ATG13A Antibody

What is ATG13 and what functional complexes does it form?

ATG13 (Autophagy-related protein 13, also known as KIAA0652) is a critical autophagy factor required for autophagosome formation and mitophagy . It functions as a key component of the ULK1 initiation complex that regulates autophagy through the target of rapamycin (TOR) kinase signaling pathway . The protein participates in two main complexes: (1) the canonical ULK1-ATG13-RB1CC1(FIP200) complex that responds to mTOR signaling, and (2) an ULK1-independent ATG13-ATG101 subcomplex that interacts with ATG9A . Through its interaction with ULK1, ATG13 plays a role in regulating mTORC1 kinase activity and cell proliferation . Recent research has also revealed ULK1-independent functions of ATG13 in basal autophagy and mitophagy, highlighting its multifaceted role in cellular homeostasis .

What are the key structural domains of ATG13 and their functional significance?

ATG13 contains several functionally significant domains, with the HORMA domain being particularly crucial for protein-protein interactions in autophagy. The HORMA domain is required for ATG13's interaction with ATG101, and research in yeast models has demonstrated its essential role in recruiting ATG9 vesicles to the pre-autophagosomal structure (PAS) . When this domain is deleted, ATG13 becomes defective in interacting with endogenous ATG9A in mammalian cells, leading to impaired autophagy . Additionally, ATG13 possesses specific binding regions for ULK1 interaction, as evidenced by the ATG13 Δ2AA mutant that fails to interact with ULK1 while maintaining other functions . ATG13 also contains lipid-binding motifs that allow membrane association, though with different biochemical properties compared to ULK1, as ATG13 does not show the tight, detergent-resistant association with membrane fractions that ULK1 does .

Which applications are ATG13 antibodies validated for in autophagy research?

Current commercially available ATG13 antibodies have been validated for multiple experimental applications essential in autophagy research. Western blotting (WB) can be performed with a recommended dilution of 1:1000, making it suitable for detecting endogenous ATG13 expression levels and phosphorylation states . Immunoprecipitation (IP) is another validated application with a recommended 1:100 dilution, allowing researchers to study protein-protein interactions involving ATG13 . Additionally, some antibodies are suitable for immunohistochemistry on paraffin-embedded tissues (IHC-P) and immunocytochemistry/immunofluorescence (ICC/IF), enabling the visualization of ATG13 localization in both tissue sections and cultured cells . These antibodies have been tested and validated across multiple species including human, mouse, and rat samples, making them versatile tools for comparative studies .

How can researchers validate the specificity of an ATG13 antibody?

Validating ATG13 antibody specificity requires a multi-faceted approach. First, perform Western blot analysis with blocking peptide controls, as demonstrated in research where rat heart tissue lysate was probed with and without a blocking peptide . The expected molecular weight for human ATG13 is approximately 56 kDa, though it may appear at around 72 kDa due to post-translational modifications . Second, include appropriate knockout or knockdown controls in your experiments to confirm signal specificity. Third, perform parallel detection using different antibodies targeting distinct epitopes of ATG13. For co-localization studies, compare the localization pattern with known ATG13 interacting partners such as ULK1 or ATG101 . Finally, verify functional aspects through rescue experiments, where ATG13 knockout cells are reconstituted with wild-type ATG13 or functional mutants (such as ATG13 Δ2AA or ΔHORMA) to confirm that the observed phenotypes are specifically related to ATG13 function .

What are the recommended protocols for studying ATG13-protein interactions?

To investigate ATG13 protein interactions, immunoprecipitation experiments represent the primary approach. Use antibodies at a 1:100 dilution for IP applications, followed by Western blotting to detect co-immunoprecipitated proteins . For studying specific interactions, such as those between ATG13 and ATG9A or ATG101, co-immunoprecipitation experiments can be designed with appropriate controls . Binary co-IP data have successfully demonstrated that ATG13 does not require FIP200 or ULK1 to interact with ATG9A, and conversely, ULK1 does not require ATG13 to interact with ATG9A .

Advanced techniques like proximity labeling (BioID) have been instrumental in revealing novel interactions, such as the ATG9A interaction with the ATG13-ATG101 subcomplex . Additionally, the split-mVenus system has been employed to visualize the ULK1-independent ATG13-ATG101 dimer in complex with ATG9A in living cells, showing that this complex often occurs in small vesicles and can accumulate at large clusters of p62/SQSTM1 and ubiquitin . These methodological approaches provide complementary evidence for protein interactions and their functional significance.

How does phosphorylation regulate ATG13 function in autophagy?

