An emerging role of non-canonical conjugation of ATG8 proteins in plant response to heat stress

ABSTRACT

Members of the ATG8 (autophagy-related protein 8) protein family can be non-canonically conjugated to single membrane-bound organelles. The exact function of ATG8 on these single membranes remains poorly understood. Recently, using Arabidopsis thaliana as a model system, we identified a non-canonical conjugation of ATG8 pathway involved in the reconstruction of the Golgi apparatus upon heat stress. Short acute heat stress resulted in rapid vesiculation of the Golgi, which was accompanied with the translocation of ATG8 proteins (ATG8a to ATG8i) to the dilated cisternae. More importantly, we found that ATG8 proteins can recruit clathrin to facilitate Golgi reassembly by stimulating the budding of ATG8-positive vesicles from dilated cisternae. These findings provide new insight into one of the possible functions of ATG8 translocation onto single membrane organelles, and will contribute to a better understanding of non-canonical conjugation of ATG8 in eukaryotic cells.

Abbreviations: ADS, AIMs docking site; AIM, ATG8-interacting motif; ATG, autophagy-related; CLC2, Clathrin light chain 2; ConcA, concanamycin A; HS, heat stress; PE, phosphatidylethanolamine; PM, plasma membrane; PS, phosphatidylserine; TGN, trans-Golgi network; V-ATPase, vacuolar-type ATPase

Macroautophagy (hereafter autophagy) involves the degradation of intracellular components through the formation of double-membrane vesicles called autophagosomes. Members of the ATG8 (autophagy-related protein 8) family act as a key component of the autophagy machinery that is critical for proper autophagosome formation, maturation, fusion and degradation, and cargo selection. Recently, a growing number of studies have revealed that ATG8 proteins can be conjugated onto various single membrane organelles, including phagosomes, endosomes, lysosomes, and entotic vacuoles, but also plasma membrane. This alternative conjugation of ATG8 displays broad functional significance in the immune response, cancer, and neurodegenerative diseases. There are some overlaps between the non-canonical and canonical ATG8 protein conjugation, but some key differences also exist as well. In most cases, the non-canonical conjugation is independent of the upstream autophagic regulators, including ATG1/ULK1 (autophagy-related protein 1/Unc-51-like kinase 1) complex, ATG9A (autophagy-related protein 9A) vesicles, and PI3K (phosphoinositide 3-kinase) complex. However, the autophagy-specific ubiquitin-like conjugation systems that catalyze the lipidation of ATG8 proteins are essential for both pathways.

In a recent study [1], we uncovered a non-canonical role of ATG8 proteins in plant response to heat stress (HS). After short exposure to acute HS conditions, 42–45°C for 3–20 min, the Golgi apparatus underwent rapid deformation, displaying swollen cisternae and loss of cis-to-trans polarity. It was observed that all nine ATG8 isoforms (ATG8a to ATG8i) could be recruited to the dilated cisternae during the morphological changes of the Golgi. Interestingly, translocation of ATG8 proteins to the swollen Golgi requires the autophagy-specific ubiquitin-like conjugation systems, but not upstream ATG regulators such as ATG1, ATG11, ATG9, and the PI3K complex, which mechanistically reflects the requirements with the non-canonical conjugation reported in mammalian cells. According to the evidence obtained in mammals, when ATG8 is anchored to single-membrane structures such as phagosomes, rather than covalently binding with phosphatidylethanolamine (PE) like canonical autophagy, it predominantly conjugates to phosphatidylserine (PS). While we have detected an increase in ATG8 lipidation in response to HS treatment, the type of covalent modification remains unknown, and further mass spectrometry analyses may help to clarify this issue.

The vacuolar-type ATPase (V-ATPase)-ATG16L1 (autophagy-related protein 16-like 1) axis has been reported as a critical mechanism of non-canonical conjugation of ATG8 in mammals. In this situation, the V-ATPase directly recruits ATG16L1 to locally direct the lipidation of ATG8 family proteins on endosomes. The V-ATPase is a highly conserved multisubunit proton pump that localizes to the trans-Golgi network (TGN) and tonoplast in Arabidopsis. Consistently, inhibition of the V-ATPase with concanamycin A (ConcA) disrupted the conjugation of ATG8s to the swollen Golgi in response to HS. However, it is worth noting that ATG8s rapidly dissociated from the Golgi after incubation with ConcA for 1 hour. Thus, it is still unclear whether ConcA inhibits ATG8s translocation onto the cisternal membrane or whether it triggers the dissociation of the ATG8s that is already on the dilated Golgi. Another important point is the subcellular location of V-ATPase, which is primarily associated with the TGN but not the Golgi, and the swollen TGN and Golgi remain separated after HS treatment. In this context, an open question is how a protein complex that resides on TGN affects the translocation of ATG8s to the Golgi. Therefore, the role of V-ATPase-ATG16 (the ATG16L1 ortholog in plants) axis in the regulation of conjugation of ATG8 onto the swollen Golgi remains to be further elucidated.

