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RESEARCH ARTICLE 1
Arabidopsis SINAT Proteins Control Autophagy by Mediating 2
Ubiquitylation and Degradation of ATG13 3
Hua Qi1†
, Juan Li1,2†
, Fan-Nv Xia1, Jin-Yu Chen
1, Xue Lei
1, Mu-Qian Han
2, Li-Juan Xie
1, 4
Qing-Ming Zhou2, and Shi Xiao
1,* 5
6 1State Key Laboratory of Biocontrol, Guangdong Provincial Key Laboratory of Plant 7
Resources, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China 8 2College of Agronomy, Hunan Agricultural University, Changsha, 410128 China 9 †These authors contributed equally to this work. 10 *Correspondence and requests for materials should be addressed to S.X. (email: 11
Running title: Regulation of ATG13s by SINAT proteins 13
One sentence summary: TRAF1a and TRAF1b have dual functions in regulating the 14
dynamics of autophagy by facilitating SINAT1/SINAT2 or SINAT6-mediated proteolysis or 15
stabilization of ATG13 proteins. 16
17
FOOTNOTE: The author responsible for distribution of materials integral to the findings 18
presented in this article in accordance with the policy described in the Instructions for Authors 19
(www.plantcell.org) is: Shi Xiao ([email protected]). 20
21
ABSTRACT 22
In eukaryotes, autophagy maintains cellular homeostasis by recycling cytoplasmic 23
components. The autophagy-related proteins (ATGs) ATG1 and ATG13 form a protein kinase 24
complex that regulates autophagosome formation; however, mechanisms regulating ATG1 25
and ATG13 remain poorly understood. Here, we show that, under different nutrient conditions, 26
the RING-type E3 ligases SINAT1 (SEVEN IN ABSENTIA OF ARABIDOPSIS THALIANA 27
1), SINAT2, and SINAT6 control ATG1 and ATG13 stability and autophagy dynamics by 28
modulating ATG13 ubiquitylation in Arabidopsis thaliana. During prolonged starvation and 29
recovery, ATG1 and ATG13 were degraded through the 26S proteasome pathway. TUMOR 30
NECROSIS FACTOR RECEPTOR ASSOCIATED FACTOR 1a (TRAF1a) and TRAF1b 31
interacted in planta with ATG13a and ATG13b and required SINAT1 and SINAT2 to 32
ubiquitylate and degrade ATG13s in vivo. Moreover, lysines K607 and K609 of ATG13a 33
protein contributed to K48-linked ubiquitylation and destabilization, and suppression of 34
autophagy. Under starvation conditions, SINAT6 competitively interacted with ATG13 and 35
induced autophagosome biogenesis. Furthermore, under starvation conditions, ATG1 36
promoted TRAF1a protein stability in vivo, suggesting feedback regulation of autophagy. 37
Consistent with ATGs functioning in autophagy, the atg1a atg1b atg1c triple knockout 38
mutants exhibited premature leaf senescence, hypersensitivity to nutrient starvation, and 39
reduction in TRAF1a stability. Therefore, these findings demonstrate that SINAT family 40
proteins facilitate ATG13 ubiquitylation and stability and thus regulate autophagy. 41
Plant Cell Advance Publication. Published on November 15, 2019, doi:10.1105/tpc.19.00413
©2019 American Society of Plant Biologists. All Rights Reserved
2
INTRODUCTION 42
Autophagy, a highly conserved cellular process in all eukaryotes, degrades 43
intracellular constituents to break down toxic materials or damaged organelles and 44
recycle essential nutrients (Bassham et al., 2006; Xie and Klionsky, 2007; Michaeli et 45
al., 2016; Marshall and Vierstra, 2018). To date, three distinct types of autophagy, 46
microautophagy, macroautophagy (referred henceforth as autophagy), and 47
mega-autophagy, have been identified in plant cells (van Doorn and Papini, 2013; 48
Marshall and Vierstra, 2018). In Arabidopsis thaliana, autophagy functions in 49
maintaining glucose-mediated root meristem activity in the root cells (Huang et al., 50
2019). In aerial tissues, autophagy is primarily inducible and activated by a variety of 51
environmental cues, such as nutrient deprivation, high salt, drought, hypoxia, 52
oxidative stress, and pathogen infection (Doelling et al., 2002; Hanaoka et al., 2002; 53
Yoshimoto et al., 2004; Liu et al., 2005; Xiong et al., 2007; Phillips et al., 2008; 54
Hayward et al., 2009; Liu et al., 2009; Chung et al., 2010; Chen et al., 2015). 55
Autophagy begins with the formation of phagophore, which expands to form a 56
double-membraned vesicle structure, termed an autophagosome. In particular, the 57
outer membrane of the autophagosome fuses with the tonoplast and releases inner 58
membrane vesicles (named autophagic bodies) containing cellular contents into the 59
vacuole, where the sequestered cargo is degraded by the resident acid hydrolases (He 60
and Klionsky, 2009; Liu and Bassham, 2012; Li and Vierstra, 2012; Zhuang et al., 61
2015; Michaeli et al., 2016). 62
Over the past few decades, a large number of autophagy-related proteins (ATGs) 63
were discovered in plants; these ATGs play essential roles in regulating the core 64
autophagic machinery (Liu et al., 2018; Soto-Burgos et al., 2018; Yoshimoto and 65
Ohsumi, 2018; Zhuang et al., 2018). Deletions of Arabidopsis ATG genes leads to 66
phenotypic changes , such as premature leaf senescence and a shortened life cycle 67
under normal growth conditions, hypersensitivity to fixed carbon or nitrogen 68
starvation, decreased tolerance to biotic and abiotic stresses, activated innate 69
immunity, and an altered cellular metabolome (Doelling et al., 2002; Xiong et al., 70
3
2007; Hayward et al., 2009; Liu et al., 2009; Chung et al., 2010; Guiboileau et al., 71
2012; Avin-Wittenberg et al., 2015; Chen et al., 2015; McLoughlin et al., 2018). 72
In plants, ATG proteins predominately assemble into four functional protein 73
complexes: 1) the ATG1–ATG13 protein kinase complex, 2) the ATG6–74
phosphatidylinositol 3-kinase (PI3K) complex, 3) a complex containing the 75
transmembrane protein ATG9, and 4) two ubiquitin-like conjugation complexes 76
ATG5–ATG12 and ATG8–PE (phosphatidylethanolamine), which regulate 77
autophagosome formation (Li and Vierstra, 2012; Liu and Bassham, 2012; Liu et al., 78
2018; Soto-Burgos et al., 2018; Yoshimoto and Ohsumi, 2018). Developmental and 79
nutritional signals promote the assembly of the ATG1–ATG13 kinase complex to 80
initiate autophagy. 81
In Arabidopsis, the ATG1–ATG13 kinase complex includes the serine/threonine 82
kinase ATG1 and its accessory proteins ATG13, ATG11, and ATG101, which are key 83
positive regulators in the induction of autophagic vesiculation (Suttangkakul et al., 84
2011; Liu and Bassham, 2012; Li et al., 2014). Through post-translational 85
phosphorylation, the Arabidopsis ATG1–ATG13 complex is regulated by the energy 86
signaling pathway and a variety of upstream kinases that affect their kinase activities 87
(Liu and Bassham, 2010; Chen et al., 2017; Pu et al., 2017; Soto-Burgos and Bassham, 88
2017). In particular, the TARGET OF RAPAMYCIN (TOR) kinase and SUCROSE 89
NONFERMENTING 1-RELATED KINASE 1 (SnRK1) are important negative and 90
positive regulators, respectively, of the ATG1–ATG13 complex. For example, 91
overexpression of TOR in Arabidopsis inhibits autophagy (Pu et al., 2017). 92
Furthermore, downregulation or overexpression of the KIN10 catalytic subunit of 93
Arabidopsis SnRK1 suppresses or enhances autophagy induction, respectively, in 94
response to nutrient starvation (Chen et al., 2017; Soto-Burgos and Bassham, 2017). 95
Increasing evidence has demonstrated that the ubiquitin modification system 96
regulates ATG protein stability during autophagosome formation in yeast, mammals, 97
and plants (Shi and Kehrl, 2010; Xia et al., 2013; Popelka and Klionsky, 2015; Xie et 98
al., 2015; Qi et al., 2017). In mammal cells, during the induction of autophagy, the E3 99
4
ligase TRAF6 (TUMOR NECROSIS FACTOR RECEPTOR ASSOCIATED 100
FACTOR 6) mediates K63-linked ubiquitylation of ULK1 (UNC-51-LIKE KINASE 1, 101
a homolog of ATG1). The ubiquitylation stabilizes ULK1, activating its 102
self-association, and kinase activity, and thereby activating autophagy (Nazio et al., 103
2013). Under prolonged nutrient starvation, ULK1 autophosphorylation promotes its 104
interaction with Cullin/KLHL20 (KELCH-LIKE PROTEIN 20), a substrate adaptor 105
of Cul3 ubiquitin ligase and binds Cul3 and substrate via its BTB domain and 106
kelch-repeat domain, for K48-linked ubiquitylation and proteasome-mediated 107
degradation (Lee et al., 2010). The degradation of ULK1 leads to the termination of 108
autophagy and thus prevents unrestrained cellular degradation (Liu et al., 2016). 109
Moreover, during the first few hours of starvation, the HECT (HOMOLOGOUS TO 110
E6-ASSOCIATED PROTEIN CARBOXYL TERMINUS) domain-containing E3 111
ubiquitin ligase NEDD4L (NEURAL PRECURSOR CELL-EXPRESSED 112
DEVELOPMENTALLY DOWN-REGULATED GENE 4-LIKE) interacts with ULK1 113
and triggers ULK1 degradation by the proteasome pathway (Nazio et al., 2016). In 114
particular, under selenite treatment in mammalian cells, ULK1 partially translocates 115
to the mitochondria, and interacts with the mitochondria-localized E3 ligase MUL1 116
(MITOCHONDRIAL UBIQUITIN LIGASE ACTIVATOR OF NFKB 1), which 117
mediates the K48-linked ubiquitylation of ULK1 for degradation in selenite-induced 118
mitophagy (Li et al., 2015). These findings suggest that the protein stabilities of the 119
ATG1–ATG13 kinase complex are tightly controlled by the ubiquitin modification 120
system to regulate autophagy in mammalian cells. 121
In Arabidopsis, the protein stabilities of ATG1–ATG13 complex are also affected 122
by the ubiquitylation system (Suttangkakul et al., 2011); however, the underlying 123
regulatory mechanism remains unknown. Our recent findings reveal that under 124
normal nutrient conditions, Arabidopsis TRAF1a and TRAF1b act as adaptors to 125
mediate the ubiquitylation and degradation of ATG6 by interacting with the 126
RING-type E3 ligases SINAT1 and SINAT2 (Qi et al., 2017). Under starvation 127
conditions, however, TRAF1a and TRAF1b recruit a starvation-inducible SINAT6 128
5
protein with a truncated RING finger domain to stabilize ATG6 and subsequently 129
activate autophagy. Here, we show that SINAT proteins regulate autophagy by 130
interacting with ATG13 proteins and modulating the stability of the ATG1–ATG13 131
kinase complex under different nutrient conditions. Moreover, we observed that the 132
ATG1s stabilize TRAF1 proteins upon nutrient starvation through a feedback 133
mechanism in the regulation of autophagy. 134
135
RESULTS 136
ATG1 and ATG13 Are Degraded by the 26S Proteasome Pathway upon 137
Starvation and During Recovery 138
Recent studies have identified the importance of ubiquitin modification in regulating 139
ATG protein stability during autophagosome formation (Popelka and Klionsky, 2015; 140
Xie et al., 2015; Qi et al., 2017). In Arabidopsis, TRAF1a and TRAF1b act as 141
adaptors to control the stability of ATG6 by competitively interacting with the RING 142
finger E3 protein ligases SINAT1/SINAT2 and SINAT5/SINAT6 under different 143
nutrient conditions (Qi et al., 2017). To further investigate the potential degradation of 144
ATG1 and ATG13 by the Ub/26S proteasome pathway, we first examined their protein 145
levels in wild-type plants upon carbon or nitrogen starvation with or without treatment 146
with the proteasome inhibitor MG132 using ATG1a- and ATG13a-specific antibodies. 147
As shown in Figure 1, ATG1a and ATG13a levels accumulated at 12 and 24 h, but 148
strongly decreased under prolonged (48 and 72 h) carbon or nitrogen starvation 149
treatments (Figures 1A and 1B; Supplemental Figures 1A and 1B). By contrast, the 150
degradation of ATG1a and ATG13a was repressed by the application of MG132 151
(Figures 1A and 1B). Interestingly, MG132 treatment also promoted the accumulation 152
of ATG8a under starvation conditions (Figures 1A and 1B). As a control, ATG7 153
showed few substantial changes in response to the carbon or nitrogen starvation and 154
MG132 application did not affect the level of ATG7 at any time point (Figure 1; 155
Supplemental Figure 1). These findings suggest that the stability of the ATG1–ATG13 156
protein complex is regulated by the 26S proteasome pathway during autophagosome 157
6
formation. 158
To confirm the role of the 26S proteasome in modulating the stability of ATG13 159
proteins, we generated the ATG13a-HA and ATG13b-HA transgenic lines 160
overexpressing HA-tagged ATG13a and ATG13b, respectively (Supplemental Figure 161
1). Genetic analyses showed that the ATG13a-HA #2 and ATG13b-HA #4 lines 162
harbored single T-DNA insertions and these lines were used for further investigation. 163
One-week-old ATG13a-HA and ATG13b-HA seedlings grown under long-day (LD) 164
conditions in Murashige and Skoog (MS) medium were transferred to medium 165
without sucrose (–C) or nitrogen (–N) for carbon or nitrogen starvation treatments. In 166
response to carbon (Figure 1C) or nitrogen (Figure 1D) starvation, ATG13a-HA and 167
ATG13b-HA accumulated at 12 h, but consistently decreased at 24 and 48 h after 168
starvation treatments. Consistent with the protein blot analyses using ATG13-specific 169
