrogen starvation

134

While glycogen and glycogen metabolism-related enzymes are non-preferred cargoes of

135

nitrogen-starvation induced autophagy, we noticed that Gsy2 was gradually degraded after 8

136

h (Figure 2A), suggesting that autophagy of glycogen (glycophagy) does occur upon

137

prolonged nitrogen starvation. TEM analysis of PATAg-stained cells subjected to prolonged

138

nitrogen starvation showed that the percentage of individual autophagic bodies that contained

139

glycogen was increased relative to samples subjected to a shorter (6 h) period of nitrogen

140

starvation (Figures 2B and 2C), and most autophagosomes contained glycogen at 48 h (Figure

141

2B). Glycophagy was in fact enhanced upon prolonged nitrogen starvation. We also concluded

142

that Gsy2 can be used as a reliable reporter of glycophagy.

143

To characterize glycophagy during prolonged nitrogen starvation, we examined whether

144

ATG

genes (

ATG2

ATG11

ATG17

, and

ATG24

) are required for the degradation of Gsy2

145

using the GFP-cleavage assay. While Atg2 is essential for all types of autophagy, the scaffold-

146

protein Atg11 is required for most forms of selective autophagy

11,40,41

and Atg17 is known to

147

be important for bulk autophagy as well as some types of selective autophagy, including

148

mitophagy and pexophagy

42,43

. In

atg2



and

atg17



cells, the degradation of Gsy2

was

149

significantly decreased (Figures 2D and 2E). In

atg11



cells, the degradation of Gsy2 was

150

slightly reduced to a similar extent to that of a representative bulk autophagy cargo protein,

151

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Pgk1 (Figures 2D and 2E), indicating that Atg11 is not specifically involved in the degradation

152

of Gsy2. Atg24 (also termed Snx4) is required for autophagy of large cargoes such as

153

organelles, ribosomes and proteasomes

–

. In

atg24



cells, the degradation of Gsy2 was

154

clearly impaired, while that of Pgk1 was only slightly decreased (Figures 2D and 2E).

155

Together, these results show that prolonged starvation-induced glycophagy does not require

156

Atg11 but does require Atg24.

157

158

Atg45 is a positive regulator of glycophagy

159

A possible explanation for the late-onset delivery of glycogen to the vacuole is that glycogen

160

may become more accessible to autophagosomes after prolonged nitrogen starvation. We

161

considered that a protein may facilitate this, thereby regulating glycophagy. To search for

162

such a protein, we focused on proteins containing the glycogen binding domain (GBD).

163

Mammalian AMPKβsubunits have a GBD called

the carbohydrate-binding module 48

164

(CBM48). We focused on six genes in yeast that encode candidate GBD domains that have

165

sequence similarity with CBM48 (Figure 3A, Pfam domain PF16561). Among these, Sip1,

166

Sip2, and Gal83 are



-subunits of yeast AMPK (Snf1), which has been reported to regulate

167

glycogen synthesis

48,49

. On the other hand, the function of Crp1, Mdg1, and Yil024c have not

168

been defined. Fluorescence microscopy showed that Crp1 and Yil024c co-localize with Gsy2

169

in the cell periphery and in the cytosol, respectively, under nitrogen starvation conditions, but

170

that Sip1, Sip2, and Gal83 do not (Figures 3B and S2A-E). Hence, we focused on Yil024c,

171

Crp1, and its paralog Mdg1.

172

We investigated the effect of the disruption of these genes on the autophagic degradation

173

of Gsy2. Degradation was slightly lower in

crp1



crp1



mdg1



cells, and was nearly the

174

same in

mdg1



cells as in WT cells (Figure 3C)

On the other hand, it was clearly lower in

175

yil024c



cells as compared with WT cells (Figure 3C), indicating that glycophagy is strongly

176

dependent on Yil024c. We then tested the contribution of Yil024c to the degradation of Gsy2

177

in the absence of glycogen. In

glg1



glg2



cells, Gsy2 degradation was not affected by Yil024c

178

deletion (Figure 3D), suggesting that Yil024c targets glycogen, not Gsy2. Taken together, we

179

conclude that Yil024c is a positive regulator of glycophagy, and therefore name this protein

180

Atg45.

181

182

Increased expression of Atg45 enhances glycophagy

183

Previous comprehensive microarray analyses have suggested that the amount of

ATG45

184

mRNA increases 1-2 d after shifting to starvation medium

. This suggests that glycophagy

185

induction by prolonged starvation may be due to the up-regulation of Atg45 expression. We

186

assessed the expression of Atg45 by western blotting in a nitrogen starvation time course

187

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experiment. We observed that the expression of Atg45 is induced during nitrogen starvation,

188

especially at 24 and 48 h after shifting to SD-N medium (Figure 4A). The amount of

ATG45

189

mRNA also gradually increased during nitrogen starvation (Figure S3A), confirming that

190

transcription of Atg45 is upregulated in this condition.

191

We next generated cells overexpressing Atg45 under the control of the

ACT1

promoter.

192

In these cells, Gsy2 was more rapidly degraded than in WT cells (Figure 4B). Moreover, the

193

degradation of Gsy2 under these conditions was dependent on Atg2, Atg17, and Atg24, but

194

only slightly reduced on Atg11 (Figure 4C), which was consistent with cells expressing Atg45

195

under the control of its own promoter (Figure 2B). We observed that bulk-autophagic activity

196

was slightly reduced in

atg11



cells even at 2 hours of nitrogen starvation (Figure S3B), as

197

previously reported

–

, suggesting that the effect of Atg11 is not specific to the degradation

198

of Gsy2 even during early-term nitrogen starvation. TEM analysis of PATAg-stained samples

199

showed that in cells overexpressing Atg45, about 40% of autophagic bodies contained

200

glycogen, while in WT cells less than 10% of autophagic bodies did (Figure 4D), providing

201

further evidence that Atg45 facilitates glycophagy. Notably, the amounts of glycogen in

202

individual autophagic bodies exhibited heterogeneity; given that the amount of glycogen is

203

rapidly increased during nitrogen starvation (Figure S1A), variation in the amounts of

204

glycogen in autophagic bodies may reflect the timing of autophagosome formation. Indeed,

205

at 6 h (i.e., when cells had accumulated glycogen), most autophagosomes contained high

206

levels of glycogen in cells overexpressing Atg45 (Figure 4D).

207

In contrast to cells overexpressing Atg45, autophagosomes contained little glycogen in

208

cells expressing Atg45 at WT levels, despite the presence of glycogen in the cytoplasm.

