# LGG-1/GABARAP lipidation is not required for autophagy and development in Caenorhabditis elegans

Romane Leboutet, Céline Largeau, Leonie Müller, Magali Prigent, Grégoire Quinet, Manuel S Rodriguez, Marie-Hélène Cuif, Thorsten Hoppe, Emmanuel Culetto, Christophe Lefebvre, et al.

► To cite this version:

Romane Leboutet, Céline Largeau, Leonie Müller, Magali Prigent, Grégoire Quinet, et al. LGG-1/GABARAP lipidation is not required for autophagy and development in Caenorhabditis elegans. eLife, inPress, 10.7554/eLife.85748. hal-04157249

HAL Id: hal-04157249

https://hal.science/hal-04157249

Submitted on 10 Jul 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
LGG-1/GABARAP lipidation is not required for autophagy
and development in *Caenorhabditis elegans*

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

Romane Leboutet¹,², Céline Largeau¹,², Leonie Müller³, Magali Prigent¹,², Grégoire Quinet⁴,
Manuel S. Rodriguez⁴, Marie-Hélène Cuif¹,², Thorsten Hoppe³,⁵, Emmanuel Culetto¹,²,
Christophe Lefebvre¹,² and Renaud Legouis¹,²*

1) Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC),
91198, Gif-sur-Yvette, France.

2) INSERM U1280, 91198, Gif-sur-Yvette, France.

3) Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany.

4) Laboratoire de Chimie de Coordination (LCC)-CNRS, UPS, 31400, Toulouse, France.

5) Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital of Cologne, Cologne, Germany.

*Correspondence: [renaud.legouis@i2bc.paris-saclay.fr](mailto:renaud.legouis@i2bc.paris-saclay.fr)

20

21

22

**Abstract**

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

The ubiquitin-like proteins Atg8/LC3/GABARAP are required for multiple steps of autophagy, such as initiation, cargo recognition and engulfment, vesicle closure and degradation. Most of LC3/GABARAP functions are considered dependent on their post-translational modifications and their association with the autophagosome membrane through a conjugation to a lipid, the phosphatidyl-ethanolamine. Contrarily to mammals, *C. elegans* possesses single homologs of LC3 and GABARAP families, named LGG-2 and LGG-1. Using site-directed mutagenesis, we inhibited the conjugation of LGG-1 to the autophagosome membrane and generated mutants that express only cytosolic forms, either the precursor or the cleaved protein. LGG-1 is an essential gene for autophagy and development in *C. elegans*, but we discovered that its functions could be fully achieved independently of its localization to the membrane. This study reveals an essential role for the cleaved form of LGG-1 in autophagy but also in an autophagy-independent embryonic function. Our data question the use of lipidated GABARAP/LC3 as the main marker of autophagic flux and highlight the high plasticity of autophagy.

**Keywords:** Atg8, LC3, GABARAP, lipidation, ubiquitin-like, di-glycine, *C. elegans*, development, CRISPR-Cas9, mitophagy, aggrephagy
41

## 42 Introduction

43

Macroautophagy is a highly dynamic vesicular degradation system that sequesters intracellular components in double membrane autophagosomes and delivers them to the lysosome (Klionsky et al., 2021). Upon induction, the successive recruitment of protein complexes triggers the phosphorylation of lipids, the transfer of lipids from various reservoirs, the recognition of cargoes, the tethering and the fusion (Galluzzi et al, 2017; Nakatogawa, 2020). One of the key players is the ubiquitin-like protein Atg8, which in yeast is required for several steps during autophagy, such as initiation, cargo recognition and engulfment, and vesicle closure (Kirisako et al., 2000; Knorr et al., 2014; Kraft et al., 2012; Nakatogawa et al., 2007; Xie et al., 2008). There are seven isoforms of Atg8 homologs in humans defining two families, the MAP-LC3 (abbreviated as LC3A-a, LC3A-b, LC3B, LC3C) and the GABARAP (GABARAP, GABARAPL1, GABARAPL2) (Shpilka et al., 2011). LC3/GABARAP proteins could have both similar and very specific functions during the autophagic flux (Alemu et al., 2012; Grunwald et al., 2020; Joachim et al., 2015; Lystad et al., 2014; Pankiv et al., 2007; Weidberg et al., 2010). LC3/GABARAP proteins can bind numerous proteins through specific motifs (LIR, LC3 interacting Region) and their interactomes are only partially overlapping (Behrends et al., 2010).

The pleiotropy of Atg8/LC3/GABARAP proteins in multiple cellular processes (Galluzzi and Green, 2019; Schaaf et al., 2016) entangles the study of their specific functions in human (Nguyen et al., 2016). Moreover, a series of post-translational modifications, similar to the ubiquitin conjugation, is involved in the membrane targeting of Atg8/LC3/GABARAP proteins. These proteins are initially synthesized as a precursor (P), then cleaved at their C-terminus after the invariant Glycine 116 (form I), and eventually conjugated to phosphatidylethanolamine (form II) at the membrane of autophagosomes (Figure 1A) (Kabeya et al., 2004, 2000; Scherz-Shouval et al., 2003). Structural analyses have shown that LC3/GABARAP can adopt an open or close configuration (Coyle et al., 2002). In addition, several other post-translational modifications have been reported, like phosphorylation (Cherra et al., 2010; Herhaus et al., 2020; Wilkinson et al., 2015), deacetylation (Huang et al., 2015) ubiquitination (Joachim et al., 2017) and oligomerization (Chen et al., 2007; Coyle et al., 2002), whose effects on LC3/GABARAP function and localization are largely unknown. The subcellular localization of Atg8/LC3/GABARAP proteins is either diffuse in the cytosol and nucleus, or associated to the membrane of various compartments or the cytoskeleton (Schaaf et al., 2016).

Due to such a versatile and pleiotropic repertoire, it is of particular interest to address the level of redundancy and specificity, including tissue specificity, of the various LC3/GABARAP members, and the possible functions of the forms P and I. In the nematode *Caenorhabditis elegans*, the presence of single homologs of LC3 and GABARAP, called respectively LGG-2 and LGG-1, represents an ideal situation to characterize their multiple functions (Chen et al., 2017; Leboutet et al., 2020).
The structure of LGG-1/GABARAP and LGG-2/LC3 is highly conserved (Wu et al., 2015) and both proteins are involved in autophagy processes during development, longevity and stress (Alberti et al., 2010; Chang et al., 2017; Chen et al., 2021; Meléndez et al., 2003; Samokhvalov et al., 2008). In particular, the elimination of paternal mitochondria upon fertilization, also called allophagy (Al Rawi et al., 2011; Sato and Sato, 2011), has become a paradigm for dissecting the molecular mechanisms of selective autophagy (Djeddi et al., 2015; Zhou et al., 2016). Genetic analyses indicated that LGG-1 and LGG-2 do not have similar functions in autophagy (Alberti et al., 2010; Jenzer et al., 2019; Manil-Ségalen et al., 2014; Wu et al., 2015). During allophagy, LGG-1 is important for the recognition of ubiquitinated cargoes through interaction with the specific receptor ALLO-1 (Sato et al., 2018) and the formation of autophagosomes, whereas LGG-2 is involved in their maturation into autolysosomes and trafficking (Djeddi et al., 2015; Manil-Ségalen et al., 2014). LGG-1 and LGG-2 are also differentially involved during physiological aggrephagy in embryo, with temporal-specific and cargo-specific functions (Wu et al., 2015). Based on the presence of LGG-1 and LGG-2, three populations of autophagosomes have been described in *C. elegans* embryo: the major part are LGG-1 only, but LGG-2 only and double positives autophagosomes are also present (Manil-Ségalen et al., 2014; Wu et al., 2015). Moreover, LGG-1 is essential for embryonic and larval development, while LGG-2 is dispensable.
Using CRISPR-Cas9 editing, we investigated the functions of the non-lipidated cytosolic forms of LGG-1/GABARAP for bulk autophagy, mitophagy and aggrephagy, but also during starvation and longevity as well as apoptotic cell engulfment and morphogenesis. Here, we demonstrate that the non-lipidated form (LGG-1 I), but not the precursor form (LGG-1 P), is sufficient to maintain LGG-1 functions during development and aging. The cleavage of LGG-1 into form I is essential for autophagosome initiation and biogenesis while form II is involved in cargo recognition and autophagosome degradation.

## Results

### The G116G117 di-Glycine motif is a substrate for cleavage of LGG-1 precursor

The LGG-1 protein is highly conserved from residue 1 to residue 116, sharing 92% and 74% similarity with the human GABARAP and the yeast Atg8, respectively (Manil-Ségalen et al., 2014). However, the GEVEKKE C-terminus of LGG-1 is unusual by its length and the presence of a non-conserved glycine residue in position 117 (Figure 1B, Figure 1- figure supplement 1). As consistent with other *Caenorhabditis* species as well as several nematodes and arthropods, the presence of a C-terminal di-glycine is reminiscent of other ubiquitin-like proteins such as SUMO and Nedd8 (Cappadocia and Lima, 2018; Jentsch and Pyrowolakis, 2000). These specificities raise the possibility that the C-terminus could confer particular functions to the precursor and the cleaved form.

To analyze the functions of LGG-1 P and LGG-1 I, a CRISPR-Cas9 approach was used to substitute the conserved glycine 116 by an alanine, and to generate three specific *Igg-1*
123 mutants with various C-terminus (Figure 1B). In theory, both *Igg-1(G116A)* and *Igg-1(G116AG117A)* mutants were expected to accumulate a P form due to the blockage of its cleavage by ATG-4 (Wu et al., 2012). Alternatively, the *Igg-1(G116AG117*)* mutant should produce a form I. Five supplementary *Igg-1* frameshift mutants were isolated during the CRISPR experiments, resulting in deletion/insertion at the C-terminus (Figure 1B and Figure 1- figure supplement 1). Among them, *Igg-1(ΔC100-123)* and *Igg-1(ΔC112-123)* have been used in the present study. The allele *Igg-1(tm3489)*, which deletes 48% of the open reading frame, was used as a negative control (Manil-Ségalen et al., 2014) as it is considered as a null mutant, and thereafter noted *Igg-1(Δ)*.

132 To assess whether *Igg-1(G116A)*, *Igg-1(G116AG117A)* and *Igg-1(G116AG117*)* alleles code for a precursor and form I, respectively, we performed a western blot analysis with two different LGG-1 antibodies (Al Rawi et al., 2011; Springhorn and Hoppe, 2019)(Figure 1C). In basal conditions the wild-type LGG-1 was mainly present as form I (13.9 kDa) with a low amount of the faster migrating form II and no detectable precursor (14.8 kDa)(Figure 1C), while no band was observed in the allele *Igg-1(tm3489)* confirming that it is a bona fide null mutant. While the *Igg-1(G116AG117*)* mutant presented a major form I, the *Igg-1(G116A)* mutant accumulated both the expected precursor form and form I. This indicated that the cleavage of the LGG-1(G116A) precursor was still present although less efficient. In both mutants, an unexpected minor form was observed migrating differently from the lipidated form II, which was no longer detected. The *Igg-1(G116AG117A)* mutant accumulated the precursor form (96% of the protein) indicating that the cleavage observed in the LGG-1(G116A) was dependent on the presence of a second glycine in position 117.

145 The respective protein substitutions were further confirmed by mass spectrometry analyses after affinity purification of LGG-1(G116A) and LGG-1(G116AG117*) (Figure 1- figure supplement 2). The identification of C-terminal peptides validated the expected precursor form in LGG-1(G116A) and its cleavage after A116, and confirmed A116 as the last residue in LGG-1(G116AG117*). These latter forms are called hereafter "cleaved form" and "truncated form", respectively.

151

152 Glycine 116 is essential for lipidation of LGG-1 after cleavage

153 To confirm western blot analyses, we next performed immunofluorescence in the embryo to analyze the subcellular localization of LGG-1 protein from the various alleles. At the 1 cell-stage and around 100 cells-stage, two selective autophagy processes have been well characterized, removing paternal mitochondria and maternal aggregates, respectively (Al Rawi et al., 2011; Sato and Sato, 2011; Zhang et al., 2009). The punctate staining, observed in the wild-type animals (Figure 1D) with two independent anti-LGG-1 antibodies, was characteristic for the autophagosomes formed during each process, and was absent in the *Igg-1(Δ)* mutant (Figure 1E). The five mutants *Igg-1(G116A)*, *Igg-1(G116AG117*)*, *Igg-1(G116AG117A)*, *Igg-1(ΔC100-123)* and *Igg-1(ΔC112-123)* presented no puncta but a diffuse cytosolic staining. (Figure 1F-K), indicating that neither the precursor nor the form I are able to conjugate to the autophagosome membrane. The increase of the diffuse signal in *Igg-
164 1(G116A) and lgg-1(G116AG117*) embryos (Figure 1L) suggests that the protein is less degraded in these mutants. Moreover, no LGG-1(G116A) puncta were observed after depleting the tethering factor EPG-5 compared to the strong accumulation of puncta in LGG-1(wt) (Figure 1- figure supplement 2) (Tian et al., 2010).

168 We performed cellular fractionation of membrane vesicles to test whether LGG-1(G116A) and LGG-1(G116AG117*) are associated with autophagosomes. Compared with ER resident 169 SEL-1 or ER-associated CDC-48, the LGG-1(wt) protein was detected in both the cytosolic 170 fraction and the membrane pellet and could only be extracted with high salt or the 171 detergent Triton X-100. In contrast to LGG-1(wt), both LGG-1(G116A) and LGG-1(G116AG117*) were absent in the membrane pellet fraction (Figure 1M), suggesting 172 defective lipidation of both LGG-1 mutant proteins. In an alternative approach, we observed 173 the localization of overexpressed GFP::LGG-1 and GFP::LGG-1(G116A) (Manil-Ségalen et al., 174 2014) after induction of autophagic flux by acute heat stress (aHS) (Chen et al., 2021; Kumsta 175 et al., 2017). After aHS, GFP::LGG-1 formed numerous puncta that further accumulated 176 when autolysosome formation was impaired by depletion of RAB-7 or EPG-5 (Figure 1- figure 177 178 supplement 3). In contrast, in GFP::LGG-1(G116A), puncta were not reduced under any 179 condition. Electron microscopy and immunogold labeling confirmed that GFP::LGG-1 was 180 frequently detected to autophagosome membranes (Manil-Ségalen et al., 2014), whereas 181 GFP::LGG-1(G116A) was rarely detected in association with autophagosomes and in these 182 rare cases was predominantly localized in the lumen (Figure 1- figure supplement 3). Taken 183 together, these results suggest that the G116A mutation does not allow conjugation of LGG-1 184 to the autophagosome membrane despite its cleavage. LGG-1(G116AG117A) represents 185 only a precursor form and LGG-1(G116AG117*) only a truncated form, whereas LGG-1(G116A) 186 produces both a precursor and a cleaved form. This allele series provides an ideal 187 situation to study the respective roles of the precursor and form I in absence of lipidated 188 form II. 189

190

191 The essential function of LGG-1 during development is dependent of its cleavage but not 192 its conjugation

193 The developmental phenotypes of the mutants lgg-1(G116A), lgg-1(G116AG117A) and lgg-1(G116AG117*) were explored in embryo, larvae and adults and compared with lgg-1(Δ) and 194 wild-type animals (Figure 2). We confirmed that lgg-1(Δ) homozygous animals present a 195 massive lethality during late embryogenesis or first larval stage (Figure 2B, H) (Manil-Ségalen 196 et al., 2014). However, few escapers, circa 8% of the progeny, were able to reach adulthood 197 and reproduce, allowing to maintain a lgg-1(Δ) homozygous population. 198