antibodies (Figures 1A and 1B), the degradation of ATG13a-HA and ATG13b-HA 170
under carbon or nitrogen starvation conditions was suppressed by the application of 171
50 µM MG132 (Figures 1C and 1D). 172
Previous studies have suggested that proteaphagy is induced by long-term 173
MG132 treatment (Marshall et al., 2015). To further explore the involvement of the 174
ATG13 protein degradation by the 26S proteasome pathway, we examined ATG13a 175
protein level upon MG132 treatment for 0, 1, 3, 6, 12 h under carbon and nitrogen 176
deprivation conditions (–C/N). ATG13a accumulated at 1 h, but decreased at 3, 6 and 177
12 h after starvation treatment. By contrast, the degradation of ATG13a was repressed 178
by the application of MG132 (Supplemental Figure 1E). The degradation of 179
ATG13a-HA under –C/N conditions was also suppressed by MG132 treatment 180
(Supplemental Figure 1F). 181
The degradation of ATG13 proteins may also occur during the recovery stages 182
following nutrient starvation to terminate autophagy, which is a key process that 183
increases survival of mammalian cells (Liu et al., 2016; Antonioli et al., 2017). To test 184
this, we monitored the protein levels of ATG13a-HA and ATG13b-HA at various 185
times (6, 12, 24, 48, and 72 h) after recovery following carbon or nitrogen starvation. 186
7
As expected, both ATG13a-HA and ATG13b-HA accumulated rapidly within 6 h 187
during recovery and declined dramatically at 72 h after recovery (Supplemental 188
Figure 1). Further, we observed that MG132 application inhibited the degradation of 189
ATG13a-HA and ATG13b-HA at recovery stages following carbon and nitrogen 190
starvation (Figures 1E and 1F). These findings indicated that the 26S proteasome 191
pathway controls the stability of ATG13a and ATG13b under both starvation 192
conditions and during recovery after starvation treatments. 193
194
TRAF1a and TRAF1b Interact with ATG13a and ATG13b 195
TRAF1a and TRAF1b mediate the degradation of ATG6 by forming a complex, 196
termed the TRAFasome, with SINAT1, SINAT2, and ATG6 under nutrient-rich 197
conditions (Qi et al., 2017). We therefore hypothesized that TRAF1 proteins may 198
contribute to the regulation of ATG1 and ATG13 protein stabilities. To test this, we 199
first examined the interactions between ATG13a/ATG13b and TRAF1a/TRAF1b by 200
the yeast two-hybrid (Y2H) assay. Only ATG13a, but not ATG1a, ATG1b, ATG1c, or 201
ATG13b interacted with TRAF1a and TRAF1b (Supplemental Figure 2A). Failure to 202
detect an interaction between TRAF1s and ATG1s is consistent with our previous 203
findings (Qi et al., 2017). Moreover, introduction of ATG13b may have detrimental 204
effects on the growth of yeast cells, an effect that is likely distinct from that of 205
ATG13a. 206
The potential associations of ATG1s and ATG13s with TRAF1a were further 207
confirmed by bimolecular fluorescence complementation (BiFC) analyses in the 208
wild-type Arabidopsis protoplast cells. To this end, protein fusions with yellow 209
fluorescent protein (YFP), ATG1a-cYFP, ATG1b-cYFP, ATG1c-cYFP, ATG13a-cYFP, 210
or ATG13b-cYFP, were transiently coexpressed with TRAF1a-nYFP in protoplast for 211
16 h under continuous light or dark conditions, followed by confocal microscopy. The 212
BiFC assays showed that for all combinations, the YFP signals were detected in the 213
cytoplasm under light conditions, but were observed as punctate structures under dark 214
conditions (Figure 2A; Supplemental Figure 3). By contrast, coexpression of the 215
8
negative controls TRAF1a-nYFP/ATG7-cYFP and nYFP/cYFP failed to reconstitute 216
an intact YFP signal in Arabidopsis leaf protoplasts under either light or dark 217
conditions (Figure 2A; Supplemental Figure 3). Further, we coexpressed 218
mCherry-ATG8a (Qi et al., 2017) as an autophagasome marker with the split 219
TRAF1a-nYFP and ATG1a-, ATG1b-, ATG1c-, ATG13a-, ATG13b-, and ATG7-cYFP 220
fusions in protoplasts under light or dark conditions. As shown in Supplemental 221
Figure 3B, starvation-inducible punctate dots primarily colocalized with 222
mCherry-ATG8a. 223
Next, we used stable transgenic lines expressing TRAF1a-FLAG (Qi et al., 2017) 224
for co-immunoprecipitation (CoIP) assays. When ATG13a-HA or ATG13b-HA was 225
transiently expressed in the protoplasts isolated from rosettes of TRAF1a-FLAG line, 226
TRAF1a-FLAG could be immunoprecipitated by ATG13a-HA and ATG13b-HA 227
(Figure 2B), but not by ATG7-HA, ATG1a-HA, ATG1b-HA, and ATG1c-HA (Figure 228
2C; Supplemental Figure 2B). We also incubated the total proteins from the 229
TRAF1a-FLAG line with FLAG magnetic beads and used anti-ATG13a-specific 230
antibodies for immunoblot analysis. The results showed that ATG13a could be 231
immunoprecipitated by TRAF1a-FLAG (Supplemental Figure 2C). These findings 232
indicate that ATG13 proteins and TRAF1a interacted in autophagosome-related 233
structures in response to starvation. 234
To investigate the functional significance of protein interaction between 235
TRAF1a/TRAF1b and ATG13a/ATG13b, we analyzed the levels of ubiquitylated 236
ATG13a in the presence or absence of TRAF1a and TRAF1b. After transient 237
expression of the ATG13a-HA plasmid in the protoplasts isolated from wild-type and 238
traf1a-1 traf1b-2 double mutant (traf1a/b) leaves (Qi et al., 2017) for 16 h, total 239
protein was extracted and co-precipitated by HA affinity agarose beads followed by 240
immunoblot analysis. As shown in Figure 2D, the total ubiquitylation in the traf1a/b 241
double mutant is almost equal to that of wild-type plants. However, the ubiquitylation 242
of ATG13a-HA was reduced in the traf1a/b mutant compared with the wild-type 243
plants (Figure 2D). To examine the role of TRAF1a/b in modulating ATG13a stability, 244
9
we detected the protein stability of ATG13a in wild-type, traf1a/b, and 245
TRAF1a-FLAG plants after a 48-h nutrient starvation treatment or recovery under 246
nutrient-rich conditions for 48 and 72 h. The results revealed that the degradation of 247
ATG13a under both nutrient-deficient and nutrient-rich recovery conditions was 248
significantly inhibited in the traf1a/b mutant and the TRAF1a-FLAG transgenic line 249
(Figure 2E), suggesting that TRAF1a is involved in regulating ATG13a stability in 250
planta. 251
252
ATG13a Is a Target of the SINAT Proteins 253
Previous studies revealed that Arabidopsis TRAF1a and TRAF1b are required for 254
SINAT1- and SINAT2-associated ubiquitylation and degradation of ATG6. However, 255
under nutrient-starvation conditions, SINAT6 is involved in inhibiting the degradation 256
of ATG6 by competitively interacting with ATG6 to induce autophagy (Qi et al., 257
2017). To further understand the molecular basis of ATG13a degradation, we used 258
Y2H assays to assess the associations of ATG13a and all six SINAT proteins, SINAT1, 259
SINAT2, SIANT3, SINAT4, SINAT5-S1 (a spliced form of the truncated SINAT5 260
lacking the RING finger domain in the Col-0 ecotype), and SINAT6. ATG13a and 261
ATG13b interacted only with SINAT5-S1 and SINAT6 in yeast cells (Figure 3A). 262
However, our CoIP assay suggested that ATG13a interacted with SINAT1, SINAT2, 263
SINAT5-S1, and SINAT6 in plant cells (Figure 3B). When ATG7 and SINATs were 264
coexpressed in the wild-type protoplasts as negative controls, ATG7 was not 265
co-precipitated by SINAT proteins (Supplemental Figure 4A). To investigate the 266
domains mediating the interaction between SINATs and ATG13a, we used two 267
alternatively spliced forms, SINAT5-S1 and SINAT5-S2 in Col-0, and a truncated 268
form of SINAT5 for the Y2H analysis. ATG13a interacted with SINAT5-S1 and 269
SINAT5-S2, instead of truncated SINAT5 without the TRAF domain, indicating that 270
the C-terminal of the TRAF domain in SINAT5 is essential for the interaction 271
between ATG13a and SINAT5 (Figure 3C). 272
The interaction between ATG13a and SINATs was further confirmed by BiFC 273
10
assays in Arabidopsis cells. When SINAT1-nYFP or SINAT2-nYFP were transiently 274
coexpressed with ATG13a-cYFP or ATG13b-cYFP, respectively, in wild-type 275
protoplasts for 16 h, punctate YFP signals were observed (Supplemental Figure 4B). 276
By contrast, uniform fluorescent BiFC signals were detected in the cytoplasm when 277
SINAT5-S1-nYFP or SINAT6-nYFP were transiently coexpressed with 278
ATG13a-cYFP or ATG13b-cYFP, respectively (Supplemental Figure 4B). As 279
controls, co-expression of SINATs-nYFP/ATG7-cYFP and nYFP/cYFP failed to 280
reconstitute intact YFP in Arabidopsis leaf protoplasts (Supplemental Figure 4B). 281
Given that SINAT1, SINAT2, SINAT5-S1, and SINAT6 interact with ATG13a 282
(Figures 3A to 3C; Supplemental Figure 4), we further asked whether these SINAT 283
proteins are involved in ubiquitylation of ATG13a and subsequently affect its protein 284
stability. To test this possibility, we coexpressed ATG13a-FLAG with green 285
fluorescent protein (GFP)-tagged SINAT fusions, GFP-SINAT1-HA, 286
GFP-SINAT2-HA, GFP-SINAT5-S1-HA, and SINAT6-GFP-HA, in wild-type 287
Arabidopsis protoplasts. The protein gel blots using the anti-HA or anti-FLAG 288
antibodies showed that the ubiquitylation of ATG13a-FLAG was induced by the 289
expression of GFP-SINAT1-HA and GFP-SINAT2-HA fusions, but very weak signals 290
were detected with the empty vector control as well as with expression of 291
GFP-SINAT5-S1-HA or SINAT6-GFP-HA fusions (Figure 3D). 292
To investigate whether SINAT proteins play a role in regulating the stability of 293
ATG13a, we identified sinat3, sinat4, and sinat5 T-DNA insertional single mutants 294
(Supplemental Figure 5), and crossed them to sinat1 sinat2 (Qi et al., 2017) or 295
sinat6-2 (Qi et al., 2017) mutants, respectively, to generate sinat1 sinat2 sinat3 sinat4 296
(sinat1/2/3/4) quadruple mutant and sinat5 sinat6 double mutant plants. Then we 297
subjected the wild-type, sinat1/2/3/4 quadruple mutant, and SINAT1-OE seedlings to 298
constant dark treatment for fixed-carbon starvation for 0, 24, 48, and 72 h, and 299
detected the ATG13 protein levels using ATG13a-specific antibodies. As shown in 300
Figures 3E and 3F, the ATG13a level clearly declined after 72 h of carbon starvation 301
treatment in the wild-type seedlings. However, the degradation of ATG13a was 302
11
inhibited in the sinat1/2/3/4 mutant and increased in the SINAT1-OE transgenic line 303
(Figure 3E). By contrast, we observed decreased levels of ATG13a in the sinat5 sinat6 304
double mutant and an increased level in the SINAT6-OE line (compared with the 305
wild-type control), in response to carbon starvation (Figure 3F). These findings 306
suggest that ATG13a is a target of SINAT1, SINAT2, SINAT5-S1, and/or SINAT6 in 307
Arabidopsis, and that SINAT1/SINAT2 and SINAT5/SINAT6 may play different roles 308
in the regulation of ATG13a protein stability. 309
To further investigate the degradation of ATG13a by SINAT1 and SINAT2 310
through the 26S proteasome pathway, we coexpressed ATG13a-HA and 311
SINATs-FLAG in protoplasts from 4-week-old wild-type and rpn10 PAG1-GFP 312
(Marshall et al., 2015) plants. Compared with the wild-type plants, the degradation of 313
ATG13a by SINAT1 and SINAT2 was impaired in the rpn10 PAG1-GFP plants 314
(Supplemental Figure 6), suggesting that SINAT1 and SINAT2 mediate degradation 315
of ATG13a by the 26S proteasome pathway. 316
317
The K607 and K609 Lysine Sites Contribute to ATG13a Ubiquitylation 318
Post-translational polyubiquitylation, such as Lys-48-linked ubiquitylation, often 319
targets substrates for proteasome degradation (Kuang et al., 2013). To investigate 320
whether ATG13a and ATG13b undergo Lys-48-linked ubiquitylation in response to 321
starvation treatment, we analyzed the ubiquitylation of ATG13a-HA and ATG13b-HA 322
using K48-linked ubiquitylation antibodies. The immunoblot analyses showed that the 323
K48-linked ubiquitylation levels of ATG13a and ATG13b increased after constant 324
dark treatment for 24 and 48 h, and were further enhanced by the application of 325
MG132 (Figures 4A and 4B), indicating that ATG13a and ATG13b are modified by 326
the K48-linked ubiquitylation in response to nutrient starvation. 327
To identify the potential ubiquitylation site of ATG13a, we used UbPred 328
(http://www.ubpred.org/), which predicted K607 and K609 as two potential 329
ubiquitylation sites of ATG13a (Figure 4C). To test this possibility, we mutated K607 330
and K609 to arginine (R) to generate ATG13aK607R-HA (ATG13a-K1-HA), 331
12
ATG13aK609R-HA (ATG13a-K2-HA), and ATG13aK607/609R-HA 332
(ATG13a-K1/2-HA) constructs. As controls, we mutated K54 and K146, which were 333
not ubiquitination sites predicted by UbPred, to R to generate ATG13aK54R-HA 334
(ATG13a-K3-HA), and ATG13aK146R-HA (ATG13a-K4-HA) constructs. We 335
evaluated the effects of the K607R, K609R, K54R, and K146R mutations on the 336
ubiquitylation and stability of ATG13a by transiently expressing ATG13a-HA, 337
ATG13a-K1-HA, ATG13a-K1/2-HA, ATG13a-K3-HA, and ATG13a-K4-HA in 338
wild-type protoplasts. The ubiquitylation of ATG13a-HA decreased in both of the 339