209

Furthermore, in cells overexpressing Atg45, we observed that glycogen was enriched on the

210

inner membrane of autophagosomes, which was also observed in WT cells after prolonged

211

nitrogen starvation (48 h, Figures 2B and 4E). This suggests that Atg45 facilitates the

212

sequestration of glycogen into autophagosomes by enhancing the binding of glycogen to

213

autophagosomal membranes.

214

215

Atg45 functions as a glycogen receptor

216

To elucidate the mechanistic bases of Atg45-mediated glycophagy, we first examined whether

217

the N-terminal half (1-96) of Atg45, which contains a putative GBD, binds to glycogen. We

218

compared the amount of Atg45 recovered in the pellet fraction of cell lysates in the presence

219

or absence of glycogen by sedimentation assay. In this assay, lysates from

glg1



glg2



cells

220

were mixed with exogenous glycogen and subjected to centrifugation. Full-length Atg45 and

221

the N-terminal region of Atg45 (Atg45

1-96

) were recovered in the pellet when sedimented in

222

the presence of glycogen (Figure 5A), while the C-terminal half of Atg45 (Atg45

97-189

) was

223

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not (Figure 5A), suggesting that Atg45 binds to glycogen through the GBD identified within

224

Atg45

1-96

225

To determine whether both regions of Atg45 are required for glycophagy, we assessed the

226

cleavage of Gsy2-GFP in cells overexpressing Atg45-, Atg45

1-96

-, or Atg45

97-189

-mScarlet

227

chimeric proteins. We found that neither Atg45

1-96

nor Atg45

97-189

facilitated glycophagy

228

(Figure 5B), confirming that both regions of Atg45 are necessary for glycophagy.

229

We next assessed the autophagic degradation of GFP-tagged Atg45, Atg45

1-96

, and

230

Atg45

97-189

under nitrogen starvation. The degradation of Atg45

97-189

was comparable to that

231

of Atg45, but Atg45

1-96

was less degraded than Atg45 (Figure S4A). In addition, Atg45 and

232

Atg45

97-189

tagged with mScarlet co-localized with GFP-Atg8, an autophagosomal marker, but

233

Atg45

1-96

-mScarlet did not (Figure S4B). Taken together, these results indicate that the C-

234

terminal half of Atg45 is required for sequestration into autophagosomes, not binding to

235

glycogen.

236

These results suggest that an AIM is present within the C-terminal half of Atg45. We

237

identified

127

YVNL

130

as the sole candidate for an AIM within the region. To investigate

238

whether this site functions as AIM, we carried out co-immunoprecipitation of GFP-Atg8 and

239

Atg45-FLAG using the GFP-Trap Magnetic Agarose system. While wild-type Atg45

240

exhibited binding to Atg8, the alanine substituted mutant of the putative AIM (Y127A and

241

L130A, herein Atg45

AIMm

) had a low binding ability (Figure 5C). In cells expressing Atg45

AIMm

242

glycophagic activity was lower than that in cells expressing WT-Atg45, even when it was

243

overexpressed (Figures 5D and S5A), confirming that

127

YVNL

130

functions as the AIM.

244

Thus, we concluded that Atg45 is a glycophagy receptor protein. Even in cells expressing

245

Atg45

AIMm

, glycophagic activity was not completely abolished, possibly due to residual Atg8

246

binding of Atg45

AIMm

through the mutant AIM or another region of the protein that allows

247

limited localization to the isolation membrane.

248

249

The C-terminal region of Atg45 is important for glycophagy

250

We next examined glycophagic activity in cells expressing the C-terminal truncated Atg45

251

mutants Atg45

1-164

and Atg45

1-123

. In the following experiments, we mainly used cells

252

harboring a single-copy plasmid encoding

ATG45

under the control of its own promoter.

253

These cells showed higher levels of both Atg45 expression and glycophagic activity compared

254

to wild-type cells (Figure S5B). Using this plasmid, we were able to develop strains with

255

elevated Atg45 expression levels, which were closer to physiological conditions compared to

256

cells containing a plasmid with the

ATG45

regulated by the

ACT1

promoter. As expected, the

257

expression of Atg45

1-123

resulted in severely impaired glycophagy as the mutant lacks an AIM

258

(Figure 6A). We also found that the Atg45

1-164

mutant was partially defective in glycophagy

259

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(Figure 6A), suggesting that the Atg45

165-189

residues at the C-terminal of Atg45 play a role in

260

glycophagy.

261

We found that these terminal 172-187 residues likely constitute an amphipathic helix

262

using JPred4 and HeliQuest (Figure 6B). It is known that some amphipathic helices bind to

263

membranes via their hydrophobic surface

. We found that Atg45

165-189

-GFP localized to

264

various membranes, including the vacuolar membrane (Figure 6C). When six hydrophobic

265

residues within Atg45

165-189

were substituted with alanine (6A; Figure 6B), membrane

266

localization was abolished (Figure 6C). To examine whether this finding is relevant to

267

glycophagy, we examined cells expressing Atg45

(6A)

-FLAG, showing clearly lower glycophagic

268

activity than cells expressing wild-type Atg45-FLAG (Figure 6D). This suggests that the

269

membrane-binding ability of the Atg45

165-189

region is important for glycophagy. When

270

Atg45

(6A)

was overexpressed using the

ACT1

promoter, glycophagic activity was recovered to

271

the level of cells overexpressing wild-type Atg45 (Figure S6A). However, cells harboring both

272

6A and AIMm mutations showed more severe defects than AIMm mutant cells (Figure S6A),

273

indicating that the C-terminal region and the AIM function synergistically. We further tested

274

the effect of the 6A and AIMm mutation on the co-localization of Atg45 with Atg8 in cells

275

expressing Atg45 under its own promotor. Both mutants of Atg45 exhibited a defect in co-

276

localization with Atg8 (Figure S6B).

277

Taken together, our findings suggest that both the C-terminal region and the AIM of

278

Atg45 collaborate in facilitating glycophagy through their involvement in binding to isolation

279

membranes.

280

281

Glycophagy is strongly induced during sporulation

282

We next sought to identify physiological conditions under which glycophagy is strongly

283

induced. While glycogen is observed to accumulate during the early stages of sporulation, it

284

has been reported that the amount of glycogen decreases later during this process

55,56

, leading

285

us to consider whether glycophagy is activated during sporulation. Because the X2180

286

background strain exhibits a low sporulation rate, we used the SEY6210/SEY6211 background

287

strain to study sporulation.

