Recombinant Arabidopsis thaliana RING-H2 finger protein ATL7 (ATL7)

What is ATL7 and how does it relate to other members of the ATL family?

ATL7 is a member of the Arabidopsis thaliana ATL (Arabidopsis Toxicos en Levadura) family of RING-H2 type E3 ubiquitin ligases. The ATL family is characterized by a specific RING-H2 finger domain that functions in protein-protein interactions, particularly with E2 ubiquitin-conjugating enzymes. Like other ATL family members, ATL7 likely contains the characteristic domains including the RING-H2 domain, a hydrophobic region that may function as a transmembrane domain, and a conserved GLD motif named for three conserved amino acids .

The ATL family in Arabidopsis is quite extensive, with numerous members including the well-studied AthATL2, AthATL5, AthATL63, and AthATL64. These proteins share common structural features but likely target different substrate proteins for ubiquitination, thereby regulating distinct cellular processes .

What are the key structural features of the ATL7 protein?

ATL7, like other members of the ATL family, likely contains several characteristic domains:

• A RING-H2 domain with a precise arrangement of 8 zinc ligands that is critical for E3 ligase activity

• A region rich in hydrophobic amino acids that likely functions as a transmembrane domain

• A GLD motif with highly conserved glycine and serine residues, though its exact function remains unknown

The RING-H2 domain directly binds to E2 ubiquitin-conjugating enzymes, particularly those of the Ubc4/Ubc5 subfamily. This interaction is crucial for the protein's E3 ligase function. The three-dimensional structure of the RING-H2 finger domain, as determined by NMR spectroscopy in other ATL proteins (such as rice EL5), maintains similar structural features to other characterized RING domains .

How is ATL7 expression regulated in Arabidopsis?

Based on studies of other ATL family members, ATL7 expression is likely regulated in response to various environmental cues and stress conditions. For example, AthATL2, the first identified member of the ATL family, shows rapid and transient induction in response to pathogen-associated molecular patterns (PAMPs). Its expression increases within 15 minutes of treatment with chitin or cellulases and continues to intensify for about 2 hours before declining .

Many ATL genes, including potentially ATL7, contain a DST element in their 3'UTR that may be involved in rapid transcript degradation, suggesting tight temporal control of expression . This characteristic rapid and transient expression pattern suggests roles in early signaling events during stress responses.

What E2 ubiquitin-conjugating enzymes interact with ATL7?

While specific E2 partners for ATL7 have not been directly identified in the provided search results, research on other ATL family members provides valuable insights. ATL proteins typically interact with members of the Ubc4/Ubc5 subfamily of E2 conjugating enzymes. In yeast-based studies, the toxic effect of expressing AthATL2 could be suppressed by mutations in the E2 enzyme Ubc4 .

Ubiquitination assays with other ATL proteins have consistently demonstrated their reliance on members of the Ubc4/Ubc5 subfamily of E2 conjugases . In Arabidopsis, this subfamily includes approximately 10 members that are likely to interact with ATL7 and other ATL proteins.

When designing experiments to identify E2 partners for ATL7, researchers should prioritize testing interactions with members of the Ubc4/Ubc5 subfamily through methods such as yeast two-hybrid assays, in vitro ubiquitination assays, or co-immunoprecipitation experiments.

How can transcriptomic databases be leveraged to understand ATL7 function?

The Test Of Arabidopsis Space Transcriptome (TOAST) database provides valuable resources for analyzing ATL7 expression patterns across various conditions. This database uses Qlik management software to aggregate and visualize plant spaceflight omics data from multiple repositories, enabling interactive comparisons between experiments .

Researchers can use TOAST to:

• Identify conditions that alter ATL7 expression

• Discover genes co-regulated with ATL7, suggesting functional relationships

• Compare ATL7 expression patterns with other ATL family members

• Investigate potential stress responses that involve ATL7

The TOAST database integrates datasets from various sources including NASA's GeneLab, NCBI-GEO, and CATdb, providing a comprehensive view of Arabidopsis transcriptomics data that can help generate new hypotheses about ATL7 function .