199 Neither lgg-1(G116A) nor lgg-1(G116AG117*) homozygous animals presented any 200 observable defect in development (Figure 2C, D, H) or adulthood and they reproduced at a 201 similar rate compared to wild-type animals (Figure 2I). In contrast, lgg-1(G116AG117A) and 202 the five independent mutants harboring various deletions and frameshifts of the C-terminus 203 presented a very strong lethality with the characteristic embryonic phenotype of lgg-1(Δ) 204 animals (Figure 2E-H). Among them lgg-1(Δ112-123) presented a premature stop codon at
205 position 112 and two others a frameshift in position 114 leading to an extension of the C-
terminus (Figure 2 and Figure 1- figure supplement 1).
206

207 These data indicate that the cleaved LGG-1(G116A) and the truncated LGG-1(G116AG117*)
208 forms, but not the precursor, are sufficient to recapitulate the normal development and
209 viability, independently of membrane conjugation. These data suggest that cleavage of the
210 C-terminus is necessary for LGG-1 developmental functions.
211

212 Autophagy is functional in LGG-1(G116A)

213 To address the functionality of LGG-1 precursor and form I, we analyzed autophagy-related
214 processes that have been well characterized during *C. elegans* life cycle (Leboutet *et al.*,
215 2020). Selective autophagy was studied in the early embryo, where a stereotyped mitophagy
216 process occurs. The degradation of selective cargos was observed in live embryos using
217 specific labeling of the paternal mitochondria (HSP-6::GFP and mitoTracker, Figure 3A-F and
218 Figure 3- figure supplement 1). In *lgg-1(Δ)* animals, the cargos accumulated while they were
219 degraded in the wild-type situation. In *lgg-1(G116A)*, but neither in *lgg-1(G116AG117*)* nor
220 in *lgg-1(G116AG117A)* mutants, paternal mitochondria were degraded, suggesting that the
221 LGG-1(G116A) protein maintained autophagic activity.
222

222 Bulk autophagy was then studied by starvation of the first stage larvae (Figure 3G). While
223 *lgg-1(G116AG117*)* and *lgg-1(G116AG117A)* mutants displayed a marked decrease of
224 survival, *lgg-1(G116A)* mutants showed no difference compared the wild-type animals.
225 Moreover, the longevity of adults, which depends on bulk autophagy, was similar for *lgg-
226 1(G116A)* and wild-type animals (Figure 3H), but strongly reduced for *lgg-1(G116AG117*)*
227 mutants.
228

228 The autophagic capacity of LGG-1(G116A) protein, but not LGG-1(G116AG117*) or LGG-
229 1(G116AG117A), was further documented by the elimination of apoptotic corpses in the
230 embryo (Figure 3I, and Figure 3- figure supplement 1) (Jenzer et al., 2019).
231

231 Overall, these data demonstrate that, despite its defect to localize to autophagosomes, LGG-
232 1(G116A) achieves both selective and bulk autophagy during physiological and stress
233 conditions. This is the first *in vivo* evidence that the autophagy functions of LGG-1/GABARAP
234 can be uncoupled from its membrane conjugation. The non-functionality of LGG-
235 1(G116AG117A) suggests that the precursor form is not responsible of LGG-1(G116A)
236 autophagy activity. Despite an identical protein sequence, the truncated LGG-
237 1(G116AG117*) is not functional in autophagy, indicating that the cleavage of the C-
238 terminus from the precursor is essential for the functionality of LGG-1(G116A). Moreover,
239 the normal development of *lgg-1(G116AG117*)* animals demonstrates that the
240 developmental functions of LGG-1 are independent of its autophagic functions. Interestingly,
241 the expression in *S. cerevisiae* of LGG-1(wt) and LGG-1(G116A), but not LGG-1(G116AG117*),
242 slightly improved the nitrogen starvation survival of *atg8Δ* mutant (Supplementary data and
243 Figure 3- figure supplement 2), suggesting that the LGG-1(G116A) retains a partial autophagy
244 functionality in the yeast.
245

245
246 Autophagy but not developmental functions of LGG-1(G116A) partially depends on LGG-2

247 Our previous study has shown a partial redundancy of LGG-1 and LGG-2 during starvation
248 survival, and longevity (Alberti et al., 2010), which raises the possibility of functional
249 compensation of lgg-1(G116A) by LGG-2. To test this possibility, we used the large deletion
250 mutant lgg-2(tm5755), which is considered as a null (Manil-Ségalen et al., 2014), and
251 constructed the double mutant strains lgg-1(G116A); lgg-2(tm5755) and lgg-
252 1(G116AG117*); lgg-2(tm5755).

253 Similar to the single mutants lgg-1(G116A) and lgg-2(tm5755), the double mutant lgg-1(G116A); lgg-2(tm5755) animals were viable and presented no morphological defect (Figure 4A-F). These data indicate that the correct development of lgg-1(G116A) is not due to a compensative mechanism involving lgg-2.

257 Next, we analyzed the autophagy functions in lgg-1(G116A); lgg-2(tm5755) animals. If LGG-2
258 compensates for LGG-1(G116A) in autophagy, lgg-1(G116A); lgg-2(tm5755) animals should
259 behave similarly to lgg-1(G116AG117*); lgg-2(tm5755) (of note lgg-1(Δ); lgg-2(tm5755)
260 animals are not viable). The lgg-1(G116A); lgg-2(tm5755) animals presented a decrease for
261 both survival to starvation and longevity compared to lgg-1(G116A) single mutant. However,
262 they survived better than lgg-1(G116AG117*); lgg-2(tm5755) animals (Figure 4G, H). These
263 results indicate that the functionality of LGG-1(G116A) in bulk autophagy partially relies on
264 LGG-2. Selective autophagy during early embryogenesis was then quantitatively analyzed in
265 the double mutant strains (Figure 4I-K). Surprisingly, paternal mitochondria were eliminated
266 in the lgg-1(G116A); lgg-2(tm5755) animals indicating that LGG-1(G116A) was sufficient for
267 the allophagy process. This suggests that paternal mitochondria could be degraded by
268 autophagosomes devoid of both LGG-1 and LGG-2. However, a delay in the degradation was
269 observed compared to lgg-1(G116A) animals suggesting that the autophagy flux is reduced.
270 These results revealed a partial redundancy between LGG-1 and LGG-2 in autophagy, but
271 demonstrated at the same time that LGG-1(G116A) fulfills developmental functions and
272 maintains some autophagy activity independent of LGG-2.

273 Interestingly, this detailed analysis also revealed a slight delay in the elimination of paternal
274 mitochondria in lgg-1(G116A) animals compared to wild-type (Figure 4K). Although the
275 cleaved LGG-1 is sufficient for autophagy, this observation suggests that loss of membrane
276 targeting could affect the dynamics of autophagy flux.

277
278 The degradation of autophagosomes is delayed in LGG-1(G116A)

279 The autophagic flux and the dynamics of autophagosome formation were compared
280 between lgg-1(G116A), lgg-1(G116AG117*) and lgg-1(Δ) animals. We first focused on the
281 early embryo where the autophagy process is stereotyped and the nature of the cargos and
282 the timing of degradation are well characterized. Moreover, the autophagosomes
283 sequestering the paternal mitochondria were clustered and positive for LGG-2 (Figure
284 5A)(Manil-Ségalen et al., 2014). In lgg-1(Δ) mutant, LGG-2 autophagosomes were not
285 detected as a cluster but were spread out in the whole embryo as single puncta that
286 persisted after the 15-cells stage (Figure 5B, E). This indicated that individual LGG-2
structures could be formed in absence of LGG-1, but were not correctly localized and not
degraded properly, presumably because of the role of LGG-1 in cargo recognition (Sato et al.,
2018) and of its latter involvement in the maturation of autophagosomes, respectively. The
pattern of LGG-2 was somehow different in *Igg-1(G116A)* and *Igg-1(G116AG117*)* mutants,
forming sparse structures of heterogeneous size, which persisted longer (Figure 5C-F). These
data suggested that the cleaved and the truncated LGG-1 could both promote the
recruitment of LGG-2 to autophagic structures, but display an altered autophagic flux. The
analysis of the colocalization between paternal mitochondria and LGG-2 did not reveal an
increase in *Igg-1(G116A)* or *Igg-1(G116AG117*)* mutants (Figure 5G-J and Figure 5- figure
supplement 1). These data suggested that the elimination of paternal mitochondria in *Igg-
1(G116A)* animals was not due to the enhanced recruitment of LGG-2. A western blot
analysis of worm lysates indicated that there was no increase of LGG-2 expression in *Igg-
1(Δ)*, *Igg-1(G116A)* and *Igg-1(G116AG117*)* mutants (Figure 5K).

The autophagic structures in *Igg-1(G116A)* and *Igg-1(G116AG117*)* embryos were further
characterized by electron microscopy and compared with wild-type and *Igg-1(Δ)* mutant
embryos (Figure 6). In wild-type animals, autophagosomes containing cytoplasmic materials
(referred as type 1) and the characteristic paternal mitochondria (Zhou et al., 2016) were
observed in early embryos (Figure 6A). At that stage, rare autophagosomes containing
partially degraded material were present (referred as type 2). As expected, almost no
autophagosome was observed in *Igg-1(Δ)* embryos and paternal mitochondria were non-
sequestered (Figure 6B). In *Igg-1(G116A)* embryos, the numbers of type 1 and type 2
autophagosomal structures increased. The autophagosomes appeared to be closed and
contained various cellular materials and membrane compartments (Figure 6C-E). This
confirmed that LGG-1(G116A) was sufficient to form functional autophagosomes but with
delayed degradation. On the other hand, *Igg-1(G116AG117*)* embryos presented non-
sequestered paternal mitochondria (Figure 6F) and multi-lamellar structures containing
cytoplasm but no membrane organelles (type 3 Figure 6G, K). The analysis of the double
mutant strains *Igg-1(G116A)*; *Igg-2(tm5755)* and *Igg-1(G116AG117*); *Igg-2(tm5755)*
revealed the presence of types 1 and 2 autophagosomes, but less frequent than in the single
*Igg-1* mutants (Figure 6H-K). This data confirmed that LGG-1(G116A) alone was able to
initiate the formation of autophagosomes but less efficiently in absence of LGG-2. Type 3
structures were only observed in *Igg-1(G116AG117*)* and *Igg-1(G116AG117*); *Igg-
2(tm5755)*, suggesting a neomorphic function of the truncated LGG-1(G116AG117*) protein
that induced a non-functional compartment.

Altogether, these data indicate that the cleaved, but not the truncated, LGG-1 form I is able
to form functional autophagosomes with a delayed degradation.

The lipidated LGG-1 is involved in the coordination between cargo recognition and
autophagosome biogenesis.

To better understand the function of LGG-1 form I during autophagy flux, we next analyzed a
developmental aggrephagy process (Figure 7). The Zhang lab has demonstrated that
aggregate-prone proteins are degraded through autophagy in *C. elegans* embryo through liquid-liquid phase separation promoted by the receptor SEPA-1 and regulated by the scaffolding protein EPG-2 (Lu et al., 2011; Tian et al., 2010; Wu et al., 2015; Zhang et al., 2018, 2009). Initiation and elongation of autophagosomes were analyzed by quantifying the colocalization between ATG-18/WIPI2 and LGG-2 (Figure 7A-E) during autophagosome formation. ATG-18, the worm homolog of the omegasome marker WIPI2 (Polson et al., 2010), acts at an early step of biogenesis (Lu et al., 2011). Puncta labelled with ATG-18 only, both ATG-18 and LGG-2, or LGG-2 only were considered as omegasomes, phagophores, and autophagosomes, respectively. In *Igg-1(RNAi)* animals the number of omegasomes increased while the proportion of phagophore decreased compared to the wild-type embryos (Figure 7A, B, E). This indicates that the initiation of autophagy was triggered in absence of LGG-1, but the biogenesis of autophagosome was defective. *Igg-1(G116A)* animals showed no difference with the wild-type (Figure 7C, E) supporting that both initiation and phagophore extension are normal with the cleaved LGG-1. Similar to the *Igg-1(RNAi)*, *Igg-1(G116AG117)* animals were defective in the phagophore extension (Figure 7D, E). These data confirmed that the cleaved LGG-1(G116A), but not the truncated LGG-1(G116AG117*), is functional for the early step of autophagosome biogenesis. Moreover, RNAi depletion demonstrated that the function of LGG-1(G116A) in aggrephagy pathway was dependent on UNC-51/Ulk1 and the scaffolding protein EPG-2 (Supplementary data and Figure 7- figure supplement 1).

Quantification of SEPA-1::GFP in late embryo showed that *Igg-1(G116A)* mutant was able to perform aggrephagy but not *Igg-1(G116AG117)* or *Igg-1(G116AG117A)* mutants (Figure 7F-K). However, the elimination was decreased compared to wild-type confirming that LGG-1(G116A) was less efficient for selective cargo degradation.

Finally, the interactions between cargoes and autophagosomes were studied in *Igg-1(G116A)* mutant and wild-type embryos by analyzing the colocalization between SEPA-1 and LGG-1 or LGG-2. In wild-type embryos, immunofluorescence analyses showed the presence of LGG-1 and LGG-2 autophagosomes in contact with SEPA-1 aggregates (Figure 7L, N). In *Igg-1(G116A)* embryos, LGG-2 positive autophagosomes were observed but no LGG-1 dots, in line with the absence of lipidation (Figure 7M, O). A part of LGG-2 puncta was present close to SEPA-1 aggregates, however, they were less numerous and the overlap between LGG-2 and SEPA-1 signals was weaker (Figure 7P). These data suggested that LGG-1(G116A) was able to maintain the function of LGG-1 for initiation and extension of autophagosomes but was partially deficient for cargo sequestering.

Altogether, the analyses of LGG-1(G116A) indicate that many of the functions of LGG-1 in autophagy can be achieved by the cleaved, non-lipidated form I. However, the lipidation of LGG-1 appears to be important for the coordination between cargo recognition and autophagosome biogenesis and for the correct degradation of the autophagosome.

## Discussion
369 The most surprising result of this study is the discovery that LGG-1(G116A) is functional for 370 many autophagy processes, covering physiological or stress conditions and selective or bulk 371 autophagy. To our knowledge, it is the first report demonstrating that different autophagy 372 processes are fully achieved in vivo in a non-lipidated LC3/GABARAP mutant. In cultured 373 cells, an elegant CRISPR strategy allowed to knock out together the six LC3/GABARAP 374 homologs, but point mutations of the conserved glycine have not been reported (Nguyen et 375 al., 2016). Most of the studies on the terminal glycine used transgenic overexpression 376 constructs (Chen et al., 2007; Kabeya et al., 2004). Interestingly, one study reported that part 377 of the autophagy functions of GABARAPL1 is independent of its lipidation (Poillet-Perez et 378 al., 2017). Several studies have used mutations in the conjugation machinery (Atg3, Atg5, 379 Atg7) or the Atg4 protease to analyze the role of the form I (Hill et al., 2019; Hirata et al., 380 2017; Nishida et al., 2009; Ohnstad et al., 2020; Vujić et al., 2021). A non-canonical 381 autophagy has been reported in Atg5, Atg7 mutants (Nishida et al., 2009), but blocking the 382 conjugation system presumably affects all LC3/GABARAP homologs. Moreover, the presence 383 of four homologs of Atg4 in mammals, which specificity versus LC3/GABARAP is unknown, 384 and the dual role in the cleavage of the precursor and the delipidation entangle the analysis 385 of the phenotypes.

386 Our data show no evidence for an intrinsic function of the LGG-1 precursor but the 387 importance of its active cleavage. This finding is not surprising because in many species the 388 Atg8 precursor is not detected, suggesting that the cleavage occurs very soon after or even 389 during translation. Moreover, phylogenetic analyses of LC3/GABARAP show no conservation 390 in sequence and length of the C-terminus but the presence of at least one residue after the 391 conserved G116. The hypothesis of a selective constraint on the cleavage but not on the C- 392 terminus sequence could explain the persistence of a precursor form. Further studies are 393 necessary to clarify the precise implication of the di-glycine G116G117 in the process.

