ER-ANT1 Antibody

What is ER-ANT1 and what is its primary function?

ER-ANT1 is an adenine nucleotide transporter that resides specifically in endoplasmic reticulum membranes and facilitates ATP/ADP exchange between the cytosol and ER lumen . The protein demonstrates high specificity for ATP/ADP antiport, as confirmed through functional integration studies in E. coli cytoplasmic membranes . This transporter plays an essential role in maintaining adequate ATP levels within the ER, which is necessary for numerous energy-dependent processes including protein folding, calcium homeostasis, and post-translational modifications . The critical nature of ER-ANT1 is evidenced by the severe phenotypic consequences that arise when the gene is disrupted in organisms like Arabidopsis thaliana, resulting in growth retardation and impaired development .

How does ER-ANT1 differ from mitochondrial adenine nucleotide transporters?

ER-ANT1 differs from mitochondrial adenine nucleotide carriers (AACs) in several key aspects. First, ER-ANT1 lacks the cleavable N-terminal transit peptide that mitochondrial AAC1-AAC3 isoforms possess, which is necessary for insertion into inner mitochondrial membranes . Second, ER-ANT1 demonstrates distinct inhibitor sensitivity profiles – it is remarkably insensitive to bongkrekic acid (BKA) and carboxyatractyloside (CAT), both of which strongly inhibit mitochondrial AACs . This biochemical distinction is partially explained by the absence of a conserved arginine residue (R279) in ER-ANT1 that is known to participate in CAT binding in mitochondrial transporters . Additionally, N-ethylmaleimide significantly inhibits ER-ANT1 (up to 71% reduction in transport) but has no effect on mitochondrial AAC1 .

What are the recommended methods for detecting ER-ANT1 protein expression?

For detecting ER-ANT1 in experimental systems, researchers have successfully employed several complementary approaches:

• Western blot analysis: Using antibodies targeting ER-ANT1 at dilutions of 1:500-1:1000 . It's important to note that different antibodies may have varying specificities for ER-ANT1 versus other ANT family members.

• Immunofluorescence assay (IFA): This approach has been used successfully with HA-tagged versions of ANT proteins to visualize subcellular localization, particularly co-localization with known ER markers such as SERCA .

• Immunogold labeling: For high-resolution localization, immunogold labeling of cryosections using peptide-specific antisera against ER-ANT1 has been successfully employed, particularly in tissues with high ER-ANT1 expression such as pollen grain tissues .

• Sucrose density gradient centrifugation: This method can verify ER localization by demonstrating the characteristic Mg²⁺-dependent shift of ER-ANT1 along with other ER markers .

What controls should be included when using ER-ANT1 antibodies in immunolocalization studies?

When conducting immunolocalization studies with ER-ANT1 antibodies, several essential controls should be incorporated to ensure reliable results:

• Preimmune serum control: Always include parallel experiments using preimmune serum to identify non-specific binding . Studies have shown that while specific ER-ANT1 antisera produce clear labeling of ER membranes, preimmune sera typically yield no detectable labeling .

• ER marker co-localization: Include established ER markers such as calreticulin or SERCA to confirm ER localization . In subcellular fractionation studies, ER-ANT1 should co-fractionate with these markers.

• Mg²⁺ manipulation in fractionation studies: When using sucrose density gradient centrifugation, perform parallel gradients with and without Mg²⁺ to observe the diagnostic shift of ER membranes . ER-ANT1 should shift with other ER markers in a Mg²⁺-dependent manner, while mitochondrial, plasma membrane, and vacuolar membrane markers will not show this shift .

• Knockout/knockdown validation: Where available, include samples from ER-ANT1 knockout or knockdown organisms to validate antibody specificity .

• Tagged protein controls: For epitope-tagged versions, include both N-terminal and C-terminal tagged constructs to ensure that tagging does not disrupt localization, particularly since ER-ANT1 contains transmembrane domains that may be affected by tagging .