288

We induced sporulation in diploid (

MAT



) cells and conducted TEM analyses of

289

PATAg-stained samples. In WT cells, glycogen accumulated in some vacuoles 12 h after

290

induction of sporulation (Figure 7A). Most glycogen was observed in the vacuolar sap, while

291

a fraction was also detected in unruptured autophagic bodies (Figure 7A). This suggests that

292

glycogen is delivered to vacuoles by autophagy, but that it is not efficiently degraded even

293

after autophagic bodies are ruptured. (Figure 7A). In

atg45



cells, the amount of glycogen in

294

vacuoles was clearly lower than in WT cells (Figure 7A). These results indicate that

295

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glycophagy is strongly induced during sporulation, and that sporulation-induced glycophagy

296

is dependent on Atg45.

297

The expression of sporulation-related genes is controlled by the

MAT

locus. Thus,

298

MAT



cells are capable of sporulation, whereas

MAT

-homozygous diploid cells (

MAT

a/a

299

cells) and haploid cells (

MAT

a and

MAT



cells) are not. To investigate whether the induction

300

of glycophagy is also regulated by the

MAT

locus, we compared the glycophagic activity of

301

MAT



MAT

a/a, and

MAT



cells. Fluorescence microscopy showed that Gsy2-GFP was

302

delivered to the vacuoles in

MAT



cells but not in

MAT

a/a or

MAT



cells 12 h after

303

induction of sporulation (Figure S7A). The accumulation of GFP signal in vacuoles was not

304

observed in

atg45



(

MAT



) cells (Figure S7B), and GFP-cleavage analyses of Gsy2-GFP

305

validated microscopic analyses (Figure 7B and S7C). These results indicate that sporulation-

306

induced glycophagy also depends on the expression of genes controlled by the

MAT

locus.

307

We next examined the expression of Atg45-GFP in

MAT



MAT

a/a, and

MAT



cells

308

by western blotting. The amount of Atg45-GFP was higher in

MAT



cells compared to

309

other cells (Figure 7C). Transcription of

ATG45

mRNA was induced in

MAT



cells (Figure

310

7D), which is consistent with a previous study showing that the expression of

ATG45

311

regulated by Ume6, the transcriptional regulator of early meiotic genes

. Therefore, the

312

expression of Atg45 is controlled by the

MAT

locus, and this increased expression of Atg45 is

313

important for the high activity of glycophagy during sporulation.

314

Finally, we examined the amount of intracellular glycogen during sporulation. The amount

315

of glycogen increased up to 12 h in both WT and

atg45



cells after shifting to sporulation

316

medium, following which a decrease was observed over time (Figure 7E). This suggests that

317

glycogen is utilized by cells from ~ 12 h following exposure to sporulation conditions. We

318

found that glycogen was consumed faster in

atg45



cells than in WT cells (Figure 7E). Given

319

that glycogen was sequestered into vacuoles in WT cells but not in

atg45



cells (Figure 7A),

320

glycophagy may play a role in the suppression of glycogen consumption, rather than

321

enhancing degradation.

322

323

324

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Discussion

325

The molecular mechanism of glycophagy in yeast has remained largely unexplored

. In this

326

study, we find for the first time that glycogen is a non-preferred cargo of bulk autophagy under

327

nitrogen starvation conditions. On the other hand, glycogen gradually becomes a target of

328

autophagy following prolonged nitrogen starvation. We found that Atg45 is important for

329

glycophagy, and that this protein binds to both glycogen and Atg8. We therefore conclude

330

that Atg45 functions as a glycophagy receptor. Furthermore, we showed that during

331

sporulation, the expression of Atg45 is increased, and that glycophagic activity is enhanced in

332

an Atg45-dependent manner under these conditions.

333

How glycogen escapes sequestration by isolation membranes during canonical nitrogen

334

starvation is an interesting question. We observed that glycogen is plentiful in regions

335

adjacent to forming autophagosomes (Figure 4E). This observation suggests that the

336

autophagosome formation site (pre-autophagosomal structure, or PAS) is not physically

337

separated from glycogen in the cytoplasm. For this reason, we speculate that the

338

physicochemical features of glycogen disfavor its sequestration by isolation membranes.

339

While glycogen granules have been reported to be approximately 10 to 40 nm in diameter

340

we observed that a large portion of glycogen in cells appears to form large assemblies, the size

341

of which can be up to ~200 nm (Figure 1E). This is possibly too large for efficient

342

sequestration by autophagosomes forming during bulk autophagy. Autophagy of large protein

343

complexes such as ribosomes and proteasomes is dependent on Atg24

45,47

; the large size of

344

glycogen assemblies might also be the reason that glycophagy requires Atg24. Given that

345

Atg45 is a glycophagy receptor, Atg45 may enhance the affinity of glycogen for isolation

346

membranes, which may, in turn, facilitate the enclosure of glycogen granules into

347

autophagosomes by releasing them from assemblies of glycogen or altering the membrane

348

morphology and/or direction of membrane expansion. Further studies are needed to uncover

349

molecular details of how glycogen escapes engulfment by the isolation membrane, as well as

350

how Atg45 mediates engulfment. Our findings provide a novel insight into the nature of

351

disfavored cargo engulfment by autophagosomes that has previously been overlooked.

352

Interestingly, we observed that Atg45 requires not only an AIM but also a C-terminal

353

region for efficient glycophagy. When the C-terminal region alone is expressed, it associates

354

with various membranes (Figure 6C), indicating that the C-terminal region of Atg45 may

355

enhance association with isolation membranes by direct binding to these membranes. We also

356

observed the dense localization of glycogen on the inner membrane of autophagosomes in

357

cells overexpressing Atg45 (Figures 4E and 7A). The tight binding of Atg45 to isolation

358

membranes via its AIM and the C-terminal region may enhance the affinity of glycogen for

359

isolation membranes, resulting in efficient glycophagy.