ATG13 phosphorylation is a key regulatory mechanism controlling autophagy initiation. Under nutrient-rich conditions, the mechanistic target of rapamycin (mTOR) phosphorylates both ATG13 and ULK1, suppressing ULK1 kinase activity and consequently inhibiting autophagy . This phosphorylation prevents the formation of functional autophagy initiation complexes. Conversely, during nutrient deprivation or other autophagy-inducing conditions, reduced mTOR activity leads to dephosphorylation of ATG13, allowing it to associate with ULK1 and promote autophagosome formation .

For studying ATG13 phosphorylation experimentally, researchers should use phospho-specific antibodies or phosphatase treatments combined with mobility shift analysis on Western blots. Additionally, phosphomimetic (e.g., serine/threonine to glutamic acid) or phospho-deficient (serine/threonine to alanine) ATG13 mutants can be employed to investigate the functional consequences of specific phosphorylation events. Mass spectrometry analysis after immunoprecipitation of ATG13 can also identify novel phosphorylation sites and their dynamics under different conditions.

What is the significance of the ULK1-independent function of ATG13 in autophagy?

Recent research has uncovered significant ULK1-independent functions of ATG13 in autophagy regulation. The ATG13-ATG101 subcomplex can function independently of ULK1 to promote autophagy, particularly in basal autophagy and mitophagy contexts . This is evidenced by studies showing that ATG13 mutants defective in ULK1 binding (ATG13 Δ2AA) can still rescue the accumulation of ATG9A puncta in ATG13 knockout cells, while mutants lacking the HORMA domain required for ATG101 interaction (ATG13 ΔHORMA) cannot .

To investigate ULK1-independent functions of ATG13, researchers can utilize ULK1 binding-deficient mutants of ATG13 and the split-mVenus ATG13-ATG101 system . These tools allow visualization of the ULK1-independent ATG13-ATG101 dimer in complex with ATG9A in living cells. Additionally, researchers can examine autophagy markers like p62/SQSTM1 in cells expressing the ULK1 binding-defective ATG13 mutant to assess whether basal autophagy is maintained despite the lack of ULK1 interaction . Comparing phenotypes between ULK1 knockout cells and cells expressing ATG13 mutants can further elucidate the distinct roles of ULK1-dependent and ULK1-independent functions of ATG13.

How does the ATG13-ATG101 interaction contribute to autophagosome formation?

The ATG13-ATG101 interaction is critical for autophagosome formation, particularly through their coordination with ATG9A-containing vesicles. The HORMA domain of ATG13 is essential for its interaction with ATG101, and this interaction is required for both proteins to bind ATG9A . Studies have shown that ATG13 is required for the interaction between ATG9A and ATG101, while ATG101 is required for the interaction between ATG9A and ATG13, supporting a model where an intact ATG13-ATG101 subcomplex interacts with ATG9A to promote its function in autophagy .

When studying this interaction, experimental approaches should include co-immunoprecipitation with wild-type and mutant forms of ATG13 (particularly the HORMA domain deletion mutant), combined with functional assays measuring autophagy activity. The split-mVenus system can visualize the ATG13-ATG101 dimer in living cells, revealing that this complex often localizes to small vesicles and can accumulate at large clusters of p62/SQSTM1 and ubiquitin . Knockout studies have shown that loss of either ATG13 or ATG101 results in similar phenotypes, including the accumulation of ATG9A at large clusters of p62/SQSTM1, further supporting their coordinated function .

Why might ATG13 appear at different molecular weights on Western blots?

ATG13 can appear at various molecular weights on Western blots due to several factors that researchers should consider when interpreting their results. While the predicted molecular weight of human ATG13 is approximately 56 kDa, it is commonly observed at around 72 kDa in experimental systems . This discrepancy is primarily due to post-translational modifications, particularly phosphorylation by mTOR and ULK1, which can significantly alter the protein's electrophoretic mobility .

When troubleshooting unexpected molecular weight observations, researchers should consider: (1) The phosphorylation state of ATG13, which varies with nutrient conditions and can be verified using phosphatase treatment before SDS-PAGE; (2) The presence of other post-translational modifications such as ubiquitination or acetylation; (3) The possibility of alternative splicing variants, which may produce isoforms of different sizes; and (4) The use of different percentage gels or running conditions, which can affect the apparent molecular weight. Including positive controls of known molecular weight and using recombinant ATG13 proteins as standards can help calibrate your experimental system.

How can researchers interpret changes in ATG13 localization during autophagy?

Changes in ATG13 localization provide crucial insights into autophagy dynamics and regulation. Under basal conditions, ATG13 shows a diffuse cytoplasmic distribution with some punctate structures representing active ULK1 complexes . Upon autophagy induction, ATG13 redistributes to form more distinct puncta that mark early autophagosomal structures.