What methodology is recommended for functional characterization of ER-ANT1?

For functional characterization of ER-ANT1, the following methodological approaches have proven effective:

• E. coli heterologous expression system: Functional integration of ER-ANT1 in E. coli cytoplasmic membranes allows for direct assessment of transport activity using radioactively labeled nucleotides . This approach has successfully demonstrated the ATP/ADP antiport function of ER-ANT1 with apparent Km values of 343.7 ± 20.4 μM for ATP and 327.3 ± 24.4 μM for ADP .

• Back-exchange experiments: To confirm the exchange mode of transport, researchers should perform experiments where cells expressing ER-ANT1 are preloaded with radioactively labeled ATP, followed by chase experiments with non-labeled substrates . This approach has demonstrated that ER-ANT1 mediates a nucleotide exchange with ~95% release of labeled nucleotides when chased with non-labeled ATP or ADP .

• Competition assays: Testing multiple potential substrates in competition assays has revealed the high specificity of ER-ANT1 for ATP and ADP, with other metabolic intermediates exerting minimal influence on transport rates .

• Inhibitor profiles: Characterizing the response to transport inhibitors (such as BKA, CAT, and N-ethylmaleimide) can provide further functional insights and distinguish ER-ANT1 from other nucleotide transporters .

• Genetic disruption studies: In planta studies using knockout or knockdown approaches provide critical information about the physiological importance of ER-ANT1 .

How can researchers investigate ER-ANT1 function across different model systems?

Investigating ER-ANT1 function across different model systems requires adapting methodologies to each organism while maintaining comparative analytical approaches:

• Plant systems (Arabidopsis thaliana):

  • ER-ANT1 knockout lines show catastrophic phenotypes including growth retardation and impaired root and seed development .

  • Transgenic ER-ANT1-promoter-β-glucuronidase lines reveal expression patterns in ER-active tissues such as pollen, seeds, root tips, and vascular bundles .

  • Knockout lines show decreased expression of ATP-dependent ER proteins, including BiP chaperones, calreticulin chaperones, Ca²⁺-dependent protein kinase, and SEC61 .

• Protozoan parasites (Toxoplasma gondii):

  • TgANT (T. gondii homolog) can be studied using CRISPR/Cas9 technology to create conditional knockdowns using the tetracycline-inducible system .

  • Phenotypic analysis focuses on parasite replication, plaque formation, and invasion ability .

  • Immunofluorescence assays with epitope-tagged proteins (HA or Ty) can confirm ER localization .

• Mammalian systems:

  • SLC35B1 (AXER) has been identified as the mammalian ER ATP/ADP transporter .

  • For comparative studies between plant and mammalian systems, researchers should note that unlike the severe phenotype in plants, mammalian ER-ANT appears to have less dramatic effects when disrupted .

• Cross-species functional complementation:

  • Introducing ER-ANT1 from one species into another's knockout background can test functional conservation .

  • Sequence alignment of conserved domains, particularly the domains identified in TgANT as essential for function, can guide mutation studies across species .

What approaches can be used to study the regulation of ER-ANT1 expression and activity?

Studying the regulation of ER-ANT1 expression and activity requires multifaceted approaches:

• Transcriptional regulation:

  • Quantitative PCR methods using SYBR Premix Ex Taq Kit or similar systems can accurately measure ER-ANT1 mRNA levels across different tissues and conditions .

  • Promoter analysis using reporter constructs (such as the ER-ANT1-promoter-β-glucuronidase system) provides insights into tissue-specific expression patterns .

• Post-translational modifications:

  • Immunoprecipitation followed by mass spectrometry can identify potential post-translational modifications of ER-ANT1 .

  • Site-directed mutagenesis of predicted modification sites can assess their functional significance.

• Protein-protein interactions:

  • Co-immunoprecipitation (CoIP) using antibodies against ER-ANT1 can identify interaction partners .