360

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Selective forms of autophagy, such as mitophagy and pexophagy, generally require

361

receptor proteins that recruit Atg11 to the cargo for isolation membrane formation site

362

However, we observed that the deletion of

ATG11

did not result in a strong effect on the

363

degradation of Gsy2, with the defect similar in extent to that of Pgk1. The reduction of Gsy2

364

degradation in

atg11

cells is likely explained by a general decrease in autophagy activity,

365

and we thus conclude that glycophagy does not require Atg11. As mentioned above, Atg45

366

possibly enhances the affinity of glycogen for the isolation membrane through its AIM and

367

the C-terminal region. Our data indicate that autophagic sequestration of glycogen, which is

368

widely distributed in the cytoplasm including around the PAS, is achieved through enhanced

369

affinity of glycogen with the isolation membrane rather than Atg11-dependent recruitment of

370

the autophagy machinery. Examination of other Atg11-independent receptor proteins

371

including Cue5 may shed light on common mechanisms that govern such forms of

372

degradation

373

While in silico (BLASTP) analyses indicate that Atg45 is conserved among the

374

Saccharomycetaceae

family and that

Candida albicans

orthologues of Atg45 also exist (such

375

as C1_01930W), direct homologs of Atg45 were not identified in other yeasts. We were also

376

unable to detect any sequence homology between the mammalian glycophagy receptor Stbd1

377

and Atg45. Stbd1 contains a predicted transmembrane domain sequence in its N-terminal

378

region and localizes to some organelles, such as the ER and lysosomes

16,59,60

. However, it has

379

been reported that glycogen is transported to lysosomes in a Stbd1-independent manner in

380

skeletal muscle

61,62

. It is possible that soluble receptors like Atg45 exist in mammalian cells in

381

addition to Stbd1. In

Drosophila melanogaster

, the glycogen synthase GlyS contains an LIR

382

motif, which is also conserved in mammals, and is important for glycophagy

. Since glycogen

383

synthase works by binding to glycogen, it is also a candidate for a soluble type of glycophagy

384

receptor in these organisms.

385

Further, a previous report has suggested that glycogen is stored in the vacuole and

386

prevented from degradation by cytosolic enzymes during stationary phase

, but this has not

387

been clearly proven. We clearly show that glycogen is accumulated in vacuoles in an Atg45-

388

dependent manner during sporulation. It has been reported that Sga1 is induced and relocated

389

to the prospore membrane from vacuoles

21,22,63

during spore maturation, which is required for

390

the efficient degradation of glycogen. Therefore, the degradation of glycogen possibly occurs

391

in the cytosol, especially around the prospore membrane, whereas vacuoles are not

392

substantially involved in the degradation of glycogen during sporulation. This implies that

393

glycophagy delivers glycogen to the vacuole, where it is protected from degradation in the

394

cytosol, as Wang et al. previously proposed

. Indeed, the amount of glycogen was decreased

395

atg45



cells compared with WT cells during sporulation. The accumulated glycogen

396

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preserved in the vacuole may subsequently be used as an energy source at a later phase, or

397

during the metabolically demanding process of spore germination.

398

399

Limitations of the study

400

Our study indicates that under nitrogen starvation, glycogen is a non-preferred cargo for

401

autophagy, and that Atg45 enhances the affinity of glycogen for isolation membranes,

402

facilitating glycophagy. However, we cannot rule out the possibility that the physicochemical

403

properties of glycogen, such as low fluidity or physical size, as well as changes between

404

nitrogen starvation and sporulation conditions, affect glycophagic activity differently under

405

each condition. The development of tools that specifically describe the state of glycogen will

406

contribute to uncovering more insights into the mechanisms of glycophagy.

407

While we found that the Gsy2-GFP cleavage assay is a suitable assay for glycophagy, the

408

assay is potentially unreliable if a portion of Gsy2-GFP does not bind glycogen. Therefore, it

409

is necessary to confirm the binding of Gsy2 with glycogen under the conditions used and to

410

validate results using other methods, such as electron microscopy.

411

412

Acknowledgments

413

We are grateful to all the members of Ohsumi laboratory for helpful discussions, the

414

Biomaterials Analysis Division (Tokyo Institute of Technology) for DNA sequencing, the

415

National Bio-Resource Project (NBRP) for providing the SEY6211 strain, Akira Matsuura for

416

providing the pYC50-GAL-HO plasmid, and Hiroshi Iwasaki for supporting my PhD research.

417

We would like to thank Alexander I May for editing the manuscript and Yui Jin for the critical

418

comments on the manuscript. This work was supported by JSPS KAKENHI (16H06375 and

419

19H05708 to Y.O., 19K16121 and 22K15101 to E.T.).

420

421

Author Contributions

422

T.I., E.T., and Y.O. designed the project. T.I., E.T., S.H., and S.H-R. performed the

423

experiments. T.I. and Y.O. wrote the manuscript. All authors discussed the results and

424

commented on the manuscript.

425

426

Declaration of Interests

427

The authors declare no competing interests.

428

429

430

Journal Pre-proof

Figure legends

431

432

Figure 1. Glycogen and glycogen metabolism-related enzymes are non-preferred cargoes of

433

nitrogen starvation-induced autophagy

434

(A) GFP-cleavage assay of Pgk1, Gsy2, Glc2, Gph1, and Trp4 in SD-N medium (upper).

435

Quantified band intensities are indicated (lower). Error bars represent SD (n=3).

436

(B) Schematic illustration of roles of Gsy2, Gph1, and Glc3 in glycogen metabolism.

437

438

Supernatant, P: Pellet. Pgk1 was used as a representative cytosolic protein.

439

(D) GFP-cleavage assay of Pgk1, Gsy2, Glc2, Gph1, and Trp4 in WT cells and

glg1



glg2



440

cells (upper). Quantified band intensities are indicated (lower). Error bars represent SD

441

(n=3).

442

(E) Electron micrographs of PATAg stained

atg15



cells and

glg1



glg2



atg15



cells after 6

443

h of nitrogen starvation. The diameters of each PATAg stained structure were roughly

444

estimated as follows; 1: 200 nm,

2: 70 nm, and 3: 40 nm. Bar: 2 µm.

445

See also Figure S7.

552

553

554

555

Journal Pre-proof

STAR METHODS

556

557

RESOURCE AVAILABILITY

558

Lead contact

559

Further information and requests for resources should be directed to the lead contact,

560

Yoshinori Ohsumi (yohsumi@iri.titech.ac.jp).

561

562

Materials availability

563

This study did not generate new unique reagents. Plasmids and cell lines generated in this

564

paper are available from the lead contact upon request.

565

566

Data and code availability

567

⚫

All data reported in this paper will be shared by the lead contact upon request.

568

⚫

This paper does not report original code.

