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# Keeping your endosymbiont under control: the enigmatic plastid membrane ATG8ylation in Apicomplexa parasites

Sébastien Besteiro

LPHI, Univ. Montpellier, CNRS, INSERM, France

## Abstract

ATG8ylation of membranes has been increasingly reported over the last few years, in various configurations and across different eukaryotic models. While the unconventional conjugation of ATG8 to the outermost membrane of the plastid in apicomplexan parasites was first observed over a decade ago, it is often overlooked in literature reviews focusing on the ATG8ylation of non-autophagosomal membranes. Here, I provide a brief overview of the current knowledge on plastid ATG8ylation in these parasites and discuss a possible parallel between the evolutionary origin of this plastid and other ATG8ylation processes, such as LC3-associated phagocytosis.

Keywords: ATG8ylation, apicoplast, organelle division, Apicomplexa, evolution,

Contact: Sébastien Besteiro, [sebastien.besteiro@inserm.fr](mailto:sebastien.besteiro@inserm.fr), LPHI, University of Montpellier, CNRS, INSERM, Place E. Bataillon, 34095 cedex 5, Montpellier, France
20 Eukaryotic parasites with a vestigial plastid

21 Apicomplexan parasites are responsible for a significant disease burden in humans
22 and other animals. Among them, the most studied research models are parasites of
23 the genus *Plasmodium*, which are responsible for about half a million deadly malaria
24 cases each year [1], and the less lethal but much more widespread parasite
25 *Toxoplasma gondii*, which can cause serious complications in immunocompromised
26 individuals [2]. Like several (but not all) apicomplexan species, *Plasmodium* and
27 *Toxoplasma* host an organelle called the apicoplast, which originated from a secondary
28 endosymbiosis (Fig. 1A). First, an early host internalized a cyanobacterium, to give
29 rise to three lineages that include red algae, green algae and land plants. This
30 potentially happened by phagocytosis, although it is still an unresolved question [3],
31 and as algal primary plastids are surrounded by two membranes (which probably
32 correspond to the cytoplasmic and the outer membranes of the original
33 cyanobacterium), it would imply that the phagosomal membrane was subsequently
34 lost. Through a second endosymbiotic event, a red alga was internalized by an
35 apicomplexan ancestor via phagocytosis (according to the traditional model, though
36 this point is also still debated [4]), to give rise the plastid that subsequently lost its ability
37 to perform photosynthesis. Due to the diverse origins of the four membranes
38 surrounding the apicoplast, and because through evolution there was a transfer of
39 genes coding for plastid proteins from the endosymbiont to the host nucleus, protein
40 import to the organelle involves a patchwork of different protein machineries [5].
41 Additionally, it should be noted that duplication and inheritance of the apicoplast during
42 cell division do not involve the proteins typically associated with plastid division in
43 plants or even red algae, but rely on an alternative machinery instead. This also
44 highlights the existence of lineage-specific adaptations related to the homeostasis and
45 maintenance of this plastid. The apicoplast hosts important metabolic pathways that
46 involve proteins largely absent from the mammalian hosts of medically-important
47 apicomplexan parasites, which is why it is considered an excellent potential drug target
48 and has drawn particular interest since its discovery in the late 1990s [6].

49

50 ATG8ylation of the apicomplexan plastid

51 In the early 2000s, several research laboratories started to explore the function of the
52 autophagy-related machinery in more diversified eukaryotic models. Bioinformatic
53 analyses looking for homologs in apicomplexan genomes revealed a partially
54 conserved machinery, the core system for membrane conjugation of the Atg8
55 (autophagy related 8) protein being the most conserved [7,8]. Atg8-family proteins
56 (MAP1LC3/LC3 and GABARAP subfamilies in mammals; referred to here as ATG8) is
57 essential for autophagosome biogenesis and maturation; it is also a primary marker of
58 autophagosomes and autophagy. Typically, membranes can be "ATG8ylated" [9–11]
59 by this ubiquitin-like protein, which is conjugated to phosphatidylethanolamine (PE)
60 through an enzymatic cascade involving E1, E2 and E3 proteins (ATG7, ATG3 and the
61 ATG12–ATG5-ATG16L complex, respectively). In both *Plasmodium* and *Toxoplasma*,
62 ATG8-decorated autophagosomes were described in stress conditions that can be
63 induced experimentally by nutrient starvation. However, early functional studies initially
64 struggled to convincingly establish the presence of a clear canonical degradative
65 autophagic pathway, which was more recently demonstrated, at least in *Toxoplasma*
[12–16]. Unexpectedly, what early studies also showed is an intriguing localization for ATG8 at the apicoplast [17–23] in absence of any external stress. This was observed in several developmental stages of *Plasmodium* (multiplying asexually in red blood cells and in the liver of the host), as well as in *Toxoplasma* [24].

