GABARAPL2 Human

What is GABARAPL2 and to which protein family does it belong?

GABARAPL2 is a member of the ATG8 ortholog family, specifically belonging to one of three distinct clusters: (i) LC3a and LC3b; (ii) GABARAP and GABARAPL1; and (iii) GABARAPL2. It functions as a ubiquitin-like protein that can be conjugated to membrane lipids, particularly phosphatidylethanolamine (PE), through a process known as lipidation. This lipidation occurs at the C-terminal glycine residue Gly116 and is essential for its role in autophagy induction and other cellular processes .

What are the primary cellular locations of GABARAPL2?

GABARAPL2 primarily localizes to autophagosomal membranes during autophagy induction. In IFN-γ-stimulated cells, GABARAPL2 accumulates around parasitophorous vacuoles (PVs) in infected cells, though it does not directly localize to the parasitophorous vacuole membrane (PVM). Rather, it appears to associate with membrane structures surrounding the PV, as demonstrated by three-dimensional confocal microscopy analysis showing some distance between GABARAPL2 and markers like GRA7 .

How is GABARAPL2 expression regulated in human cells?

GABARAPL2 expression is significantly upregulated in response to IFN-γ stimulation. Research demonstrates that in HeLa cells treated with IFN-γ, GABARAPL2 recruitment to parasitophorous vacuoles increases from approximately 5% in unstimulated cells to about 22% in IFN-γ-treated cells. This regulation is part of the cell's innate immune response system against intracellular pathogens like Toxoplasma gondii .

What specific function does GABARAPL2 perform in the autophagy pathway?

GABARAPL2 plays a critical role in regulating autophagosome formation and autophagosome-lysosome fusion processes. Its lipidation at Gly116 is essential for autophagy induction. Unlike other LC3 family proteins, GABARAPL2 appears to have specialized functions in non-canonical autophagy pathways, particularly in immune-related contexts. Research indicates that it regulates lysosome-autophagosome fusion and participates in IFN-γ-induced clearance of pathogens, suggesting a distinct role from other ATG8 homologs in specific autophagy contexts .

How do GABARAPL2 deficiencies affect autophagy processes?

GABARAPL2 deficiency significantly impacts autophagy flux, leading to accumulation of autophagosomes. This is evidenced by increased LC3-II levels in GABARAPL2-deficient cells, indicating either enhanced autophagosome formation or impaired degradation. In experimental models, GABARAPL2 knockout cells show defects in the non-acidification-dependent restriction of pathogen growth, suggesting its role in specialized autophagy pathways distinct from the canonical degradative autophagy .

What experimental approaches are most effective for studying GABARAPL2's role in autophagy?

Effective experimental approaches include:

• CRISPR/Cas9-mediated knockout models to generate GABARAPL2-deficient cell lines

• Complementation studies with wild-type and mutant (e.g., G116A lipidation-deficient) GABARAPL2

• GFP-tagged GABARAPL2 expression for localization studies using confocal microscopy

• LC3-II quantification via immunoblotting to assess autophagosome accumulation

• Co-immunoprecipitation assays to identify interaction partners

• Three-dimensional confocal microscopy to determine precise subcellular localization

These approaches have successfully demonstrated GABARAPL2's specific role in non-canonical autophagy processes, particularly in response to IFN-γ stimulation .

How does GABARAPL2 contribute to IFN-γ-mediated immune responses?

GABARAPL2 serves as a critical effector in IFN-γ-mediated immune responses against intracellular pathogens. In IFN-γ-stimulated HeLa cells, GABARAPL2 accumulates around parasitophorous vacuoles containing Toxoplasma gondii, forming membrane structures that participate in pathogen restriction. Unlike in mouse cells where GBPs (Guanylate Binding Proteins) are essential for control, human cells utilize GABARAPL2 as a dominant effector for controlling parasite growth. The protein functions through a non-acidification-dependent growth restriction mechanism that differs from conventional lysosomal degradation pathways .

How do autophagy adaptors mediate GABARAPL2 function in pathogen restriction?

Autophagy adaptors including p62, NDP52, and OPTN (optineurin) play crucial roles in bridging GABARAPL2 to pathogen-containing vacuoles. In triple knockout cells lacking these adaptors, GABARAPL2 recruitment to parasitophorous vacuoles drops from approximately 24% to less than 10%, even after IFN-γ stimulation. Complementary expression of NDP52 or OPTN rescues this localization defect. Importantly, while autophagy adaptors are necessary for GABARAPL2 recruitment, GABARAPL2 deficiency does not affect the localization of adaptors like NDP52. This indicates a unidirectional pathway where adaptors function upstream of GABARAPL2 in the pathogen restriction process .

What is known about the GABARAPL2-GIMAP complex?

GABARAPL2 forms a functional complex with GTPase of the immune-associated protein 6 (GIMAP6) and GIMAP7. This complex plays important roles in autophagy and immune function. Human GIMAP6 shows stronger binding to GABARAPL2 compared to mouse GIMAP6, suggesting species-specific interaction patterns. The GIMAP6/GABARAPL2 complex is involved in autophagosome formation, and deficiencies in this complex result in impaired autophagy and immune dysfunction, including increased susceptibility to bacterial infections .

How do experimental methodologies distinguish between GABARAPL2 and other ATG8 family proteins?

Distinguishing between GABARAPL2 and other ATG8 family proteins requires:

• Specific antibodies validated for selective detection of GABARAPL2 versus LC3 or GABARAP

• Gene-specific knockout models using CRISPR/Cas9 targeting unique regions of GABARAPL2

• Complementation studies with tagged versions of specific ATG8 family members

• Functional assays comparing phenotypes of cells deficient in different ATG8 proteins

• Co-immunoprecipitation studies with potential interacting partners that preferentially bind specific ATG8 family members

Research has shown that even within the same family, these proteins have distinct functions, with GABARAPL2 being specifically critical for IFN-γ-induced restriction of Toxoplasma gondii, while other family members like LC3a or LC3b are not essential for this function .