569

⚫

Any additional information required to reanalyze the data reported in this paper is

570

available from the lead contact upon request

571

572

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

573

Yeast strains and media

574

Strains used in this study are listed in Table S1. Gene disruption and tagging were performed

575

using a standard PCR-based method with specific primers, as listed in Table S2

64,65

. Cells were

576

grown in rich medium (YPD medium: 1% yeast extract, 2% peptone, and 2% glucose),

577

synthetic defined medium (SD medium: 0.17% Difco yeast nitrogen base without amino acids

578

and ammonium sulfate, 0.5% ammonium sulfate, and 2% glucose) or SDCA medium which

579

is SD medium containing 0.5% BactoTM casamino acid (Difco). To induce autophagy, yeast

580

cells were grown in liquid medium at 30

C to a density of around OD

600

= 1.0, were washed

581

twice with sterile water and then shifted to SD-N medium (0.17% Difco yeast nitrogen base

582

without amino acids and ammonium sulfate, and 2% glucose). To induce sporulation, cells

583

were grown in YPD medium at 30

C for 24 h and shifted to YPA medium (1% CH

COOK,

584

1% yeast extract, 2% Peptone) at OD

600

= 0.2, then cultured for 12 h before being transferred

585

to sporulation medium (1% CH

COOK) at OD

600

= 1.0 after washing twice with sterile water.

586

587

Plasmids

588

Plasmids used in this study are listed in Table S3. For the construction of pTA1, the full length

589

ATG45

, including the 500 bp upstream and 500 bp downstream regions, was cloned into

590

the pRS416 vector. A 3xFLAG-tag sequence was then inserted downstream of

ATG45

591

Journal Pre-proof

inverse PCR. For the construction of pTA2, the Tyr127 and Leu130 residues of Atg45 were

592

replaced with Ala, using the PrimeSTAR Mutagenesis Basal Kit (Takara) and pTA1 as the

593

template. For the construction of pTA5 and pTA6, the

ACT1

promoter and

ATG45

- or

594

ATG45

AIMm

-3xFLAG were amplified from pET103 and pTA1 or pTA2, respectively, with the

595

resulting DNA fragments then assembled by the SLiCE method

. For the construction of

596

pTA7, the Leu173, Leu176, Ile179, Val183, Tyr186, and Trp187 residues of Atg45 were

597

replaced with Ala, using pTA5 as the template. For the construction of pTA8, the Tyr127 and

598

Leu130 residues of Atg45 were replaced with Ala, using pTA7 as the template. For the

599

construction of pTA3 and pTA4, the full length of

ATG45,

including the 500 bp upstream

600

region, and ATG45

(6A)

- or ATG45

AIMm(6A)

-3xFLAG were amplified from pTA1 and pTA7 or

601

pTA8, respectively, and assembled by the SLiCE method. For the construction of pTA9, GFP

602

was amplified from pFA6a-GFP-natNT2 and a DNA fragment from pRS416-

ACT1

pr-

603

ATG45

-3xFLAG (pTA5) without the 3xFLAG region was amplified from pTA5. pRS416-

604

ACT1

pr-

ATG45

-GFP was constructed by the SLiCE method using these DNA fragments.

605

pTA9 was amplified by a standard PCR mutagenesis technique using plasmid pRS416-

606

ACT1

pr-

ATG45

-GFP as the template DNA. For the construction of pTA10, GFP was

607

amplified from pFA6a-mGFPhy-natNT2, and the DNA fragment containing pRS416-

608

ACT1

pr-

ATG45

(6A)

-3xFLAG (pTA7) without the 3xFLAG sequence was amplified from

609

pTA7. pRS416-

ACT1

pr-

ATG45

(6A)

-GFP was constructed by the SLiCE method using these

610

DNA fragments. For the construction of pTA11, mScarlet was amplified from pFA6a-

611

mScarlet-hphNT1 and a DNA fragment from pRS416-

ATG45

pr-

ATG45

-3xFLAG without

612

the 3xFLAG region was amplified from pTA1. pRS416-

ATG45

pr-

ATG45

-mScarlet was

613

constructed by the SLiCE method using these DNA fragments. For the construction of pTA12,

614

mScarlet was amplified from pFA6a-mScarlet-hphNT1 and a DNA fragment from pRS416-

615

ATG45

pr-

ATG45

AIMm

-3xFLAG (pTA2) without the 3xFLAG region was amplified from

616

pTA2. pRS416-

ATG45

pr-

ATG45

AIMm

-mScarlet was constructed by the SLiCE method using

617

these DNA fragments. For the construction of pTA12, mScarlet was amplified from pFA6a-

618

mScarlet-hphNT1 and a DNA fragment from pRS416-

ATG45

pr-

ATG45

(6A)

-3xFLAG

619

(pTA3) without the 3xFLAG region was amplified from pTA3. pRS416-

ATG45

pr-

620

ATG45

(6A)

-mScarlet was constructed by the SLiCE method using these DNA fragments.

621

622

METHOD DETAILS

623

Protein extraction

624

1 OD

600

unit of cells was treated with 10% trichloroacetic acid (TCA) for at least 30 min on

625

ice. Samples were centrifuged at 20,000

at 4

C for 15 min. The supernatant was removed,

626

and the pellet was washed with ice-cold acetone and resuspended in HU sample buffer (0.5

627

Journal Pre-proof

M NaH

-Na

HPO

pH 6.8, 50 mM EDTA, 8 M urea, 10% SDS, 10% glycerol, 25 mM

628

dithiothreitol [DTT], 0.2 mg/mL bromophenol blue). Samples were homogenized using 0.5

629

mm zirconia beads (Yasui Kikai, YZB05) and a FastPrep-24 (MP Biomedicals, 6004-500)

630

with a setting of 60 s at 6.0 m/s, before incubation at 65

C for 15 min.