394 Albeit a similar sequence, the difference of functionality between the cleaved LGG-1(G116A) 395 and the truncated LGG-1(G116AG117*) suggests that the cleavage allows a first level of 396 specificity for LGG-1 functions. The normal development of lgg-1(G116AG117*) animals is 397 the first evidence that LGG-1 function in development relies on the cleavage but is 398 independent of autophagy and conjugation. Our results could explain the embryonic 399 lethality reported upon depletion of the two Atg4 homologs precursors in C. elegans (Wu et 400 al., 2012). While the cleavage is sufficient for developmental functions, autophagy functions 401 of LGG-1 form I seem to require a further modification to be efficient. Our data suggest that 402 this modification is dependent on and possibly associated to the cleavage. The presence of a 403 new minority band for LGG-1(G116A) could reflect an intermediary transient processing 404 state but should not correspond to a functional form because it was also detected for LGG- 405 1(G116AG117*) and LGG-1(G116AG117A).

406 Our observations in yeast also support an autophagy independent function of Atg8 form I in 407 vacuolar shaping. Non-autophagic functions for LC3/GABARAP have been identified in yeast 408 and higher eukaryotes (Ishii et al., 2019; Liu et al., 2018; Schaaf et al., 2016; Wesch et al., 409 2020), but the roles of the cytosolic forms are poorly documented especially in the context
410 of the development. The two Atg8 homologs of drosophila are involved in several
departmental processes independently of canonical lipidation (Chang et al., 2013) or
411 autophagy (Jipa et al., 2020). They are highly similar and both correspond to GABARAP
412 homologs (Manil-Ségalen et al., 2014). It is possible that duplication of Atg8 during evolution
413 allowed the acquisition of specific developmental functions by GABARAP proteins but
414 reports in apicomplex parasites (Lévêque et al., 2015; Mizushima and Sahani, 2014) rather
415 support a non-autophagy ancestral function of Atg8.
416

417 The major goal of this study was to bring new insights concerning the implication of LGG-1
418 form I in various steps of autophagy. Numerous studies identified interacting partners of
419 Atg8/LC3/GABARAP family during autophagy but its mechanistic function for
420 autophagosome biogenesis is still debated. In yeast, the amount of Atg8 regulates the level
421 of autophagy and controls phagophore expansion, but is mainly released from the
422 phagophore assembly site during autophagosome formation (Xie et al., 2008). *In vitro*
423 studies using liposomes or nanodiscs suggested that Atg8 is a membrane-tethering factor
424 and promotes hemifusion (Nakatogawa et al., 2007), membrane tubulation (Knorr et al.,
425 2014), or membrane-area expansion and fragmentation (Maruyama et al., 2021). Another
426 study showed that Atg8-PE assembles with Atg12-Atg5-Atg16 into a membrane scaffold
427 that is recycled by Atg4 (Kaufmann et al., 2014). A similar approach with LGG-1 supports a
428 role in tethering and fusion activity (Wu et al., 2015). *In vivo*, the functions of these proteins
429 could depend on their amount, their posttranslational modifications, and the local
430 composition of the membrane. For instance, an excess of lipidation of the overexpressed
431 LGG-1 form I mediates the formation of enlarged protein aggregates and impedes the
432 degradation process (Wu et al., 2015). A recent report showed that the phosphorylation of
433 LC3C and GABARAP-L2 impedes their binding to ATG4 and influences their conjugation and
434 de-conjugation (Herhaus et al., 2020).

435 Our genetic data suggest that form I of LGG-1 is sufficient for initiation, elongation and
436 closure of autophagosomes but that lipidated LGG-1 is important for the cargo sequestering
437 and the dynamics of degradation. However, the partial redundancy with LGG-2 is
438 presumably an important factor during these processes. If the main functions of LGG-1
439 reside in its capacity to bind multiple proteins, the localization to autophagosome
440 membrane through lipidation is an efficient but not unique way to gather cargoes and
441 autophagy complexes. Furthermore, the possibility that non-positive LGG-1/LGG-2
442 autophagosomes could mediate cargo degradation questions the use of Atg8/GABARAP/LC3
443 family as a universal marker for autophagosomes. Overall, our results confirm the high level
444 of plasticity and robustness of autophagosome biogenesis.
445
446

447 Acknowledgments

448 The authors would like to thank Fulvio Reggiori for yeast plasmids, and the Caenorhabditis
449 Genetic Center, which is funded by the NIH National Center for Research Resources (NCRR),
450 for strains. We are grateful to Laïla Sago and Virginie Redeker for help with mass
451 spectrometry. The present work has benefited from the facilities and expertise of the I2BC
452 proteomic platform (Proteomic-Gif, SICaPS) supported by IBiSA, Ile de France Region, Plan
453 Cancer, CNRS and Paris-Sud University as well as the core facilities of Imagerie-Gif, member
454 of IBiSA), supported by "France-BioImaging" and the Labex "Saclay Plant Science".

455 This work was supported by the Agence Nationale de la Recherche (project EAT, ANR-12-
456 BSV2-018), the Association pour la Recherche contre le Cancer (SFI20111203826) and the
457 Ligue contre le Cancer (MM). RoL received a fellowship from Fondation pour la Recherche
458 Médicale.

459

460

461 Declaration of interests

462 The authors declare no competing interests

463

464

465 Materials and Methods

466

<table><caption>Key Resources Table</caption><thead><tr><th>Reagent type (species) or resource</th><th>Designation</th><th>Source or reference</th><th>Identifiers</th><th>Additional information</th></tr></thead><tbody><tr><td>gene (C. elegans)</td><td>lgg-1</td><td>Wormbase</td><td>WBGene0002980</td><td></td></tr><tr><td>strain, strain background (C. elegans)</td><td>N2</td><td>CGC</td><td></td><td>Wild-type strain</td></tr><tr><td>genetic reagent (C. elegans)</td><td>DA2123</td><td>CGC</td><td></td><td>adls2122[gfp::lgg-1;rol-6(su1006)]</td></tr><tr><td>genetic reagent (C. elegans)</td><td>GK1057</td><td>Sato and Sato, 2011</td><td></td><td>Pspe-11-hsp-6::GFP</td></tr><tr><td>genetic reagent (C. elegans)</td><td>HZ455</td><td>CGC</td><td></td><td>him-5(e1490) V; bpIs131[sepa-1::gfp]</td></tr><tr><td>genetic reagent (C. elegans)</td><td>HZ1685</td><td>CGC</td><td></td><td>atg-4.1(bp501)</td></tr><tr><td>genetic reagent (C. elegans)</td><td>MAH247</td><td>CGC</td><td></td><td>sqIs25[atg-18p::atg-18::gfp+rol-6(su1006)]</td></tr><tr><td>genetic reagent (C. elegans)</td><td>RD202</td><td>Legouis lab</td><td></td><td>Is202[unc-119(ed3)III;plgg-1::GFP::LGG-1 G-&gt;A]</td></tr><tr><td>genetic reagent (C. elegans)</td><td>lgg-1(&Delta;)</td><td>Mitani lab</td><td>NBRP: tm3489</td><td>lgg-1(tm3489)</td></tr><tr><td>genetic reagent (C. elegans)</td><td>lgg-2(tm5755)</td><td>Mitani lab</td><td>NBRP: tm5755</td><td>lgg-2(tm5755)</td></tr><tr><td>genetic reagent (C. elegans)</td><td>RD363 ; lgg-1(&Delta;112-123)</td><td>This paper</td><td></td><td>lgg-1(pp22)dpy-10(pp157) Legouis lab</td></tr><tr><td>genetic reagent (C. elegans)</td><td>RD367 ;</td><td>This paper</td><td></td><td>lgg-1(pp65[G116A]) Legouis lab</td></tr></tbody></table>
<table><tr><td>(C. elegans)</td><td>Igg-1(G116A)</td><td></td><td></td></tr><tr><td>genetic reagent (C. elegans)</td><td>RD368 ; Igg-1(Δ100-123)</td><td>This paper</td><td>Igg-1(pp66) Legouis lab</td></tr><tr><td>genetic reagent (C. elegans)</td><td>RD420 ; Igg-1(G116AG117*)</td><td>This paper</td><td>Igg-1(pp141[G116AG117stop]) Legouis lab</td></tr><tr><td>genetic reagent (C. elegans)</td><td>RD421 ; Igg-1(G116AG117A)</td><td>This paper</td><td>dpy-10(pp163)Igg-1(pp142[G116AG117A]) Legouis lab</td></tr><tr><td>genetic reagent (C. elegans)</td><td>RD425</td><td>This paper</td><td>dpy-10(pp163)Igg1(pp142)/+; SEPA-1::gfp Legouis lab</td></tr><tr><td>genetic reagent (C. elegans)</td><td>RD435</td><td>This paper</td><td>Igg-1(pp141[G116AG117stop]); atg-18p::atg-18::gfp+rol-6(su1006)] Legouis lab</td></tr><tr><td>genetic reagent (C. elegans)</td><td>RD436</td><td>This paper</td><td>Igg-1(pp65[G116A]); atg-18p::atg-18::gfp+rol-6(su1006)] Legouis lab</td></tr><tr><td>genetic reagent (C. elegans)</td><td>RD440</td><td>This paper</td><td>Igg-1(pp141[G116AG117stop]); Igg-2(tm5755) Legouis lab</td></tr><tr><td>genetic reagent (C. elegans)</td><td>RD446</td><td>This paper</td><td>Igg-1(pp65[G116A]); Igg-2(tm5755) Legouis lab</td></tr><tr><td>genetic reagent (C. elegans)</td><td>RD447</td><td>This paper</td><td>Igg-1(tm3489); atg-18p::atg-18::gfp+rol-6(su1006)] Legouis lab</td></tr><tr><td>genetic reagent (C. elegans)</td><td>RD448</td><td>This paper</td><td>Igg-1(pp65[G116A]); SEPA-1::gfp Legouis lab</td></tr><tr><td>genetic reagent (C. elegans)</td><td>RD449</td><td>This paper</td><td>Igg-1(pp141[G116AG117stop]); SEPA-1::gfp Legouis lab</td></tr><tr><td>genetic reagent (C. elegans)</td><td>RD450</td><td>This paper</td><td>Igg-1(tm3489)II; SEPA-1::gfp Legouis lab</td></tr><tr><td>strain, strain background (S. cerevisiae)</td><td>BY4742</td><td>Euroscarf</td><td>Mat alpha ura3Δ0, his3Δ1, leu2Δ0, lys2Δ0</td></tr><tr><td>genetic reagent (S. cerevisiae)</td><td>OC513</td><td>YKO collection</td><td>BY4742, atg1::KanMX4</td></tr><tr><td>genetic reagent (S. cerevisiae)</td><td>OC612</td><td>YKO collection</td><td>BY4742, atg8::KanMX4</td></tr><tr><td>genetic reagent (S. cerevisiae)</td><td>OC608-OC609</td><td>This paper</td><td>BY4742, atg8G116A Legouis lab</td></tr><tr><td>genetic reagent (S. cerevisiae)</td><td>OC610-OC611</td><td>This paper</td><td>BY4742, atg8G116A-R117* Legouis lab</td></tr><tr><td>genetic reagent (S. cerevisiae)</td><td>OC613</td><td>This paper</td><td>BY4742, pho8::pho8Δ60-URA3KL Legouis lab</td></tr><tr><td>genetic reagent (S. cerevisiae)</td><td>OC614</td><td>This paper</td><td>BY4742, atg1::KanMX4, pho8::pho8Δ60-URA3KL Legouis lab</td></tr></table>
<table><tr><td>genetic reagent (S. cerevisiae)</td><td>OC615</td><td>This paper</td><td>BY4742, atg8::KanMX4, pho8::pho8Δ60-URA3KL Legouis lab</td></tr><tr><td>genetic reagent (S. cerevisiae)</td><td>OC616-OC617</td><td>This paper</td><td>BY4742, atg8G116A, pho8::pho8Δ60-URA3KL Legouis lab</td></tr><tr><td>genetic reagent (S. cerevisiae)</td><td>OC618-OC619</td><td>This paper</td><td>BY4742, atg8G116A-R117*, pho8::pho8Δ60-URA3KL Legouis lab</td></tr><tr><td>Strain strain background (E. coli)</td><td>OP50</td><td>CGC</td><td>see Material and Methods</td></tr><tr><td>genetic reagent (E. coli)</td><td>JA-C32D5.9</td><td>Open Biosystem</td><td>Igg-1 RNAi feeding bacterial clone</td></tr><tr><td>genetic reagent (E. coli)</td><td>JA-C56C10.12</td><td>Open Biosystem</td><td>epg-5 RNAi feeding bacterial clone</td></tr><tr><td>genetic reagent (E. coli)</td><td>JA-Y55F3AM.4</td><td>Open Biosystem</td><td>atg-3 RNAi feeding bacterial clone;</td></tr><tr><td>genetic reagent (E. coli)</td><td>JA-M7.5</td><td>Open Biosystem</td><td>atg-7 RNAi feeding bacterial clone</td></tr><tr><td>genetic reagent (E. coli)</td><td>JA-W03C9.3</td><td>Open Biosystem</td><td>rab-7 RNAi feeding bacterial clone</td></tr><tr><td>genetic reagent (E. coli)</td><td>JA-Y39G10AR.10</td><td>Open Biosystem</td><td>epg-2 RNAi feeding bacterial clone</td></tr><tr><td>sequenced-based reagent</td><td>CrRNA(s)</td><td>Paix et al 2015</td><td>dpy-10: 5'GCUACCAUAGGCACCACGAGGUUUUAGAG CUAUGCUGUUUUG3'</td></tr><tr><td>sequenced-based reagent</td><td>CrRNA(s)</td><td>This paper</td><td>Igg-1 Legouis lab 5'UACAGUGACGAAAGUGUGUAGUUUUAGA GCUAUGCUGUUUUG3'</td></tr><tr><td>sequenced-based reagent</td><td>Repair template</td><td>Paix et al., 2015;</td><td>dpy-10: 5'CACTTGAACTTCAATACGGCAAGATGAGAAT GACTGGAAACCGTACCGCATGCGGTGCCTATG GTAGCGGAGCTTCACATGGCTTCAGACCAACA GCCTAT3'</td></tr><tr><td>sequenced-based reagent</td><td>Repair template</td><td>This paper</td><td>Igg-1 (G116A): Legouis lab 5'CTTTACATCGCGTACAGTGACGAAAGTGTCT ACGCCGGAGAGGTCGAAAAGAAGGAATAAAG TGTCATGTAT3'</td></tr><tr><td>sequenced-based reagent</td><td>Repair template</td><td>This paper</td><td>Igg-1 (G116AG117 *): Legouis lab 5'TTCCTTTACATCGCCTACAGTGACGAAAGTGT GTACGCCTAAGAATTCGAAAAGAAGGAATAAA GTGTCATGTATTATCCG3'</td></tr></table>
<table><tr><td>sequenced-based reagent</td><td>Repair template</td><td>This paper</td><td>Igg-1 (G116AG117A): Legouis lab<br>5'TTCCTTTACATCGCCTACAGTGACGAAAGTGT<br>GTACGCCGCAGAGGTCGAAAAGAAGGAATAA<br>GAATTCAGTGTCATGTATTATCCGCCGACGAAT<br>GTGTATAC3'</td></tr><tr><td>sequenced-based reagent</td><td>Universal<br>tracrRNA</td><td>Dharmacon<br>GE</td><td>U-002000-05<br>5'AACAGCAUAGCAAGUUAAAAAUAAGGCUAG<br>UCCGUUAUCAACUUGAAAAAGUGGCACCGAG<br>UCGGUGCUUUUUUUU3'</td></tr><tr><td>peptide, recombinant<br>protein</td><td>S. pyogenes<br>Cas9</td><td>Dharmacon</td><td>CAS11201<br>Edit-R Cas9 Nuclease Protein, 1000 pmol</td></tr><tr><td>Antibody</td><td>anti-LGG-1<br>(rabbit<br>polyclonal)</td><td>Springhorn<br>& Hoppe,<br>2019</td><td>Ab#3 WB (1:3000)</td></tr><tr><td>Antibody</td><td>anti-LGG-1<br>(rabbit<br>polyclonal)</td><td>Al Rawi et<br>al., 2011</td><td>Ab#1 WB (1:200) IF(1:100)</td></tr><tr><td>Antibody</td><td>anti-LGG-2<br>(rabbit<br>polyclonal)</td><td>Manil-Ségalen et<br>al., 2014</td><td>WB (1:200) IF (1:200)</td></tr><tr><td>Antibody</td><td>anti-Tubulin<br>(mouse<br>monoclonal)</td><td>Sigma</td><td>WB (1:1000)</td></tr><tr><td>Antibody</td><td>anti-SEL-1<br>(rabbit<br>polyclonal)</td><td>Hoppe's lab</td><td>WB (1:8000)</td></tr><tr><td>Antibody</td><td>anti-CDC-48.1<br>(rabbit<br>polyclonal)</td><td>Hoppe's lab</td><td>WB (1:5000)</td></tr><tr><td>Antibody</td><td>Anti-Rabbit HRP<br>(goat<br>polyclonal)</td><td>Promega</td><td>WB (1:5000)</td></tr><tr><td>Antibody</td><td>Anti-mouse HRP<br>(goat<br>polyclonal)</td><td>Promega</td><td>WB (1:10000)</td></tr><tr><td>Antibody</td><td>anti-GABARAP<br>(rabbit<br>polyclonal)</td><td>Chemicon</td><td>IF (1:200)</td></tr><tr><td>Antibody</td><td>anti-GFP<br>(mouse<br>monoclonal)</td><td>Roche</td><td>IF (1:250)</td></tr><tr><td>Antibody</td><td>anti-mouse IgG<br>Alexa Fluor®488<br>(goat polyclonal)</td><td>Molecular<br>Probes</td><td>IF (1:500 to 1:1000)</td></tr><tr><td>Antibody</td><td>anti-rabbit IgG<br>Alexa Fluor®488</td><td>Molecular<br>Probes</td><td>IF (1:500 to 1:1000)</td></tr></table>
<table><tr><td></td><td>(goat polyclonal)</td><td></td><td></td><td></td></tr><tr><td>Antibody</td><td>anti-rabbit IgG Alexa Fluor®568 (goat polyclonal)</td><td>Sigma-Aldrich</td><td>A11036</td><td>IF (1:500 to 1:1000)</td></tr><tr><td>Antibody</td><td>anti-GFP (rabbit polyclonal)</td><td>Abcam</td><td>ab6556</td><td>(Immunogold 1:10)</td></tr><tr><td>Antibody</td><td>anti-rabbit IgG goat polyclonal)</td><td>Biovalley</td><td>810.011</td><td>Coupled to 10-nm colloidal gold particles (Immunogold 1:20)</td></tr><tr><td>chemical compound, drug</td><td>EPON</td><td>Agar Scientific</td><td>R1165</td><td>see Material and Methods</td></tr><tr><td>chemical compound, drug</td><td>lead citrate</td><td>Sigma-Aldrich</td><td>15326</td><td>see Material and Methods</td></tr><tr><td>chemical compound, drug</td><td>LRWHITE</td><td>Electron Microscopy Sciences</td><td>14381</td><td>see Material and Methods</td></tr><tr><td>peptide, recombinant protein</td><td>LC3 traps</td><td>Quinet et al., 2022</td><td></td><td>Molecular traps for LGG-1</td></tr><tr><td>commercial assay or kit</td><td>Super Signal Pico Chemiluminescent Substrate</td><td>Thermo Scientific</td><td>34579</td><td>see Material and Methods</td></tr><tr><td>commercial assay or kit</td><td>NuPAGE 4%-12% Bis-Tris gel</td><td>Life Technologies</td><td>NP0321 BOX</td><td>see Material and Methods</td></tr><tr><td>software, algorithm</td><td>ImageJ</td><td>http://image.j.nih.gov/ij</td><td></td><td>see Material and Methods</td></tr><tr><td>software, algorithm</td><td>Fidji</td><td>https://fiji.sc/</td><td></td><td>see Material and Methods</td></tr><tr><td>software, algorithm</td><td>Prism</td><td>GraphPad</td><td></td><td>see Material and Methods</td></tr><tr><td>software, algorithm</td><td>R software</td><td>www.r-project.org</td><td></td><td>see Material and Methods</td></tr><tr><td>software, algorithm</td><td>Crispr</td><td>http://Crispr.mit.edu</td><td></td><td>see Material and Methods</td></tr><tr><td>software, algorithm</td><td>Crispor</td><td>http://crispor.org</td><td></td><td>see Material and Methods</td></tr><tr><td>other</td><td>MitoTracker Red CMXRos</td><td>Molecular Probes</td><td>M7512</td><td>see Material and Methods</td></tr></table>

