Customize FREE1 Antibody

Description

FREE1 Protein: Core Functions and Mechanisms

FREE1 is an ESCRT-III-associated protein uniquely conserved in plants. Key findings from Arabidopsis studies include:

Property	Description	Source
Autophagosome Closure	FREE1 links the ATG conjugation system (ATG8a, ATG8i) to ESCRT-III machinery.
Phosphorylation	Phosphorylation by KIN10 (SnRK1α1) regulates its recruitment under nutrient stress.
AIM Motif	Contains a classical Atg8-interacting motif (W438, I441) critical for ATG8 binding.
Mutant Phenotype	free1 mutants accumulate unclosed autophagosomes, impairing vacuolar fusion.

Antibody Development Challenges and Opportunities

While no validated FREE1 antibodies are explicitly documented in the provided sources, the structural and functional data from FREE1 studies suggest potential strategies:

Target Epitopes

AIM Motif: The W438I441 sequence (required for ATG8 interaction) could serve as a peptide epitope for antibody generation.
Phosphorylated Residues: Sites phosphorylated by KIN10 may enable phospho-specific antibodies to study nutrient-responsive signaling.

Related Antibody Precedents

The Addgene database lists antibodies for autophagy-related proteins (e.g., ATG8, GPC1, APP) but none for FREE1 . This highlights a gap in reagent availability for plant-specific autophagy regulators.

Research Applications for Hypothetical FREE1 Antibodies

If developed, FREE1 antibodies would enable:

Application	Method	Expected Outcome
Immunoprecipitation	Co-IP with ESCRT-III or ATG8 proteins	Validate protein-protein interactions .
Immunoblotting	Detect FREE1 abundance in free1 mutants	Confirm knockout efficiency .
Subcellular Localization	Confocal microscopy with ATG8 markers	Map FREE1 dynamics during phagophore closure
Phosphorylation Analysis	Kinase assays with KIN10	Study nutrient-dependent post-translational modifications .

Critical Gaps and Future Directions

Reagent Availability: No commercial FREE1 antibodies are documented; custom antibody production would require validated epitopes.
Cross-Species Reactivity: Plant-specific proteins often lack mammalian homologs, complicating antibody cross-reactivity.
Functional Assays: Antibodies could clarify whether FREE1’s ESCRT-III recruitment is conserved in other plant lineages.

Product Specs

Buffer

Preservative: 0.03% Proclin 300
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4

Form

Liquid

Lead Time

Made-to-order (14-16 weeks)

Synonyms

FREE1 antibody; FYVE1 antibody; At1g20110 antibody; T20H2.10Protein FREE1 antibody; FYVE domain protein required for endosomal sorting 1 antibody; FYVE domain-containing protein 1 antibody

Target Names

FREE1

Uniprot No.

Q9ASS2

Target Background

Function

FREE1 Antibody is an antibody targeting the FYVE domain protein required for endosomal sorting 1 (FREE1), an essential component of the endosomal sorting complex required for transport (ESCRT) machinery. FREE1 plays a crucial role in regulating multivesicular body (MVB) protein sorting, impacting various cellular processes including plant growth. It facilitates the formation of intra-luminal vesicles (ILVs) within MVBs, a process mediated by its binding to phosphatidylinositol-3-phosphate (PI3P) and ubiquitin. FREE1 further controls the recycling of the iron transporter IRT1 to the plasma membrane, contributing to the targeted delivery of this transporter to the outer plasma membrane domain. Beyond MVB biogenesis, FREE1 exerts influence on ubiquitin-dependent membrane protein degradation, vacuolar transport, autophagy, and vacuole biogenesis. It also functions as an ESCRT component that binds ubiquitin and orchestrates the vacuolar sorting of proteins. FREE1 notably attenuates abscisic acid (ABA) signaling by promoting the degradation of the ABA receptors PYR1 and PYL4 through the RSL1 pathway. This interaction involves binding to PYL4 and PYR1, ultimately leading to the delivery of ubiquitinated ABA receptors for vacuolar degradation. In response to ABA, FREE1 undergoes phosphorylation by SnRK2 kinases, triggering its nuclear import. Within the nucleus, FREE1 interacts with the ABA-responsive transcription factors ABF4 and ABI5, hindering their ability to bind to their cis-regulatory sequences. This interaction ultimately leads to transcriptional inhibition of the ABA signaling pathway. Notably, FREE1 negatively regulates salt stress tolerance through a feedback loop involving the ABA signaling pathway.