During parasite division, apicoplast replication involves elongation and branching of the organelle prior to fission and repartition into daughter cells [25]. The machinery associated with this process in apicomplexan parasites is quite different from the one used for division of plastids in algae and plants, with the involvement of a different class of dynamins. Functional studies showed that ATG8 depletion leads to apicoplast segregation defects in *Plasmodium* and *Toxoplasma* [26,27], and live imaging in *Toxoplasma* showed that ATG8 recruitment to the organelle was dynamic and happening predominantly during its elongation [26]. Conversely, ATG8 overexpression in *Plasmodium* liver stages led to an abnormally fast expansion of the apicoplast [28]. While converging evidence points to an important and original role for ATG8 in driving the proper replication and segregation of the apicoplast, the precise function of the protein in this process is still not completely elucidated at the molecular level. Particularly, the structural determinants underlying the specific effector function of apicomplexan ATG8 at the organelle membrane remain largely uncharacterized [29]. ATG8 may be involved in apicoplast membrane elongation and dynamics during division or, as it was localized close to the centrosomes that drive organelle inheritance [26], it may also serve as an adapter for proper anchoring of the organelle in the final stages of segregation.

## Similarities with other non-canonical membrane ATGylation

The data gathered over the years in Apicomplexa demonstrated that ATG8 associates with the outermost membrane of the apicoplast in a highly regulated manner. In contrast to most eukaryotes, apicomplexan ATG8s already end with a free C-terminal glycine necessary for phospholipid conjugation. So, while the ATG4 cysteine protease is unnecessary for ATG8 maturation prior to membrane conjugation, it was nevertheless shown to be important for recycling of the protein and maintaining the homeostasis of the apicoplast [19,30]. This is also in line with a potential dynamic and transient role for ATG8 at the apicoplast membrane.

The molecular machinery needed for ATG8ylation of the outermost apicoplast membrane was characterized through a series of functional studies (Fig. 1A). The class III phosphatidylinositol 3-kinase (PtdIns 3-kinase) VPS34 (called PIK3C3 in mammals), and its regulatory subunit VPS15 (PIK3R4 in mammals), generate the PtdIns3P lipid at the apicoplast [31–33]. Typically, in yeast and mammalian cells most PtdIns3P is associated with endosomal membranes and with the autophagosome preassembly machinery [34]. In *Plasmodium* blood stages while this lipid is present at the lysosome-like digestive vacuole of the parasite, it also localizes at the apicoplast [31]. Similarly, in *Toxoplasma* PtdIns3P is in the vicinity of the apicoplast and is essential for maintaining the homeostasis of the organelle [32]. PtdIns3P is key for protein recruitment initiating the ATG8 membrane conjugation machinery. Among PtdIns3P-binding proteins are members of the PROPPIN (β-propellers that bind polyphosphoinositides)/Atg18 family (called WIPI and WDR45 in mammals).
Interestingly, while *Plasmodium* only expresses a single PROPPIN, *Toxoplasma* has two, PROP1 and PROP2, and while the former is most likely associated with canonical autophagy, the latter is associated with the apicoplast-specific ATG8ylation [14,35,36]. Protein ATG16L is then recruited to bring together the ATG12–ATG5 complex [37], which is the ubiquitin-protein ligase E3-like enzyme and specifies the site of ATG8–PE conjugation. The final step of ATG8 involves the action of E1 (ATG7) and E2 (ATG3) enzymes, which were also shown to be important for ATG8 conjugation to the apicoplast [38,30,39].

There is a considerable eukaryotic diversity in the ATG conjugation systems beyond canonical models [40], which might reflect lineage-specific adaptations and unusual membrane ATG8ylation needs in specific organisms. For instance, there is some evolutionary divergence for the ATG12 system within the apicomplexan lineages: *Plasmodium* and *Toxoplasma* have evolved a non-covalent ATG12-ATG5 complex in place of the covalent conjugation [41], and some Apicomplexa have even possibly lost the ATG12 complex [42]. Like mammalian ATG16L1, apicomplexan homologs of this protein contain coiled-coil domains and C-terminal WD-repeats [37]. It was shown that the WD-repeats domain of mammalian ATG16L1 is typically not required for canonical autophagy, but is important for the non-canonical lipidation of LC3 on single membranes [43]. However, intriguingly, experimental deletions demonstrated that the corresponding domain in the apicomplexan homolog is not necessary for ATG8ylation of the apicoplast outermost membrane [37].