467 Further information and requests for resources and reagents should be directed to the

468 corresponding author, Renaud Legouis (renaud.legouis@i2bc.paris-saclay.fr).

469

470

471 *C. elegans* culture and strains
472 Nematode strains were grown on nematode growth media [for 500ml H2O: 1.5g NaCl (Sigma-Aldrich, 60142), 1.5g bactopeptone (Becton-Dickinson, 211677), 0.5ml cholesterol (Sigma-Aldrich, C8667) 5mg/ml, 10g bacto agar (Becton-Dickinson, 214010) supplemented with 500µl CaCl2 (Sigma-Aldrich, C3306) 1M, 500µl MgSO4 (Sigma-Aldrich, M5921) 1M, 10ml KH2PO4 (Sigma-Aldrich, P5655) 1M, 1650µl K2HPO4 (Sigma-Aldrich, 60356) 1M] and fed with *Escherichia coli* strain OP50.

478

479 **CRISPR-Cas9**

480 A CRISPR-Cas9 approach optimized for *C. elegans* was used, based on a *dpy-10* co-CRISPR protocol (Paix et al., 2015). All reagents are in 5mM Tris-HCl pH 7.5. Crispr.mit.edu and CRISPOR (http://crispor.org) web tools were used to choose a Cas9 cleavage site (NGG) close to the edit site, the best sequence of the crRNAs (50 to 75% of GC content), and for off-target prediction. 1µL of CrRNA(s) (8µg/µL or 0.6nmole/µL) and repair template(s) (1µg/µL) designed for *lgg-1* and *dpy-10* genes were mixed with 4.1 µL of *S. pyrogenes* Cas9 (20 pmole/µL) and 5 µL of universal tracrRNA (4µg/µL 4µg/µL or 0.17 nmol/µL molarity) in 0.75 µL Hepes (200 mM) 0.5µL KCl (1M) and water up to 20 µL. The mix was heated for 10 min at 37°C and injected in the gonad of young adult hermaphrodites. Progenies of injected animals were cloned and genotyped by PCR. Mutants were outcrossed three times and *lgg-1* gene was sequenced to check for the specific mutations.

491

492 **Nematode starvation and lifespan**

493 For starvation experiments, adult hermaphrodites were bleached to obtain synchronized L1 larvae. L1 were incubated in 0.5 mL sterilized M9 at 20°C on spinning wheel. At each time point, an aliquot from each sample tube was placed on a plate seeded with *E. coli* OP50. The number of worms surviving to adulthood was counted 2 or 4 days after. Life span was performed on more than 100 animals for each genotype with independent duplicates and analyzes using Kaplan-Meier method and Log-Rank (Mantel-Cox) test.

499

500 **RNA mediated interference**

501 RNAi by feeding was performed as described (Kamath et al., 2003). Fourth-larval stage (L4) animals or embryos were raised onto 1 mM isopropyl-D-β-thiogalactopyranoside (IPTG)-containing nematode growth media (NGM) plates seeded with bacteria (*E. coli* HT115[DE3]) carrying the empty vector L4440 (pPD129.36) as a control or the bacterial clones from the J. Ahringer library, Open Biosystem).

506

507 **Western blot and cellular fractionation**

508 The worms were collected after centrifugation in M9 and then mixed with the lysis buffer described previously (Springhorn and Hoppe, 2019) (25mM tris-HCl, pH7.6; 150mM NaCl; 1mM ethylenediaminetetraacetic acid (EDTA) 1% Triton X-100; 1% sodium deoxycholate (w/v); 1% SDS (w/v)) containing glass beads (Sigma-Aldrich 425-600um G8772100G). They were then denatured using Precellys 24 machine at 6000 rpm with incubation for about 5

512
min twice to cool down the sample. The protein extracts are then centrifuged at 15000 rpm and separated on a NuPAGE 4%-12% Bis-Tris gel (Life Technologies, NP0321BOX). The non-specific sites are then blocked after the incubation for one hour with PBS Tween 0.1% (Tris Base NaCl, Tween20) BSA 2%. Blots were probed with anti-LGG-1 (1:3000 rabbit Ab#3 (Springhorn and Hoppe, 2019) or 1:200 Ab#1 (Al Rawi et al., 2011)), anti-LGG-2 (1:200 rabbit), anti-Tubulin (1:1000 mouse; Sigma, 078K4763), anti-SEL-1 (1:8000, rabbit), anti-CDC-48.1 (1:5000, rabbit) and revealed using HRP-conjugated antibodies (1: 5000 promega W401B and 1:10000 promega W4021) and the Super Signal Pico Chemiluminescent Substrate (Thermo Scientific, 34579). Signals were revealed on a Las3000 photoimager (Fuji) and quantified with Image Lab software. For cellular fractionation, 4000 age-synchronized worms (day 1 of adulthood) were collected from NGM/OP-50 plates, washed three times with M9 buffer and transferred to NGM plates without OP-50 to induce starvation. Worms were starved at 20°C for 7 hours, and then transferred to fractionation buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM DTT, 1 mM PMSF, and protease inhibitor cocktail). For cell lysis, worms were homogenized 50 times using a Dounce homogenizer and sonicated for 20 seconds at 60% amplitude. Cell lysates were centrifuged at 500 RCF and 4°C for 5 minutes to remove cell debris and the nuclear fraction. The supernatant was centrifuged again at 20,000 RCF and 4°C for 90 minutes to separate soluble (cytosolic) and insoluble (membrane) fractions. Supernatant and pellet were separated and the pellet was resuspended in 150 µL of fractionation buffer. From this step, input samples were prepared for Western blot analysis. Subsequently, 30 µL of the pellet sample was mixed with 3 µL each of fractionation buffer, 3 µL 5 M NaCl, and 3 µL Triton X-100. Treated pellet samples were incubated on ice for 1 hour and then centrifuged at 20,000 RCF and 4 °C for 60 minutes. The resulting supernatants and pellets were again separated and analyzed by Western blotting.

537

## 538 Immunofluorescence and light microscopy

Fifty adult hermaphrodites were cut to release the early embryos on a previously poly-L-lysinated slide (0.1%). Late embryos were deposited using a flattened platinum wire and bacteria as glue. Embryos were prepared for immunofluorescence staining by freeze-fracture and methanol fixation 30 min at -20°C, incubated 40 min in 0.5% Tween, 3% BSA, PBS solution, and washed twice 30 min in 0.5% Tween PBS solution. Incubation overnight at 4°C overnight with the primary antibodies anti-LGG-1(rabbit 1:100) anti-GABARAP (rabbit 1:200) (1:200), anti-LGG-2 (rabbit 1:100) was followed by 2 washes, 2 hours incubation at room temperature with the secondary antibodies, Alexa488 and Alexa 568 (1:1000), and two washes. Embryos were mounted in DABCO and imaged on an AxiolmagerM2 microscope (Zeiss) equipped with Nomarski optics, coupled to a camera (AxioCam506mono) or a confocal Leica TCS SP8 microscope with serial z sections of 0.5 to 1 µm. Images were analyzed, quantified and processed using ImageJ or Fiji softwares.

For live imaging samples were mounted on a 2% agarose pad and larvae were immobilized by 40 mM sodium azide. For MitoTracker staining, adult worms were transferred to NGM agar plates containing 3.7 µM of Red CMXRos (Molecular Probes, Invitrogen) and incubated for overnight in the dark.
555

### 556 Electronic microscopy

557 One-day adults were transferred to M9 20% BSA (Sigma-Aldrich, A7030) on 1% phosphatidylcholine (Sigma-Aldrich) pre-coated 200 µm deep flat carriers (Leica Biosystems), followed by cryo-immobilization in the EMPACT-2 HPF apparatus (Leica Microsystems; Vienna Austria) as described (Largeau & Legouis, 2019). Cryo-substitution was performed using an Automated Freeze-substitution System (AFS2) with integrated binocular lens, and incubating chamber (Leica Microsystems; Wetzlar, Germany) with acetone. Blocks were infiltrated with 100% EPON, and embedded in fresh EPON (Agar Scientific, R1165). Ultrathin sections of 80 nm were cut on an ultramicrotome (Leica Microsystems, EM UC7) and collected on a formvar and carbon-coated copper slot grid (LFG, FCF-2010-CU-50). Sections were contrasted with 0,05% Oolong tea extract (OTE) for 30 minutes and 0.08 M lead citrate (Sigma-Aldrich, 15326) for 8 minutes. Sections were observed with a Jeol 1400 TEM at 120 kV and images acquired with a Gatan 11 Mpixels SC1000 Orius CCD camera.

569

### 570 Affinity purification of LGG-1

571 1 mg of total proteins from *C. elegans* lysate were incubated on ice 10 minutes in 800 µL of TUBE lysis buffer [50 mM sodium fluoride, 5 mM tetra-sodium pyrophosphate, 10 mM β-glyceropyrophosphate, 1% Igepal CA-630, 2 mM EDTA, 20 mM Na₂HPO₄, 20 mM NaH₂PO₄, and 1.2 mg/ml complete protease inhibitor cocktail (Roche, Basel, Switzerland)] supplemented with 200 µg of purified LC3 traps or GST control (Quinet et al., 2022). After cold centrifugation at 16200 g for 30 minutes, supernatant was harvested and added to 400 µl of prewashed glutathione-agarose beads (Sigma), and incubated for 6 hours rotating at 4°C. Beads were centrifugated at 1000 g for 5 min at 4°C (Beckman Coulter Microfuge 22R, Fullerton, CA, USA), washed five times using 10 column volumes of PBS-tween 0.05%. Elution was done in 100 µL of [Tris pH7.5, 150mM NaCl, 1% Triton, 1% SDS] at 95°C during 10 minutes, and supernatant was harvested.