Gene References Into Functions

FYVE1 is essential for plant growth and development, positioning it as a key regulator of intracellular trafficking and vacuole biogenesis. PMID: 25699591
FREE1 binds to phosphatidylinositol-3-phosphate (PI3P) and ubiquitin and specifically interacts with Vps23 via PTAP-like tetrapeptide motifs to be incorporated into the ESCRT-I complex. PMID: 25438943
Beyond regulating MVB biogenesis, the ESCRT component FYVE domain protein required for endosomal sorting 1 (FREE1) also plays dual roles in vacuolar protein transport and autophagic degradation. PMID: 25624505

Database Links

KEGG: ath:AT1G20110

STRING: 3702.AT1G20110.1

UniGene: At.15444

Subcellular Location

Cytoplasm. Prevacuolar compartment membrane; Peripheral membrane protein. Late endosome. Endosome, multivesicular body. Nucleus.

Tissue Specificity

Ubiquitous. Lowest expression in mature seeds.

Q&A

Here’s a structured collection of FAQs for researchers working with FREE1 antibody in academic settings, synthesized from peer-reviewed studies and technical guidelines:

Advanced Research Questions

How do I resolve contradictory data on FREE1’s role in autophagosome closure?

Approach:
- Compare 3D electron tomography of free1 mutants vs. wild-type plants to assess autophagosome sealing .
- Quantify ATG8-PE levels under nutrient-starvation vs. repletion conditions to isolate fusion defects .
- Use AIM motif mutants (e.g., FREE1 W438A/I441A) to test ATG8 interaction dependency .

What genetic tools are available to study FREE1-related trafficking defects?

Tool	Application	Outcome
FREE1-RNAi (DEX-inducible)	Conditional silencing	Seedling lethality, vacuole fragmentation
sof suppressors	Identify genetic interactors	84 FREE1-related suppressors isolated
EYFP-ATG8e/free1 lines	Autophagosome imaging	Accumulated unsealed autophagosomes

How do I troubleshoot nonspecific bands in FREE1 Western blots?

Solutions:
- Pre-adsorb antibodies with plant lysates from free1 mutants.
- Optimize membrane blocking with 5% BSA + 0.1% Tween-20.
- Validate with tandem epitope tags (e.g., FLAG/HA) .

What advanced techniques confirm FREE1-ATG8 interaction dynamics?

Yeast two-hybrid mutagenesis: Map AIM motifs (W438/I441) critical for binding .
Live-cell imaging: Use Arabidopsis protoplasts expressing FREE1-GFP and mCherry-ATG8 to monitor co-localization .
Immunogold EM: Localize FREE1 on autophagosomes in root tips .

Data Contradiction Analysis

Why do some studies report FREE1 as essential for MVB formation while others emphasize autophagy roles?

Resolution: FREE1 has dual functions:
- MVB biogenesis: Mediates intraluminal vesicle formation via ESCRT-I .
- Autophagy: Facilitates autophagosome-vacuole fusion via SH3DOMAIN2 interaction .
Experimental context (e.g., tissue type, stress conditions) influences observed phenotypes.

How to interpret weak FREE1 antibody signals in mutant screens?

Consider:
- RNAi off-target effects (validate with CRISPR lines) .
- Epistatic interactions (e.g., sof suppressors rescuing lethality) .
- Post-translational modifications altering epitope accessibility .

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FREE1 Antibody