K607R and K609R single mutants, and was further reduced in the ATG13a-K1/2-HA 340
double mutant compared with the wild-type ATG13a-HA control (Figure 4D; 341
Supplemental Figure 7A ), suggesting that K607 and K609
are necessary for the 342
ubiquitylation of ATG13a. Consistent with the corresponding ubiquitylation levels, 343
ATG13a-HA accumulated in the ATG13a-K1/2-HA mutant (Figure 4D). By contrast, 344
the ubiquitylation and stability of ATG13a-HA showed little difference in the K54R 345
and K146R single mutants compared with the wild-type ATG13a-HA control 346
(Supplemental Figure 7A). Together, these findings suggest that K607 and K609
are 347
required for ubiquitylation and degradation of ATG13a in planta. 348
Mutations in the ubiquitylation sites of animal ATG1s autophagy proteins lead to 349
a longer half-life compared with that of the control, and therefore the mutant proteins 350
are more stable in response to the protein translation inhibitor cycloheximide (CHX; 351
Nazio et al., 2016; 2017). We accordingly used CHX to monitor the effects of 352
ATG13a-K1/2 mutations on ATG13a stability. We introduced the ATG13a-K1/2-HA 353
construct into wild-type Arabidopsis to generate two independent transgenic lines 354
ATG13a-K1/2 #1 and ATG13a-K1/2 #2 (Supplemental Figure 7B to 7E). We 355
compared the ATG13a protein stabilities in the ATG13a-HA and ATG13a-K1/2-HA 356
mutant lines in the presence or absence of CHX under carbon starvation conditions. 357
Under constant darkness with CHX for 6 and 12 h, the ATG13a-K1/2-HA was more 358
stable with longer half-life compared to that of ATG13a-HA protein (Supplemental 359
Figure 7F). This result supports the conclusion that the K607 and K609 residues play 360
13
a primary role in mediating the stability of ATG13a protein. 361
To test the significance of ATG13a ubiquitylation in autophagy-mediated nutrient 362
starvation stress tolerance in Arabidopsis, we further analyzed the phenotypes of the 363
ATG13a-K1/2-HA mutant in response to nutrient starvation treatment. The 364
1-week-old wild-type, ATG13a-OE, ATG13a-K1/2 #1, and ATG13a-K1/2 #2 seedlings 365
were grown on MS and transferred to liquid MS medium (N+) or nitrogen-deficient 366
liquid MS medium (N–) for 4 days. All the plants had very similar phenotypes to the 367
wild-type plants under nitrogen-sufficient (N+) conditions (Figure 4E). However, the 368
ATG13a-OE, ATG13a-K1/2 #1, and ATG13a-K1/2 #2 lines exhibited increased 369
tolerance to nitrogen starvation (Figure 4E). In particular, the two mutant lines 370
ATG13a-K1/2 #1 and ATG13a-K1/2 #2 were more tolerant than the ATG13a-OE line, 371
as calculated by the relative chlorophyll contents of the seedlings (Figures 4F). 372
Similarly, when 1-week-old wild-type, ATG13a-OE, ATG13a-K1/2 #1, and 373
ATG13a-K1/2 #2 seedlings were subjected to constant dark treatment for fixed-carbon 374
starvation (C–) for 9 days, the ATG13a-OE, ATG13a-K1/2 #1, and ATG13a-K1/2 #2 375
seedlings showed improved survival with green true leaves compared to the wild type 376
(Figures 4G and Supplemental Figure 8A). Consistent with the nitrogen starvation, 377
the ATG13a-K1/2 #1 and ATG13a-K1/2 #2 showed better tolerance than that of the 378
ATG13a-OE line, and the three lines had significantly higher relative chlorophyll 379
contents and fresh weights than the wild type (Figure 4H; Supplemental Figure 8B). 380
To further investigate the functional relevance of ATG13a ubiquitylation in 381
autophagy-associated nutrient starvation tolerance, we performed a complementation 382
test by introducing ATG13a-K1/2-HA into the atg13a atg13b (atg13a/b) double 383
mutant (Suttangkakul et al., 2011) to generate the ATG13a-K1/2-HA atg13a/b lines 384
(ATG13a-K1/2-HA atg13a/b #1 and ATG13a-K1/2-HA atg13a/b #3). When 385
1-week-old seedlings of wild type, the atg13a/b double mutant, and the 386
ATG13a-K1/2-HA atg13a/b lines were subjected to 4 d nitrogen starvation, the double 387
mutants showed increased sensitivity, with yellowing seedlings and significantly 388
lower chlorophyll contents (Supplemental Figures 8C and 8D). However, the 389
14
sensitivity of atg13a/b double mutant to nitrogen starvation was completely 390
complemented by ATG13a-K1/2-HA and showed comparable relative chlorophyll 391
contents to the wild-type seedlings (Supplemental Figures 8C and 8D). 392
393
TRAF1s Are Required for SINAT1- and SINAT2-Mediated Ubiquitylation and 394
Degradation of ATG13 Proteins 395
To determine the involvement of TRAF1a and TRAF1b in the SINAT-mediated 396
ubiquitylation and degradation of ATG13a, we coexpressed ATG13a-FLAG and 397
GFP-SINAT1-HA in the protoplasts isolated from rosettes of the wild type and the 398
traf1a/b double mutant. As shown in Figure 5A, ATG13a-FLAG ubiquitylation was 399
enhanced by the presence of GFP-SINAT1-HA in the wild-type background. However, 400
it strongly declined in the traf1a/b mutant either in the presence or in the absence of 401
GFP-SINAT1-HA (Figure 5A), suggesting that TRAF1a and TRAF1b are required for 402
SINAT-mediated ubiquitylation of ATG13a. Furthermore, we observed that the 403
degradation of ATG13a-FLAG induced by the expression of GFP-SINAT1-HA was 404
impaired in the traf1a/b mutant (Figure 5B). Together, these findings indicate that 405
TRAF1a and TRAF1b contribute to SINAT1-mediated ubiquitylation and degradation 406
of ATG13a. 407
In Arabidopsis, SINAT6 plays an opposite role to SINAT1 in ubiquitylation and 408
destabilization of ATG6 by competitively interacting with ATG6 to form a different 409
TRAFasome, TRAF1-SINAT6-ATG6 (Qi et al., 2017). Given the evidence that 410
SINAT6 promoted the accumulation of ATG13a (Figure 3F), we therefore 411
hypothesized that SINAT6 may be involved in maintaining the stability of ATG13a by 412
associating with ATG13a under certain growth conditions. To test this possibility, we 413
transiently expressed ATG13a-FLAG with GFP-SINAT1-HA alone or 414
GFP-SINAT1-HA together with SINAT6-GFP-HA in wild-type protoplasts and 415
detected the ubiquitylation and degradation of ATG13a by immunoblot analyses. 416
Competition analyses showed that the SINAT1-mediated ubiquitylation of ATG13a 417
was strongly reduced by coexpression of SINAT6 (Figure 5C). Furthermore, 418
15
SINAT1-mediated degradation of ATG13a was strongly inhibited by the addition of 419
SINAT6 in a dose-dependent manner (Figure 5D), implying that SINAT1 and SINAT6 420
may play opposing roles in the regulation of the stability of ATG13 proteins. 421
422
ATG1s Stabilize TRAF1a in vivo 423
The ATG1 Arabidopsis serine/threonine kinases proteins interact with ATG13s and 424
other regulatory proteins, such as ATG11 and ATG101, to form a protein kinase 425
complex that stimulates autophagic vesiculation (Liu and Bassham, 2010; 426
Suttangkakul et al., 2011; Li et al., 2014). Although ATG1s did not interact with 427
TRAF1 proteins in the Y2H and CoIP assays, they reconstituted an intact YFP signal 428
in the BiFC analysis (Figure 2; Supplemental Figure 2; Supplemental Figure 3). In the 429
CoIP assay, particularly, when TRAF1a-FLAG and ATG1a-HA, ATG1b-HA, or 430
ATG1c-HA were transiently coexpressed, TRAF1a-FLAG mobility was clearly 431
shifted by ATG1 proteins (Figure 2C); this prompted us to ask whether ATG1s affect 432
the patterns of TRAF1 proteins. Given that ATG1s function as serine/threonine 433
kinases in response to autophagy induction, the shifted mobility of TRAF1a-FLAG is 434
likely due to the phosphorylation by ATG1s. To confirm this, we expressed 435
ATG1a-HA, ATG1b-HA, or ATG1c-HA in protoplast cells from the rosettes of stable 436
TRAF1a-FLAG transgenic plants. The immunoblot analysis detected two bands of 437
TRAF1a-FLAG using anti-FLAG antibodies, and the higher molecular weight band 438
appeared with the co-expression of ATG1 proteins (Figure 6A). TRAF1a-FLAG 439
patterns were evaluated by adding lambda protein phosphatase and the phosphatase 440
inhibitor PhosSTOP to the total proteins (Suttangkakul et al., 2011). As shown in 441
Figure 6B, the lambda phosphatase treatment reduced the levels of the higher 442
molecular weight species of TRAF1a-FLAG, and PhosSTOP blocked this shift, 443
implying that ATG1 proteins are involved in the phosphorylation-like modification of 444
TRAF1a in vivo. 445
To investigate the function of ATG1a in modulating TRAF1a stability, we crossed 446
TRAF1a-FLAG to YFP-ATG1a stable transgenic plants (Chen et al., 2017) to generate 447
16
a TRAF1a-FLAG YFP-ATG1a double combination line and determined the 448
TRAF1a-FLAG stability in response to nutrient starvation treatment at various times. 449
TRAF1a-FLAG degraded after exposure to constant darkness at 16 and 24 h. By 450
contrast, in the YFP-ATG1a line, TRAF1a-FLAG accumulated continuously from 0 to 451
24 h after carbon starvation treatment (Figure 6C), suggesting that ATG1a plays a 452
crucial role in maintaining the stability of TRAF1a in response to nutrient starvation. 453
In particular, in the presence of YFP-ATG1a, potential phosphorylated 454
TRAF1a-FLAG (pTRAF1a-FLAG) was the predominant form at 24 h after carbon 455
starvation (Figure 6C). 456
457
ATG1 Null Mutants Are Hypersensitive to Nutrient Deprivation 458
To further investigate the potential roles of ATG1s in modulating the stability of 459
TRAF1 proteins, we identified four T-DNA insertional mutants, atg1a-2, atg1b-1, 460
atg1c-1, and atg1t-1, which compromise the expression of ATG1a, ATG1b, ATG1c, 461
and ATG1t, respectively (Supplemental Figure 9; Suttangkakul et al., 2011). Reverse 462
transcription PCR (RT-PCR) analyses showed that these four T-DNA insertions 463
blocked the transcription of ATG1a, ATG1b, ATG1c, and ATG1t, respectively 464
(Supplemental Figure 9C), indicating that all of these lines are null mutants. 465
Previous studies demonstrate that the classic autophagy-defective mutants 466
exhibit premature leaf senescence and hypersensitivity to nutrient deprivation 467
(Doelling et al., 2002; Hanaoka et al., 2002; Yoshimoto et al., 2004; Thompson et al., 468
2005; Xiong et al., 2005; Phillips et al., 2008; Chung et al., 2010; Suttangkakul et al., 469
2011; Qi et al., 2017). All of the atg1 single mutants showed little or no phenotypic 470
change compared to wild-type plants grown in either nutrient-rich or 471
nutrient-deprived conditions (Supplemental Figure 10), confirming previous findings 472
(Suttangkakul et al., 2011). 473
To test their functional redundancy, we crossed the atg1a-2, atg1b-1, atg1c-1, 474
and atg1t-1 mutants to generate atg1a atg1c and atg1b atg1t double mutants, the 475
atg1a atg1b atg1c (atg1abc) triple mutant, and the atg1a atg1b atg1c atg1t (atg1abct) 476
17
quadruple mutant for further phenotypic analyses. The atg1a atg1c and atg1b atg1t 477
double mutants also showed few morphological differences from the wild-type plants 478
under normal growth conditions and after starvation treatments (Supplemental Figure 479
10). Similar to the other identified autophagy-deficient atg mutants, the atg1abc triple 480
and atg1abct quadruple mutants did not display obvious phenotypic differences from 481
the wild-type plants under nutrient-rich conditions for up to 3 weeks of growth 482
(Figure 7; Supplemental Figure 11; Supplemental Figure 12). 483
In contrast to their phenotypes on nutrient-rich medium, the atg1abc and 484
atg1abct mutants showed significant hypersensitivity when grown in fixed-carbon- or 485
nitrogen-deficient medium (Figure 7; Supplemental Figure 11; Supplemental Figure 486
12). Following fixed-carbon starvation induced by constant darkness for 7 d, the 487
mutants showed yellowing leaves, in contrast to the green leaves and significantly 488
higher chlorophyll contents in the wild-type plants (Figures 7A to 7C; Supplemental 489
Figure 11). Following a 7-d recovery under normal light/dark conditions, 60% of the 490
wild-type plants survived, while only 20–30% of the triple or quadruple mutants 491
survived (Figure 7D). 492
To confirm the response of the atg1abc and atg1abct mutants to fixed-carbon 493
starvation, 3-week-old soil-grown mutants were subjected to constant dark treatment. 494
Indeed, the atg1abc and atg1abct mutants displayed hypersensitivity to carbon 495
deprivation with lower relative chlorophyll contents and survival rates than that of the 496
wild-type plants (Supplemental Figures 12A to 12C). When 1-week-old wild-type, 497
atg1abc triple mutant, and atg1abct quadruple mutant plants were subjected to a 498
nitrogen-deprivation treatment on solid medium for 5 d or in liquid medium for 4 d, 499
all cotyledons of the triple and quadruple mutants exhibited increased yellowing, as 500
calculated by the relative chlorophyll contents of the plants (Figures 7E to 7G; 501
Supplemental Figure 11; Supplemental Figures 12D and 12H). The sensitivities of the 502
atg1abc and atg1abct mutants to nutrient starvations observed in this study were 503
similar to that of the atg13a atg13b double mutant (Supplemental Figure 12F to 12H; 504