631

632

Western blotting

633

10% polyacrylamide gels (375 mM Tris-HCl pH 8.8, 10% acrylamide/bis mixed solution

634

[nacalai tesque], 0.1% SDS, 0.16% APS, and 0.09% TEMED) were overlayed with a stacking

635

gel (125 mM Tris-HCl pH 6.8, 5% acrylamide/bis mixed solution, 0.1% SDS, 0.16% APS,

636

and 0.09% TEMED). Samples (except for those used for the detection of Pgk1-GFP) were

637

loaded into wells directly following the preparation described above; Pgk1-GFP samples were

638

diluted 1:25 before loading into wells. In Figure 4A, the protein concentrations were measured

639

and samples loaded at equivalent concentrations. Samples were run at 40 mA (per gel) for 70

640

min using a myPower

Ⅱ

300 power supply (ATTO, AE-8135). Separated proteins were then

641

transferred onto 0.45



m PVDF membranes (Millipore, IPVH00010) using a Trans-Blot

642

Turbo transfer system (Bio-Rad). Membranes were stained with 1% Ponceau S (nacalai

643

tesque, 283-22-25G) dissolved in 5% CH

COOH and digitized using a scanner (EPSON,

644

GT-X970). Membranes were washed with TBS-T three times and blocked in 1% skim milk

645

in TBS-T for 30 min. Membranes were then incubated with the relevant primary antibody at

646

C overnight, or at room temperature for 1 h, were washed three times with TBS-T before

647

incubation with a relevant secondary antibody at room temperature for 30 min. After washing

648

with TBS-T three times, Femtoglow HRP Substrate (Michigan Diagnostics, 21008) was used

649

to initiate chemiluminescence and blots were visualized using a FUSION-FX7 (Vilber-

650

Lourmat) imaging system.

651

652

GFP-cleavage assay

653

Cells expressing proteins tagged with GFP were incubated in SD-N medium or sporulation

654

medium. The amounts of GFP-tagged proteins and free GFP were assessed by western

655

blotting using anti-GFP antibodies. The density of blots was quantified by using Vision-Capt

656

(Vilber-Lourmat), and the ratio of free GFP to the sum of full-length fusion protein and GFP

657

were calculated.

658

659

Antibodies

660

Anti-FLAG (Sigma-Aldrich, F-4042, 1:1000), anti-Pgk1 (Thermo Fisher Scientific, 459250,

661

1:2000), anti-

-actin (Fujifilm Wako Pure Chemical Corporation, 011-24554, 1:500), anti-

662

GFP (Roche,11814460001, 1:1000), and anti-mCherry (Abcam, ab125096, 1:1000)

663

Journal Pre-proof

antibodies were used as primary antibodies. Anti-mouse IgG, HRP-linked antibody (Jackson

664

Immuno Research, 315-035-003, 1:5000) was used as the secondary antibody.

665

666

Electron microscopy

667

Electron microscopy was performed by Tokai-EMT Inc. Cells were sandwiched between

668

copper disks and quickly frozen in liquid propane at

−

175

C. The frozen samples were

669

substituted with 2% glutaraldehyde, and 1% tannic acid in ethanol at

−

C for 2 days and

670

then kept at

−

C for 2 h, followed by a warm-up to 4

C for 2 h. These samples were

671

dehydrated through ethanol three times for 30 min each, and then dehydrated with ethanol at

672

room temperature overnight. The samples were infiltrated with propylene oxide (PO) twice

673

for 30 min each and were put into a 50:50 mixture of PO and resin (Quetol-651; Nisshin EM

674

Co.) for 3 h. Samples were then transferred to 100% resin and incubated overnight. These

675

samples were polymerized at 60

℃

for 48 h. After polymerization, the blocks were ultra-thin

676

sectioned at 80 nm with a diamond knife using an ultramicrotome (ULTRACUT, UCT,

677

Leica). These sections were placed on nickel grids, and incubated with 1% periodic acid for

678

20 min, followed by washing with distilled water. These grids were incubated with 0.5%

679

thiocarbohydrazide in 20% acetic acid for 60 min, and were then washed with 10% acetic acid

680

and distilled water. Finally, grids were incubated in 1% silver proteinate for 60 min and then

681

washed with distilled water. The grids were observed by a transmission electron microscope

682

(JEM-1400Plus; JEOL Ltd.) at an acceleration voltage of 100 kV. Digital images were taken

683

with a CCD camera (EM-14830RUBY2; JEOL Ltd.).

684

685

Fluorescence microscopy

686

Cells were cultured in SD, SD-N, or sporulation medium for the indicated times. Fluorescence

687

microscopy was carried out using a confocal microscope (IXplore SpinSR10) equipped with

688

an sCMOS camera (ORCA-Flash4.0, Hamamatsu), a 100x objective lens (UPLAPO

689

100xOHR, NA/1.50; Olympus) and appropriate lasers and filters. Images were acquired and

690

processed using CellSens (Olympus) software and Fiji distribution of ImageJ

691

In Figures S2B-E, fluorescence intensities were obtained using the Plot Profile function

692

in Fiji. The average of the value of 2 pixels was calculated, and the minimum and maximum

693

values from individual fluorescent channels were normalized to 0 and 100, respectively.

694

695

Co-immunoprecipitation

696

Cells were cultured in SD-N medium at 30

C for 1 h and 50 OD

600

units of cells were

697

harvested. The cells were disrupted in lysis buffer (10 mM Tris-HCl pH 7.5, 30 mM NaCl, 1

698

mM EDTA, 2% glycerol, 50 mM NaF, 1 mM DTT, 2 mM phenylmethylsulfonyl fluoride

699

Journal Pre-proof

[PMSF], 2× cOmplete protease inhibitor cocktail

[Roche], and 0.1× PhosSTOP

700

phosphatase inhibitor cocktail [Roche]), using a Multi-beads Shocker (Yasui Kikai) and 0.5-

701

mm zirconia beads. The lysates were solubilized by rotation at 4

C for 15 min with 0.5%

702

Triton X-100, and were then centrifuged twice at 500

for 5 min each. The supernatants were

703

incubated with GFP-Trap_M beads (ChromoTek) at 4

C for 2 h. The beads were washed

704

with lysis buffer containing 0.3% Triton X-100 three times, and then incubated in SDS sample

705

buffer at 95

C for 5 min to elute bound proteins.

706

707

Glycogen pellet assay

708

WT or

glg1



glg2



cells expressing each GFP-tagged protein were grown in SD medium at

709

C to a density of around OD

600

= 1.0, and 50 OD

600

units of cells were harvested for sample

710

preparation. Samples were disrupted in lysis buffer, as described for co-immunoprecipitation,

711

using a Multi-beads Shocker and 0.5-mm zirconia beads. In Figure 5A, the lysates were

712

centrifuged twice at 20,000

for 5 min at 4

C. The supernatants were mixed with the same

713

volume of the lysis buffer or the lysis buffer containing 20 mg/mL glycogen by rotation at 4

714

for 1 h. In Figure 1C, the lysates were centrifuged twice at 2,000

for 5 min at 4

C. In both

715

figures, a portion of lysates was collected as total lysate, and the remaining samples were

716

centrifuged at 100,000

and 4

C for 1 h. the supernatants were collected, and the pellets

717

were resuspended in an equal volume of lysis buffer to the supernatants. Each sample was

718

mixed with the same volume of 2x SDS sample buffer and incubated at 95

C for 5 min.