What experimental approaches can identify ATL7 substrates?

Identifying the substrates of ATL7 is crucial for understanding its biological function. Several complementary approaches can be employed:

• Yeast two-hybrid screening: This method can identify proteins that directly interact with ATL7, potentially including its substrates. Using the RING-H2 domain or full-length ATL7 as bait, researchers can screen Arabidopsis cDNA libraries to identify interacting partners.

• Co-immunoprecipitation coupled with mass spectrometry: By expressing tagged versions of ATL7 in Arabidopsis or in heterologous systems, researchers can pull down ATL7 along with associated proteins and identify them through mass spectrometry.

• Ubiquitination assays: In vitro ubiquitination assays using purified recombinant ATL7, E1, E2 enzymes, and potential substrate proteins can directly test ATL7's ability to ubiquitinate specific targets.

• Quantitative proteomics: Comparing protein levels in wild-type plants versus ATL7 knockout or overexpression lines can identify proteins whose stability is affected by ATL7 activity.

For these approaches, it's important to consider that ATL7 may be membrane-associated due to its hydrophobic region, which could affect experimental design and interpretation .

What are the optimal systems for recombinant ATL7 production?

Arabidopsis thaliana itself offers an excellent system for homologous expression of recombinant ATL7. A recently established Arabidopsis-based super-expression system has proven effective for producing preparative-scale amounts of recombinant proteins . This system has several advantages for ATL7 expression:

• Proper post-translational modifications: As a homologous system, Arabidopsis ensures appropriate post-translational modifications of ATL7.

• Complex formation with endogenous partners: This system allows ATL7 to form complexes with its native interaction partners, which is particularly important for studying E3 ligases that function in multi-protein complexes.

• High yields: The system can yield up to 0.4 mg of purified protein per gram fresh weight, making it suitable for biochemical and structural studies .

• Compatibility with membrane proteins: The system has been successfully used for integral membrane protein complexes, which is relevant for ATL7 given its predicted transmembrane domain .

For heterologous expression, E. coli systems may be challenging due to the potential for improper folding of the RING-H2 domain, which requires precise zinc coordination. If E. coli expression is attempted, it's advisable to use strains optimized for expression of proteins containing zinc-finger domains.

What purification strategies are most effective for recombinant ATL7?

When purifying recombinant ATL7, several considerations should guide your approach:

• Affinity tags: Incorporating affinity tags such as His6, GST, or FLAG at either the N- or C-terminus can facilitate purification. The tag position should be carefully chosen to avoid interfering with the RING-H2 domain function.

• Membrane protein considerations: Due to the hydrophobic region that may function as a transmembrane domain, purification buffers should include appropriate detergents to solubilize ATL7. Commonly used detergents include n-dodecyl-β-D-maltoside (DDM), digitonin, or CHAPS.

• Metal chelation chromatography: If using a His-tagged version, immobilized metal affinity chromatography (IMAC) can be employed, but care must be taken with buffers to avoid chelating the zinc ions essential for the RING-H2 domain structure.

• Protein stability: Including zinc ions (ZnCl₂ or ZnSO₄) in purification buffers at low concentrations (10-50 μM) can help maintain the integrity of the RING-H2 domain during purification.

• Size exclusion chromatography: As a final purification step, size exclusion chromatography can separate properly folded ATL7 from aggregates and other contaminants while also providing information about its oligomeric state.

The Arabidopsis super-expression system mentioned earlier has been successfully used to purify various proteins, including integral membrane protein complexes, making it a promising approach for ATL7 purification .

How can the ubiquitin ligase activity of purified ATL7 be assessed?