  • Proximity labeling approaches (BioID or APEX) can reveal the wider interaction network within the ER membrane environment.

• Response to cellular stresses:

  • Monitoring ER-ANT1 expression and activity under various stress conditions (ER stress, energy depletion, oxidative stress) can reveal regulatory mechanisms.

  • In plant systems, high external CO₂ concentration has been shown to revert phenotypes of ER-ANT1 mutants, suggesting environmental regulation of ATP-dependent ER processes .

What critical domains in ER-ANT1 are essential for function and antibody recognition?

Several critical domains in ER-ANT1 have been identified as essential for function and should be considered when selecting or developing antibodies:

• Transmembrane domains:

  • The first two transmembrane domains (TMDs) often act as a noncleavable signal sequence directing cotranslational protein synthesis and membrane anchoring .

  • The first TMD of ER-ANT1 exhibits substantially higher hydrophobicity compared to mitochondrial AAC isoforms and contains four N-terminal charged amino acids (K6, E8, R9, and D13) that may serve as a signal anchor .

• Conserved functional domains:

  • Studies in Toxoplasma gondii have identified two domains in TgANT that, when mutated, lead to complete loss of function .

  • These domains are conserved across multiple species, suggesting a shared transport mechanism .

• C-terminal regions:

  • Unlike soluble ER proteins with KDEL/HDEL retention signals, ER-ANT1 as a multispanning membrane protein lacks these motifs .

  • In T. gondii, TgANT has a C-terminal polylysine motif (-KKQC) potentially responsible for ER retention .

• Epitope accessibility considerations:

  • When designing experiments with antibodies, researchers should consider the topology of ER-ANT1, as both N-terminal and C-terminal regions face the ER lumen in some species .

  • This topology can affect epitope accessibility in intact versus permeabilized cells and may influence the detection success in different experimental contexts.

How should researchers interpret contradictory localization data for ER-ANT1?

When faced with contradictory localization data for ER-ANT1, researchers should consider several factors:

• Antibody specificity concerns:

  • Cross-reactivity with other ANT family members can lead to mixed localization signals.

  • Verify antibody specificity against knockout/knockdown controls .

  • Consider using multiple antibodies targeting different epitopes to confirm localization.

• Fixation and sample preparation effects:

  • Different fixation methods can affect epitope accessibility and membrane integrity.

  • Compare results from multiple fixation protocols (paraformaldehyde, glutaraldehyde, methanol).

  • For immunogold labeling, cryosection preparation methods can significantly impact results .

• Expression level artifacts:

  • Overexpression can sometimes lead to mislocalization or aggregation.

  • Compare native expression versus overexpression systems.

  • Use inducible expression systems to control expression levels.

• Species and tissue-specific differences:

  • ER-ANT1 localization and abundance varies across tissues, with highest expression in ER-active tissues like pollen in plants .

  • Different species may have unique localization patterns for ER-ANT homologs.

  • Use the appropriate positive controls for each species (e.g., established ER markers for that species).

• Resolution limitations:

  • Light microscopy may not clearly distinguish between ER and closely associated membranes.

  • Complement immunofluorescence with higher resolution techniques like immunogold electron microscopy .

  • Consider super-resolution microscopy methods for more precise localization.

What are the recommended dilutions and protocols for ER-ANT1 antibody applications?

Based on available antibody data for ANT family members, the following recommendations apply to ER-ANT1 antibody applications:

Note: These recommendations serve as starting points and should be optimized for specific experimental systems and antibodies. Researchers should always perform titration experiments to determine optimal dilutions for their particular application and sample type .

How can researchers distinguish between ER-ANT1 and other adenine nucleotide transporter family members?

Distinguishing between ER-ANT1 and other adenine nucleotide transporter family members requires multiple complementary approaches:

• Sequence-specific antibodies:

  • Use antibodies raised against unique peptide sequences that differ between ANT family members.

  • Validate antibody specificity using overexpression systems and knockout controls.