Suttangkakul et al., 2011), but slightly weaker than that of the atg10-1 mutant (Figure 505
18
7; Supplemental Figure 11 ; Supplemental Figure 12; Phillips et al., 2008). 506
We monitored the natural senescence of these mutants, observing that the 507
rosettes of the atg1abc and atg1abct mutants were green like the wild-type plants 508
during the first four weeks (Figure 7H). However, similar to the atg10-1 mutant, the 509
cotyledons and some true leaves of the 5-week-old atg1abc and atg1abct mutants 510
were yellowing, indicating the onset of senescence (Figure 7H). By contrast, all of the 511
leaves in the wild-type plants were still green at the same stage. By measuring the 512
chlorophyll contents, we found that the relative chlorophyll contents in the rosette 513
leaves of the atg1abc and atg1abct mutants at 5 and 6 weeks old were significantly 514
lower than that of wild-type plants (Figure 7I). Together, these results indicate that, 515
similar to other autophagy-deficient mutants, the atg1abc triple and atg1abct 516
quadruple mutants showed enhanced sensitivities to nutrient starvation and premature 517
leaf senescence. 518
519
ATG1s Are Required for the Regulation of TRAF1 Stability 520
To investigate the role of ATG1s in ATG protein turnover and TRAF1 maintenance, 521
we first examined the levels of ATG1a, ATG13a, and ATG8a, in the atg1abc and 522
atg1abct mutants using the corresponding specific antibodies. Protein gel blot 523
analyses revealed that compared to the wild-type seedlings, ATG1a decreased 524
significantly, but ATG8a and ATG13a accumulated to high levels in the atg1abc and 525
atg1abct mutants under either nutrient-rich or starvation conditions (Figure 8A), 526
suggesting that loss of ATG1s prevents starvation-induced autophagy protein turnover. 527
Moreover, we coexpressed TRAF1a-HA and ATG1a-HA in wild-type to detect the 528
possible phosphorylation and protein stability of TRAF1a. As shown in Figure 8B, the 529
phosphorylation-like modification of TRAF1a-HA (pTRAF1a-HA) was 530
predominately increased by constant darkness for 16 h and disappeared after 6 h of 531
recovery under light conditions in the wild-type plants (lanes 1-3), while the wild-type 532
(lanes 4-6) and atg1abc mutant (lanes 7-9) cells expressing only TRAF1a-HA did not 533
show this increase. Compared to the TRAF1a-HA levels in the wild-type cells under 534
19
either light or dark conditions, the deletions of ATG1 proteins in the atg1abc mutant 535
significantly destabilized TRAF1a-HA (lanes 7-9, Figure 8B). Taken together, these 536
findings suggest that ATG1 proteins are redundantly essential for the regulation of 537
TRAF1a stability. 538
539
DISCUSSION 540
Autophagy is required for the degradation of cellular nutrients, toxic materials, and 541
damaged organelles to promote survival and cellular homeostasis at certain 542
developmental stages or in response to biotic and abiotic stresses and is an 543
evolutionarily conserved process in all eukaryotes (Xie and Klionsky, 2007; Michaeli 544
et al., 2016). 545
Thus far, more than 40 ATG proteins and their associated regulatory proteins 546
have been identified in plants. Among these, ATG1 and ATG13 form a protein kinase 547
complex that plays crucial roles in the initiation of autophagy and autophagic vesicle 548
assembly by interacting with the regulatory proteins ATG11 and ATG101 549
(Suttangkakul et al., 2011; Liu and Bassham, 2012; Li et al., 2014). 550
ATG13 is one of the core components of the ATG1–ATG13 complex, which is 551
regulated by a series of upstream effectors dependent on nutrient availability. In yeast 552
cells, ATG13 is structurally divided into an N-terminal globular domain (Jao et al., 553
2013), and a C-terminal region, that is predicted to be an intrinsically disordered 554
region (IDR) (Kamada et al., 2000). The C-terminal intrinsically disordered region of 555
ATG13 is dephosphorylated in response to starvation and interacts with ATG1 and 556
ATG17, thereby leading to the formation of the ATG1–ATG13 complex (Fujioka et 557
al., 2014; Yamamoto et al., 2016). In yeast, the TOR kinase acts as a key negative 558
regulator to phosphorylate ATG13 under nutrient-rich conditions, which reduces the 559
ability of the ATG1–ATG13 complex to alleviate autophagy (Rabinowitz and White, 560
2010). 561
Previous findings revealed that in response to nutrient starvation, Arabidopsis 562
ATG1a and ATG13a are dramatically degraded in the vacuole in an 563
20
autophagy-dependent manner, demonstrating a feedback turnover mechanism during 564
the biogenesis of starvation-induced autophagy (Suttangkakul et al., 2011; Li et al., 565
2014). We further discovered herein that ATG13a and ATG13b are subject to 566
ubiquitylation and proteasomal degradation upon prolonged nutrient starvation and 567
during recovery following starvation (Figure 1; Supplemental Figure 1) implying that, 568
similar to mammalian ULK1 and ATG13, Arabidopsis ATG13 turnover is also 569
controlled by ubiquitylation-mediated proteolysis. Moreover, this process involves the 570
RING-type E3 ligases SINAT1 and SINAT2, and RING-finger-truncated SINAT6 571
(Figure 3) to maintain the autophagy dynamics under different nutrient conditions. 572
Particularly, we observed that ATG1a and ATG13a levels declined at 48 h after 573
MG132 treatment compared to those of 12 or 24 h (Figure 1), confirming the 574
regulation of these two proteins by alternative pathway such as autophagy 575
(Suttangkakul et al., 2011). 576
Based on these findings, it is conceivable that: 1) under nutrient-rich conditions, 577
SINAT1 and SINAT2 accumulate to ubiquitylate and destabilize ATG13 proteins to 578
maintain a relatively low autophagy level; 2) in response to prolonged nutrient 579
starvation, SINAT1 and SINAT2 likely contribute by targeting ATG13 proteins for 580
ubiquitylation and degradation to modulate the highly activated autophagy to proper 581
cellular levels; and 3) during recovery after starvation, the proteolysis of ATG13 582
proteins by the action of SINAT1 and SINAT2 is necessary for the termination of 583
activate autophagy. By contrast, SINAT6 is likely to play an opposing role in 584
suppressing the ubiquitylation and degradation of the ATG1/ATG13 complex by 585
competitively interacting with ATG13 proteins to promote autophagy in response to 586
nutrient deprivation. 587
Consistent with the autophagy-associated phenotypes and autophagosome 588
formation in the root cells of their knockout mutants (Qi et al., 2017), we further 589
showed that SINAT1/SINAT2 and SINAT6 act as negative and positive regulators, 590
respectively, in the regulation of autophagy by modulating ATG1 and ATG13 591
stabilities (Figure 5). Our previous findings have suggested that in response to carbon 592
21
starvation, the GFP-SINAT1 and GFP-SINAT2 fusion proteins are rapidly degraded, 593
while that of SINAT6-GFP is accumulated from 6 to 24 h upon treatment (Qi et al., 594
2017). Genetically, we observed that the sinat1 sinat2 double mutant and 595
SINAT1/SINAT2-Cas9 deletion lines showed increased tolerance and enhanced 596
autophagosome formation under carbon and nitrogen starvation conditions, while the 597
sinat6 knockout mutants displayed opposite phenotypes (Qi et al., 2017), suggesting 598
SINAT1/SINAT2 and SINAT6 play opposing roles in autophagy. 599
In this study, we provided further evidence to support the idea that 600
SINAT1/SINAT2 and SINAT6 proteins differentially modulate ATG13 stabilities 601
under various nutrient conditions. Particularly, SINAT1 and SINAT2 were involved 602
in ubiquitylation and degradation of ATG13, which were strongly decreased in the 603
presence of SINAT6 (Figures 3 and 5). We thus propose that under nutrient-rich 604
conditions, TRAF1a/TRAF1b proteins could interact with SINAT1/SINAT2 for 605
ubiquitylation and degradation of ATG13s. Instead, under nutrient-starvation 606
conditions, TRAF1a/TRAF1b proteins promote the stabilization of ATG13s by 607
interacting with SINAT6, which acts as a positive regulator in maintaining ATG13 608
stability and autophagy induction. 609
Besides the TRAF-domain-containing SINAT proteins, we and other groups 610
have identified two Arabidopsis TRAF proteins, TRAF1a and TRAF1b (also termed 611
MUSE14 and MUSE13, respectively), which contain only an N-terminal TRAF 612
domain and serve as molecular adaptors rather than E3 ligases to regulate plant 613
immunity, development, and abiotic stress tolerance (Huang et al., 2016; Qi et al., 614
2017). In Arabidopsis, TRAF1a and TRAF1b regulate plant autoimmunity and 615
pathogen resistance by interacting with the E3 ubiquitin ligase SCFCPR1
complex to 616
form a plant-type TRAFasome that modulates the ubiquitylation and degradation of 617
the NLR immune sensors SNC1 (suppressor of npr1-1, constitutive 1) and RPS2 618
(resistant to P. syringae 2) (Huang et al., 2016). Moreover, Arabidopsis TRAF1 619
proteins regulate autophagy by interacting with the E3 ligases SINAT1, SINAT2, and 620
SINAT6 to modulate the stability of ATG6 under differential nutrient conditions (Qi 621
22
et al., 2017). 622
In this study, we further showed that Arabidopsis TRAF1a and TRAF1b interact 623
with SINAT1, SINAT2, and SINAT6 to modulate autophagy dynamics and form two 624
different plant TRAFasomes, TRAF1–SINAT1/SINAT2–ATG13 and TRAF1–625
SINAT6–ATG13, providing evidence to demonstrate the central roles of 626
TRAF1-mediated ubiquitylation of the ATG1–ATG13 complex in autophagy 627
initiation in plant cells. Consistent with this notion, we suggested that ATG13a 628
degradation was strongly inhibited in both the traf1a/b double mutant and the 629
TRAF1a-FLAG overexpressing transgenic lines (Figure 2E), indicating that TRAF1a 630
and TRAF1b act as both positive and negative regulators in modulating the protein 631
stabilities of the ATG1–ATG13 complex. 632
Although we did not detect an interaction between ATG1s and TRAF1 proteins 633
by Y2H and CoIP assays, we observed by the BiFC assay that ATG1s and TRAF1 634
proteins were associated in the autophagosome-related punctate structures (Figure 2; 635
Supplemental Figure 2; Supplemental Figure 3). Interestingly, our data revealed that 636
ATG1 proteins prevent the degradation of TRAF1a, possibly by phosphorylation, in 637
response to carbon starvation (Figure 6A to 6C), suggestive of a feedback regulatory 638
mechanism between the ATG1–ATG13 kinase complex and TRAF1 proteins. 639
Given that the potential role of Arabidopsis ATG1s in autophagosome formation 640
has not been well understood, we further characterized the atg1abc triple mutant and 641
the atg1abct quadruple mutant. Similar to other autophagy-defective atg mutants, a 642
previous finding reported that the Arabidopsis atg13a atg13b double mutants exhibit 643
premature leaf senescence and hypersensitivity to fixed carbon and nitrogen 644
limitations (Suttangkakul et al., 2011). In this study, we showed that the atg1abc 645
triple mutant and the atg1abct quadruple mutant were similar to the atg13a atg13b 646
double mutants in their phenotypes of age-dependent and starvation-induced leaf 647
senescence (Figure 7; Supplemental Figure 11; Supplemental Figure 12). Moreover, 648
the turnover of ATG13a and ATG8a was repressed in the atg1abc and atg1abct 649
mutants compared with the wild-type seedlings (Figure 8A), suggesting that ATG1a, 650
23
ATG1b, and ATG1c function redundantly in the regulation of autophagy in 651
Arabidopsis. Because the atg1abct quadruple mutant did not show an enhanced 652
senescence phenotype compared to the atg1abc triple mutant (Figure 7), the roles of 653
ATG1t in this process are still obscure. 654
More interestingly, a recent work validated the role of ATG1s in autophagosome 655
formation by using GFP-ATG8e transgenic lines. Specifically, Huang et al. found that 656
in response to nutrient starvation, the formation of GFP-ATG8e-labeled punctuate 657
structures (autophagosomes or their intermediates) was markedly induced in the 658
wild-type root cells (Huang et al., 2019). However, such accumulation was not 659
evident in the atg1abc and atg1abct backgrounds under either nutrient-rich or 660
starvation conditions (Huang et al., 2019), further confirming that deletion of ATG1s 661
leads to deficiency of autophagosome formation. 662
As expected, the phosphorylation-like modification and stability of TRAF1a 663
were significantly reduced in the atg1abc triple mutant (Figure 8B), confirming the 664
involvement of TRAF1a regulation by ATG1s for modulating its stability. Although 665
we have confirmed that ATG1s were involved in the phosphorylation-like 666
modification of TRAF1a in vitro, the shift molecular weight of TRAF1a may also be 667
caused by other larger post-translational modifications. Consistent with this, a recent 668
study revealed that the degradation of both TRAF1a and TRAF1b are mediated by the 669
SCFSNIPER4
complex to control the turnover of TRAF1 proteins in plant cells (Huang 670