582

### 583 Mass spectrometry

584 Protein samples affinity purification were prepared using the single-pot, solid-phase-enhanced sample-preparation (SP3) approach as described (Hughes et al., 2019). Samples were mixed with 10 µl of 10 µg/µl solution of Sera-Mag SpeedBeadsTM hydrophilic and hydrophobic magnetics beads (GE healthcare, ref 45152105050250 and 65152105050250) with a bead to sample ratio of 10:1. After a binding step in 50% ethanol in water, and three successive washes with 80% ethanol in water, sample were digested with 100 µl of a 5 ng/ul sequencing grade modified trypsin solution (PROMEGA). 50 µl of Trypsin-generated peptides were vacuum dried, resuspended in 10 µl of loading buffer (2% acetonitrile and 0.05% Trifluoroacetic acid in water) and analyzed by nanoLC-MSMS using a nanoElute liquid chromatography system (Bruker) coupled to a timsTOF Pro mass spectrometer (Bruker). Briefly, peptides were loaded on an Aurora analytical column (ION OPTIK, 25cm x75µm, C18, 1.6µm) and eluted with a gradient of 0-35% of solvent B for 100 min. Solvent A was 0.1 %
formic acid and 2% acetonitrile in water, and solvent B was 99.9% acetonitrile with 0.1%
596
formic acid. MS and MS/MS spectra were recorded and converted into mgf files. Proteins
597
identification were performed with Mascot search engine (Matrix science, London, UK)
598
against a database composed of all LGG-1 sequences including the wild-type and mutant
599
sequences. Database searches were performed using semi-trypsin cleavage specificity with
600
five possible miscleavages. Methionine oxidation was set as variable modification. Peptide
601
and fragment tolerances were set at 15 ppm and 0.05 Da, respectively. A peptide mascot
602
score threshold of 13 was set for peptide identification. C-terminal peptides were further
603
validated manually.
604

605

Quantification and statistical analysis
606

All experiments were done at least three times. All data summarization and statistical
607
analyses were performed by using either the GraphPad-Prism or the R software (www.r-
608
project.org). The Shapiro-Wilk's test was used to evaluate the normal distribution of the
609
values and the Hartley Fmax test for similar variance analysis. Data derived from different
610
genetic backgrounds were compared by Student t test, Anova, Kruskal-Wallis or Wilcoxon-
611
Mann-Whitney tests. The Fisher's exact test was used for nominal variables. Longevity was
612
assessed using Log-Rank (Mantel-Cox) test. Error bars are standard deviations and boxplot
613
representations indicate the minimum and maximum, the first (Q1/25th percentile), median
614
(Q2/50th percentile) and the third (Q3/75th percentile) quartiles. NS (Not Significant p>0.05;
615
* 0.05>p>0.01, **0.01>p>0.001, *** 0.001>p>0.0001 and **** p<0.0001. Exact values of n
616
and statistical tests used can be found in the figure legends.
617

618

619 References

Al Rawi S, Louvet-Vallée S, Djeddi A, Sachse M, Culetto E, Hajjar C, Boyd L, Legouis R, Galy V. 2011.

Postfertilization autophagy of sperm organelles prevents paternal mitochondrial DNA

transmission. *Science* **334**:1144–1147. doi:10.1126/science.1211878

Alberti A, Michelet X, Djeddi A, Legouis R. 2010. The autophagosomal protein LGG-2 acts

synergistically with LGG-1 in dauer formation and longevity in *C. elegans*. *Autophagy* **6**:622–

633. doi:10.4161/auto.6.5.12252

Alemu EA, Lamark T, Torgersen KM, Birgisdottir AB, Larsen KB, Jain A, Olsvik H, Øvervatn A, Kirkin V,

Johansen T. 2012. ATG8 Family Proteins Act as Scaffolds for Assembly of the ULK Complex:

SEQUENCE REQUIREMENTS FOR LC3-INTERACTING REGION (LIR) MOTIFS. *Journal of*

Biological Chemistry **287**:39275–39290. doi:10.1074/jbc.M112.378109

Banta LM, Robinson JS, Klionsky DJ, Emr SD. 1988. Organelle assembly in yeast:

characterization of yeast mutants defective in vacuolar biogenesis and protein

sorting. *J Cell Biol* **107**:1369–1383. doi:10.1083/jcb.107.4.1369

Behrends C, Sowa ME, Gygi SP, Harper JW. 2010. Network organization of the human autophagy

system. *Nature* **466**:68–76. doi:10.1038/nature09204

Cappadocia L, Lima CD. 2018. Ubiquitin-like Protein Conjugation: Structures, Chemistry, and

Mechanism. *Chem Rev* **118**:889–918. doi:10.1021/acs.chemrev.6b00737

Chang JT, Kumsta C, Hellman AB, Adams LM, Hansen M. 2017. Spatiotemporal regulation of

autophagy during Caenorhabditis elegans aging. *Elife* **6**. doi:10.7554/eLife.18459
639 Chang T-K, Shravage BV, Hayes SD, Powers CM, Simin RT, Wade Harper J, Baehrecke EH. 2013. Uba1

640 functions in Atg7- and Atg3-independent autophagy. *Nat Cell Biol* **15**:1067–1078.

641 doi:10.1038/ncb2804

642 Chen Y, Leboutet R, Largeau C, Zentout S, Lefebvre C, Delahodde A, Culetto E, Legouis R. 2021.

643 Autophagy facilitates mitochondrial rebuilding after acute heat stress via a DRP-1-dependent
644 process. *J Cell Biol* **220**:e201909139. doi:10.1083/jcb.201909139

645 Chen Y, Scarcelli V, Legouis R. 2017. Approaches for Studying Autophagy in Caenorhabditis elegans.

646 *Cells* **6**. doi:10.3390/cells6030027

647 Chen Z-W, Chang C-SS, Leil TA, Olsen RW. 2007. C-terminal modification is required for GABARAP-

648 mediated GABA(A) receptor trafficking. *J Neurosci* **27**:6655–6663.

649 doi:10.1523/JNEUROSCI.0919-07.2007

650 Cherra SJ, Kulich SM, Uechi G, Balasubramani M, Mountzouris J, Day BW, Chu CT. 2010. Regulation of

651 the autophagy protein LC3 by phosphorylation. *J Cell Biol* **190**:533–539.

652 doi:10.1083/jcb.201002108

653 Coyle JE, Qamar S, Rajashankar KR, Nikolov DB. 2002. Structure of GABARAP in two conformations:

654 implications for GABA(A) receptor localization and tubulin binding. *Neuron* **33**:63–74.

655 doi:10.1016/s0896-6273(01)00558-x

656 Djeddi A, Al Rawi S, Deuve JL, Perrois C, Liu Y-Y, Russeau M, Sachse M, Galy V. 2015. Sperm-inherited

657 organelle clearance in C. elegans relies on LC3-dependent autophagosome targeting to the

658 pericentrosomal area. *Development* **142**:1705–1716. doi:10.1242/dev.117879

659 Galluzzi L, Baehrecke EH, Ballabio A, Boya P, Bravo-San Pedro JM, Cecconi F, Choi AM, Chu CT,

660 Codogno P, Colombo MI, Cuervo AM, Debnath J, Deretic V, Dikic I, Eskelinen E-L, Fimia GM,

661 Fulda S, Gewirtz DA, Green DR, Hansen M, Harper JW, Jäättelä M, Johansen T, Juhasz G,

662 Kimmelman AC, Kraft C, Ktistakis NT, Kumar S, Levine B, Lopez-Otin C, Madeo F, Martens S,

663 Martinez J, Melendez A, Mizushima N, Münz C, Murphy LO, Penninger JM, Piacentini M,

664 Reggiori F, Rubinsztein DC, Ryan KM, Santambrogio L, Scorrano L, Simon AK, Simon H-U,

665 Simonsen A, Tavernarakis N, Tooze SA, Yoshimori T, Yuan J, Yue Z, Zhong Q, Kroemer G. 2017.

666 Molecular definitions of autophagy and related processes. *EMBO J* **36**:1811–1836.

667 doi:10.15252/embj.201796697

668 Galluzzi L, Green DR. 2019. Autophagy-Independent Functions of the Autophagy Machinery. *Cell*

669 **177**:1682–1699. doi:10.1016/j.cell.2019.05.026

670 Grunwald DS, Otto NM, Park J-M, Song D, Kim D-H. 2020. GABARAPs and LC3s have opposite roles in

671 regulating ULK1 for autophagy induction. *Autophagy* **16**:600–614.

672 doi:10.1080/15548627.2019.1632620

673 Herhaus L, Bhaskara RM, Lystad AH, Gestal-Mato U, Covarrubias-Pinto A, Bonn F, Simonsen A,

674 Hummer G, Dikic I. 2020. TBK1-mediated phosphorylation of LC3C and GABARAP-L2 controls

675 autophagosome shedding by ATG4 protease. *EMBO Rep* **21**:e48317.

676 doi:10.15252/embr.201948317

677 Hill SE, Kauffman KJ, Krout M, Richmond JE, Melia TJ, Colón-Ramos DA. 2019. Maturation and

678 Clearance of Autophagosomes in Neurons Depends on a Specific Cysteine Protease Isoform,

679 *ATG-4-2. Developmental Cell* **49**:251-266.e8. doi:10.1016/j.devcel.2019.02.013

680 Hirata E, Ohya Y, Suzuki K. 2017. Atg4 plays an important role in efficient expansion of autophagic

681 isolation membranes by cleaving lipidated Atg8 in Saccharomyces cerevisiae. *PLoS One*

682 **12**:e0181047. doi:10.1371/journal.pone.0181047

683 Huang R, Xu Y, Wan W, Shou X, Qian J, You Z, Liu B, Chang C, Zhou T, Lippincott-Schwartz J, Liu W.

684 2015. Deacetylation of nuclear LC3 drives autophagy initiation under starvation. *Mol Cell*

685 **57**:456–466. doi:10.1016/j.molcel.2014.12.013

686 Hughes CS, Moggridge S, Müller T, Sorensen PH, Morin GB, Krijgsveld J. 2019. Single-pot, solid-phase-

687 enhanced sample preparation for proteomics experiments. *Nat Protoc* **14**:68–85.

688 doi:10.1038/s41596-018-0082-x
689 Ishii A, Kurokawa K, Hotta M, Yoshizaki S, Kurita M, Koyama A, Nakano A, Kimura Y. 2019. Role of Atg8 in the regulation of vacuolar membrane invagination. *Sci Rep* **9**:14828.
690 doi:10.1038/s41598-019-51254-1
691
692 Jentsch S, Pyrowolakis G. 2000. Ubiquitin and its kin: how close are the family ties? *Trends in Cell Biology* **10**:335-342. doi:10.1016/S0962-8924(00)01785-2
693
694 Jenzer C, Simionato E, Largeau C, Scarcelli V, Lefebvre C, Legouis R. 2019. Autophagy mediates phosphatidylserine exposure and phagosome degradation during apoptosis through specific functions of GABARAP/LGG-1 and LC3/LGG-2. *Autophagy* **15**:228-241.
695
696 doi:10.1080/15548627.2018.1512452
697
698 Jipa A, Vedelek V, Merényi Z, Ürmösi A, Takáts S, Kovács AL, Horváth GV, Sinka R, Juhász G. 2020. Analysis of Drosophila Atg8 proteins reveals multiple lipidation-independent roles. *Autophagy* 1-11. doi:10.1080/15548627.2020.1856494
699
700
701 Joachim J, Jefferies HBJ, Razi M, Frith D, Snijders AP, Chakravarty P, Judith D, Tooze SA. 2015. Activation of ULK Kinase and Autophagy by GABARAP Trafficking from the Centrosome Is Regulated by WAC and GM130. *Molecular Cell* **60**:899-913.
702
703
704 doi:10.1016/j.molcel.2015.11.018
705 Joachim J, Razi M, Judith D, Wirth M, Calamita E, Encheva V, Dynlacht BD, Snijders AP, O'Reilly N, Jefferies HBJ, Tooze SA. 2017. Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy. *Current Biology* **27**:2123-2136.e7.
706
707
708 doi:10.1016/j.cub.2017.06.021
709
710
711 Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T. 2000. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. *EMBO J* **19**:5720-5728. doi:10.1093/emboj/19.21.5720
712
713
714 Kabeya Y, Mizushima N, Yamamoto A, Oshitani-Okamoto S, Ohsumi Y, Yoshimori T. 2004. LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation. *J Cell Sci* **117**:2805-2812. doi:10.1242/jcs.01131
715
716
717
718 Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, Gotta M, Kanapin A, Le Bot N, Moreno S, Sohrmann M, Welchman DP, Zipperlen P, Ahringer J. 2003. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. *Nature* **421**:231-237.
719 doi:10.1038/nature01278
720
721 Kaufmann A, Beier V, Franquelim HG, Wollert T. 2014. Molecular mechanism of autophagic membrane-scaffold assembly and disassembly. *Cell* **156**:469-481.
722 doi:10.1016/j.cell.2013.12.022
723
724
725 Kirisako T, Ichimura Y, Okada H, Kabeya Y, Mizushima N, Yoshimori T, Ohsumi M, Takao T, Noda T, Ohsumi Y. 2000. The Reversible Modification Regulates the Membrane-Binding State of Apg8/Aut7 Essential for Autophagy and the Cytoplasm to Vacuole Targeting Pathway. *Journal of Cell Biology* **151**:263-276. doi:10.1083/jcb.151.2.263
726
727
728 Klionsky DJ, Abdel-Aziz AK, Abdelfatah S, Abdellatif M, Abdoli A, Abel S et al. 2021. Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1. *Autophagy* **17**:1-382. doi:10.1080/15548627.2020.1797280
729
730
731 Knorr RL, Nakatogawa H, Ohsumi Y, Lipowsky R, Baumgart T, Dimova R. 2014. Membrane morphology is actively transformed by covalent binding of the protein Atg8 to PE-lipids. *PLoS One* **9**:e115357. doi:10.1371/journal.pone.0115357
732
733
734
735 Kraft C, Kijanska M, Kalie E, Siergiejuk E, Lee SS, Semplicio G, Stoffel I, Brezovich A, Verma M, Hansmann I, Ammerer G, Hofmann K, Tooze S, Peter M. 2012. Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy. *EMBO J* **31**:3691-3703. doi:10.1038/emboj.2012.225
736
737
738 Kumsta C, Chang JT, Schmalz J, Hansen M. 2017. Hormetic heat stress and HSF-1 induce autophagy to improve survival and proteostasis in C. elegans. *Nat Commun* **8**:14337. doi:10.1038/ncomms14337
739 Leboutet R, Chen Y, Legouis R, Culetto E. 2020. Mitophagy during development and stress in C.

740 elegans. *Mechanisms of Ageing and Development* **189**:111266.

