An essential role for the ATG8 ortholog LC3C in antibacterial autophagy

Abstract

Autophagy defends the mammalian cytosol against bacterial invasion. Efficient bacterial engulfment by autophagy requires cargo receptors that bind (a) homolog(s) of the ubiquitin-like protein Atg8 on the phagophore membrane. The existence of multiple ATG8 orthologs in higher eukaryotes suggests that they may perform distinct functions. However, no specific role has been assigned to any mammalian ATG8 ortholog. We recently discovered that the autophagy receptor CALCOCO2/NDP52, which detects cytosol-invading Salmonella enterica serovar Typhimurium (S. Typhimurium), preferentially binds LC3C. The CALCOCO2/NDP52-LC3C interaction is essential for cell-autonomous immunity against cytosol-exposed S. Typhimurium, because cells lacking either protein fail to target bacteria into the autophagy pathway. The selectivity of CALCOCO2/NDP52 for LC3C is determined by a novel LC3C interacting region (CLIR), in which the lack of the key aromatic residue of canonical LIRs is compensated by LC3C-specific interactions. Our findings provide a new layer of regulation to selective autophagy, suggesting that specific interactions between autophagy receptors and the ATG8 orthologs are of biological importance.

Macroautophagy (hereafter autophagy) is an evolutionarily conserved process that captures cytoplasmic components into double-membrane vesicles known as autophagosomes. The biogenesis of autophagosomes starts when a portion of the cytoplasm is encapsulated by a phagophore, which subsequently elongates to form an autophagosome. This process requires more than thirty AuTophaGy (ATG)-related proteins, including ATG8, a ubiquitin-like protein conjugated to phosphatidylethanolamine at the phagophore/autophagosomal membrane. Higher eukaryotes encode multiple ATG8 orthologs that form the GABARAP-like and LC3-like subfamilies. No specific function for any individual family member had been determined until our recent discovery that LC3C is essential for antibacterial autophagy in human cells.

The uptake of cargo into autophagosomes proceeds either nonselectively, when during periods of starvation cytosolic components are recycled indiscriminately, or selectively, when aggregated proteins, organelles or pathogens are targeted preferentially compared with bulk cytosol. Selective autophagy is mediated by cargo receptors, a.k.a. autophagy adaptors, which direct autophagosomes toward cargo by bridging the phagophore membrane to ‘eat-me’ signals on cargo. Selective autophagy is essential for the cell-autonomous defense against bacteria attempting to colonize the host cytosol. In contrast to professional cytosol-dwelling bacteria that have evolved to avoid autophagy, selective autophagy efficiently restricts bacteria that accidentally encounter the cytosol, as may occur when bacteria fail to establish their replicative niche inside phagosomes. Infection with the Gram-negative bacterium S. Typhimurium has become a model system for antibacterial autophagy since S. Typhimurium frequently damages the limiting membrane of its vacuole and thereafter proliferates rapidly in the cytosol of epithelial cells unless restricted by autophagy (Fig. 1). Membrane remnants of Salmonella-containing vacuoles attract LGALS8/galectin-8, a cytosolic β-galactoside-binding lectin that detects glycans previously hidden within the vesicle. This LGALS8-dependent ‘eat me’ signal is detected by the autophagy receptor CALCOCO2/NDP52. In addition, cytosol-exposed bacteria together with the damaged membranes become labeled with polyubiquitin, an ‘eat-me’ signal distinct from LGALS8 that is detected by three cargo receptors, CALCOCO2/NDP52, SQSTM1/p62 and OPTN/optineurin. Each of these receptors is crucial for the restriction of bacterial proliferation, because cells lacking any of these proteins fail to target bacteria to autophagy. Notwithstanding the unique role of CALCOCO2/NDP52 as cargo receptor in the LGALS8-dependent pathway, the nonredundant contributions of SQSTM1, OPTN and CALCOCO2/NDP52 to antibacterial autophagy remain poorly understood.

Figure 1. CALCOCO2/NDP52-LC3C-dependent autophagy restricts the proliferation of S. Typhimurium. Salmonella enterica serovar Typhimurium, a Gram-negative pathogen, invades human cells to replicate within a modified phagosome known as the Salmonella-containing vacuole. Damage to the limiting membrane of this vacuole exposes glycans, which were previously hidden inside the vacuole and which are detected by the danger receptor LGALS8/galectin-8. In addition, membrane remnants and/or cytosolic bacteria are decorated with polyubiquitinated proteins. The LGALS8- and polyubiquitin-dependent ‘eat me’ signals are recognized by the autophagy receptor CALCOCO2/NDP52. CALCOCO2/NDP52 triggers autophagy by preferentially binding to LC3C via a noncanonical LIR motif. CALCOCO2/NDP52 and LC3C are required for efficient antibacterial autophagy, because in the absence of either protein other ATG8 orthologs are not recruited to invading bacteria, resulting in hyperproliferation of bacteria in the host cell cytosol. GBR, GABARAP.