et al., 2018). Thus, further investigations of the interaction of post-translational 671
modifications, such as ubiquitylation and phosphorylation, in determining the stability 672
of TRAF1 proteins will be needed to better understand the molecular mechanism of 673
TRAF1 proteins in the regulation of autophagy initiation. 674
In conclusion, our observations present strong evidence that, under normal 675
nutrient conditions, the RING-type E3 ligases SINAT1 and SINAT2 regulate the 676
ubiquitylation and degradation of ATG13a, leading to disassociation of the ATG1–677
ATG13 complex, and therefore suppressing autophagy (Figure 9). Under starvation 678
conditions, however, the ATG1s stabilize TRAF1 proteins by a feedback regulatory 679
24
mechanism (Figure 9). Given that in response to nutrient starvation, SINAT1 and 680
SINAT2 are degraded, but SINAT6 is accumulated (Qi et al., 2017), the 681
TRAF1-SINAT6-ATG13 TRAFasome is therefore predominant under starvation 682
conditions to promote autophagy induction in Arabidopsis (Figure 9). The SINATs 683
competitively interact with ATG13 and ATG6 under different nutrient conditions 684
reminiscent of the role of HvARM1 (Hordeum vulgare Armadillo 1) and HvPUB15 685
(H. vulgare Plant U-Box 15) in regulating powdery mildew infection in barley 686
(Rajaraman et al., 2018). This suggests that this regulatory strategy may be widely 687
used by plants (Rajaraman et al., 2018). 688
689
METHODS 690
Plant Materials, Growth Conditions, and Treatments 691
All wild-type, mutants, and transgenic Arabidopsis thaliana plants used in this study 692
are in the Columbia (Col-0) background. The T-DNA insertional mutants described in 693
this study were obtained from The Arabidopsis Information Resource 694
(http://www.arabidopsis.org), with the locus names atg1a-2 (SALK-054351), atg1b-1 695
(CS446939), atg1c-1 (CS920806), atg1t-1 (SALK-062634), sinat3 (SALK-125517), 696
sinat4 (CS415212), and sinat5 (SALK-069496). The mutants were identified by PCR 697
using a gene-specific primer paired with a T-DNA border-specific primer 698
(Supplemental Data Set 1). The atg1a-2, atg1b-1, atg1c-1, and atg1t-1 mutants were 699
crossed to each other to generate the atg1ac and atg1bt double mutants, the atg1abc 700
triple mutant, and the atg1abct quadruple mutant. The sinat1 sinat2 double mutant (Qi 701
et al., 2017) was crossed to sinat3 and sinat4 single mutant to generate sinat1/2/3/4 702
quadruple mutant. The sinat5 single mutant was crossed to sinat6-2 (Qi et al., 2017) 703
to generate sinat5 sinat6 double mutant. The atg10-1 single mutant, atg13ab double 704
mutant, rpn10 PAG1-GFP, and traf1a traf1b double mutant were described by 705
Phillips et al. (2008), Suttangkakul et al. (2011), Marshall et al. (2015) and Qi et al. 706
(2017), respectively. The mutants and transgenic lines generated in this study are 707
listed in Supplemental Table 1 and Supplemental Table 2. All Arabidopsis seeds were 708
25
surface sterilized with 20% bleach containing 0.1% Tween 20 for 20 min and washed 709
with sterilized water 5 times. The seeds were sown on Murashige and Skoog (MS) 710
medium (Sigma-Aldrich) containing 2% sucrose (w/v) and 0.8% agar (w/v). 711
Following cold treatment under dark conditions for 3 days, the plates were incubated 712
at 22°C under long-day (LD) (16-h-light/8-h-dark) or short-day (SD) 713
(8-h-light/16-h-dark) photoperiods with a light intensity of 170 mmol/m2/s using 714
fluorescent bulbs (Philips F17T8/ TL841 17W). After germination for 7 days, the 715
seedlings were transferred to soil for further growth. 716
For the carbon-deprivation treatment, 1-week-old seedlings grown on MS 717
medium or 3-week-old soil-grown plants were transferred to continuous darkness for 718
the indicated duration, followed by recovery under normal growth conditions for 7 719
days. Samples were photographed at the indicated time points. The ratio of surviving 720
plants, as defined by the growth of new leaves, to dead plants was calculated from 10 721
plants per genotype. 722
The effects of nitrogen starvation on plant growth were determined according to 723
Qi et al. (2017). Briefly, 1-week-old seedlings grown on MS medium were transferred 724
to MS or nitrogen-deficient MS medium (solid or liquid) and grown under normal 725
growth conditions for the indicated times. 726
Arabidopsis seedlings (1-week-old) grown on solid MS with 2% sucrose for 727
biochemical analysis were transferred to the sterile 12-well plates with liquid MS 728
medium (–C, –N, or –C/N) and 50µM MG132 or 0.5mM CHX for treatment. After 729
the specified treatments, seedlings were dried on paper, flash-frozen in liquid nitrogen 730
for protein extraction before protein blot analysis. 731
732
Plasmid Construction 733
All plasmids used in this study were generated using an In-Fusion method. The 734
gene-specific primers with 15-bp extensions homologous to the corresponding vectors 735
are listed in Supplemental Data Set 1. Plasmids for transient expression analyses were 736
derived from the pUC119 vector (Li et al., 2013). For the ATG13a-HA, 737
26
ATG13a-FLAG, ATG13b-HA, ATG13a-K1-HA, ATG13a-K2-HA, ATG13a-K1/2-HA, 738
ATG13a-K3-HA, ATG13a-K4-HA, ATG7-HA, and ATG7-FLAG constructs, the 739
full-length coding regions of ATG13a, ATG13b, ATG13a-K1, ATG13a-K2, 740
ATG13a-K1/2, ATG13a-K3, ATG13a-K4, and ATG7 were inserted into BamHI- and 741
StuI-digested pUC119 plasmids. SINAT1, SINAT2, SINAT5-S1, and SINAT6 were 742
cloned into StuI (SINAT1, SINAT2, and SINAT5-S1) or BamHI (SINAT6) digested 743
pUC119 to generate GFP and HA-tagged SINAT constructs. TRAF1a-HA, 744
SINAT1-FLAG, SINAT2-FLAG, SINAT5-S1-FLAG and SINAT6-FLAG were 745
constructed as previously described (Qi et al., 2017). To generate stable transgenic 746
plants expressing ATG13a-HA, ATG13b-HA, ATG13a-K1/2-HA, GFP-SINAT1-HA, 747
and SINAT6-GFP-HA, the UBQ10pro:ATG13a-HA, UBQ10pro:ATG13b-HA, 748
UBQ10pro:ATG13a-K1/2-HA, UBQ10pro:GFP-SINAT1-HA, and 749
UBQ10pro:SINAT6-GFP-HA fragments derived from pUC119 constructs were 750
digested by AscI and cloned into the binary vector pFGC-RCS (Li et al., 2013). The 751
expression cassettes were subsequently introduced into wild-type Arabidopsis (Col-0) 752
by Agrobacterium tumefaciens-medium transformation via the floral dip method 753
(Clough and Bent, 1998). The TRAF1a-FLAG transgenic plants were described by Qi 754
et al. (2017). To generate plasmids for Y2H analysis, full-length coding sequence 755
fragments of ATG13a, ATG13b, ATG1a, ATG1b, ATG1c, and SINAT5-S1 were 756
amplified and inserted into pGADT7 and pGBKT7 vectors digested by EcoRI. The 757
fragments of SINAT1/SINAT2/SINAT3/SINAT4/SINAT6 were inserted into pGADT7 758
vectors digested with EcoRI and BamHI to generate 759
SINAT1/SINAT2/SINAT3/SINAT4/SINAT6-AD plasmids for Y2H. The SINAT5-BD 760
plasmids for Y2H were constructed as previously described (Qi et al., 2017). To 761
generate plasmids for the BiFC assay, the full-length coding sequence fragments of 762
ATG13a, ATG13b, ATG1a, ATG1b, ATG1c, ATG7, SINAT1, SINAT2, SINAT5-S1, 763
SINAT6, and TRAF1a were inserted into BamHI-digested pHBT-YC (Qi et al., 2017) 764
or pHBT-YN (Qi et al., 2017) respectively, to generate fusions with nYFP or cYFP. 765
766
27
Measurement of Chlorophyll Contents 767
Measurement of chlorophyll contents was performed as previously described (Porra et 768
al., 1989; Xiao et al., 2010). Arabidopsis leaves were harvested after nitrogen or 769
carbon starvation or at different growth period. Arabidopsis total chlorophyll was 770
extracted by immersing the samples in 2 mL of N, N-dimethylformamide for 48 h in 771
the dark at 4°C. Absorbance was determined at 664 and 647 nm, and the total 772
chlorophyll content was measured and normalized to fresh weight per sample. 773
774
Protein Isolation and Immunoblot Analysis 775
For total protein extraction, 1-week-old Arabidopsis seedlings grown on MS medium 776
or after nutrient starvation were ground in liquid nitrogen and homogenized in 777
ice-cold protein extraction buffer (50 mM sodium phosphate, pH 7.0, 200 mM NaCl, 778
10 mM MgCl2, 0.2% β-mercaptoethanol, and 10% glycerol) supplemented with 779
protease inhibitor cocktail (Roche 04693132001). The samples were placed on ice for 780
30 min and centrifuged at 4°C at 12,000 g for 30 min. The supernatant was transferred 781
to a new microfuge tube prior to electrophoresis. 782
For immunoblot analysis, clarified extracts were subjected to sodium dodecyl 783
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a 784
Hybond-C membrane (Amersham). Specific anti-ATG1a (Suttangkakul et al., 2011; 785
1:8000), anti-ATG13a (Suttangkakul et al., 2011; 1:5000), anti-ATG7 (Abcam, cat. no. 786
ab99001; 1:2000), anti-ATG8a (Abcam, cat. no. ab77003; 1:1500), anti-HA 787
(Sigma-Aldrich, cat. no. H6533; 1:5000), anti-FLAG (Sigma-Aldrich, cat. no. A8592; 788
1:5000), anti-Ub (Proteintech, cat. no. 10201-2-AP; 1:2000), anti-K48Ub (Cell 789
Signaling Technology, cat. no.12930; 1:1000), anti-GFP (Cell Signaling Technology, 790
cat. no.2955; 1:1000), anti-YFP (Abcam, cat. no. ab290; 1:2500), and anti-ACTIN 791
(Cell Signaling Technology, cat. no. 58169; 1:1000) antibodies were used in the 792
protein blotting analysis. Quantification of the protein immunoblot signal was 793
determined with Image J software. 794
795
28
Lambda Protein Phosphatase Treatment 796
ATG1-HA proteins were expressed in the protoplasts isolated from the TRAF1-FLAG 797
transgenic line for 16 h, followed by extraction of total protein by 798
immunoprecipitation (IP) buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM 799
EDTA, and 10% glycerol) with 0.5% Triton X-100 supplemented with protease 800
inhibitor cocktail (Roche 4693132001). After the samples were centrifuged at 4°C at 801
12,000 g for 30 min, the supernatants were incubated with Lambda protein 802
phosphatase (New England Biolabs) according to the instructions, with or without 803
application of a 1× concentration of the phosphatase inhibitor PhosSTOP (Roche) for 804
30 min at 30°C. The reactions were heated to 95°C for 5 min before immunoblot 805
analysis. 806
807
Y2H, CoIP, and BiFC Assays 808
Determination of protein–protein interactions by the Y2H assay was conducted as 809
previously described (Chen et al., 1992) with minor modifications. Both plasmids 810
with AD and BD were transformed into yeast strain YH109. The protein interactions 811
were identified by growth after 2 d on medium lacking His, Leu, and Trp. To avoid 812
self-activation of the transformants, 5 mM 3-amino-1,2,4-triazole was added to the 813
medium. 814
Preparation and transfection of Arabidopsis mesophyll protoplasts were 815
performed according to Yoo et al. (2007). Protoplasts isolated from 4-week-old 816
rosettes were transfected with the indicated plasmids and cultured for 16 h before 817
protein extraction. For the CoIP assays, the cells were collected and lysed in IP buffer 818
(10 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM EDTA, and 10% glycerol) with 0.5% 819
Triton X-100. A 10% volume of total lysis (10%) was used for input, and the 820
remainder was incubated with GFP, HA, or FLAG affinity beads (Sigma-Aldrich) for 821
4 h at 4°C. The beads were then collected and washed five times with IP buffer 822
containing 0.1% Triton X-100, followed by adding 5× SDS-PAGE sample buffer and 823
heated at 95°C for 5 min before protein blot analysis. 824
29
For the BiFC assay, the split nYFP and cYFP plasmids or with mCherry-ATG8a 825
were coexpressed in leaf protoplasts prepared from the wild-type plants for 16 h under 826
light or dark conditions, and the YFP and mCherry signals were detected by confocal 827
microscopy irradiated with 488-nm (YFP) or 516-nm (mCherry) light, and visualized 828
with the band-pass 500- to 530-nm (YFP) or 560- to 610-nm (mCherry) IR filters. 829
830
In vivo Ubiquitylation Assay 831
For the in vivo ubiquitylation assay, the ATG13a-HA plasmid was either expressed in 832
Arabidopsis mesophyll protoplasts isolated from wild-type or traf1a/b-1 double 833
mutant protoplasts, or ATG13a-FLAG co-expressed with 834
GFP-SINAT1/SINAT2/SINAT5-S1-HA or SINAT6-GFP-HA plasmids in wild-type 835
protoplasts. After a 16 h incubation, the cells were collected and lysed in the IP buffer 836
containing 0.5% Triton X-100 with vigorous vortexing. The supernatants were 837
incubated with HA affinity agarose beads or FLAG magnetic beads before 838
immunoblot analysis. The empty vector pUC119-UBQ10-GFP-HA was co-expressed 839
with other constructs for determining the expression efficiency. The ubiquitylation 840
patterns of ATG13a-HA or ATG13a-FLAG were detected by anti-ubiquitin antibodies 841
(Proteintech, cat. no. 10201-2-AP, 1:2000). 842
843
RNA Extraction and RT-qPCR 844
Total RNA was extracted from 3-week-old Arabidopsis leaves using a HiPure Plant 845
RNA Mini kit (E.Z.N.A. Omega bio-tek) according to the manufacturer’s instructions. 846
One µg of total RNA was used to convert into cDNA with the HiScript II Q RT Super 847