When interpreting ATG13 localization experiments, researchers should analyze: (1) Co-localization with other autophagy markers such as LC3, ULK1, or FIP200 to confirm authentic autophagy structures; (2) The presence of ATG13 at ATG9A-positive vesicles, which suggests active vesicle trafficking during autophagosome formation ; (3) In advanced autophagy, ATG13-ATG101 complexes may accumulate at large clusters of p62/SQSTM1 and ubiquitin, indicating their role in targeting ubiquitinated substrates for selective autophagy .

For accurate interpretation, use fluorescence confocal microscopy with appropriate controls including ATG13 knockout cells to establish background signals. The split-mVenus system can be particularly valuable for visualizing the ULK1-independent ATG13-ATG101 dimer in living cells, revealing that this complex often localizes to small vesicles that may traffic with ATG9A .

What experimental controls are essential when studying ATG13 in autophagy research?

When investigating ATG13 function in autophagy, several essential controls must be included to ensure result validity and interpretability. First, include positive and negative controls for antibody specificity, such as ATG13 knockout or knockdown cells alongside wild-type cells . For functional studies involving ATG13 mutants, include rescue experiments with wild-type ATG13 expression in knockout backgrounds to confirm phenotype specificity .

When studying protein interactions, use reciprocal co-immunoprecipitation approaches and include appropriate controls such as IgG controls and input samples. For mutant studies, include both ULK1 binding-deficient mutants (ATG13 Δ2AA) and HORMA domain deletion mutants (ATG13 ΔHORMA) to distinguish between ULK1-dependent and ULK1-independent functions .

For autophagy flux measurements, include treatments with lysosomal inhibitors (such as bafilomycin A1 or chloroquine) to differentiate between increased autophagosome formation and impaired autophagosome degradation. Finally, when examining ATG13 localization, use multiple markers of autophagy structures (LC3, WIPI2, FIP200) to confirm the identity of the observed puncta and include starvation or rapamycin treatment as a positive control for autophagy induction.

How can researchers investigate ATG13's role in selective autophagy processes?

Investigating ATG13's role in selective autophagy requires specialized experimental approaches. For mitophagy studies, researchers can examine the ULK1-independent functions of ATG13, as recent work suggests that ATG13 contributes to mitophagy independently of ULK1 . This can be achieved by using mitophagy-inducing treatments (such as CCCP or antimycin A/oligomycin) in cells expressing ULK1 binding-deficient ATG13 mutants, followed by assessment of mitochondrial clearance through immunofluorescence or biochemical approaches.

For studying ATG13's role in aggrephagy (the selective autophagy of protein aggregates), examine its interaction with p62/SQSTM1 and ubiquitin. Research has shown that ATG13-ATG101-ATG9A complexes accumulate at large clusters of p62/SQSTM1 and ubiquitin . This can be investigated through co-localization studies and proximity labeling approaches. Additionally, researchers can generate artificial protein aggregates (using treatments like puromycin or expression of aggregate-prone proteins) and assess whether ATG13 is recruited to these structures and whether this recruitment depends on its interaction with ATG101 or ULK1.

What techniques can be used to study ATG13 in non-mammalian model systems?

ATG13 is evolutionarily conserved across species, making comparative studies in non-mammalian models valuable for understanding its fundamental functions. In invertebrate models such as blood clams, quantitative real-time PCR has been successfully employed to measure ATG13 expression levels in various tissues and in response to immune challenges . This approach revealed that blood clam ATG13 (TgATG13) is universally expressed across tissues, with highest expression in hemocytes, and its expression is robustly increased following exposure to pathogens like Vibrio alginolyticus or lipopolysaccharide (LPS) .

When studying ATG13 in non-mammalian systems, researchers should first identify the orthologous sequence through phylogenetic analysis to confirm its evolutionary relationship with mammalian ATG13 . Following identification, expression analysis can be performed using qRT-PCR with appropriate internal controls (such as 18S rRNA in invertebrates) . Functional studies can utilize fluorescence confocal microscopy with GFP-LC3 transfection to track autophagy processes, as demonstrated in blood clam research showing TgATG13's involvement in autophagosome formation .

For genetic manipulation in non-mammalian models, CRISPR-Cas9 or RNAi approaches can be employed to knock out or knock down ATG13, followed by assessment of autophagy markers and challenge assays (such as pathogen exposure) to determine functional consequences. These approaches provide valuable insights into the conserved and divergent aspects of ATG13 function across evolutionary lineages.