741 doi:10.1016/j.mad.2020.111266

742 Lévêque MF, Berry L, Cipriano MJ, Nguyen H-M, Striepen B, Besteiro S. 2015. Autophagy-Related

743 Protein ATG8 Has a Noncanonical Function for Apicoplast Inheritance in Toxoplasma gondii.

744 *mBio* **6**:e01446-01415. doi:10.1128/mBio.01446-15

745 Liu X-M, Yamasaki A, Du X-M, Coffman VC, Ohsumi Y, Nakatogawa H, Wu J-Q, Noda NN, Du L-L. 2018.

746 Lipidation-independent vacuolar functions of Atg8 rely on its noncanonical interaction with a vacuole membrane protein. *Elife* **7**:e41237. doi:10.7554/eLife.41237

748 Lu Q, Yang P, Huang X, Hu W, Guo B, Wu F, Lin L, Kovács AL, Yu L, Zhang H. 2011. The WD40 Repeat

749 PtdIns(3)P-Binding Protein EPG-6 Regulates Progression of Omegasomes to

750 Autophagosomes. *Developmental Cell* **21**:343-357. doi:10.1016/j.devcel.2011.06.024

751 Lystad AH, Ichimura Y, Takagi K, Yang Y, Pankiv S, Kanegae Y, Kageyama S, Suzuki M, Saito I,

752 Mizushima T, Komatsu M, Simonsen A. 2014. Structural determinants in GABARAP required

753 for the selective binding and recruitment of ALFY to LC3B-positive structures. *EMBO Rep*

754 **15**:557-565. doi:10.1002/embr.201338003

755 Manil-Ségalen M, Christophe Lefebvre, Jenzer C, Trichet M, Boulogne C, Satiat-Jeunemaitre B,

756 Legouis R. 2014. The C. elegans LC3 Acts Downstream of GABARAP to Degrade

757 Autophagosomes by Interacting with the HOPS Subunit VPS39. *Developmental Cell* **28**:43-55.

758 doi:10.1016/j.devcel.2013.11.022

759 Maruyama T, Alam JM, Fukuda T, Kageyama S, Kirisako H, Ishii Y, Shimada I, Ohsumi Y, Komatsu M,

760 Kanki T, Nakatogawa H, Noda NN. 2021. Membrane perturbation by lipidated Atg8 underlies

761 autophagosome biogenesis. *Nat Struct Mol Biol* **28**:583-593. doi:10.1038/s41594-021-00614-5

762

763 Meléndez A, Tallóczy Z, Seaman M, Eskelinen E-L, Hall DH, Levine B. 2003. Autophagy genes are

764 essential for dauer development and life-span extension in C. elegans. *Science* **301**:1387-1391. doi:10.1126/science.1087782

765

766 Mizushima N, Sahani MH. 2014. ATG8 localization in apicomplexan parasites: apicoplast and more?

767 *Autophagy* **10**:1487-1494. doi:10.4161/auto.32183

768 Nakatogawa H. 2020. Mechanisms governing autophagosome biogenesis. *Nat Rev Mol Cell Biol*

769 **21**:439-458. doi:10.1038/s41580-020-0241-0

770 Nakatogawa H, Ichimura Y, Ohsumi Y. 2007. Atg8, a ubiquitin-like protein required for

771 autophagosome formation, mediates membrane tethering and hemifusion. *Cell* **130**:165-178. doi:10.1016/j.cell.2007.05.021

772

773 Nakatogawa H, Ohbayashi S, Sakoh-Nakatogawa M, Kakuta S, Suzuki SW, Kirisako H, Kondo-

774 Kakuta C, Noda NN, Yamamoto H, Ohsumi Y. 2012. The autophagy-related protein

775 kinase Atg1 interacts with the ubiquitin-like protein Atg8 via the Atg8 family

776 interacting motif to facilitate autophagosome formation. *J Biol Chem* **287**:28503-28507. doi:10.1074/jbc.C112.387514

777

778 Nguyen TN, Padman BS, Usher J, Oorschot V, Ramm G, Lazarou M. 2016. Atg8 family LC3/GABARAP

779 proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation

780 during PINK1/Parkin mitophagy and starvation. *The Journal of Cell Biology* **215**:857-874.

781 doi:10.1083/jcb.201607039

782 Nishida Y, Arakawa S, Fujitani K, Yamaguchi H, Mizuta T, Kanaseki T, Komatsu M, Otsu K, Tsujimoto Y,

783 Shimizu S. 2009. Discovery of Atg5/Atg7-independent alternative macroautophagy. *Nature*

784 **461**:654-658. doi:10.1038/nature08455

785 Noda T, Klionsky DJ. 2008. The quantitative Pho8Delta60 assay of nonspecific autophagy.

786 *Methods Enzymol* **451**:33-42. doi:10.1016/S0076-6879(08)03203-5

787 Ohnstad AE, Delgado JM, North BJ, Nasa I, Kettenbach AN, Schultz SW, Shoemaker CJ. 2020.

788 Receptor-mediated clustering of FIP200 bypasses the role of LC3 lipidation in autophagy.

789 *EMBO J* **39**:e104948. doi:10.15252/embj.2020104948
790 Paix A, Folkmann A, Rasoloson D, Seydoux G. 2015. High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes. *Genetics* **201**:47–54. doi:10.1534/genetics.115.179382

792 Pankiv S, Clausen TH, Lamark T, Brech A, Bruun J-A, Outzen H, Øvervatn A, Bjørkøy G, Johansen T. 2007. p62/SQSTM1 Binds Directly to Atg8/LC3 to Facilitate Degradation of Ubiquitinated Protein Aggregates by Autophagy. *J Biol Chem* **282**:24131–24145. doi:10.1074/jbc.M702824200

797 Poillet-Perez L, Jacquet M, Hervouet E, Gauthier T, Fraichard A, Borg C, Pallandre J-R, Gonzalez BJ, Ramdani Y, Boyer-Guittaut M, Delage-Mourroux R, Despouy G. 2017. GABARAPL1 tumor suppressive function is independent of its conjugation to autophagosomes in MCF-7 breast cancer cells. *Oncotarget* **8**. doi:10.18632/oncotarget.19639

801 Polson HEJ, de Lartigue J, Rigden DJ, Reedijk M, Urbé S, Clague MJ, Tooze SA. 2010. Mammalian Atg18 (WIPI2) localizes to omegasome-anchored phagophores and positively regulates LC3 lipidation. *Autophagy* **6**:506–522. doi:10.4161/auto.6.4.11863

803 Quinet G, Génin P, Ozturk O, Belgareh-Touzé N, Courtot L, Legouis R, Weil R, Cohen MM, Rodriguez MS. 2022. Exploring selective autophagy events in multiple biologic models using LC3-interacting regions (LIR)-based molecular traps. *Sci Rep* **12**:7652. doi:10.1038/s41598-022-11417-z

808 Samokhvalov V, Scott BA, Crowder CM. 2008. Autophagy protects against hypoxic injury in C. elegans. *Autophagy* **4**:1034–1041. doi:10.4161/auto.6994

810 Sato M, Sato K. 2011. Degradation of paternal mitochondria by fertilization-triggered autophagy in C. elegans embryos. *Science* **334**:1141–1144. doi:10.1126/science.1210333

812 Sato M, Sato Katsuya, Tomura K, Kosako H, Sato Ken. 2018. The autophagy receptor ALLO-1 and the IKKE-1 kinase control clearance of paternal mitochondria in Caenorhabditis elegans. *Nat Cell Biol* **20**:81–91. doi:10.1038/s41556-017-0008-9

815 Schaaf MBE, Keulers TG, Vooijs MA, Rouschop KMA. 2016. LC3/GABARAP family proteins: autophagy-(un)related functions. *The FASEB Journal* **30**:3961–3978. doi:10.1096/fj.201600698R

817 Scherz-Shouval R, Sagiv Y, Shorer H, Elazar Z. 2003. The COOH terminus of GATE-16, an intra-Golgi transport modulator, is cleaved by the human cysteine protease HsApg4A. *J Biol Chem* **278**:14053–14058. doi:10.1074/jbc.M212108200

820 Shpilka T, Weidberg H, Pietrokovski S, Elazar Z. 2011. Atg8: an autophagy-related ubiquitin-like protein family. *Genome Biol* **12**:226. doi:10.1186/gb-2011-12-7-226

822 Springhorn A, Hoppe T. 2019. Western blot analysis of the autophagosomal membrane protein LGG-1/LC3 in Caenorhabditis elegans. *Methods Enzymol* **619**:319–336. doi:10.1016/bs.mie.2018.12.034

825 Tian Y, Li Z, Hu W, Ren H, Tian E, Zhao Y, Lu Q, Huang X, Yang P, Li X, Wang X, Kovács AL, Yu L, Zhang H. 2010. C. elegans screen identifies autophagy genes specific to multicellular organisms. *Cell* **141**:1042–1055. doi:10.1016/j.cell.2010.04.034

828 Vujić N, Bradić I, Goeritzer M, Kuentzel KB, Rainer S, Kratky D, Radović B. 2021. ATG7 is dispensable for LC3-PE conjugation in thioglycolate-elicited mouse peritoneal macrophages. *Autophagy* **1**:6. doi:10.1080/15548627.2021.1874132

831 Weidberg H, Shvets E, Shpilka T, Shimron F, Shinder V, Elazar Z. 2010. LC3 and GATE-16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis. *The EMBO Journal* **29**:1792–1802. doi:10.1038/emboj.2010.74

834 Wesch N, Kirkin V, Rogov VV. 2020. Atg8-Family Proteins-Structural Features and Molecular Interactions in Autophagy and Beyond. *Cells* **9**:E2008. doi:10.3390/cells9092008

836 Wilkinson DS, Jariwala JS, Anderson E, Mitra K, Meisenhelder J, Chang JT, Ideker T, Hunter T, Nizet V, Dillin A, Hansen M. 2015. Phosphorylation of LC3 by the Hippo Kinases STK3/STK4 Is Essential for Autophagy. *Molecular Cell* **57**:55–68. doi:10.1016/j.molcel.2014.11.019

839 Wu F, Li Y, Wang F, Noda NN, Zhang H. 2012. Differential Function of the Two Atg4 Homologues in the Aggrephagy Pathway in Caenorhabditis elegans. *J Biol Chem* **287**:29457–29467. doi:10.1074/jbc.M112.365676
842 Wu F, Watanabe Y, Guo X-Y, Qi X, Wang P, Zhao H-Y, Wang Z, Fujioka Y, Zhang Hui, Ren J-Q, Fang T-C,
843 Shen Y-X, Feng W, Hu J-J, Noda NN, Zhang Hong. 2015. Structural Basis of the Differential
844 Function of the Two C. elegans Atg8 Homologs, LGG-1 and LGG-2, in Autophagy. *Molecular
845 Cell* **60**:914–929. doi:10.1016/j.molcel.2015.11.019

846 Xie Z, Nair U, Klionsky DJ. 2008. Atg8 controls phagophore expansion during autophagosome
847 formation. *Mol Biol Cell* **19**:3290–3298. doi:10.1091/mbc.e07-12-1292

848 Zhang G, Wang Z, Du Z, Zhang H. 2018. mTOR Regulates Phase Separation of PGL Granules to
849 Modulate Their Autophagic Degradation. *Cell* **174**:1492-1506.e22.
850 doi:10.1016/j.cell.2018.08.006

851 Zhang Y, Yan L, Zhou Z, Yang P, Tian E, Zhang K, Zhao Y, Li Z, Song B, Han J, Miao L, Zhang H. 2009.
852 SEPA-1 mediates the specific recognition and degradation of P granule components by
853 autophagy in C. elegans. *Cell* **136**:308–321. doi:10.1016/j.cell.2008.12.022

854 Zhou Q, Li Haimin, Li Hanzeng, Nakagawa A, Lin JLJ, Lee E-S, Harry BL, Skeen-Gaar RR, Suehiro Y,
855 William D, Mitani S, Yuan HS, Kang B-H, Xue D. 2016. Mitochondrial endonuclease G
856 mediates breakdown of paternal mitochondria upon fertilization. *Science* **353**:394–399.
857 doi:10.1126/science.aaf4777

858

859

860

861 **Figure legends**

862

863 **Figure 1 - G116A abolishes the conjugation of LGG-1 to the membrane but not its cleavage**
864 (A) Schematic representation of the various isoforms of Atg8s proteins after cleavage of the
865 precursor and reversible conjugation to a phosphatidylethanolamine (PE).

866 (B) Diagram of the theoretical proteins produced by the allelic *lgg-1* series used in this study.
867 LGG-1(Δ) protein corresponds to the reference allele *lgg-1(tm3489)*, considered as a null, all
868 others mutants have been generated using CRISP-Cas9. Black arrows point to the di-glycine
869 residues which are mutated in alanine or stop codon (*). Other deletion mutants of the C-
870 terminus result from non-homologous end joining. The mapping of the epitopes recognized
871 by the LGG-1 antibodies (Ab#1, 2, 3) used in this study are indicated by horizontal grey
872 arrows.

873 (C) Western blot analysis of endogenous LGG-1 from total protein extracts of *wild-type*, *lgg-1(G116A)*, *lgg-1(G116AG117*)*, *lgg-1(G116AG117A)*, *lgg-1(Δ)* young adults. The data shown
874 is representative of three experiments using Ab#3 and was confirmed with Ab#1. The
875 theoretic molecular mass of the precursor, and the form I are 14.8kDa and 14.0 kDa,
876 respectively, while the lipidated form II migrates faster. The asterisk indicates an unknown
877 band. The quantification of each LGG-1 isoforms was normalized using tubulin.
878

879 (D-L) Immunofluorescence analysis of endogenous LGG-1 (Ab#1 or Ab#2) in early and late
880 embryos in *wild-type* (D), *lgg-1(Δ)* (E), *lgg-1(G116A)* (F), *lgg-1(Δ112-123)* (G), *lgg-
881 1(G116AG117*)* (H), *lgg-1(G116AG117A)* (I), *lgg-1(Δ100-123)* (J). Inset in E shows the
882 corresponding DAPI staining of nuclei. Box-plots quantification showing the absence of
883 puncta in all *lgg-1* mutants (K, left n= 19, 13, 11, 10, 6, 7, 6; right n= 18, 14, 12, 10, 10, 9, 12)
884 and the increase of cytosolic staining in *lgg-1(G116A)* and *lgg-1(G116AG117*)* (L, n= 19, 13,
885 11, 10). Kruskal Wallis test, p-value *<0.05, **<0.01, ****<0.0001, NS non-significant. Scale
886 bar is 10 µm.
(M) Cellular fractionation of membrane vesicles. Western blot analysis for detection of LGG-1 together with LGG-2 (autophagosome marker), SEL-1 (ER marker), and CDC-48 (ER-associated and cytosol) using supernatant (S) and pellet (P) fractions of *Igg-1 wild-type*, *Igg-1(G116A)*, and *Igg-1(G116AG117*)* worm lysates treated with fractionation buffer (-), sodium chloride (NaCl) or Triton X-100 (TX-100) after subcellular fractionation. Proteins associated with membranes are solubilized by NaCl, and resident proteins in membrane-bound organelles are released only by dissolving the membrane with detergents. While wild-type LGG-1 is detected in the cytosolic fraction (input S) and in the various membrane fractions, mutant LGG-1 is almost exclusively present in the cytosolic fraction in *Igg-1(G116A)* and *Igg-1(G116AG117*)*.

**Figure 2 - *Igg-1(G116A)* and *Igg-1(G116AG117*)* mutants are viable with no developmental defect**

(A-G) DIC images of embryos after morphogenesis in *wild-type* (A), *Igg-1(Δ)* (B), *Igg-1(G116A)* (C), *Igg-1(G116AG117*)* (D), *Igg-1(G116AG117A)* (E), *Igg-1(Δ112-123)* (F), *Igg-1(Δ100-123)* (G). *Igg-1(G116AG117A)*, *Igg-1(Δ112-123)*, *Igg-1(Δ100-123)*, *Igg-1(Δ)* mutant embryos present severe developmental defects. Short and long white arrows point to the anterior (a) and posterior (p) part of the pharynx, respectively. Scale bar is 10 µm.

(H) The viability, expressed as the percentage of embryos reaching adulthood, is not affected in *Igg-1(G116A)* and *Igg-1(G116AG117*)* mutants (42<n<103).

(I) The fertility, total number of progenies, of *Igg-1(G116A)* and *Igg-1(G116AG117*)* adults is similar to *wild-type* (n=20).

**Figure 3 - Autophagy is functional in *Igg-1(G116A)* but not in *Igg-1(G116AG117*)* and *Igg-1(G116AG117A)**

(A-E) *In vivo* epifluorescence images of paternal mitochondria (HSP-6::GFP) at the 1-cell and 15-cells stages in *wild-type* (A), *Igg-1(Δ)* (B), *Igg-1(G116A)* (C), *Igg-1(G116AG117*)*(D), *Igg-1(G116AG117A)* (E) embryos showing an effective degradation of paternal mitochondria in *wt* and *Igg-1(G116A)* but not in *Igg-1(Δ)* *Igg-1(G116AG117*)* and *Igg-1(G116AG117A)*. Quantification are shown in (F).

(G, H) Bulk autophagy during aging and stress was assessed by worm longevity (G, log rank test n > 100 animals, p-value ****<0.001) and starvation survival (H, Chi-square test at day 15 p-value ****<0.001). The survival is significantly reduced in *Igg-1(Δ)*, *Igg-1(G116AG117*)* and *Igg-1(G116AG117A)* compared to *wt* and *Igg-1(G116A)*. NS non-significant.

