ATG8D Antibody

What is ATG8D and what role does it play in autophagy?

ATG8D belongs to the ATG8 family of ubiquitin-like proteins that play essential roles in autophagosome formation. In plants, the ATG8 proteins are encoded by multiple genes (ATG8a through ATG8i) that are ubiquitously expressed in all organs and further induced under nitrogen starvation conditions . The ATG8 proteins undergo post-translational modification, including C-terminal processing by ATG4 proteases and subsequent conjugation to phosphatidylethanolamine (PE), which allows them to associate with autophagosomal membranes . This lipidation process is critical for autophagosome formation and maturation, making ATG8D an important marker for monitoring autophagy.

How is ATG8D processed within the cell?

ATG8D undergoes a specific processing pathway that involves several key steps:

• Initial cleavage of the C-terminal extension by the cysteine protease ATG4, exposing a glycine residue at the C-terminus (resulting in ATG8D-G)

• Activation by the E1-like enzyme ATG7 through formation of a thioester bond between ATG8D's glycine residue and ATG7's cysteine residue

• Transfer to the E2-like enzyme ATG3 via another thioester bond

• Final conjugation to phosphatidylethanolamine (PE) through an amide bond between ATG8D's glycine residue and PE's amino group

This processed form (ATG8D-PE) localizes to preautophagosomal structures and plays a role in autophagosome formation. Notably, both the lipidation and subsequent deconjugation by ATG4 are essential for proper autophagosome formation .

What are the cross-reactivity considerations when selecting an ATG8D antibody?

Due to the high sequence conservation among ATG8 family members, antibodies raised against one ATG8 isoform often cross-react with other isoforms. For example, antibodies against ATG8a can recognize all ATG8 proteins with varying degrees of reactivity, while anti-ATG8i antibodies primarily recognize ATG8i with weaker detection of ATG8h . When selecting an ATG8D antibody, researchers should carefully review the specificity and cross-reaction profile to ensure it will detect their target of interest . The PhytoAB ATG8D antibody, for instance, shows cross-reactivity with ATG8D from multiple plant species including Oryza sativa, Hordeum vulgare, Panicum virgatum, Triticum aestivum, and Setaria viridis .

What is the optimal sample preparation protocol for detecting ATG8D in plant tissues?

For effective detection of ATG8D in plant tissues, researchers should follow this optimized protocol:

• Harvest fresh plant material and immediately flash-freeze in liquid nitrogen

• Grind tissues to a fine powder while maintaining frozen conditions

• Extract proteins using a buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • Protease inhibitor cocktail

• Fractionate the sample to separate membrane-associated and soluble fractions:

  • Centrifuge at 1,000g to remove cellular debris

  • Further centrifuge supernatant at 100,000g to separate high-speed pellet (HSP) and high-speed supernatant (HSS) fractions

When analyzing ATG8D, it's important to note that most ATG8 proteins are predominantly detected in the pellet fraction, particularly in the low-speed pellet (LSP), with smaller amounts in the HSP and HSS fractions . This distribution reflects their membrane association following lipidation.

How can I distinguish between lipidated and non-lipidated forms of ATG8D?

Distinguishing between lipidated (ATG8D-PE) and non-lipidated forms is crucial for autophagy research. The most effective method utilizes a urea-containing SDS-PAGE system:

• Prepare 13.5% acrylamide gels containing 6M urea

• Load protein samples alongside appropriate markers

• Run electrophoresis at a constant voltage (120V)

• Transfer to PVDF membrane and probe with anti-ATG8D antibody

In this system, the lipidated form (ATG8D-PE) migrates faster than the non-lipidated form, appearing as a lower band on the immunoblot . This mobility shift is consistent across various organisms using the Atg8/LC3 system and serves as a reliable indicator of autophagy activation. The membrane-associated fractions (particularly the LSP) typically contain predominantly the lipidated form, while the cytosolic fraction (HSS) contains mainly the non-lipidated form .

What controls should be included when using ATG8D antibody in autophagy studies?