Mix kit with gDNA Wiper (Vazyme). cDNA samples were diluted 1:10 in water 848
before use. 849
RT-qPCR was performed in 10 µL reaction volumes with gene-specific primers 850
(Supplemental Data Set 1), and conducted with a StepOne Plus Real-time PCR 851
System (Applied Biosystems) using ChamQ SYBR Color qPCR Master Mix 852
(Vazyme). The RT-qPCR was performed with the following protocol: 95°C for 5 min 853
30
followed by 40 cycles of 95°C for 15 s, 55°C for 150 s, and 72°C for 30 s, and a 854
subsequent standard dissociation protocol to validate the presence of a unique PCR 855
product. 856
For calculation of relative transcription levels, the delta of threshold cycle (△Ct) 857
values were calculated by subtracting the arithmetic mean Ct values of the target 858
ATG13a from the normalizing ACTIN2. The relative transcription level (2−ΔΔCt
) was 859
calculated from three independent experiments. 860
861
Statistical Analysis 862
Data reported in this study are means ± SD of three independent experiments unless 863
otherwise indicated. The significance of the differences between groups was 864
determined by One-Way ANOVA. The P values < 0.05 or < 0.01 were used to 865
determine significance. The One-Way ANOVA results are listed in Supplemental Data 866
Set 2. 867
868
Accession Numbers 869
Sequence data from this article can be found in the Arabidopsis Genome Initiative or 870
GenBank/EMBL databases under the following accession numbers: TRAF1a 871
(At5g43560), TRAF1b (At1g04300), SINAT1 (At2g41980), SINAT2 (At3g58040), 872
SINAT3 (At3g61790), SINAT4 (At4g27880), SINAT5 (At5g53360), SINAT6 873
(At3g13672), ATG13a (At3g49590), ATG13b (At3g18770), ATG1a (At3g61960), 874
ATG1b (At3g53930), ATG1c (At2g37840), ATG1t (At1g49780), ATG8a (At4g21980), 875
ATG7 (At5g45900), ATG10 (At3g07525). 876
877
SUPPLEMENTAL DATA 878
Supplemental Figure 1. Degradation of ATG13 Proteins during Recovery Following 879
Starvation. (Supports Figure 1) 880
Supplemental Figure 2. Interaction of ATG1/ATG13 and TRAF1a in vivo and in 881
vitro. (Supports Figure 2) 882
31
Supplemental Figure 3. Interaction of ATG1/ ATG13 and TRAF1a by BiFC assay. 883
(Supports Figure 2) 884
Supplemental Figure 4. Interaction of ATG13a/b and SINATs in vivo. (Supports 885
Figure 3) 886
Supplemental Figure 5. Identification of sinat Single Mutants. (Supports Figure 3) 887
Supplemental Figure 6. ATG13a Is Degraded via the 26S Proteasome Pathway. 888
(Supports Figure 3) 889
Supplemental Figure 7. Identification of ATG13a ubiquitylation Site Mutants. 890
(Supports Figure 4) 891
Supplemental Figure 8. Phenotypic Analyses of ATG13a ubiquitylation mutants in 892
the atg13ab double mutant background. (Supports Figure 4) 893
Supplemental Figure 9. Isolation of atg1 Single Mutants. (Supports Figure 7) 894
Supplemental Figure 10. Phenotypic Analyses of atg1 Single and Double Mutants. 895
(Supports Figure 7) 896
Supplemental Figure 11. Phenotypic Analyses of atg1abc and atg1abct Mutants. 897
(Supports Figure 7) 898
Supplemental Table 1. Mutants Generated in This Study 899
Supplemental Table 2. Transgenic Arabidopsis thaliana Plants Generated in This 900
Study 901
Supplemental Data Set 1. Sequence of Primers Used in This Study. 902
Supplemental Data Set 2. ANOVA Analysis in This Study. 903
904
ACKNOWLEDGEMENTS 905
We thank the ABRC (www.arabidopsis.org) for providing atg1a-2, atg1b-1, atg1c-1, 906
and atg1t-1 mutant seeds. This work was supported by the National Natural Science 907
Foundation of China (Projects 31725004, 31670276, and 31461143001 to S.X.; 908
Project 31800217 to H.Q.), and the Major Project of China on New Varieties of GMO 909
Cultivation (2018zx08011-01B to J.L.). 910
911
32
AUTHOR CONTRIBUTIONS 912
S.X. designed the research. H.Q., J.L., F.N.X., J.Y.C., X.L., and L.J.X. carried out the913
experiments. S.X., H.Q., and Q.M.Z. analyzed the data. S.X., H.Q., and J.L. wrote the 914
manuscript. 915
916
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1107
39
FIGURE LEGENDS 1108
Figure 1. The ATG1 and ATG13 Proteins are Degraded through the 26S Proteasome 1109
Pathway upon Starvation and During Recovery. 1110
(A) and (B) ATG1a, ATG13a, ATG8a, and ATG7 protein levels in the wild-type (WT)1111
plants upon carbon (–C; A) or nitrogen (–N; B) starvation with or without MG132. 1112
One-week-old WT seedlings were subjected to carbon or nitrogen starvation with or 1113
without 50 µM MG132 for 0, 12, 24, and 48 h. Relative intensity of each protein 1114
normalized to the loading control is shown below. 1115
(C) and (D) ATG13a-HA and ATG13b-HA levels in response to carbon starvation (–C;1116
C) or nitrogen starvation (–N; D) with or without MG132. One-week-old transgenic1117
lines expressing ATG13a-HA and ATG13b-HA were treated with constant darkness or 1118
nitrogen starvation with or without 50 µM MG132 for 0, 12, 24, and 48 h. 1119
(E) and (F) ATG13a-HA and ATG13b-HA levels upon carbon (–C; E) or nitrogen (–N;1120
F) starvation or during recovery (R) or in the presence of MG132 (R+MG132) for the1121
indicated times. One-week-old transgenic lines expressing ATG13a-HA and 1122
ATG13b-HA were treated with constant darkness or nitrogen starvation, and moved 1123
back to MS medium under normal light/dark conditions in the absence or presence of 1124
50 µM MG132 to recover for 24, 48, and 72 h. 1125
Anti-ACTIN antibodies and Ponceau S-stained Rubisco bands are shown below the 1126
blots to indicate the amount of protein loaded per lane. Numbers on the left indicate 1127
the molecular weight (kD) of each band. hpt, hours post treatment. 1128
1129
Figure 2. TRAF1s Interact with ATG13a and ATG13b in vivo. 1130
(A) BiFC assay of ATG1/ATG13 proteins (ATG1a and ATG13a) and TRAF1a in1131
Arabidopsis. The split cYFP fusions ATG13a-cYFP, ATG1a-cYFP or ATG7-cYFP and 1132
TRAF1a-nYFP were co-expressed in leaf protoplasts and incubated for 16 h under 1133
light or dark conditions. The TRAF1a-nYFP/ATG7-cYFP and nYFP/cYFP vectors 1134
were co-expressed as negative controls. Confocal micrographs obtained from YFP, 1135
auto-fluorescent chlorophyll, bright-field, and merged images are shown. The 1136
40
numbers in the cells of ATG13a or ATG1a-cYFP + TRAF1a-nYFP/dark indicate the 1137
average number of autophagosomes ± SD (n = 3) calculated from three independent 1138
experiments. For each experiment, 15 images were used for the calculation for each 1139
coexpression combination. Bars = 10 μm. 1140
(B) In vivo CoIP assay of the association between ATG13 (ATG13a and ATG13b) and1141
TRAF1a. HA-tagged ATG13a or ATG13b (ATG13a-HA or ATG13b-HA) was 1142
transiently expressed in protoplasts from transgenic plants expressing TRAF1a-FLAG 1143
under light conditions for 16 h, and immunoprecipitated with HA affinity agarose 1144
beads. 1145
(C) In vivo CoIP assay of the interaction between TRAF1a and ATG1 (ATG1a, ATG1b,1146
and ATG1c) proteins. HA-tagged ATG1a, ATG1b, and ATG1c (ATG1a-HA, 1147
ATG1b-HA, and ATG1c-HA) were transiently expressed in protoplasts from 1148
transgenic plants expressing TRAF1a-FLAG for 16 h under continuous dark 1149
conditions and immunoprecipitated with HA affinity agarose beads. 1150
(D) The ubiquitylation of ATG13a in the traf1a/b mutant. ATG13a-HA was transiently1151
expressed in Arabidopsis protoplasts isolated from four-week-old wild-type (WT) and 1152
traf1a/b mutant plants, and the ubiquitylation of ATG13a was detected by 1153
immunoprecipitation and immunoblot analysis. Proteins were extracted at 16 h after 1154
expression under constant light conditions and then incubated with HA affinity 1155
agarose beads. The blots were probed with anti-HA and anti-Ub antibodies. 1156
(E) The degradation of ATG13a in traf1a/b-1 and TRAF1a-FLAG plants.1157
One-week-old wild-type, traf1a/b, and TRAF1a-FLAG plants were subjected to 1158
carbon (–C; the upper images) or nitrogen starvation (–N; the lower images) treatment 1159
for 48 h, following by recovery (R) for 48 and 72 h. The blots were probed with 1160
anti-ATG13a-specific antibodies. hpt, hours post treatment. 1161
Numbers on the left indicate the molecular weight (kD) of each band. Anti-ACTIN 1162
antibodies and Ponceau S-stained Rubisco bands are shown below the blots to 1163
indicate the amount of protein loaded per lane. The expression of GFP-HA shows the 1164
expression efficiency of each sample. 1165
41
1166
Figure 3. Interaction of ATG13s with SINAT Proteins. 1167
(A) Y2H analysis showing the interaction between ATG13a/b and SINAT proteins.1168
ATG13a/b-BD and SINAT-AD (SINAT1-AD, SINAT2-AD, SINAT3-AD, 1169
SINAT4-AD, SINAT5-S1-AD, and SINAT6-AD) were co-expressed in the YH109 1170
yeast strain and selected on SD/ –Trp-Leu-His-Ade medium (–LWH). AD indicates 1171
the empty AD plasmid. 1172
(B) In-vivo Co-IP analysis showing the interaction between ATG13a and SINATs.1173
FLAG-tagged ATG13a (ATG13a-FLAG) was co-expressed with GFP-HA-tagged 1174
SINATs (GFP-SINAT1-HA, GFP-SINAT2-HA, GFP-SINAT5-S1-HA, and 1175
SINAT6-GFP-HA) in Arabidopsis protoplasts and immunoprecipitated by GFP 1176
agarose beads. 1177
(C) Truncation analysis of SINAT5 to identify the functional domain mediating the1178
ATG13a/b-SINAT5 association. Full-length SINAT5 was amplified from ecotype Ler 1179
containing a RING finger (RING), a zinc finger (ZINC), and a TRAF domain (TRAF). 1180
SINAT5-S1 and SINAT5-S2 are two alternatively spliced products produced in 1181
ecotype Col-0 and containing a TRAF domain with impaired RING or ZINC domains. 1182
△183-309 is an artificially truncated protein without the TRAF domain. Truncated1183
SINAT5 was fused to the BD domain and co-expressed with ATG13a/b-AD in yeast. 1184
Positive clones were selected on SD medium lacking Trp, Leu, His, and Ade (–LWH). 1185
AD indicate the empty AD plasmid. 1186
(D) In vivo ubiquitylation of ATG13a by SINAT1, SINAT2, SINAT5-S1, and SINAT6.1187
ATG13a-FLAG was co-expressed with GFP-SINAT1-HA, GFP-SINAT2-HA, 1188
GFP-SINAT5-S1-HA, or SINAT6-GFP-HA in Arabidopsis protoplasts for 16 h under 1189
continuous light conditions, and its ubiquitylation was detected by immunoblot 1190
analysis. 1191
(E) ATG13a protein level in the sinat1/2/3/4 mutant and SINAT1-OE line in response1192
to carbon starvation. One-week-old wild type (WT), sinat1/2/3/4 mutant, and 1193
SINAT1-OE lines were treated with darkness for 0, 24, 48, and 72 h. The blots were 1194
42
probed with anti-ATG13a-specific antibodies. 1195
(F) ATG13a accumulation following treatment with constant darkness in the sinat5 1196
sinat6 mutant and SINAT6-OE line. One-week-old WT, sinat5/6 mutant, and 1197
SINAT6-OE lines were treated with darkness for 0, 24, 48, and 72 h. The blots were 1198
probed with anti-ATG13a antibodies. 1199
Numbers on the left indicate the molecular weight (kD) of each band. The blot 1200
expression of GFP-HA shows the expression efficiency of each sample. Anti-ACTIN 1201
antibodies, or Ponceau S-stained Rubisco bands are shown below the blots to indicate 1202
the amount of protein loaded per lane. Relative intensity of each protein normalized to 1203
the loading control is shown below. hpt, hours post treatment. 1204
1205
Figure 4. Identification of ATG13a/b Ubiquitylation Sites. 1206
(A) and (B) K48-linked ubiquitylation in response to carbon starvation in 1207
ATG13a-HA (A) and ATG13b-HA (B). One-week-old transgenic lines expressing 1208
ATG13a-HA and ATG13b-HA were treated with darkness or 50 µM MG132 for 0, 12, 1209
24, and 48 h. Total protein was immunoprecipitated by HA affinity agarose beads and 1210
immunoblotted with Lys48
anti-ubiquitin-specific antibodies. 1211
(C) Alignment of ATG13a and the ATG13a ubiquitylation site mutant. The alignment 1212
was analyzed using T-Coffee website (http://tcoffee.crg.cat/apps/tcoffee/index.html). 1213
ATG13a-K1 indicates the K607R mutation, ATG13a-K2 indicates the K609R 1214
mutation, and ATG13a-K1/2 indicates both the K607 and K609 point mutations to R. 1215
(D) ATG13a levels in the ubiquitylation site mutants. HA-tagged ATG13a 1216
(ATG13a-HA), and ATG13a ubiquitylation site mutants (ATG13a-K1-HA, 1217
ATG13a-K2-HA, and ATG13a-K1/2-HA) were expressed in wild-type (WT) 1218
protoplast. Total protein was extracted after incubation for 16 h under constant light 1219
conditions, and immunoprecipitated by HA affinity agarose beads. The blots were 1220
probed with anti-HA and anti-Ub antibodies. 1221
Numbers on the left indicate the molecular weight (kD) of each band. hpt, hours post 1222
treatment. Anti-ACTIN antibodies and Ponceau S-stained membranes are shown 1223
43
below the blots to indicate the amount of protein loaded per lane. 1224
(E) Response of the ATG13a ubiquitylation mutant to nitrogen-starvation treatment.1225
One-week-old seedlings grown on MS medium were transferred to nitrogen-rich (N+) 1226
or nitrogen-deficient (N–) liquid medium for an additional 5 days before 1227
photographing and chlorophyll measurement. 1228
(F) Relative chlorophyll contents of seedlings with or without nitrogen-deficient1229
treatment shown in (E). The relative chlorophyll contents were calculated by 1230
