Beyond Autophagy: The Expanding Roles of ATG8 Proteins

Abstract

The ATG8 family proteins are critical players in autophagy, a cytoprotective process that mediates degradation of cytosolic cargo. During autophagy, ATG8s conjugate to autophagosome membranes to facilitate cargo recruitment, autophagosome biogenesis, transport, and fusion with lysosomes, for cargo degradation. In addition to these canonical functions, recent reports demonstrate that ATG8s are also delivered to single-membrane organelles, which leads to highly divergent degradative or secretory fates, vesicle maturation, and cargo specification. The association of ATG8s with different vesicles involves complex regulatory mechanisms still to be fully elucidated. Whether individual ATG8 family members play unique canonical or non-canonical roles, also remains unclear. This review summarizes the many open molecular questions regarding ATG8s that are only beginning to be unraveled.

Keywords: GABARAP; LC3; LC3-associated phagocytosis; multivesicular bodies; non-canonical autophagy; unconventional secretion.

Figure 1. Key Figure: Overview of degradative and secretory roles of ATG8 proteins described in this review.

ATG8s (green circles) incorporate into intracellular vesicles with degrative fate, including autophagosomes (Panel A) and diverse vesicles of endosomal origin (Panels B and C). During macroautophagy (Panel A), ATG8s associate with the outer- and inner membrane of autophagosomes via canonical conjugation mechanisms (orange text boxes). In the inner membrane, ATG8s interact with receptors to recruit cytosolic cargo, such as defective mitochondria and protein aggregates. On the outer membrane, ATG8s bind adaptor proteins that bring the transport and fusion machinery, allowing directional retrograde transport of autophagosomes and fusion with lysosomes for cargo degradation. Via non-canonical conjugation mechanisms (purple boxes), ATG8 also incorporate into phagosomes containing components of extracellular origin such as bacteria or apoptotic bodies (Panel B). During this process, called LC3-associated phagocytosis (LAP) ATG8s associate to the cytosol-facing part of the membrane and may play roles in fusion and transport for lysosomal degradation. In addition, ATG8s associate to multivesicular bodies (MVB) to recruit receptors and facilitate cargo degradation in a process called endosomal microautophagy (eMI). Interestingly, ATG8s can also associate with secretory vesicles: small extracellular vesicles and secretory autophagosomes, via potentially non-canonical and canonical conjugation machineries, respectively. Within MVB, ATG8s participate in the inward budding of small extracellular vesicles and recruit RNA-binding proteins by direct interaction, to allow their subsequent secretion upon fusion of the MVB with the plasma membrane, during a process called LC3-dependent extracellular vesicle loading and secretion (LDELS) (Panel D). Within secretory autophagosomes, ATG8s may mostly participate in the recruitment of specific transport and fusion machinery, which can facilitate directional anterograde trafficking towards the plasma membrane for fusion and cargo release to the extracellular media. Some cargo, such as IL-1β can be incorporated in between the inner- and outer membranes of autophagosomes for unconventional secretion (Panel E). Finally, ATG8s incorporate into amyloid-β-containing endosomes (Panel C). During LC3-associated endocytosis (LANDO), endosomes containing amyloid-β are endocytosed (1), then amyloid-β is delivering to lysosomes for degradation (2) and finally fusion of endosomes with the plasma membrane allows the recycling of amyloid-β receptor back to the cell surface (3). See text for further details.

Figure 2.. Proposed model of LC3-dependent extracellular vesicle loading and secretion (LDELS).

During LDELS, LC3B (green circles) and possibly other ATG8 proteins are delivered to the limiting membrane of multivesicular bodies (MVBs) where they specify the loading of cargoes including RNA binding proteins (RBPs; blue) into extracellular vesicles for secretion outside the cell. This pathway requires neutral sphingomylinase-2 (nSMase2; yellow) and LC3B-dependent recruitment of Factor-associated with nSMase2 activity (FAN; purple), which may facilitate intraluminal budding via localized ceramide production.

Box 1 Figure I:. 3-D structure of LC3B.

Crystal structure of LC3B showing the typical ubiquitin-like folding described for ATG8s (PDB ID 3VTU). The two consecutive N-terminal alpha helices are highlighted by an orange dotted line. Basic amino acids shown in blue form part of the LIR-docking site (LDS) and participate in the binding of LIR-containing proteins. Highlighted in red is LC3B Threonine 50, a key residue located within the LDS, whose phosphorylation regulates protein-protein interactions and is crucial for the autophagy process (see section on Regulatory mechanisms operating on ATG8 proteins that specify canonical versus non-canonical functions). Figure created with the software PyMOL.

Box 2 Figure I:. Viral subversion of ATG8 proteins for exocytosis.

Viruses hijack LC3B and possibly other ATG8 family members (green circles) to facilitate egress and transmission between cells. Non-enveloped RNA viruses including poliovirus and coxsackievirus B (CVB3) are released in autophagosome-like vesicles bearing LC3B and phosphatidylserine. Enveloped double stranded DNA (dsDNA) viruses including Epstein-Barr virus (EBV), human cytomegalovirus (HCMV) and varicella zoster virus (VZV) are released via mechanisms regulated by ATGs and contain lipidated LC3B. Dengue virus (DENV) and hepatitis C virus (HCV) are released in small extracellular vesicles harboring lipid-modified LC3B that may originate from multivesicular bodies (MVBs). Finally, influenza virus filamentous budding involves matrix protein 2 (M2)-dependent targeting of LC3B to the plasma membrane; however, LC3B does not appear to be packaged into bud influenza virions. See text for more details.