(I) Box-plots quantification of apoptotic corpses showing a defective degradation in *Igg-1(G116AG117*)* and *Igg-1(G116AG117A)* but not in *Igg-1(G116A)* (n=22, 40, 46, 14, 21 Kruskal Wallis test ***<0.001, ****<0.0001, NS non-significant).

**Figure 4 - Autophagy but not developmental function of LGG-1(G116A) partially depends on LGG-2**
927 (A-F) DIC images of embryos and bright field images of adults in wild-type (A), lgg-2(tm5755)
928 (B, E), lgg-1(G116A); lgg-2(tm5755) (C, F). The double mutant lgg-1(G116A); lgg-2(tm5755)
929 animals have no morphogenetic defects and no decrease in viability compare to single
930 mutants or the lgg-1(Δ) (quantification in D).

931 (G-H) Bulk autophagy during stress and aging was assessed by starvation survival (G, Chi-
932 square test at day 9 ***p-value<0.001) and worm longevity (H, log rank test n>100 animals,
933 ***p-value<0.001, ****p-value<0.0001). The survival of double mutants lgg-1(G116A); lgg-
934 2(tm5755) and lgg-1(G116AG117*); lgg-2(5755) is reduced compared to wild-type and single
935 mutant lgg-1(G116A) and lgg-2(tm5755). lgg-1(G116A); lgg-2(tm5755) animals survive to
936 starvation better than lgg-1(G116AG117*); lgg-2(5755) and present a slightly higher lifespan.
937 (I-K) In vivo epifluorescence imaging of paternal mitochondria (HSP-6::GFP) at the 1-cell, 15-
938 cells and 30-cells stages in lgg-2(tm5755), (I) lgg-1(G116A); lgg-2(tm5755) (J) embryos and
939 quantification (n=50, 39, 35, 45, 46 Chi-square test ****<0.0001) (K). Elimination of
940 mitochondria is efficient but delayed in lgg-1(G116A); lgg-2(5755) compared to lgg-
941 1(G116A). Insets show the corresponding DIC pictures.

942 Scale bar is 10 µm (A-C, I, J) or 100 µm (E, F).

943

944 **Figure 5 - The degradation of autophagosomes is delayed in lgg-1(G116A)**

945 (A-F) Confocal images of LGG-2 immunofluorescence in 2 cells, 4 cells and 15 cells in wild-
946 type (A), lgg-1(Δ) (B), lgg-1(G116A) (C), lgg-1(G116AG117*) (D) and quantification of the
947 number (E) and size of puncta (F) (embryo analyzed 19, 37, 28, 14; Mann-Whitney test, p-
948 value ****<0.0001). In lgg-1(G116A) and lgg-1(G116AG117*) mutants LGG-2 is detected as
949 heterogeneous sparse structures that persist.

950 (G-J) Colocalization analysis of paternal mitochondria (HSP-6::GFP) and LGG-2 puncta (H)
951 from confocal images of wild-type (H), lgg-1(G116A) (I) and lgg-1(G116AG117*) (J) early
952 embryos. (Mean + SD, n = 16, 20, 12, Kruskal Wallis test p-value*<0.05). The clustering of
953 paternal mitochondria and LGG-2 autophagosomes are absent in lgg-1(G116A) and lgg-
954 1(G116AG117*) where HSP-6::GFP and LGG-2 puncta are mainly separated with rare
955 colocalization events (yellow arrows).

956 (K) Western blot analysis of endogenous LGG-2 from total protein extracts from wild-type,
957 lgg-1(G116A), lgg-1(G116AG117*), lgg-1(Δ) young adults. The quantification of LGG-2 upper
958 and lower bands was normalized using tubulin.

959

960 **Figure 6 - The cleaved LGG-1 is sufficient for autophagosome biogenesis**

961 (A-J) Electron microscopy images of autophagosomes in wild-type (A), lgg-1(Δ) (B), lgg-
962 1(G116A) (C-E), lgg-1(G116AG117*) (F-G), lgg-1(G116A); lgg-2(tm5755) (H), lgg-
963 1(G116AG117*) lgg-2(tm5755) (I) and lgg-2(tm5755) (J) early embryos. Type 1
964 autophagosomes (A, C, D) appear as closed structures containing various membrane
965 organelles. Among those, sequestered paternal mitochondria (black arrows) are observed in
966 wild-type and lgg-1(G116A) embryos but remain unsequestered in lgg-1(Δ) and lgg-
967 1(G116AG117*) embryos. Type 2 autophagosomes (E, white arrow in H, J) appear as closed
structures containing unidentified or degraded materials. Type 3 structures (G, I) are multi-lamellar structures only detected in *Igg-1(G116AG117*)* embryos. Scale bar is 200 nm.

(K) Quantification of type 1, type 2 and type 3 structures in early embryo (1 to 12 cells). In *Igg-1(G116A)* embryos, the numbers of type 1 and type 2 autophagosomal structures increase supporting a retarded degradation. The formation of autophagosomes in *Igg-1(G116A)* and *Igg-1(G116AG117*)* embryos is partially dependent of LGG-2 (n sections= 32, 62, 32, 19, 32, 26, 52).

975

**Figure 7 - The lipidated LGG-1 is involved in the coordination between cargo recognition and autophagosome biogenesis.**

(A-E) Confocal images of ATG-18::GFP (green) and LGG-2 (red) immunofluorescence in *wild-type* (H), *Igg-1(RNAi)* (I), *Igg-1(G116A)* (J), *Igg-1(G116AG117*)* (K) 100-cells embryos. Insets are 2-fold magnification of the white boxed regions. (E) Compared to ATG-18 puncta the number of colocalization is decreased in *Igg-1(RNAi)* (p-value <0.05) and *Igg-1(G116AG117*)* (p-value*<0.001) but not *Igg-1(G116A)* embryos (mean + SD, n= 10, 10, 10, 10; Kruskal Wallis p-value*<0.05**<0.01).

(F-K) Quantification (F) and maximum projections of epifluorescence images of the aggrephagy cargo SEPA-1::GFP in 1.5 fold embryos for *wild-type* (G), *Igg-1(Δ)* (H), *Igg-1(G116A)* (I), *Igg-1(G116AG117*)* (J) and *Igg-1(G116AG117A)* (K). Boxplots of SEPA-1::GFP dots (n=10) (F) indicate that the degradation is stronger in *Igg-1(G116A)* embryos than in *Igg-1(RNAi)*, *Igg-1(G116AG117*)* and *Igg-1(G116AG117A)* but weaker than in *wt*.

(L-P) Confocal images of SEPA-1::GFP (green) and LGG-1 (L, M) or LGG-2 (N, O) (red) immunofluorescence in *wild-type* (L, N) and *Igg-1(G116A)* (M, O) 100-cells embryos. Insets are 2.5-fold magnification of the white boxed regions. In *Igg-1(G116A)* embryos LGG-2-positive/ LGG-1-negative autophagosomes are detected close to SEPA-1::GFP cargos but with a decreased overlap. P) Box-plots of the overlap between green and red pixels (Manders coefficient) in *wild-type* and *Igg-1(G116A)* (n=11, 13; Mann-Whitney test **<0.01). Scale bar is 10 µm (A-K) or 5 µm (L-O).

996

997

**Supplementary figures**

999

**Figure 1-figure supplement 1 - Description of Igg-1 alleles**

(A) Schematic representation of *Igg-1* locus indicating the exon/intron composition and the localization of the PAM used for CRISPR-Cas9. The names of the alleles and the theoretical sequences of the proteins series are indicated. Residues in red are the results of the point mutations and residues in blue italics are the theoretical consequence of frameshifts. The di-glycine motif in position 116-117 is shown in green and the red arrow points to the conserved glycine 116. The grey box shows the aligned sequence of *S. cerevisiae* Atg8

(B) Comparison of Atg8/GABARAP homologs in worm (Ce), fly (Dm), human (Hs), mouse (Mm), zebrafish (Dr), plants (At, Gm), yeasts (Sp, Sc), amoeba (Dd) and parasite protozoa
1009 (Ld), identified in the eukaryotic proteomes from NCBI landmark Blast database. The
1010 conserved core sequence (blue box) is represented using a LOGO analysis (after removal of a
1011 small insertion in At Atg8D and in two Gm uncharacterized homologs) and the variable N-
1012 and C-terminus sequences are indicated in light beige. Green arrows points to the invariable
1013 glycine 116.
1014

1015 **Figure 1-figure supplement 2 - Identification of LGG-1(G116A) and LGG-1(G116AG117*) forms**

1016 (A) Western blot analysis of affinity purified samples using LC3 traps. Molecular weight
1017 markers (kDa) are indicated on the right.

1018 (B) Protein sequence, and peptides coverage of LGG-1(G116AG117*) identified by mass
1019 spectrometry analyses after trypsin treatment (blue underline). The MS/MS fragmentation of
1020 the C-terminal peptide 89-116 (green) identifying the truncated form is shown below.

1021 (C) Protein sequence and peptides coverage of LGG-1(G116A) identified by mass
1022 spectrometry analyses after trypsin treatment (blue underline). The yellow underline
1023 indicates a C-terminal 111-123 peptide identified after trypsin and AspN digestion. The
1024 MS/MS fragmentation of the C-terminal peptide 107-121 (red box) identifying the precursor
1025 form and peptide 89-116 (green) identifying the cleaved form are shown below.

1026 (D-G) Mutant for the protease atg-4.1(bp501) (D) still presents few small puncta (arrows) in
1027 wild-type due to the presence of a paralog, atg-4.2. Autophagosome maturation defective
1028 epg-5(RNAi) embryos accumulate LGG-1 positive autophagosomes (E) but do not show
1029 puncta in lgg-1(G116A) and lgg-1(G116AG117*) embryos (F, G). Scale bar is 10 µm.

1030 (H) Schematic representation of the forms present in the wild-type and lgg-1 mutants based
1031 on western-blot, immunofluorescence and mass spectrometry analyses.

1032 **Figure 1-figure supplement 3 - GFP::LGG-1(G116A) does not localize to autophagosomes**

1033 (A) Western blot analysis of GFP::LGG-1 and GFP::LGG-1(G116A) using Ab#3, from total
1034 protein extracts from young adults. Accumulation of the precursor but absence of form II is
1035 observed. Molecular weight markers (kDa) are indicated on the left.

1036 (B) Schema of the experimental approach indicating the GFP reporters and the autophagy
1037 steps affected upon specific RNAi depletion. A 60 minutes heat-stress at 37°C induces an
1038 autophagy flux.

1040 (C-D) *In vivo* epifluorescence images of GFP::LGG-1(wt) (left columns) and GFP::LGG-
1041 1(G116A) (right columns) in the epidermis of *control*, *atg-3(RNAi)*, *atg-7(RNAi)*, *epg-
1042 5(RNAi)*,and *rab-7(RNAi)* 4th stage larvae. GFP::LGG-1(wt) puncta indicate the
1043 autophagosomes formed upon autophagy induction. The alteration of the conjugation
1044 process (*atg-3*, *atg-7*) or the fusion with the lysosome (*epg-5*, *rab-7*) results in a decrease or
1045 an accumulation of the puncta, respectively. GFP::LGG-1(G116A) does not localize to
1046 autophagosomes even in the conditions of strong accumulation. The zoomed images are 3
1047 fold magnifications of the dotted rectangles. Scale bar is 50 µm. Quantifications are shown in
1048 (D) (n=10, 10, 6, 7, 10, 10, 4, 8, 10, 10, 7, 10, 10, 10, 4, 6, 10, 10, 5, 6).
1049 (E-F) Electron microscopy images of GFP-immunolabeled GFP::LGG-1(wt) and GFP::LGG-1(G116A) in the epidermis of 4th stage larvae after heat stress. Arrows indicates gold beads present on autophagosomes and quantifications are shown in (F). In GFP::LGG-1(G116A) animals labelled autophagosomes are very rare and beads are located in the lumen (n=110, 116, from 4 independent immunostainings). Scale bar is 200 nm.

1054

1055 **Figure 3-figure supplement 1 - Autophagy is functional in lgg-1(G116A) but not in lgg-1(G116AG117*)**

1056 (A-B) *In vivo* epifluorescence images of paternal mitochondria (left, mitotracker) and corresponding DIC images (right) at the 1-4 cells and 20-50 cells stages in *wild-type*, *lgg-1(Δ)*, *lgg-1(G116A)*, *lgg-1(G116AG117*)* embryos showing an effective degradation of paternal mitochondria in *wt* and *lgg-1(G116A)* but not in *lgg-1(Δ) lgg-1(G116AG117*)*. Quantifications are shown in (B).

1061 (C) DIC images of 1.5 fold stage *wild-type*, *lgg-1(Δ)*, *lgg-1(G116A)*, *lgg-1(G116AG117*)* and *lgg-1(G116AG117A)* embryos. Apoptotic corpses in the head region are indicated by yellow arrows. The quantification of apoptotic corpses is shown in Figure 3I. Scale bar is 10 µm.

1065

1066 **Figure 3-figure supplement 2 - Atg8(G116A) and Atg8(G116AR117*) are functional for vacuolar shaping but not for autophagy in S. cerevisiae**

1067 (A) Schematic representation of wild-type and mutant Atg8 proteins.

1068 (B) Autophagic activity is abolished in *S. cerevisiae* mutant strains *atg8G116A* or *atg8G116AR117Δ* forms of Atg8. Autophagic activity of wild-type, *atg1Δ*, *atg8Δ*, *atg8G116A* and *atg8G116AR117*\* strains was measured using the quantitative Pho8Δ60 assay, before (+N) and after (-N) 4 hours of nitrogen starvation. The data shown are the mean ± s.e.m. of 3 independent experiments (Mann-Whitney test, p-value****<0.0001).

1069 (C) *S. cerevisiae* mutants *atg8Δ*, *atg8G116A* or *atg8G116AR117*\* have a similar cell viability defect upon nitrogen starvation.

1074 (D) Epifluorescence image of FM4-64 staining of the vacuole of *wild-type* and *atg8* mutant strains (single focal plan).

1078 (E) Quantification of vacuoles in wild-type and *atg8* mutants. *atg8G116A* and *atg8G116AR117*\* mutants present a less severe vacuolar phenotype than *atg8Δ* mutant (n > 200 cells, Chi-square test, p-value *<0.05, ***<0.001, ****<0.0001).

1081 (F) Rescue assays of *atg8Δ* by *lgg-1(wt)*, *lgg-1(G116A)* and *lgg-1(G116AG117*)*. In the upper part, the survival in nitrogen starvation of *atg8Δ* cells transformed with *lgg-1(wt)* or *ATG8* is compared with *atg8Δ* and *wild-type*. In the lower part, the survival in nitrogen starvation of *atg8Δ* cells transformed with *lgg-1(wt)*, *lgg-1(G116A)*, *lgg-1(G116AG117*)* are compared with *atg8Δ*. The percentage of surviving cells was calculated in comparison with day 0. *lgg-1(G116A)* expression improves the survival but less efficiently than *lgg-1(wt)*. Black and red stars show statistical difference with wild-type and *atg8Δ*, respectively, Mann-Whitney test, p-value **<0.001).

1089
1090 **Figure 5- figure supplement 1 - Colocalization quantification of HSP-6::GFP and LGG-2**

1091 Quantification of puncta positive for the paternal mitochondria marker HSP-6::GFP, the
1092 autophagosomal marker LGG-2 and both, in early embryos wild-type, *Igg-1(G116A)* and *Igg-1(G116AG117*)* (Mean + SD, n= 16, 20, 12 Kruskal Wallis p-value*<0.05, **<0.01).