comparing the values in N– seedlings versus N+ seedlings. 1231
(G) Phenotypes of the ATG13a ubiquitylation mutant in response to carbon starvation.1232
One-week-old WT, ATG13a-OE, ATG13a-K1/2 #1, and ATG13a-K1/2 #2 seedlings 1233
grown on MS solid medium were transferred to MS plates with sucrose (C+) or 1234
without sucrose followed by constant dark treatment (C–) for 9 d. The images were 1235
recorded after a 7-d recovery. 1236
(H) Relative chlorophyll contents of WT, ATG13a-OE, ATG13a-K1/2 #1, and1237
ATG13a-K1/2 #2 seedlings described in (G) following recovery. The relative 1238
chlorophyll contents were calculated by comparing the values of C–treated versus C+ 1239
treated seedlings. 1240
Relative chlorophyll contents are average values ± SD (n = 3) calculated from three 1241
independent experiments. For each experiment, five technical replicates pooled with 1242
at least 15 seedlings were used per genotype. Asterisks indicate significant differences 1243
from WT (a) or ATG13a-OE (b) (*P < 0.05; **P < 0.01 by One-Way ANOVA). 1244
1245
Figure 5. TRAF1a Is Required for SINAT-Mediated ubiquitylation and Degradation 1246
of ATG13a. 1247
(A) SINAT-mediated ATG13a ubiquitylation in the traf1a/b mutant. ATG13a-FLAG1248
and GFP-SINAT1-HA were transiently co-expressed in Arabidopsis protoplasts 1249
prepared from wild-type (WT) or traf1a/b plants for 16 h under constant light 1250
conditions. 1251
(B) SINAT1-associated degradation of ATG13a is dependent on TRAF1a.1252
44
ATG13a-FLAG and GFP-SINAT1-HA were transiently co-expressed in Arabidopsis 1253
protoplasts prepared from WT and traf1a/b plants for 16 h under continuous light 1254
conditions. 1255
(C) SINAT1-mediated ATG13a ubiquitylation in response to co-expression of 1256
SINAT6. ATG13a-FLAG was co-expressed with SINAT1 or SINAT1/SINAT6 in 1257
Arabidopsis protoplasts for 16 h under constant light conditions. 1258
(D) SINAT1-associated degradation of ATG13a in response to SINAT6. 1259
ATG13a-FLAG was co-expressed with GFP-SINAT1-HA in the presence of various 1260
amounts (0, 10, 20, and 30 µg) of SINAT6-GFP-HA in Arabidopsis protoplasts for 16 1261
h under continuous light conditions. 1262
The blot expression of GFP-HA shows the expression efficiency of each sample. 1263
Ponceau S-stained Rubisco bands are shown below the blots to indicate the amount of 1264
protein loaded per lane. The numbers on the left indicate the molecular mass (kD) of 1265
each size marker. Relative intensity of each protein normalized to the loading control 1266
is shown below. 1267
1268
Figure 6. Phosphorylation of TRAF1 by ATG1s. 1269
(A) Migration of TRAF1a in cells expressing ATG1 proteins. HA-tagged ATG1a, 1270
ATG1b, and ATG1c (ATG1a-HA, ATG1b-HA, and ATG1c-HA) were transiently 1271
expressed in protoplasts from TRAF1a-FLAG transgenic plants. Proteins were 1272
extracted at 16 h after expression under continuous dark conditions, and the blots 1273
were probed with anti-HA and anti-FLAG antibodies. 1274
(B) TRAF1a phosphorylation by ATG1a. HA-tagged ATG1a (ATG1a-HA) was 1275
transiently expressed in protoplasts from TRAF1a-FLAG transgenic plants. Proteins 1276
were extracted at 16 h after expression under constant dark conditions. The 1277
phosphorylation of TRAF1a was confirmed by digestion with phosphatase and 1278
phosphatase inhibitor, and the blots were probed with anti-HA and anti-FLAG 1279
antibodies. 1280
(C) Degradation of TRAF1a in the YFP-ATG1a transgenic line. One-week-old 1281
45
TRAF1a-FLAG and TRAF1a-FLAG/YFP-ATG1a seedlings were transferred to MS 1282
medium without sucrose followed by dark treatment for the indicated times. 1283
Anti-FLAG and anti-YFP were used for immunoblot analysis. The arrowhead 1284
indicates the YFP-ATG1a bands. 1285
Numbers on the left indicate the molecular weight (kD) of each band. Anti-ACTIN 1286
antibodies and Ponceau S-stained Rubisco bands are shown below the blots to 1287
indicate the amount of protein loaded per lane. hpt, hours post treatment. 1288
1289
Figure 7. Deletion of ATG1a, ATG1b, and ATG1c Confers Hypersensitivity to Carbon 1290
and Nitrogen Starvation. 1291
(A) and (B) Phenotypes of the atg1abc triple mutant and atg1abct quadruple mutant1292
in response to carbon starvation. One-week-old wild-type (WT), atg1abc, atg1abct, 1293
and atg10-1 seedlings were grown on MS solid medium for 1 week. The seedlings 1294
were transferred to MS agar with sucrose (C+) or MS agar plates without sucrose 1295
followed by constant dark treatment (C–) for 7 d. The images were recorded after a 1296
7-d recovery.1297
(C) and (D) (C) Relative chlorophyll contents and (D) survival rates of WT, atg1abc,1298
atg1abct, and atg10-1 seedlings described in (A) following recovery. The relative 1299
chlorophyll contents were calculated by comparing the values of C– treated seedlings 1300
versus C+ treated seedlings. Data are average values ± SD (n = 3) calculated from 1301
three independent experiments. For each experiment, five technical replicates pooled 1302
with 15 seedlings were used per genotype. Asterisks indicate significant differences 1303
from the wild type (*P < 0.05; **P < 0.01 by One-Way ANOVA). 1304
(E) and (F) Phenotype of the atg1abc triple mutant and atg1abct quadruple mutant in1305
response to nitrogen starvation. One-week-old WT, atg1abc, atg1abct, and atg10-1 1306
seedlings were grown on MS agar for 1 week. The seedlings were transferred to 1307
N-rich (N+) or N-deficient (N–) medium and photographed at 7 d after treatment.1308
(G) Relative chlorophyll contents of WT, atg1abc, atg1abct, and atg10-1 seedlings1309
with or without nitrogen starvation shown in (E). The relative chlorophyll contents 1310
46
were calculated by comparing the values of N– treated seedlings versus N+ treated 1311
seedlings. Data are average values ± SD (n = 4) calculated from four independent 1312
experiments. For each experiment, five technical replicates pooled with 15 seedlings 1313
were used per genotype. Asterisks indicate significant differences from the wild type 1314
(**P < 0.01 by One-Way ANOVA). 1315
(H) Images showing the onset of leaf senescence in the WT, atg1abc triple mutant,1316
atg1abct quadruple mutant, and atg10-1 mutant plants grown under normal light/dark 1317
growth conditions. Photographs were taken at 4, 5, and 6 weeks after germination. 1318
Arrows indicate senescent leaves. 1319
(I) Relative chlorophyll content of plants grown under normal light/dark conditions1320
for the indicated times in (H). The values of 4-week-old WT, atg1abc triple mutant, 1321
atg1abct quadruple mutant, and atg10-1 mutant plants were set at 100%, and the 1322
relative chlorophyll contents of WT and atg1abc and atg1abct mutant leaves at 5- and 1323
6-weeks-old were calculated accordingly. Data are average values ± SD (n = 3)1324
calculated from three independent experiments. For each experiment, 5 whole plants 1325
(technical replicates) were used per genotype. Asterisks indicate significant 1326
differences from the WT (**P < 0.01 by One-Way ANOVA). 1327
1328
Figure 8. ATG1s Are Required for ATG Protein Turnover and TRAF1 1329
Phosphorylation. 1330
(A) ATG1a, ATG13a, and ATG8 levels in the wild-type (WT) and the atg1abc and1331
atg1abct mutants after carbon starvation treatments for the indicated times. An 1332
immunoblot with anti-ACTIN antibodies is shown below the blots to indicate the 1333
amount of protein loaded per lane. hpt, hours post treatment 1334
(B) Phosphorylation and stability of TRAF1a in the WT and atg1abc mutant.1335
TRAF1a-HA and ATG1a-HA were expressed in the WT and atg1abc mutant 1336
protoplasts for 16 h under light (L) or dark conditions (D) or followed by recovery 1337
under light conditions for 6 h (R). Ponceau S-stained membranes are shown below the 1338
blots to indicate the amount of protein loaded per lane. The expression of GFP-HA 1339
47
indicates the expression efficiency of each sample. Relative intensity of TRAF1a-HA 1340
and ATG1a-HA normalized to the loading control is shown below. Numbers on the 1341
left indicate the molecular weight (kD) of each band. 1342
1343
Figure 9. Working Model for Two Distinct TRAFasomes, 1344
TRAF1s-SINAT1/SINAT2-ATG13s and TRAF1s-SINAT6-ATG13s in the Regulation 1345
of Autophagy Dynamics in Arabidopsis. 1346
In response to different nutrient signals, the RING-type E3 ligases SINAT1, SINAT2, 1347
and SINAT6 control the stability of ATG13 proteins and the dynamics of autophagy 1348
by modulating the ubiquitylation of ATG13s. Under normal conditions, TRAF1a and 1349
TRAF1b interact in planta with ATG13a and ATG13b and require the presence of 1350
SINAT1 and SINAT2 to ubiquitylate and degrade ATG13s in vivo. Under nutrient 1351
starvation conditions, SINAT6 competitively interact with ATG13 and induce 1352
autophagy. Furthermore, under starvation conditions, the ATG1 kinase phosphorylated 1353
TRAF1a and promoted its protein stability in vivo, suggesting a feedback mechanism 1354
regulating autophagy. 1355
1356
Figure 1. The ATG1 and ATG13 Proteins are Degraded through the 26S Proteasome Pathway upon Starvation and During Recovery. (A) and (B) ATG1a, ATG13a,ATG8a, and ATG7 proteinlevels in the wild-type (WT)plants upon carbon (–C; A) ornitrogen (–N; B) starvation withor without MG132. One-week-old WT seedlings weresubjected to carbon or nitrogenstarvation with or without 50µM MG132 for 0, 12, 24, and 48h. Relative intensity of eachprotein normalized to theloading control is shown below.(C) and (D) ATG13a-HA andATG13b-HA levels in responseto carbon starvation (–C; C) ornitrogen starvation (–N; D) withor without MG132. One-week-old transgenic lines expressingATG13a-HA and ATG13b-HA were treated with constantdarkness or nitrogen starvation
with or without 50 µM MG132 for 0, 12, 24, and 48 h. (E) and (F) ATG13a-HA and ATG13b-HA levels upon carbon (–C; E) or nitrogen (–N; F) starvation or during recovery(R) or in the presence of MG132 (R+MG132) for the indicated times. One-week-old transgenic lines expressingATG13a-HA and ATG13b-HA were treated with constant darkness or nitrogen starvation, and moved back to MSmedium under normal light/dark conditions in the absence or presence of 50 µM MG132 to recover for 24, 48, and 72h.Anti-ACTIN antibodies and Ponceau S-stained Rubisco bands are shown below the blots to indicate the amount ofprotein loaded per lane. Numbers on the left indicate the molecular weight (kD) of each band. hpt, hours post treatment.
Figure 2. TRAF1s Interact with ATG13a and ATG13b in vivo. (A) BiFC assay of ATG1/ATG13 proteins (ATG1a and ATG13a) and TRAF1a in Arabidopsis. The split cYFP fusionsATG13a-cYFP, ATG1a-cYFP or ATG7-cYFP and TRAF1a-nYFP were co-expressed in leaf protoplasts and incubatedfor 16 h under light or dark conditions. The TRAF1a-nYFP/ATG7-cYFP and nYFP/cYFP vectors were co-expressed asnegative controls. Confocal micrographs obtained from YFP, auto-fluorescent chlorophyll, bright-field, and mergedimages are shown. The numbers in the cells of ATG13a or ATG1a-cYFP + TRAF1a-nYFP/dark indicate the averagenumber of autophagosomes ± SD (n = 3) calculated from three independent experiments. For each experiment, 15images were used for the calculation for each coexpression combination. Bars = 10 μm.(B) In vivo CoIP assay of the association between ATG13 (ATG13a and ATG13b) and TRAF1a. HA-tagged ATG13a orATG13b (ATG13a-HA or ATG13b-HA) was transiently expressed in protoplasts from transgenic plants expressingTRAF1a-FLAG under light conditions for 16 h, and immunoprecipitated with HA affinity agarose beads.(C) In vivo CoIP assay of the interaction between TRAF1a and ATG1 (ATG1a, ATG1b, and ATG1c) proteins. HA-tagged
ATG1a, ATG1b, and ATG1c (ATG1a-HA, ATG1b-HA, and ATG1c-HA) were transiently expressed in protoplasts from transgenic plants expressing TRAF1a-FLAG for 16 h under continuous dark conditions and immunoprecipitated with HA affinity agarose beads. (D) The ubiquitylation of ATG13a in the traf1a/b mutant. ATG13a-HA was transiently expressed in Arabidopsisprotoplasts isolated from four-week-old wild-type (WT) and traf1a/b mutant plants, and the ubiquitylation of ATG13awas detected by immunoprecipitation and immunoblot analysis. Proteins were extracted at 16 h after expression underconstant light conditions and then incubated with HA affinity agarose beads. The blots were probed with anti-HA andanti-Ub antibodies.(E) The degradation of ATG13a in traf1a/b-1 and TRAF1a-FLAG plants. One-week-old wild-type, traf1a/b, andTRAF1a-FLAG plants were subjected to carbon (–C; the upper images) or nitrogen starvation (–N; the lower images)treatment for 48 h, following by recovery (R) for 48 and 72 h. The blots were probed with anti-ATG13a-specificantibodies. hpt, hours post treatment.Numbers on the left indicate the molecular weight (kD) of each band. Anti-ACTIN antibodies and Ponceau S-stainedRubisco bands are shown below the blots to indicate the amount of protein loaded per lane. The expression of GFP-HA shows the expression efficiency of each sample.
