Recombinant Arabidopsis thaliana RING-H2 finger protein ATL1 (ATL1)

What is ATL1 and how does it fit within the ATL family of proteins?

ATL1 (ARABIDOPSIS TOXICOS EN LEVADURA1) is a member of the ATL family of RING-H2 ubiquitin ligases in Arabidopsis thaliana. The ATL family consists of 91 members that contain a characteristic RING-H2 variation and a hydrophobic domain located at the N-terminal end . These proteins function as E3 ubiquitin ligases within the ubiquitin proteasome system (UPS), coordinating the transfer of ubiquitin to target proteins for degradation or signaling modification . The ATL family name originates from the first identified member, AthATL2, which was selected as a conditionally toxic A. thaliana clone when overexpressed in yeast (hence "Arabidopsis Tóxicos en Levadura" or "Arabidopsis genes toxic to yeast") .

What is the structural characterization of ATL1?

ATL1 contains several key structural elements:

• A RING-H2 finger domain that binds directly to E2 ubiquitin-conjugating enzymes

• A hydrophobic/transmembrane domain at the N-terminal end

• No other previously described domains typical to other RING finger E3 ligases

Unlike many other RING-finger E3 ligases that contain additional domains such as coiled-coils, ankyrin repeats, BRCT, or zinc-fingers, ATL1 and other ATL family members are characterized by their minimal domain structure consisting primarily of the RING-H2 domain and transmembrane region .

What is the subcellular localization of ATL1?

ATL1 has been found to localize to trans-Golgi network/early endosome (TGN/EE) vesicles in Arabidopsis thaliana . This subcellular localization is significant for its function, as ATL1 interacts with the EDR1 protein kinase at these vesicles . The transmembrane domain of ATL1 facilitates its insertion into these membrane structures, positioning it to play roles in vesicular trafficking and protein quality control systems .

How does ATL1 function as an E3 ubiquitin ligase?

ATL1 functions as a single-subunit RING finger E3 ubiquitin ligase that contains both the substrate recognition sequences and the catalytic RING-H2 domain in the same polypeptide . The RING-H2 finger domain of ATL1 directly binds to E2 ubiquitin-conjugating enzymes . Specifically, studies using yeast models suggest that ATL family proteins like ATL2 interact with the Ubc4/Ubc5 subfamily of E2 enzymes, which includes 10 members in A. thaliana .

The general mechanism involves:

• Binding of the RING-H2 domain to an E2 enzyme

• Recognition of a substrate protein

• Facilitation of ubiquitin transfer from the E2 to the substrate

• Targeting of the ubiquitinated protein for degradation or signaling modification

What is the relationship between ATL1 and EDR1?

ATL1 interacts with the Arabidopsis thaliana ENHANCED DISEASE RESISTANCE1 (EDR1) protein kinase, which negatively regulates ATL1's activity . This interaction occurs on trans-Golgi network/early endosome (TGN/EE) vesicles . EDR1 functions as a suppressor of ATL1-mediated cell death in both Nicotiana benthamiana and Arabidopsis . This regulatory relationship is critically important for modulating stress responses and programmed cell death.

The interaction can be represented in this simplified pathway:

How does ATL1 interact with the ubiquitination machinery?

ATL1 interacts with E2 ubiquitin-conjugating enzymes through its RING-H2 domain . Evidence from studies on the ATL family suggests that these proteins require specific E2 enzymes for their function . For instance, AthATL2 toxicity in yeast can be suppressed by mutation in the E2 enzyme Ubc4, which belongs to the conserved Ubc4/Ubc5 subfamily . Only members of the A. thaliana Ubc4/Ubc5 subfamily are able to complement the yeast ubc4 mutant for AthATL2 toxicity, suggesting specificity in the E2-E3 interaction .

What role does ATL1 play in programmed cell death?

ATL1 functions as a positive regulator of programmed cell death in Arabidopsis thaliana . Research findings indicate:

• Overexpression of ATL1 in transgenic Arabidopsis induces severe growth inhibition and patches of cell death

• Transient overexpression of ATL1 in Nicotiana benthamiana leaves induces cell death and tissue collapse

• The E3 ligase activity of ATL1 is required for these cell death processes

• Knockdown of ATL1 expression suppresses cell death phenotypes associated with the edr1 mutant

These findings collectively establish ATL1 as a critical mediator of programmed cell death, likely through its ubiquitination of specific target proteins that regulate cell death pathways.

How is ATL1 involved in plant disease resistance?