What techniques are most effective for studying GABARAPL2 lipidation status?

Effective techniques for studying GABARAPL2 lipidation include:

• SDS-PAGE migration analysis to distinguish lipidated (GABARAPL2-II) from non-lipidated (GABARAPL2-I) forms

• Site-directed mutagenesis of the lipidation site (Gly116) followed by functional complementation studies

• Immunoblotting with phospholipase treatment to confirm lipid conjugation

• Subcellular fractionation to separate membrane-bound (lipidated) from cytosolic (non-lipidated) forms

• Fluorescence microscopy with lipidation-dependent membrane localization as readout

These approaches have demonstrated that lipidation at Gly116 is essential for GABARAPL2 function in autophagy and pathogen restriction, as mutant forms lacking this lipidation site (G116A) fail to rescue defects in GABARAPL2-deficient cells .

How does GABARAPL2 function differ between human and mouse cells in pathogen restriction?

In human cells, GABARAPL2 serves as a dominant effector in IFN-γ-induced restriction of Toxoplasma gondii, functioning through a non-acidification-dependent mechanism. Unlike mouse cells which rely heavily on Immunity Related GTPases (IRGs) and Guanylate Binding Proteins (GBPs), human cells utilize GABARAPL2 in a distinct pathway. While mouse GABARAPL2 has been implicated in LC3-associated phagocytosis (LAP) that recruits GBPs via interaction with ADP ribosylation factor 1 (Arf1), human GABARAPL2 appears to function independently of GBPs, which are dispensable for parasite control in human cells. This highlights species-specific evolutionary adaptations in immunity pathways .

What are the implications of GABARAPL2 dysfunction in human disease?

GABARAPL2 dysfunction has been linked to immune dysregulation and susceptibility to infections. Research on proteins that interact with GABARAPL2, such as GIMAP6, has shown that mutations affecting this interaction network can lead to inborn errors of immunity (IEIs) characterized by infections, lymphoproliferation, autoimmunity, and multiorgan vasculitis. Defects in GABARAPL2-dependent pathways impair autophagy, redox regulation, and polyunsaturated fatty acid (PUFA)–containing lipid metabolism. These findings suggest that GABARAPL2 dysfunction may contribute to a spectrum of immunological and inflammatory disorders, though direct mutations in GABARAPL2 itself are less well characterized than those in its interaction partners .

What experimental strategies can resolve contradictory findings about GABARAPL2 function?

To resolve contradictory findings about GABARAPL2 function, researchers should consider:

• Cell type-specific analyses to account for tissue-dependent functions

• Temporal studies examining GABARAPL2 activation at different time points following stimulation

• Comparative studies between species (human vs. mouse models) to identify evolutionary divergence

• Combinatorial knockout approaches targeting multiple ATG8 family members to identify redundant functions

• Structural studies of GABARAPL2 protein complexes to elucidate molecular interactions

• Pathogen strain-specific responses to determine variable requirements across different infectious agents

These approaches can help clarify whether GABARAPL2 functions primarily in canonical autophagy, non-canonical pathways, or both, depending on cellular context and stimulation conditions .

What are the key considerations when designing CRISPR/Cas9 knockout models for GABARAPL2 research?

When designing CRISPR/Cas9 knockout models for GABARAPL2 research, key considerations include:

• Guide RNA selection targeting unique regions to avoid off-target effects on other ATG8 family members

• Validation of knockout through multiple methods (immunoblotting, genomic sequencing, RT-PCR)

• Generation of multiple independent knockout clones to control for clonal effects

• Inclusion of rescue experiments with wild-type GABARAPL2 to confirm phenotype specificity

• Development of conditional knockout systems for studying essential functions

• Creation of knock-in mutants (e.g., G116A) to study specific functional domains

Effective knockout models should demonstrate complete absence of the protein while maintaining cell viability for subsequent functional studies .

How can researchers effectively measure GABARAPL2 recruitment to pathogen-containing vacuoles?

To effectively measure GABARAPL2 recruitment to pathogen-containing vacuoles, researchers should employ:

• Stable expression of fluorescently tagged GABARAPL2 (e.g., GFP-GABARAPL2) at near-endogenous levels

• Co-staining with pathogen vacuole markers (e.g., anti-GRA7 for Toxoplasma gondii)

• Three-dimensional confocal microscopy to precisely determine spatial relationships

• Quantification of the percentage of vacuoles positive for GABARAPL2 recruitment

• Time-course analysis to determine recruitment kinetics following infection

• Comparative analysis between wild-type and stimulated conditions (e.g., IFN-γ treatment)

Research has shown that in IFN-γ-stimulated HeLa cells, approximately 22% of parasitophorous vacuoles show GABARAPL2 recruitment compared to only 5% in unstimulated cells .

What controls are essential when studying GABARAPL2 lipidation and its functional significance?

Essential controls for studying GABARAPL2 lipidation include:

• Comparison between wild-type GABARAPL2 and lipidation-deficient mutants (G116A)

• Pharmacological inhibitors of the lipidation machinery (e.g., ATG7 or ATG3 inhibitors)

• Nutrient starvation conditions as positive controls for autophagy induction

• Treatment with Bafilomycin A1 to block autophagosome-lysosome fusion and accumulate lipidated forms

• Cell-free lipidation assays with recombinant components to confirm direct mechanisms

• Comparison with other ATG8 family members to identify lipidation-dependent functional differences

These controls help establish whether observed phenotypes depend specifically on GABARAPL2 lipidation rather than other functions of the protein or experimental artifacts .