719

720

RNA extraction

721

Total cellular RNA was extracted by a hot phenol method, as described previously

. 10 OD

600

722

units of cells were harvested and frozen. 400



L of AE buffer (50 mM CH

COONa and 10

723

mM EDTA pH 5.0) with 1% SDS were added to cell pellets on ice, and 0.5-mm zirconia beads

724

and 500



L of AE buffer-saturated hot phenol, pre-warmed at 65

C, were added. The samples

725

were homogenized using a FastPrep-24 for 30 sec at 5.5 m/s in four cycles. The homogenized

726

samples were incubated on ice for 1 min and centrifuged. The supernatants were mixed with

727

ANE buffer (10 mM sodium acetate, 100 mM NaCl, and 2 mM EDTA)-saturated phenol:

728

chloroform and vortexed for 1 min and then centrifuged for 5 min. The supernatants were

729

mixed with chloroform: isoamyl alcohol and vortexed for 1 min. Samples were centrifuged for

730

5 min, and the supernatants were mixed with 2.5 times the volume of ethanol with 300 mM

731

COONa (pH 5.3), and then stored overnight at

−

C. The samples were centrifuged

732

at 4

C and supernatants were removed. 500



L of 70% ethanol was added to pellets and

733

samples were centrifuged at 4

C. After removing the supernatant, pellets were resuspended

734

in RNase-free water.

735

Journal Pre-proof

736

qPCR

737

Complementary DNAs (cDNAs) were synthesized from 1 mg of RNA using the PrimeScript

738

RT reagent kit with gDNA eraser (TAKARA, RR047). A random hexamer was used for cDNA

739

synthesis. qPCR was performed using TB Green Premix Ex Taq II (Tli RNase H Plus)

740

(TAKARA, RR820) with the target mRNA-specific primers listed in Table S2.

741

Serial dilutions of cDNA were used for the qPCR standard. Melting-curve analyses confirmed

742

the amplification of a single product for each mRNA. The relative amount of

ATG45

mRNA

743

and

TAF10

mRNA

ACT1

mRNA (internal control) was calculated from CT value, using

744

the qPCR standard. The expression level of

ATG45

was normalized by calculating the relative

745

amount of

ATG45

mRNA to that of

TAF10

in Figure 7D. In Figure S3A, transcripts of

ACT1

746

and TAF10

mRNA were markedly reduced after nitrogen starvation induction compared to 0

747

hours. Therefore, the relative amount of

ATG45

and these internal controls was shown

748

together rather than the normalized expression level of

ATG45

mRNA .

749

750

Quantification of intracellular glycogen

751

The amount of glycogen was measured by a method previously reported

. 5 OD

600

units of

752

cells were harvested. The cells were lysed by incubation in 125



L of 0.25 M Na

753

°C

for 4 h. 75



L of 1 M CH

COOH and 300



L of CH

COONa were added to each

754

sample, and the samples were vortexed for 10 s. 250



L of each sample was mixed with 10

755



L of 20 mg/mL amyloglucosidase from

Aspergillus niger

(Sigma-Aldrich, 10115-1G-F),

756

dissolved in 0.2 M CH

COONa (pH 5.2) and was then incubated at 58

°C

for 17 h. These

757

samples were centrifuged and supernatants were collected. The amount of glucose of the

758

supernatants was measured using a Glucose (GO) Assay Kit (Sigma-Aldrich, GAGO20-

759

1KT). Note that the removal of intracellular glucose is unnecessary because most

760

intracellular glucose is converted to a phosphorylated form (e. g. glucose-6-phosphate) that

761

is not detected in this assay.

762

763

QUANTIFICATION AND STATISTICAL ANALYSES

764

Error bars represent SD as indicated in the figure legends. Data were processed in Excel 2016,

765

R, or JMP 16. Statistical analysis of differences between two groups was performed using

766

Wilcoxon rank sum test or Welch’s

-test, and three or more groups were One-way ANOVA

767

with Dunnett

’s test, One

way ANOVA with Tukey’s multiple comparisons test, or Steel

test

768

as indicated in figure legends.

769

770

Journal Pre-proof

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0.9

1.2

0.6

0.3
0.0

Gsy2

GFP’

/ Gsy2-GFP+GFP’

15D の電顕
N-6h

-N 6 h

-N 24 h

-N 48 h

=0.0026

=0.1137

% of ABs containig glycogen

48 h

(n=20)

(n=55)

6 h

24 h

(n=32)

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Crp1-GFP

Gsy2-mScarlet

merge

Sip1

Yil024c (Atg45)

Gal83

Crp1

Mdg1

561

505

Sip2

164

248

163

246

: putative GBD

Atg45-GFP

Gsy2-mScarlet

merge

Mdg1-GFP

Gsy2-mScarlet

merge

Long exposure

Gsy2-GFP

GFP’

-GFP

atg45

∆

glg1

∆

glg2

∆

atg45

∆

glg1

∆

glg2

∆

Gsy2-GFP

GFP’

-GFP

atg45

∆

crp1

∆

mdg1

∆

crp1

∆

mdg1

∆

Long

exposure

’/Gsy2-GFP+

’

Ponceau S

<0.0001

=0.0033

=0.4092

=0.0070

GFP’

/ Gsy2-GFP+GFP’

1.2
0.9
0.6

0.0

0.3

’/Gsy2-GFP+

’

0.0

0.5

1.0

1.5

0.0

0.5

1.0

1.5

=0.0250

=0.2837

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Gsy2-GFP
GFP’

0 4 8 24

-N (h)

0 4 8 24

atg45

∆

0 4 8 24

ATG45

o.e.

-GFP

Long exposure

-GFP

Gsy2-GFP

0 8 24 0 8 24 0 8 24 0 8 24 0 8 24

atg2

∆

atg11

∆

atg17

∆

atg24

∆

atg15

∆

ATG45

o.e.

atg15

∆

ATG45

o.e.

atg15

∆

ATG45

o.e.

atg15

∆

% of ABs containig glycogen

(n=30)

-FLAG

Atg45-Flag

atg15

∆

ATG45

o.e.

atg15

∆

Long

exposure

-GFP

-N (h)

<0.0001

GFP’

-N (h)

Atg45-GFP

GFP’

-GFP

-beta actin

0.0

1.0

2.0

3.0

Atg45-GFP+

’/actin

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Atg45

1-96

Atg45

97-189

8 24

-N (h)

0 8 24 0 8 24 0 8 24

Atg45-

mScarlet

Atg45-

mScarlet

Atg45

97-189

-mScarlet

Atg45

1-96

-mScarlet

Gsy2-GFP

GFP’

Atg45-mScarlet

-GFP

-mScarlet

o.e.