• Subcellular fractionation:

  • Employ differential centrifugation and gradient techniques to separate organelles.

  • The characteristic Mg²⁺-dependent shift in sucrose gradients is a hallmark of ER membranes and can help distinguish ER-ANT1 from mitochondrial or other ANTs .

• Inhibitor profiles:

  • ER-ANT1 shows distinctive insensitivity to BKA and CAT, which strongly inhibit mitochondrial AACs .

  • N-ethylmaleimide inhibits ER-ANT1 but not mitochondrial AAC1 .

• Co-localization studies:

  • Combine ANT family antibodies with established organelle markers.

  • ER-ANT1 should co-localize with ER markers like calreticulin or SERCA .

  • Mitochondrial ANTs should co-localize with mitochondrial markers.

• Functional characterization:

  • Transport kinetics and substrate specificities can differ between ANT family members.

  • Back-exchange experiments and competition assays can reveal functional differences .

How does ER-ANT1 dysfunction contribute to disease pathology in different organisms?

The role of ER-ANT1 dysfunction in disease pathology varies across organisms and warrants further investigation:

• Plant systems:

  • In Arabidopsis, ER-ANT1 disruption causes catastrophic phenotypes including growth retardation and impaired development .

  • The ability of high CO₂ to rescue these phenotypes suggests connections to carbon metabolism and potential implications for plant adaptation to changing environmental conditions .

  • Decreased expression of ATP-dependent ER proteins in ER-ANT1 knockout lines indicates potential impacts on protein folding and ER stress pathways .

• Protozoan parasites:

  • In Toxoplasma gondii, TgANT depletion leads to fatal growth defects, including slowed replication, absence of plaque formation, and reduced invasion ability .

  • This suggests TgANT could be a potential therapeutic target for antiparasitic interventions.

  • The lack of ATP in the ER has been identified as the direct cause of parasite death in these systems .

• Mammalian systems:

  • Studies with Ant1 (Slc25a4) mutant mice have established connections between mitochondrial dysfunction and serotonergic defects that may contribute to bipolar disorder vulnerability .

  • While these studies focus primarily on mitochondrial Ant1, they suggest that nucleotide transport dysfunction broadly can impact neurotransmitter systems and contribute to neuropsychiatric conditions .

  • The relatively recent identification of SLC35B1 (AXER) as the mammalian ER ATP/ADP transporter opens new avenues for investigating ER energy homeostasis in mammalian disease contexts .

What emerging techniques are enhancing the study of ER-ANT1 function and regulation?

Several emerging techniques are advancing ER-ANT1 research:

• CRISPR/Cas9 genome editing:

  • Precise modification of ER-ANT1 genes enables creation of conditional knockdowns, specific point mutations, and epitope tagging at endogenous loci .

  • This approach has been successfully employed in T. gondii to insert HA epitopes within transmembrane regions of TgANT while maintaining proper localization .

• Real-time ATP sensors:

  • Genetically encoded fluorescent ATP sensors targeted to the ER lumen can provide dynamic measurements of ATP levels.

  • These tools can assess ER-ANT1 function in living cells and tissues under various conditions.

• Single-molecule techniques:

  • Single-molecule localization microscopy can provide nanoscale resolution of ER-ANT1 distribution within ER subdomains.

  • Single-molecule tracking could reveal dynamics of ER-ANT1 movement within ER membranes.

• Proteomics approaches:

  • Proximity labeling methods (BioID, APEX) can identify the ER-ANT1 interactome.

  • Quantitative proteomics can assess changes in ER protein composition in response to ER-ANT1 disruption.

• Structural biology:

  • Cryo-electron microscopy techniques are advancing membrane protein structural determination.

  • Structural insights into ER-ANT1 could reveal the molecular basis for its substrate specificity and transport mechanism.

  • Comparative structural analysis with mitochondrial AACs could explain the distinct inhibitor sensitivity profiles .