ATL1 plays a significant role in plant disease resistance mechanisms:

• Knockdown of ATL1 expression makes Arabidopsis hypersusceptible to powdery mildew infection

• ATL1 appears to be involved in stress responses initiated by ATL1-mediated ubiquitination events

• The interaction between ATL1 and EDR1 controls stress responses at the TGN/EE

The regulatory relationship between ATL1 and EDR1 appears to be critical for balancing disease resistance and cell death responses. While ATL1 promotes cell death as part of defense responses, EDR1 negatively regulates this activity to prevent excessive cell death .

What phenotypes are observed with ATL1 overexpression and knockdown?

The effects of manipulating ATL1 expression levels have been experimentally documented:

These phenotypes demonstrate that ATL1 expression must be precisely regulated to balance plant growth, stress responses, and disease resistance.

What experimental methods are most effective for studying ATL1 function?

Several methodological approaches have proven valuable for investigating ATL1 function:

• Genetic manipulation techniques:

  • Overexpression studies in both Arabidopsis and Nicotiana benthamiana

  • Knockdown expression using RNAi or antisense approaches

  • Mutation analysis of the E3 ligase domain to assess enzymatic requirements

• Protein interaction studies:

  • Co-localization studies at TGN/EE vesicles

  • Analysis of interactions with E2 enzymes and regulatory proteins like EDR1

• Phenotypic analyses:

  • Assessment of cell death phenotypes

  • Pathogen susceptibility testing (particularly powdery mildew)

  • Growth inhibition measurements

• Biochemical approaches:

  • In vitro ubiquitination assays to confirm E3 ligase activity

  • Vesicle isolation to study TGN/EE localization and function

How do ATL1 and other ATL family members differ in their functions?

The ATL family shows functional diversity despite structural similarities:

• While ATL1 is associated with programmed cell death and stress responses , other ATL family members have been implicated in:

  • Defense responses to pathogens

  • Regulation of carbon/nitrogen responses during post-germinative seedling growth transition

  • Regulation of cell death during root development

  • Endosperm development

  • Transition to flowering under short day conditions

• Toxicity in yeast exhibits variation among ATL family members:

  • From 25 AthATLs expressed in yeast, only ATL2 and ATL63 showed toxic phenotypes

  • ATL2 and ATL63 share similar domain architecture, suggesting functional similarity

This functional diversity makes the ATL family an excellent model for studying how structurally related E3 ligases can evolve distinct biological roles.

What is the mechanism by which ATL1 promotes programmed cell death?

The mechanism of ATL1-mediated programmed cell death likely involves:

• Ubiquitination of specific target proteins that regulate cell death pathways

• Potential competition with EDR1 for binding partners or substrates at TGN/EE vesicles

• Modulation of stress signal transduction pathways

• Possible regulation of vesicle trafficking to control delivery of defense-related proteins

Research suggests that EDR1 suppresses ATL1-mediated cell death, indicating that the balance between these two proteins is critical for determining cell fate during stress responses .

What are the specific ubiquitination targets of ATL1?

A critical area for future research is the identification of the specific proteins targeted by ATL1 for ubiquitination. While it's established that ATL1 functions as an E3 ubiquitin ligase , the specific substrates remain largely unknown. Identifying these targets would provide significant insights into the molecular mechanisms by which ATL1 regulates programmed cell death and stress responses.

Potential experimental approaches include:

• Immunoprecipitation coupled with mass spectrometry

• Yeast two-hybrid screening for ATL1 interactors

• Differential proteomics comparing wild-type and ATL1 overexpression/knockdown plants

• In vitro ubiquitination assays with candidate substrates

How is ATL1 activity regulated beyond EDR1 suppression?

While EDR1 has been identified as a negative regulator of ATL1 , other regulatory mechanisms likely exist. Future research could explore:

• Post-translational modifications of ATL1 that affect its activity

• Transcriptional and translational regulation of ATL1 expression

• Protein-protein interactions that modulate ATL1 function

• Subcellular trafficking and localization dynamics that affect ATL1 availability

Understanding these regulatory mechanisms would provide a more comprehensive picture of how ATL1 function is controlled in different developmental contexts and stress conditions.

What is the evolutionary significance of the ATL family in plant adaptation?

The ATL family has been instrumental in evolution studies for showing how gene families are expanded in plant genomes . Future research could investigate:

• The evolutionary history of the ATL family across plant species

• Functional diversification of ATLs in different plant lineages

• Selection pressures that have shaped ATL diversity

• The relationship between ATL evolution and adaptation to different environmental stresses

This evolutionary perspective could provide insights into how plants have adapted their stress response and programmed cell death mechanisms across evolutionary time.