1094

1095 **Figure 7- figure supplement 1 - LGG-1(G116A) function in aggrephagy is dependent on**
1096 **UNC-51 and EPG-2**

1097 (A-H) *In vivo* confocal images of ATG-18::GFP, showing the initiation of autophagosome
1098 biogenesis, in 50-100 cells embryos for *wild-type* (A), *Igg-1(Δ)(B)*, *Igg-1(G116A)* (C), *Igg-1(G116AG117*)* (D), *unc-51* (*RNAi*)(*E*), *unc-51(RNAI)*;*Igg-1(Δ)(F)*, *unc-51(RNAI)*;*Igg-1(G116A)*(*G*) and *unc-51(RNAI)*;*Igg-1(G116AG117*)*(*H*).

1101 (I, J) Boxplots of the number of ATG-18::GFP puncta (P) (n=13, 24, 10, 14, 28, 36, 27, 20) and the mean fluorescent intensity (Q) (n= 42, 45, 55, 140, 145, 130,115, 100) (Kruskal-Wallis test p-value *<0.05, **p<0.01 ***p<0.001, ****p<0.0001, NS non-significant)

1104 (K-O) Maximum projections of epifluorescence images of the aggrephagy cargo SEPA-1::GFP
1105 in 1.5 fold embryos for *epg-2(RNAi)* (K), *Igg-1(G116A)* *epg-2(RNAi)* (L), *Igg-1(G116AG117*)*
1106 *epg-2(RNAi)* (M) and *Igg-1(G116AG117A)* *epg-2(RNAi)* (N). Boxplots of SEPA-1::GFP dots
1107 (n=10, Kruskal Wallis p-value***<0.001, NS non-significant) (O). The degradation of SEPA-
1108 1::GFP in *Igg-1(G116A)* animals is dependent on EPG-2. Scale bar is 10 µm.

1109

1110 **Source data**

1111

1112 **Figure1-Source_Data1**

1113 Folder containing original microscopy pictures, quantification data and western blots shown
1114 in Figure 1

1115 **Figure2-Source_Data1**

1116 Folder containing original microscopy pictures and quantification data shown in Figure 2

1117 **Figure3-Source_Data1**

1118 Folder containing original microscopy pictures and quantification data shown in Figure 3

1119 **Figure4-Source_Data1**

1120 Folder containing original microscopy pictures and quantification data shown in Figure 4

1121 **Figure5-Source_Data1**

1122 Folder containing original microscopy pictures, quantification data and western blots shown
1123 in Figure 5

1124 **Figure6-Source_Data1**

1125 Folder containing original microscopy pictures and quantification data shown in Figure 6

1126 **Figure7-Source_Data1**

1127 Folder containing original microscopy pictures and quantification data shown in Figure 7

1128 **Figure1-figure-supplement2-Source_Data1**

1129 Folder containing original microscopy pictures and quantification data shown in Figure1-
1130 figure-supplement2
1131 **Figure1-figure-supplement3-Source_Data1**

1132 Folder containing original microscopy pictures and quantification data shown in Figure1-

1133 figure-supplement3

1134 **Figure3-figure-supplement1-Source_Data1**

1135 Folder containing original microscopy pictures and quantification data shown in Figure3-

1136 figure-supplement1

1137 **Figure3-figure-supplement2-Source_Data1**

1138 Folder containing original microscopy pictures and quantification data shown in Figure3-

1139 figure-supplement2

1140 **Figure5-figure-supplement1-Source_Data1**

1141 Folder containing quantification data shown in Figure5-figure-supplement1

1142 **Figure7-figure-supplement1-Source_Data1**

1143 Folder containing original microscopy pictures and quantification data shown in Figure7-

1144 figure-supplement1

1145
# Appendix 1

1

2

3 LGG-1(wt) and LGG-1(G116A) partially restore the survival to nitrogen starvation of yeast

4 *atg8(Δ)*

5 Our results demonstrate that the localization of LGG-1 to the membrane is dispensable for
6 autophagy and other functions. To address whether it is a particularity of *C. elegans*, a similar
7 strategy was performed for Atg8 in the yeast *S. cerevisiae*. Atg8 precursor ends with an
8 arginine at position 117 (Figure 1-figure supplement 3A) and using mutant proteins expressed
9 from centromeric plasmids the Oshumi lab (Kirisako et al., 2000; Nakatogawa et al., 2012) has
10 shown that G116 is essential for autophagy. The endogenous *ATG8* was modified by an
11 homologous recombination strategy to generate *atg8(G116A)* and *atg8(G116AR117*)* alleles,
12 and the autophagy flux was assessed using the Pho8Δ60 reporter (Noda and Klionsky, 2008).
13 Both *atg8(G116A)* and *atg8(G116AR117*)* mutants were unable to achieve a functional
14 autophagy and behave similarly to *atg8(Δ)* or *atg1(Δ)* null mutants (Figure 1-figure
15 supplement 3B). The analysis of nitrogen starvation survival showed that both *atg8(G116A)*
16 and *atg8(G116AR117*)* strains were unable to recover after a 4 days starvation, similarly to
17 *atg1Δ* and *atg8Δ* (Figure 1-figure supplement 3C).

18 The non-autophagy function of *atg8(G116A)* and *atg8(G116AR117*)* mutants was assessed by
19 analyzing the shape of the vacuole (Banta et al., 1988). During exponential growth, *atg8(Δ)*
20 cells frequently presented multiple small vacuoles compared to wild-type cells which harbor
21 usually less than 4 vacuoles (Figure 1-figure supplement 3D, E). The incidence of defective
22 vacuolar shape decreased in *atg8(G116A)* and *atg8(G116AR117*)* cells indicating that the non-
23 autophagy functions were partially maintained. These data suggest that in *S. cerevisiae* the
24 non-autophagy functions of Atg8 are partially independent of its cleavage and conjugation.
25 *Atg8* mutant was then used to investigate whether the functionality of LGG-1(G116A) in
26 autophagy was restricted to *C. elegans*. The capacity of LGG-1(G116A) to restore nitrogen
27 starvation survival to *atg8(Δ)* mutant cells was compared with LGG-1(wt) and LGG-
28 1(G116AG117*). The corresponding cDNAs were cloned in a centromeric vector and the
29 proteins were expressed in *atg8(Δ)* mutants. The expression of LGG-1(wt) and LGG-1(G116A),
30 but not LGG-1(G116AG117*), improved weakly the nitrogen starvation survival indicating a
31 partial complementation (Figure 1-figure supplement 3F). This suggests that the functionality
32 of LGG-1(G116A) is not restricted to *C. elegans*, supporting an intrinsic property of LGG-1 form
33 I.

34

35

36 LGG-1(G116A) function in aggrephagy is dependent on UNC-51 and EPG-2

37

38 Data in yeast and mammals have revealed that Atg8/LC3/GABARAP can interact with
39 Atg1/ULK1 and modify the kinase activity of the ULK1 complex (Alemu et al., 2012; Grunwald
40 et al., 2020; Kraft et al., 2012; Nakatogawa et al., 2012). In *C. elegans*, LGG-1 can directly binds
UNC-51/ULK1 (Wu et al., 2015) and the cargo SEPA-1 (Zhang et al., 2009) and could have an early function for initiating aggrephagy (Lu et al., 2011). To decipher the function of LGG-1 form I in the induction of autophagy we performed a genetic approach. Using RNAi we depleted UNC-51 and quantified the initiation events in vivo in wild-type, *lgg-1(Δ)*, *lgg-1(G116A)*, and *lgg-1(G116AG117*)* embryos (Figure 7-figure supplement 1A-J). As expected, depleting UNC-51 resulted in the decrease of ATG-18 puncta while depleting LGG-1 lead to the increase of ATG-18 intensity and number of puncta. The decrease of ATG-18::GFP puncta after co-depletion of UNC-51 and LGG-1 indicated that LGG-1 functions depends on UNC-51. For *lgg-1(G116A)* animals, a small decrease was observed in the number of puncta compared to the wild-type animals but no change in the total signal of ATG-18::GFP. Moreover, the depletion of UNC-51 further decreased ATG-18::GFP puncta and intensity confirming that LGG-1(G116A) almost behaves like the wild-type LGG-1 for the initiation (Figure 7-figure supplement 1C, G, I, J). In *lgg-1(G116AG117*)* embryos, a marked increase of ATG-18 puncta was observed, but contrarily to *lgg-1(Δ)*, the number did not decrease when UNC-51 was depleted (Figure 7-figure supplement 1D, H, I, J). These data confirm electron microscopy observations and support a neomorphic function for the truncated LGG-1(G116AG117*), independent of ULK1 complex.

Finally, we analyzed whether the cargoes degradation by LGG-1(G116A) was dependent of the scaffolding protein EPG-2 (Figure 7-figure supplement 1K-O). Aggregate-prone proteins are degraded through autophagy in *C. elegans* embryo through liquid-liquid phase separation promoted by the receptor SEPA-1 and regulated by the scaffolding protein EPG-2 (Zhang et al., 2018). The depletion of EPG-2 induced the persistence of SEPA-1::GFP aggregates in wild-type and in *lgg-1(G116A)* embryos. This data indicates that the function of LGG-1(G116A) for degrading SEPA-1 is dependent of EPG-2.

## Supplementary Material and Methods

67

## Immunostaining and electron microscopy

200 µm-deep flat carriers (Leica Biosystems) were incubated few minutes in 1% phosphatidylcholine (Sigma-Aldrich,61755) in chloroform. Young adults were transferred to the carriers containing 20% BSA (Sigma-Aldrich, A7030) in M9 buffer, followed by cryo-immobilization in the EMPACT-2 HPF apparatus (Leica Microsystems) and cryo-substitution with Automated Freeze-substitution System (AFS2, Leica Microsystems). Cryosubstitution medium, composed by 0.1% acetate uranyl in acetone, for 3 days with a slow increase of the temperature from -90°C to -15°C. After several washes of acetone and ethanol at -15°C, samples were incubated successively in 25% to 100% LRWHITE resin (Electron Microscopy Sciences, 14381) in ethanol, then UV-polymerized 24 hours at -15°C. 80 nm thin sections were collected on a Nickel 100-mesh grids (Electron Microscopy Sciences, FCF-100-Ni) and immunostained with the immunogold labelling system (IGL, Leica microsystem). Samples were labelled during 1 h with the primary rabbit anti-GFP antibody (Abcam, ab6556; 1:10 dilution in 0.1% BSA in PBS), washed 4 times for 2min with PBS, and twice for 5 min with 0.1% BSA in
82

PBS. Samples where then labelled during 30 min with the secondary goat anti-rabbit antibody coupled to 10-nm colloidal gold particles (Biovalley, 810.011) at 1:20 dilution in 0.1% BSA in PBS. Sections were contrasted with 2% uranyl acetate (Merck, 8473) for 8 min and 0.08 M lead citrate (Sigma-Aldrich, 15326) for 2 min, and were observed with a Jeol 1400 TEM at 120 kV equipped with a Gatan SC1000 Orius CCD camera (Roper Industries).

83

84

85

86

87

88

**S. cerevisiae culture and strains**

89

Yeast cells were grown to log phase in YPD (1% yeast extract, 2% bactopeptone and 2% glucose) or complete synthetic medium (CSM) without uracil or leucine. The reference strain is BY4742. Other strain and genotypes are listed in the Key Resources Table.

90

91

92

93

**S. cerevisiae culture and autophagy assays**

94

The quantitative Pho8Δ60 assay for bulk autophagy, was performed as described (Noda and Klionsky, 2008). Cells were grown to log phase in YPD medium then were transferred to nitrogen starvation medium for 4h. At different time point, 5 OD₆₀₀ units of cells were collected, washed and resuspended in ice-cold assay buffer (250mM Tris-HCl, pH 9; 10mM MgSO₄ and 10 µM ZnSO₄) with 1mM PMSF. Then cells were broken using glass beads. For the assay, 10 µl of lysed cells are added to 500 µl of ice-cold assay buffer, placed at 30°C for 5 min before tadding 50 µl of 55 mM α-naphthyl phosphate disodium salt for 20 min at 30°C. The reaction was stopped with 500 µl of 2M glycine-NaOH, pH 11 and the fluorescence measured (345 nm excitation /472 nm emission). The Pho8Δ60 activity corresponds to light emission per amount of protein in the reaction (mg) and reaction time (min).

95

96

97

98

99

100

101

102

103

104

The number of vacuoles was counted after incubation of exponentially growing cells with FM4-64 (33 µM) in YPD medium at 30°C for one hour, washing and imaging.

105

106

For survival to nitrogen starvation cells were grown to log phase in appropriate complete synthetic medium (CSM) and transferred to nitrogen starvation medium (0.17% yeast nitrogen base and 2% glucose). After 0 to 6 days of starvation, cells were spread on YPD plates and colonies were counted after 2 days at 30°C. For LGG-1 rescue assays, LGG-1(G116A) and LGG-1(G116AG117*) were generated by PCR amplification from cDNA LGG-1 and cloned in pRS416 vector under the control of GPD promoter.

107

108

109

110

111

112
<figure><img src="image_2.png" /><figcaption>Figure 1 Leboutet et al.</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>**Figure 2** Leboutet et al.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 3 Leboutet et al</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Figure 4** Leboutet et al.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Figure 5** Leboutet et al.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Figure 6** Leboutet et al.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Figure 7** Leboutet et al.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>**Figure 1-figure supplement 1** Leboutet et al.</figcaption></figure>
A

<figure><img src="image_11.png" /></figure>

C

LGG-1 (G116A)

Protein sequence coverage 73%

1. MKWAYKEENNEFEKRRAEGDKIRRKYPDRIPVIVEKAPKSKLHDLDKKKYL

51. VPSDLTVGQFYFLIRKRIQLRPEDALFFFVNNVIPQTMTTMGQLYQDHHEE

101. DLFLYIAYSDESVYAGEVEKKE

Fragmentation of peptide 107-121: IAYSDESVYAGEVEK Precursor form

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

m/z=830.3981 (2+), experimental monoprotonated mass MH+ 1658.7817 Da,
theoretical mass MH+ 1658.7675 Da (delta ppm=8.53)

Fragmentation of peptide 89-116: TTMGQLYQDHHEEDLFLYIAYSDESVYA Cleaved form

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

m/z=1113.5049 (3+), experimental monoprotonated mass MH+ 3337.4929 Da,
theoretical mass MH+ 3337.4761 Da (delta ppm=5.04)

B

LGG-1 (G116AG117*)

Protein sequence coverage 78%

1. MKWAYKEENNEFEKRRAEGDKIRRKYPDRIPVIVEKAPKSKLHDLDKKKYL

51. VPSDLTVGQFYFLIRKRIQLRPEDALFFFVNNVIPQTMTTMGQLYQDHHEE

101. DLFLYIAYSDESVYAGEVEKKE

Fragmentation of peptide 89-116: TTMGQLYQDHHEEDLFLYIAYSDESVYGA Truncated form

<figure><img src="image_16.png" /></figure>

m/z=1113.4997 (3+), experimental monoprotonated mass MH+ 3337.4925 Da,
theoretical mass MH+ 3337.4761 Da (delta ppm=4.90)

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /><figcaption>**Figure 1-figure supplement 2** Leboutet et al.</figcaption></figure>
<figure><img src="image_22.png" /><figcaption>Figure 1-figure supplement 3 Leboutet et al.</figcaption></figure>
<figure><img src="image_23.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_25.png" /></figure>

**Figure 3-figure supplement 1** Leboutet et al.
<figure><img src="image_26.png" /><figcaption>Figure 3-figure supplement 2 Leboutet et al.</figcaption></figure>
<figure><img src="image_27.png" /><figcaption>Figure 5-figure supplement 1 Leboutet et al.</figcaption></figure>
<figure><img src="image_28.png" /><figcaption>**Figure 7-figure supplement 1** Leboutet et al.</figcaption></figure>