Figure 3. Interaction of ATG13s with SINAT Proteins. (A) Y2H analysis showing the interaction between ATG13a/b and SINAT proteins. ATG13a/b-BD and SINAT-AD(SINAT1-AD, SINAT2-AD, SINAT3-AD, SINAT4-AD, SINAT5-S1-AD, and SINAT6-AD) were co-expressed in theYH109 yeast strain and selected on SD/ –Trp-Leu-His-Ade medium (–LWH). AD indicates the empty AD plasmid.(B) In-vivo Co-IP analysis showing the interaction between ATG13a and SINATs. FLAG-tagged ATG13a (ATG13a-FLAG) was co-expressed with GFP-HA-tagged SINATs (GFP-SINAT1-HA, GFP-SINAT2-HA, GFP-SINAT5-S1-HA,and SINAT6-GFP-HA) in Arabidopsis protoplasts and immunoprecipitated by GFP agarose beads.(C) Truncation analysis of SINAT5 to identify the functional domain mediating the ATG13a/b-SINAT5 association.Full-length SINAT5 was amplified from ecotype Ler containing a RING finger (RING), a zinc finger (ZINC), and a
TRAF domain (TRAF). SINAT5-S1 and SINAT5-S2 are two alternatively spliced products produced in ecotype Col-0 and containing a TRAF domain with impaired RING or ZINC domains. △183-309 is an artificially truncated protein without the TRAF domain. Truncated SINAT5 was fused to the BD domain and co-expressed with ATG13a/b-AD in yeast. Positive clones were selected on SD medium lacking Trp, Leu, His, and Ade (–LWH). AD indicate the empty AD plasmid. (D) In vivo ubiquitylation of ATG13a by SINAT1, SINAT2, SINAT5-S1, and SINAT6. ATG13a-FLAG was co-expressedwith GFP-SINAT1-HA, GFP-SINAT2-HA, GFP-SINAT5-S1-HA, or SINAT6-GFP-HA in Arabidopsis protoplasts for16 h under continuous light conditions, and its ubiquitylation was detected by immunoblot analysis.(E) ATG13a protein level in the sinat1/2/3/4 mutant and SINAT1-OE line in response to carbon starvation. One-week-old wild type (WT), sinat1/2/3/4 mutant, and SINAT1-OE lines were treated with darkness for 0, 24, 48, and 72 h. Theblots were probed with anti-ATG13a-specific antibodies.(F) ATG13a accumulation following treatment with constant darkness in the sinat5 sinat6 mutant and SINAT6-OE line.One-week-old WT, sinat5/6 mutant, and SINAT6-OE lines were treated with darkness for 0, 24, 48, and 72 h. The blotswere probed with anti-ATG13a antibodies.Numbers on the left indicate the molecular weight (kD) of each band. The blot expression of GFP-HA shows theexpression efficiency of each sample. Anti-ACTIN antibodies, or Ponceau S-stained Rubisco bands are shown belowthe blots to indicate the amount of protein loaded per lane. Relative intensity of each protein normalized to the loadingcontrol is shown below. hpt, hours post treatment.
Figure 4. Identification of ATG13a/b Ubiquitylation Sites. (A) and (B) K48-linked ubiquitylation in response to carbon starvation in ATG13a-HA (A) and ATG13b-HA (B). One-week-old transgenic lines expressing ATG13a-HA and ATG13b-HA were treated with darkness or 50 µM MG132 for 0,12, 24, and 48 h. Total protein was immunoprecipitated by HA affinity agarose beads and immunoblotted with Lys48
anti-ubiquitin-specific antibodies.
(C) Alignment of ATG13a and the ATG13a ubiquitylation site mutant. The alignment was analyzed using T-Coffeewebsite (http://tcoffee.crg.cat/apps/tcoffee/index.html). ATG13a-K1 indicates the K607R mutation, ATG13a-K2indicates the K609R mutation, and ATG13a-K1/2 indicates both the K607 and K609 point mutations to R.(D) ATG13a levels in the ubiquitylation site mutants. HA-tagged ATG13a (ATG13a-HA), and ATG13a ubiquitylationsite mutants (ATG13a-K1-HA, ATG13a-K2-HA, and ATG13a-K1/2-HA) were expressed in wild-type (WT) protoplast.Total protein was extracted after incubation for 16 h under constant light conditions, and immunoprecipitated by HAaffinity agarose beads. The blots were probed with anti-HA and anti-Ub antibodies.Numbers on the left indicate the molecular weight (kD) of each band. hpt, hours post treatment. Anti-ACTIN antibodiesand Ponceau S-stained membranes are shown below the blots to indicate the amount of protein loaded per lane.(E) Response of the ATG13a ubiquitylation mutant to nitrogen-starvation treatment. One-week-old seedlings grown onMS medium were transferred to nitrogen-rich (N+) or nitrogen-deficient (N–) liquid medium for an additional 5 daysbefore photographing and chlorophyll measurement.(F) Relative chlorophyll contents of seedlings with or without nitrogen-deficient treatment shown in (E). The relativechlorophyll contents were calculated by comparing the values in N– seedlings versus N+ seedlings.(G) Phenotypes of the ATG13a ubiquitylation mutant in response to carbon starvation. One-week-old WT, ATG13a-OE,ATG13a-K1/2 #1, and ATG13a-K1/2 #2 seedlings grown on MS solid medium were transferred to MS plates withsucrose (C+) or without sucrose followed by constant dark treatment (C–) for 9 d. The images were recorded after a 7-d recovery.(H) Relative chlorophyll contents of WT, ATG13a-OE, ATG13a-K1/2 #1, and ATG13a-K1/2 #2 seedlings described in(G) following recovery. The relative chlorophyll contents were calculated by comparing the values of C–treated versusC+ treated seedlings.Relative chlorophyll contents are average values ± SD (n = 3) calculated from three independent experiments. For eachexperiment, five technical replicates pooled with at least 15 seedlings were used per genotype. Asterisks indicatesignificant differences from WT (a) or ATG13a-OE (b) (*P < 0.05; **P < 0.01 by One-Way ANOVA).
Figure 5. TRAF1a Is Required for SINAT-Mediated ubiquitylation and Degradation of ATG13a. (A) SINAT-mediated ATG13a ubiquitylation in the traf1a/b mutant. ATG13a-FLAG and GFP-SINAT1-HA weretransiently co-expressed in Arabidopsis protoplasts prepared from wild-type (WT) or traf1a/b plants for 16 h underconstant light conditions.(B) SINAT1-associated degradation of ATG13a is dependent on TRAF1a. ATG13a-FLAG and GFP-SINAT1-HA weretransiently co-expressed in Arabidopsis protoplasts prepared from WT and traf1a/b plants for 16 h under continuouslight conditions.(C) SINAT1-mediated ATG13a ubiquitylation in response to co-expression of SINAT6. ATG13a-FLAG was co-expressed with SINAT1 or SINAT1/SINAT6 in Arabidopsis protoplasts for 16 h under constant light conditions.(D) SINAT1-associated degradation of ATG13a in response to SINAT6. ATG13a-FLAG was co-expressed with GFP-SINAT1-HA in the presence of various amounts (0, 10, 20, and 30 µg) of SINAT6-GFP-HA in Arabidopsis protoplastsfor 16 h under continuous light conditions.The blot expression of GFP-HA shows the expression efficiency of each sample. Ponceau S-stained Rubisco bands areshown below the blots to indicate the amount of protein loaded per lane. The numbers on the left indicate the molecularmass (kD) of each size marker. Relative intensity of each protein normalized to the loading control is shown below.
Figure 6. Phosphorylation of TRAF1 by ATG1s. (A) Migration of TRAF1a in cellsexpressing ATG1 proteins. HA-taggedATG1a, ATG1b, and ATG1c (ATG1a-HA, ATG1b-HA, and ATG1c-HA) weretransiently expressed in protoplastsfrom TRAF1a-FLAG transgenic plants.Proteins were extracted at 16 h afterexpression under continuous darkconditions, and the blots were probedwith anti-HA and anti-FLAG antibodies.(B) TRAF1a phosphorylation byATG1a. HA-tagged ATG1a (ATG1a-HA) was transiently expressed inprotoplasts from TRAF1a-FLAGtransgenic plants. Proteins wereextracted at 16 h after expression underconstant dark conditions. Thephosphorylation of TRAF1a wasconfirmed by digestion withphosphatase and phosphatase inhibitor,and the blots were probed with anti-HA and anti-FLAG antibodies.(C) Degradation of TRAF1a in theYFP-ATG1a transgenic line. One-week-old TRAF1a-FLAG and TRAF1a-FLAG/YFP-ATG1a seedlings weretransferred to MS medium withoutsucrose followed by dark treatment forthe indicated times. Anti-FLAG andanti-YFP were used for immunoblotanalysis. The arrowhead indicates the
YFP-ATG1a bands. Numbers on the left indicate the molecular weight (kD) of each band. Anti-ACTIN antibodies and Ponceau S-stained Rubisco bands are shown below the blots to indicate the amount of protein loaded per lane. hpt, hours post treatment.
Figure 7. Deletion of ATG1a, ATG1b, and ATG1c
Confers Hypersensitivity
to Carbon and Nitrogen
Starvation. (A) and (B) Phenotypes of the atg1abc triple mutant and atg1abct quadruple mutant in response to carbon starvation. One-week-old wild-type (WT), atg1abc, atg1abct, and atg10-1 seedlings were grown on MS solid medium for 1 week. The seedlings were transferred to MS agar with sucrose (C+) or MS agar plates without sucrose followed by constant dark treatment (C–) for 7 d. The images wererecorded after a 7-drecovery.
(C) and (D) (C) Relative chlorophyll contents and (D) survival rates of WT, atg1abc, atg1abct, and atg10-1 seedlingsdescribed in (A) following recovery. The relative chlorophyll contents were calculated by comparing the values of C–treated seedlings versus C+ treated seedlings. Data are average values ± SD (n = 3) calculated from three independentexperiments. For each experiment, five technical replicates pooled with 15 seedlings were used per genotype. Asterisksindicate significant differences from the wild type (*P < 0.05; **P < 0.01 by One-Way ANOVA).(E) and (F) Phenotype of the atg1abc triple mutant and atg1abct quadruple mutant in response to nitrogen starvation.One-week-old WT, atg1abc, atg1abct, and atg10-1 seedlings were grown on MS agar for 1 week. The seedlings weretransferred to N-rich (N+) or N-deficient (N–) medium and photographed at 7 d after treatment.(G) Relative chlorophyll contents of WT, atg1abc, atg1abct, and atg10-1 seedlings with or without nitrogen starvationshown in (E). The relative chlorophyll contents were calculated by comparing the values of N– treated seedlings versusN+ treated seedlings. Data are average values ± SD (n = 4) calculated from four independent experiments. For eachexperiment, five technical replicates pooled with 15 seedlings were used per genotype. Asterisks indicate significant
differences from the wild type (**P < 0.01 by One-Way ANOVA). (H) Images showing the onset of leaf senescence in the WT, atg1abc triple mutant, atg1abct quadruple mutant, andatg10-1 mutant plants grown under normal light/dark growth conditions. Photographs were taken at 4, 5, and 6 weeksafter germination. Arrows indicate senescent leaves.(I) Relative chlorophyll content of plants grown under normal light/dark conditions for the indicated times in (H). Thevalues of 4-week-old WT, atg1abc triple mutant, atg1abct quadruple mutant, and atg10-1 mutant plants were set at100%, and the relative chlorophyll contents of WT and atg1abc and atg1abct mutant leaves at 5- and 6-weeks-old werecalculated accordingly. Data are average values ± SD (n = 3) calculated from three independent experiments. For eachexperiment, 5 whole plants (technical replicates) were used per genotype. Asterisks indicate significant differences fromthe WT (**P < 0.01 by One-Way ANOVA).
Figure 8. ATG1s Are Required for ATG Protein Turnover and TRAF1 Phosphorylation. (A) ATG1a, ATG13a, and ATG8 levels in the wild-type (WT) and the atg1abc and atg1abct mutants after carbonstarvation treatments for the indicated times. An immunoblot with anti-ACTIN antibodies is shown below the blots toindicate the amount of protein loaded per lane. hpt, hours post treatment(B) Phosphorylation and stability of TRAF1a in the WT and atg1abc mutant. TRAF1a-HA and ATG1a-HA wereexpressed in the WT and atg1abc mutant protoplasts for 16 h under light (L) or dark conditions (D) or followed byrecovery under light conditions for 6 h (R). Ponceau S-stained membranes are shown below the blots to indicate theamount of protein loaded per lane. The expression of GFP-HA indicates the expression efficiency of each sample.Relative intensity of TRAF1a-HA and ATG1a-HA normalized to the loading control is shown below. Numbers on theleft indicate the molecular weight (kD) of each band.
Figure 9. Working Model for Two Distinct TRAFasomes, TRAF1s-SINAT1/SINAT2-ATG13s and TRAF1s-SINAT6-ATG13s in the Regulation of Autophagy Dynamics in Arabidopsis. In response to different nutrient signals, the RING-type E3 ligases SINAT1, SINAT2, and SINAT6 control the stability of ATG13 proteins and the dynamics of autophagy by modulating the ubiquitylation of ATG13s. Under normal conditions, TRAF1a and TRAF1b interact in planta with ATG13a and ATG13b and require the presence of SINAT1 and SINAT2 to ubiquitylate and degrade ATG13s in vivo. Under nutrient starvation conditions, SINAT6 competitively interact with ATG13 and induce autophagy. Furthermore, under starvation conditions, the ATG1 kinase phosphorylated TRAF1a and promoted its protein stability in vivo, suggesting a feedback mechanism regulating autophagy.
DOI 10.1105/tpc.19.00413; originally published online November 15, 2019;Plant Cell
Shi XiaoHua Qi, Juan Li, Fan-Nv Xia, Jin-Yu Chen, Xue Lei, Mu-Qian Han, Li-Juan Xie, Qing-Ming Zhou and
of ATG13Arabidopsis SINAT Proteins Control Autophagy by Mediating Ubiquitylation and Degradation
This information is current as of March 16, 2020
Supplemental Data /content/suppl/2019/11/14/tpc.19.00413.DC1.html
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