To ensure reliable interpretation of ATG8D antibody results, include the following controls:

• Positive control: Samples from nitrogen-starved plants, as nitrogen starvation induces ATG8 expression and lipidation

• Negative control:

  • Samples treated with phospholipase D, which cleaves the PE moiety from ATG8D-PE

  • When available, atg8 mutant or ATG8-silenced lines

• Loading control: Probing with antibodies against housekeeping proteins like tubulin

• Specificity control: Pre-absorption of the antibody with recombinant ATG8D protein to confirm signal specificity

Including these controls helps validate the specificity of the antibody and ensures proper interpretation of autophagy dynamics in your experimental system.

How can ATG8D antibodies be used to study differential expression of ATG8 isoforms across tissues and developmental stages?

ATG8D antibodies can be powerful tools for studying tissue-specific and developmental expression patterns of ATG8 isoforms. Research has shown that ATG8 proteins are ubiquitously expressed in all plant organs, but with varying protein levels—higher in roots, flowers, and siliques compared to other organs . Additionally, the expression patterns change throughout plant development, with protein levels increasing until plants reach approximately 4 weeks of age, followed by a gradual decrease .

To investigate isoform-specific expression:

• Collect various tissue types and developmental stages

• Prepare protein extracts from each sample

• Perform immunoblot analysis using anti-ATG8D antibody

• Analyze both total ATG8 levels and the proportion of lipidated forms

• Use urea gels to separate different ATG8 isoforms based on their electrophoretic mobility

This approach reveals not only the presence of ATG8 proteins but also their modification state. Interestingly, the banding patterns of lipidated ATG8 forms (ATG8*) differ slightly among organs, suggesting that different ATG8 isoforms may be functionally assigned to specific organs .

What approaches can be used to study ATG8D interaction partners in autophagy and beyond?

ATG8D interacts with various proteins through ATG8-interacting motifs (AIMs). Several complementary approaches can be used to identify and characterize these interactions:

• Yeast Two-Hybrid (Y2H) analysis:

  • Use truncated ATG8D constructs to prevent self-activation

  • Screen against candidate interacting proteins or libraries

  • Verify interactions using growth assays on selective media

• Co-immunoprecipitation (Co-IP):

  • Express tagged versions of ATG8D (e.g., YFP-ATG8D) with potential interacting partners

  • Perform IP with anti-GFP antibodies (GFP-Trap)

  • Detect co-precipitated proteins by immunoblotting

• Fluorescence Resonance Energy Transfer (FRET):

  • Co-express CFP-tagged candidate proteins with YFP-ATG8D

  • Measure energy transfer between fluorophores

  • Compare FRET efficiency to appropriate controls (CFP/YFP alone and CFP-YFP fusion)

• Confocal microscopy and co-localization studies:

  • Express fluorescently-tagged ATG8D and candidate partners

  • Analyze co-localization patterns under normal and autophagy-inducing conditions

  • Use super-resolution techniques like Structured Illumination Microscopy (SIM) for detailed analysis

These approaches have been successfully used to identify interactions between ATG8 isoforms and proteins like FREE1, revealing differential binding preferences among ATG8 paralogs .

What factors can affect ATG8D detection and how can these issues be resolved?

Several factors can impact the detection of ATG8D using antibodies:

Additionally, ATG8D is predominantly membrane-associated after lipidation and cannot be extracted by salt, urea, or alkali treatments, but is solubilized by Triton X-100 or deoxycholate . Consider this when optimizing extraction protocols.

How can researchers distinguish between ATG8D's roles in autophagy versus non-autophagic functions?

ATG8D and other ATG8 proteins participate in both canonical autophagy and non-autophagic processes like phagosome biogenesis. To distinguish between these roles:

• Use multiple autophagy markers: Combine ATG8D detection with other autophagy markers like ATG5 to confirm canonical autophagy

• Perform co-localization studies: Examine whether ATG8D colocalizes with:

  • Autophagosome markers (for autophagy function)

  • Endosomal/phagosomal markers (for non-autophagic functions)

• Employ function-specific assays:

  • For autophagy: Monitor long-lived protein degradation or autophagic cargo receptors

  • For phagocytosis: Assess phagosome formation and maturation using specialized assays

• Comparative proteomics: Analyze protein composition of ATG8D-positive structures to identify autophagy-specific versus non-autophagic components

Research in Entamoeba histolytica has shown that ATG8 plays a role in phagosome biogenesis distinct from its role in autophagy, with ATG8-silenced strains showing specific defects in phagosome acidification and altered recruitment of proteins to phagosomes .