In contrast to the diverse ‘eat-me’ signals detected by autophagy receptors on prospective cargo, the recognition of the phagophore membrane was thought to depend on a uniform mechanism, namely the binding of ATG8/LC3 by the Atg8-interacting motif/LC3-interacting region (AIM/LIR) of the autophagy receptor. Known LIRs comprise the consensus motif W/FxxI/L/V, which forms an intermolecular β-sheet with ATG8/LC3 upon binding. However, while yeast encodes only one ATG8 gene, the genomes of plants, metazoans and certain protists contain multiple ATG8 orthologs. The human genome in particular harbors six expressed ATG8 orthologs, which all associate with phagophores/autophagosomes and comprise two subfamilies. The LC3-like group consists of LC3A, LC3B, and LC3C, while the GABARAP-like group comprises GABARAP, GABARAPL1, and GABARAPL2/GATE-16. The LC3 and GABARAP subfamilies are required for the elongation and closure of the autophagosomal membrane, respectively. However, no specific role for individual family members had been identified until we recently discovered that CALCOCO2/NDP52 binds preferentially to LC3C and that cells lacking either protein fail to restrict the proliferation of S. Typhimurium because of inefficient antibacterial autophagy. LC3C therefore serves an essential role in antibacterial autophagy that other ATG8 orthologs cannot execute. In addition, our work revealed an unexpected hierarchy within the ATG8 family, since in the absence of LC3C, none of the remaining ATG8 orthologs are recruited efficiently to bacteria.

Our structural studies showed that CALCOCO2/NDP52 achieves specificity for LC3C via a noncanonical LIR motif that consists of the tripeptide Leu-Val-Val and which we refer to as CLIR (LC3C-specific LIR). Similar to the canonical LIR-ATG8 interaction, the CLIR forms an intermolecular β-sheet with the β2-strand of LC3C. However, the CLIR does not occupy the major binding pocket of canonical LIRs, the so-called aromatic pocket present in all ATG8 orthologs. The resulting impairment in binding of the CLIR peptide to ATG8 family members is an important prerequisite for its specific interaction with LC3C, since compensatory hydrophobic contacts can be established only between these two ligands. The CALCOCO2/NDP52-LC3C interaction is crucial for efficient antibacterial autophagy, since cells expressing CALCOCO2/NDP52 or LC3C mutants unable to interact with each other fail to execute antibacterial autophagy. LC3C therefore has a specialized nonredundant role, namely the autophagy of CALCOCO2/NDP52-selected bacterial cargo.

Although the role of LC3C outside antibacterial autophagy has not been tested yet, we speculate that antibacterial autophagy may benefit from a dedicated set of proteins, as the execution of antibacterial autophagy is probably more urgent than that of other forms of autophagy. Antibacterial autophagy must contain the bacterium before proliferation begins and must occur in situ, i.e., wherever the bacterium resides. In addition, bacteria-engulfing autophagosomes are larger than conventional autophagosomes and often enwrap their cargo multiple times, therefore requiring a significant supply of membrane. The potential to outcompete other membrane demands might therefore be advantageous for antibacterial autophagy. In this regard, binding of CALCOCO2/NDP52 to LC3C may recruit phagophores pre-charged with other ATG8 orthologs to the bacterium. The CALCOCO2/NDP52-LC3C pathway may also be required for the preferential handling of CALCOCO2/NDP52-selected cargo downstream of autophagosome biogenesis such as the recruitment of effectors to kill the infectious cargo or the generation of antigenic peptides for presentation to T-lymphocytes.

The selective interaction between CALCOCO2/NDP52 and LC3C suggests that ATG8 orthologs provide an extra layer of specificity to selective autophagy. It will be interesting to investigate whether other types of selective autophagy, such as the elimination of misfolded proteins or damaged organelles, require selective interactions between ATG8 orthologs and autophagy receptors. Recent studies show that in vivo a number of autophagy receptors may distinguish between ATG8 orthologs. The selective handling of cargo may therefore not be limited to antibacterial autophagy. Further studies will be necessary to determine whether these specific interactions are of biological significance and whether they use similar or unrelated mechanisms to the CLIR-LC3C interaction to achieve their specificity.

Glossary

Abbreviations:

CLIR

LC3C-interacting region

GBR

GABARAP

LGALS8

galectin-8

LIR

LC3-interacting region

NDP52

nuclear dot protein 52 kDa

S. Typhimurium

Salmonella enterica serovar Typhimurium

von Muhlinen N, Akutsu M, Ravenhill BJ, Foeglein Á, Bloor S, Rutherford TJ, et al. LC3C, bound selectively by a noncanonical LIR motif in NDP52, is required for antibacterial autophagy. Mol Cell. 2012;48:329–42. doi: 10.1016/j.molcel.2012.08.024.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.