The association of ATG8 with the apicoplast involves a number of factors shared with the conjugation of the protein to autophagosomes in the context of canonical autophagy, but also with non-canonical ATG8ylation of membranes. For instance, in the last few years it has been shown that, besides autophagosomes, a variety of intracellular membranes can be ATG8ylated, which include pathogen-containing phagosomes, endosomal compartments, damaged lysosomes, exocytic vesicles, and even lipid droplets (although they are only surrounded by a lipid monolayer) [9–11]. Among these, LC3-associated phagocytosis (LAP) is a form of phagocytosis that uses components of the autophagy pathway to enhance degradation of extracellular pathogens [44]. It is distinct from xenophagy, which is a specialized form of autophagy that degrades intracellular pathogens through autophagosomes. Although it involves a single membrane, LAP shares several key regulators with canonical autophagy throughout various stages, ultimately leading to lysosomal degradation of the pathogen (Fig. 1B). However, there are also noticeable differences between the two processes. One of the main differences is that unlike canonical autophagy the activation of LAP does not rely on the stress response-triggered signaling pathway driven by MTORC1 and ULK1 kinases. Instead, quite logically, its activation is driven by phagocytosis and the recognition of pathogen-specific ligands. Upon activation, the class III PtdIns 3-kinase complex, that includes PIK3C3/VPS34 and beclin1 (BECN1), generates PtdIns3P on the phagosome membrane. The rubicon (RUBCN) protein, a key LAP regulator, enhances PtdIns 3-kinase activity while it typically inhibits canonical autophagy. This leads to the recruitment of ATG12–ATG5-ATG16L1, which facilitates LC3 lipidation and its conjugation to the phagosome.

There are some differences between the molecular machinery underlying ATG8 conjugation at the apicoplast and LAP: for instance RUBCN has no known homolog in Apicomplexa, and the *Toxoplasma* BECN1 homologue does not seem essential for
158 apicoplast homeostasis [37]. Thus, synthesis of PtdIns3P at the apicoplast may be
159 regulated differently in apicomplexan parasites. Otherwise the two processes involve
160 similar steps (Fig. 1). Given the origin of the apicoplast as an endosymbiotic organelle,
161 likely involving the phagocytosis of a bacterial ancestor, it is tempting to draw a parallel
162 between ATG8ylation of the phagosomal membrane in the context of LAP, and the one
163 of the outermost apicoplast membrane. Autophagy is considered an ancestral pathway
164 that may already have existed in the last eukaryotic common ancestor, and
165 phagocytosis is commonly thought to be central to the very origin of the eukaryotic cell,
166 so LAP is at the crossroads of these two ancient evolutionary pathways [45]. Of course,
167 the precise involvement of phagocytosis in the acquisition of the apicoplast remains
168 experimentally unproven and phagocytosis itself may have evolved independently
169 several times within eukaryotes, so it does not imply that apicoplast and phagosomal
170 ATG8ylation share a common evolutionary origin. Yet, more generally, the relationship
171 between the autophagy machinery and endosymbiotic organelles remains fascinating,
172 and it was even suggested that autophagy may have evolved specifically as a
173 response to the selective pressures imposed by endosymbiosis [46]. Moreover, the
174 outermost membrane of complex plastids has been alternatively suggested to derive
175 from an autophagosomal membrane or a phagosomal membrane [5].

176

177

**Outstanding questions**

178 Several key outstanding questions remain to fully elucidate the role and the process of
179 apicoplast membrane ATG8ylation. First, while ATG8 conjugation to the apicoplast
180 seems to be a dynamic process which is timely regulated during parasite division [26],
181 the recruitment signals remain to be identified. How the outermost apicoplast
182 membrane is specifically recognized for ATG8ylation is another outstanding question.
183 The precise lipid composition of this membrane is currently unknown, and recent
184 findings in *Toxoplasma* have suggested in that ATG8 could be translocated to the
185 organelle via vesicles, in a SNARE-dependent manner [37], rather than through direct
186 conjugation at the apicoplast membrane. Finally, while ATG8 seems important for
187 driving the division and/or the inheritance of the apicoplast during division, the exact
188 molecular function of ATG8 and its putative interacting partners remain a major
189 conundrum. The membrane-remodeling properties of ATG8 [47] might be used for the
190 drastic change in shape displayed by the organelle, illustrated by the extensively
191 elongated and branched aspect of the apicoplast during division of several
192 *Plasmodium* developmental stages [48]. Of course, elucidating these specific points
193 will require further investigation. In this regard, apicomplexan models may prove
194 invaluable for gaining insights into the diversity and primary functions of the autophagy
195 machinery.

196

197

**Disclosure statement**

198

No potential conflict of interest was reported by the author

199
200 Funding

201 Research in my laboratory is currently funded by the Agence Nationale de la Recherche (grant ANR-24-CE15-3620).

202

203

204 ORCID

205 Sébastien Besteiro [0000-0003-1853-1494](https://orcid.org/0000-0003-1853-1494)

206

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

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**Figure 1.** Schematic representation of (A) the endosymbiotic events leading to the acquisition of the apicoplast in apicomplexan parasites and (B) LC3-associated phagocytosis (LAP). Insets are showing the molecular players for the different steps of ATG8/LC3 conjugation to the membrane of the apicoplast and the LAPosome (LC3-decorated phagosome), respectively. See corresponding text for details.