How conserved is ATG8D across species and what implications does this have for antibody selection?

ATG8 proteins show remarkable evolutionary conservation across eukaryotes, though with varying numbers of isoforms in different organisms. In Arabidopsis, there are nine ATG8 isoforms (ATG8a-i) , while other species may have different numbers. Phylogenetic analysis reveals distinct clades of ATG8 proteins, with important implications for antibody selection .

When selecting antibodies for cross-species studies:

• Consider the evolutionary relationships between ATG8 isoforms

• Verify sequence conservation in the epitope region

• Test antibody reactivity against recombinant proteins from target species

The anti-ATG8 antibody from Agrisera recognizes multiple ATG8 isoforms from Arabidopsis thaliana with 70-80% conservation, including ATG8a, ATG8b, ATG8c, ATG8d, and ATG8e . Similarly, the PhytoAB ATG8D antibody shows cross-reactivity with ATG8D from multiple plant species including rice, barley, and wheat .

How do different experimental systems affect the interpretation of ATG8D antibody results?

Different experimental systems present unique considerations for ATG8D antibody use:

• Plant systems:

  • Consider the presence of multiple ATG8 isoforms

  • Account for tissue-specific expression patterns

  • Be aware that nitrogen starvation strongly induces ATG8 expression

• Cell culture systems:

  • Monitor cell density and nutrient status, which affect basal autophagy

  • Consider transfection efficiency when using tagged constructs

• Transgenic systems:

  • Expression level differences between endogenous and transgenic proteins

  • Potential artifacts from fusion tags affecting localization or function

• Stress responses:

  • Different stressors induce autophagy through distinct pathways

  • H2S levels can affect ATG8 protein accumulation in some systems

When comparing results across different experimental systems, researchers should be cautious about direct extrapolations and ensure appropriate controls specific to each system.

What emerging technologies are enhancing ATG8D-based autophagy research?

Several cutting-edge technologies are advancing ATG8D-based autophagy research:

• Super-resolution microscopy:

  • Structured Illumination Microscopy (SIM) allows visualization of ATG8D dynamics at sub-diffraction resolution

  • Single-molecule localization microscopy provides insights into the molecular organization of ATG8D on autophagic membranes

• Proximity labeling techniques:

  • BioID or APEX2 fused to ATG8D can identify proximal proteins in living cells

  • These approaches capture transient interactions missed by traditional co-IP methods

• Engineered ATG8D sensors:

  • Split fluorescent protein systems fused to ATG8D enable real-time monitoring of autophagy

  • pH-sensitive ATG8D reporters distinguish between autophagosome formation and fusion events

• Mass spectrometry-based proteomics:

  • Quantitative proteomics of isolated ATG8D-positive structures

  • Analysis of ATG8D-dependent recruitment of proteins to autophagosomes or phagosomes

These technologies will help researchers gain deeper insights into the dynamic roles of ATG8D in autophagy and beyond.

How can researchers effectively study differential functions of ATG8 isoforms using antibodies?

Studying the specific functions of ATG8D versus other ATG8 isoforms presents a significant challenge due to their high sequence similarity. Effective strategies include:

• Isoform-specific antibodies:

  • Generate antibodies against unique epitopes (often in N-terminal regions or C-terminal extensions)

  • Validate specificity using recombinant proteins and knockout/knockdown lines

• Complementary genetic approaches:

  • Create isoform-specific knockouts or knockdowns

  • Perform complementation studies with other ATG8 isoforms

• Interaction profiling:

  • Compare binding partners of different ATG8 isoforms using Y2H, co-IP, or FRET approaches

  • Identify isoform-specific interactors that may explain functional differences

• Domain swapping experiments:

  • Create chimeric proteins between ATG8D and other isoforms

  • Identify regions responsible for isoform-specific functions

These approaches have revealed that different ATG8 paralogs may have distinct functions, as evidenced by their differential binding to proteins like FREE1 and their varied expression patterns across tissues and developmental stages .