8 24 0

8 24

-N (h)

Gsy2-GFP

atg45

∆

Atg45

97-189

-mScarlet

Atg45

1-96

-mScarlet

-FLAG

GFP’

GBD

-GFP

189

Atg45

GBD

189

DSLND

SSHSD

127 130

-GFP

-FLAG

Atg45-Flag

IP:GFP

Input

●

●
●

Atg45-Flag

GFP-Atg8

●

●
●

GFP-Atg8

Atg45-Flag

α−

GFP

α−

Pgk1

T S P

Gsy2-GFP

Atg45-GFP

Atg45

1-96

-GFP

T S P T S P T S P T S P T S P T S P T S P

Gsy2-GFP

Atg45-GFP

Atg45

97-189

-GFP

Atg45

1-96

-GFP

−

glycogen

−

glycogen

−

glycogen

−

Atg45

97-189

-GFP

Long exposure

ACT1pr-

ATG45-FLAG

AIMm

-GFP

Long exposure

Atg45

AIMm

-Flag

atg15

∆

atg45

∆

−

glycogen

0.000

0.025

0.050

0.075

0.100

’

/ Gsy2-GFP+GFP’

8 24 0

8 24

-N (h)

AIMm

−

ACT1pr-

ATG45-FLAG

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Gsy2-GFP

GFP’

Atg45

1-123

-mScarlet

0 8 24

-N (h)

0 8 24 0 8 24 0 8 24

Atg45-

mScarlet

Atg45-

mScarlet

Atg45

1-123

-mScarlet

Atg45

1-164

-mScarlet

o.e.

-GFP

-mScarlet

Atg45

GBD

189

AIM

GBD

164

Atg45

1-164

Atg45

1-123

GBD 122

AIM

Atg45

GBD

189

AIM

172 SLNGLMCIAKKVKTYW 187

Atg45

165-189

-GFP

Vph1-mCherry

merge

Vph1-mCherry

merge

Gsy2-GFP

GFP’

Atg45-Flag

-GFP

Long exposure

-FLAG

0 8 24

-N (h)

0 8 24 0 8 24 0 8 24

AIMm

atg45

∆

AIMm(6A)

ATG45pr-

ATG45-FLAG

-GFP

Long exposure

0 8 24

Atg45-mScarlet

Atg45

1-164

-mScarlet

−

Atg45

165-189(6A)

-GFP

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Spo (h)

0 6

Gsy2-GFP
GFP’

0 6

(

MAT

)

(

MAT

)

(

MAT

)

atg45

∆

(

MAT

)

Long exposure

-GFP

MAT

-GFP

Atg45

-GFP

GFP’

(

MAT

)

atg45

∆

(

MAT

)

glycogen (

g/OD

600

)

Spo medium (h)

WT (

MAT

)

atg45

∆

(

MAT

)

Ponceau S

MAT

0.0

0.5

1.0

1.5

2.0

2.5

Nomarized amount

of Atg45

MAT

=0.0363

=0.0005

=0.0085

0.0

0.5

1.0

1.5

2.0

2.5

MAT

=0.6504

=0.0342

=0.1069

Nomarized expression level

ATG45

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Key resources table

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies

Monoclonal ANTI-FLAG

M5 antibody

Sigma

–Aldrich

Cat# F-4042; RRID:

AB_439686

PGK1 Monoclonal Antibody (22C5D8)

Thermo Fisher Scientific

Cat# 459250;

RRID:

AB_2532235

Anti β-Actin, Monoclonal Antibody

Fujifilm Wako Pure Chemical

Corporation

Cat# 011-24554

Anti-GFP

Roche

Cat#

11814460001;

RRID: AB_390913

Anti-mCherry antibody

Abcam

Cat#

ab125096

;

RRID: AB_11133266

Anti-mouse IgG, HRP-linked antibody

Jackson Immuno Research

Cat#

315-035-003;

RRID: AB_2340061

Chemicals, peptides, and recombinant proteins

PrimeScript™ RT reagent Kit with gDNA Eraser

TaKaRa

Cat# RR047

PrimeSTAR Mutagenesis Basal Kit

TaKaRa

Cat# R046A

Quetol-651

Nisshin EM Co.

Cat# 371

Ponceau S

nacalai tesque

Cat# 283-22-25G

cOmplete protease inhibitor cocktail

Roche

Cat# 11836170001

PhosSTOP phosphatase inhibitor cocktail

Roche

Cat# 4906845001

TB Green Premix Ex Taq II (Tli RNase H Plus)

TaKaRa

Cat# RR820

amyloglucosidase from

Aspergillus niger

Sigma-Aldrich

Cat# 10115-1G-F

Glycogen from Oyster

Sigma-Aldrich

Cat# G8751-5G

Femtoglow HRP Substrat

Michigan Diagnostics,

Cat# 21008

Critical commercial assays

Glucose (GO) Assay Kit

Sigma-Aldrich

Cat# GAGO20-1KT

Experimental models: Organisms/strains

S. cerevisiae

strains, see Table S1

This study

N/A

Oligonucleotides

Primers, see Table S2

This study

N/A

Recombinant DNA

Journal Pre-proof

Plasmids, see Table S3

This study

N/A

Software and algorithms

imageJ/Fiji

National

Institutes of Health

https://imagej.net/Fiji

CellSens

Olympus

https://www.olympus-

lifescience.com/en/sof

tware/cellsens/

Vision-Capt

Vilber-Lourmat

N/A

JMP 16

JMP Statistical Discovery,

North Carolina, United States

https://www.jmp.com/

en_us/home.html

Other

GFP-Trap_M beads

ChromoTek

Cat# gtma-20

PrimeScript™ RT reagent Kit with gDNA Eraser

TaKaRa

Cat# RR047

PrimeSTAR Mutagenesis Basal Kit

TaKaRa

Cat# R046A

0.5 mm zirconia beads

Yasui Kikai

Cat# YZB05

0.45



m PVDF membranes

Millipore

Cat# IPVH00010

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