Assessing the E3 ubiquitin ligase activity of purified ATL7 is crucial for functional studies. Several complementary approaches can be employed:

Table 1: Methods for Assessing ATL7 E3 Ligase Activity

When designing these assays, it's important to include appropriate controls:

• Negative controls should include reactions without ATP or with catalytically inactive ATL7 (with mutations in key residues of the RING-H2 domain)

• Positive controls could include well-characterized E3 ligases such as AthATL2 with known activity

• E2 enzyme controls should test multiple members of the Ubc4/Ubc5 subfamily to identify the optimal partner for ATL7

The choice of E2 enzyme is particularly important, as ATL proteins typically show specificity for members of the Ubc4/Ubc5 subfamily of E2 conjugases .

What genetic resources are available for studying ATL7 function in planta?

Several genetic resources and approaches are available for studying ATL7 function in Arabidopsis:

• T-DNA insertion lines: Collections of T-DNA insertion mutants are available through stock centers like the Arabidopsis Biological Resource Center (ABRC). These lines may contain insertions in the ATL7 gene that disrupt its function.

• CRISPR/Cas9 gene editing: For generating targeted mutations in ATL7, CRISPR/Cas9 technology can be employed to create specific knockout or knock-in lines.

• Near-isogenic line (NIL) populations: NIL populations with genomic regions introgressed from different accessions (like Cape Verde Islands into Landsberg erecta background) can be valuable for studying natural variation in ATL7 function and for QTL mapping of traits influenced by ATL7 .

• Overexpression lines: Creating transgenic lines overexpressing ATL7 under constitutive promoters like 35S or inducible promoters can provide insights into gain-of-function phenotypes.

• Promoter-reporter fusions: ATL7 promoter fused to reporter genes like GUS or fluorescent proteins can help study its expression patterns in different tissues and under various conditions, similar to what has been done for AthATL2 .

How do ATL7 expression patterns correlate with specific stress responses?

Based on studies of other ATL family members, ATL7 may play roles in early stress responses. Many ATL proteins show rapid and transient induction in response to various stimuli. For example, AthATL2 is rapidly induced by PAMPs like chitin and cellulases within 15-30 minutes, with expression levels decreasing thereafter .

To characterize ATL7's role in stress responses, researchers should:

• Monitor ATL7 expression using qRT-PCR or promoter-reporter fusions under various stress conditions (biotic stresses like pathogen infection, abiotic stresses like drought, salt, or oxidative stress)

• Compare expression kinetics with known early PAMP-responsive genes like AthATL2

• Utilize transcriptome databases like TOAST to identify conditions that alter ATL7 expression

• Assess whether ATL7 expression is affected by cycloheximide treatment, which would indicate whether its induction is independent of de novo protein synthesis, a characteristic of early response genes like AthATL2

• Examine the ATL7 3'UTR for the presence of DST elements, which are associated with rapid transcript degradation in AthATL2 and other early response genes

What phenotypic assays are most informative for characterizing ATL7 function?

When characterizing the function of ATL7 through phenotypic analysis of knockout, knockdown, or overexpression lines, several types of assays should be considered:

• Stress response assays: Given the likely involvement of ATL7 in stress responses, testing plant performance under various stress conditions is essential. This includes biotic stress (pathogen infection), abiotic stresses (drought, salt, oxidative stress), and exposure to PAMPs.

• Growth and development measurements: Basic growth parameters (plant height, leaf size, flowering time) should be assessed under both normal and stress conditions to identify subtle phenotypes.

• Protein degradation assays: Since ATL7 likely functions in protein ubiquitination and degradation, monitoring the stability of potential substrate proteins in ATL7 mutant backgrounds can provide functional insights.

• Hormone sensitivity tests: Many E3 ligases play roles in hormone signaling pathways. Testing sensitivity to plant hormones (auxin, jasmonate, ethylene, abscisic acid) may reveal pathway-specific roles for ATL7.

• Subcellular localization studies: Determining the subcellular localization of ATL7 using fluorescent protein fusions can provide clues about its function and the cellular processes it regulates.

When performing these assays, it's important to include appropriate controls, such as complementation lines where the ATL7 gene is reintroduced into knockout backgrounds to confirm that observed phenotypes are directly attributable to ATL7 function.