In canonical autophagy, one of the critical functions of ATG8 proteins is the recognition and binding of cargoes and autophagy receptors, which is usually mediated by the ATG8-interacting motif (AIM) that binds to the AIM docking site (ADS) present in the ATG8 proteins. Topologically, ATG8 on single-membrane structures is unlikely to sequester cargoes as it does in classical autophagosomes, in which the concerned ATG8 pool is present in the interior of this intermediate. Using a proximity labeling-based proteomic mapping strategy, we identified clathrin light chain 2 (CLC2) as a direct interacting partner of ATG8s, and showed that the AIM-ADS interface is required for this interaction. Immuno-EM and three-dimensional (3D) electron tomography analyses revealed that CLC2 is recruited to vesicles budding from swollen Golgi cisternae. We have thus speculated that ATG8s might function as an adaptor to recruit clathrin at the budding site of dilated cisternae. Indeed, a large number of ATG8-positive vesicles derived from the vesicular cisternae were separated from the Golgi after HS recovery for 6 h. Of note, wortmannin, which enhances vacuolar fusion in plant, triggered ATG8 accumulation at the tonoplast, suggesting that the final destination of these ATG8-positive vesicles may be the vacuole, possibly helping to remove blocked cargoes and damaged resident proteins in the dilated cisternae during heat stress.

Recruiting ATG8 proteins to single membrane vesicles yields significant implications, notably in facilitating their degradation. This widely accepted notion prompts inquiry into the reason why ATG8 present on swollen Golgi fails to directly induce its degradation. Some clues may be provided in the mechanistic details of the process of Golgi disassembly and reassembly during mitosis in mammalian cells. At the beginning of mitosis, the Golgi undergoes extensive fragmentation, allowing the equal distribution of this organelle between the dividing cells. The fragmented cisternae in the daughter cells serve as a “seed” to regrowth a new Golgi complex. In this context, we hypothesized that selective degradation of the budded ATG8-positive vesicles, rather than degradation of the entire swollen Golgi, would be a more efficient way for subsequent Golgi recovery in plant cells.

Collectively, this study leads us to a model of non-canonical ATG8 conjugation pathway involved in the reestablishment of Golgi apparatus upon heat stress (Figure 1). Our work opens up a fascinating research area of non-canonical autophagy in plants, and several interesting questions remain to be addressed. For example, how do plant cells sense heat stress to rapidly initiate Golgi disassembly? Does the V-ATPase-ATG16 axis play a role in ATG8 translocation to the swollen Golgi? How do ATG8-positive vesicles generation contribute to the Golgi cisternal restacking? Future investigations will greatly broaden our understanding of the function of non-canonical ATG8 conjugation in eukaryotic cells.

Figure 1.

Working model of the regulation of Golgi reconstruction by non-canonical ATG8 conjugation pathway upon heat stress. Short acute heat stress at 42–45°C for 3–20 min causes vacuolation of the Golgi apparatus, resulting in cis-to-trans polarity loss and disruption of post-Golgi trafficking. ATG conjugation systems catalyze the lipidation of ATG8s, possibly with phosphatidylserine (PS) form, binding to the dilated Golgi cisternal membranes. ATG8s recruit clathrin to generate ATG8-positive vesicles, which may eventually fuse with the vacuole to eliminate cargoes accumulate in the Golgi upon heat stress. PE, phosphatidylethanolamine; PM, plasma membrane; TGN, trans-Golgi network.

Funding Statement

This work was supported by grants from the National Natural Science Foundation of China (32061160467, 32270291, 31870171) and Fok Ying-Tong Education Foundation for Young Teachers in the Higher Education Institutions of China (171014) to CG, the National Science Foundation of China (31600288) and the Basic Research Program of Guangzhou (202201010508) to JZ

Disclosure statement

No potential conflict of interest was reported by the authors.