SPAC26A3.14c Antibody

What is SPAC26A3.14c and what is its function in cells?

SPAC26A3.14c, now designated as atg44+, is a gene in Schizosaccharomyces pombe (fission yeast) that encodes a 73-amino acid protein essential for mitophagy (selective autophagy of mitochondria). The protein localizes to the mitochondrial intermembrane space (IMS) and has been found to play a crucial role in mitochondrial fission processes . Research has revealed that it contains no known functional domains yet is specifically required for mitophagy while being dispensable for other forms of selective autophagy or bulk autophagy . Atg44 has a functional ortholog (YIL156W-B) in Saccharomyces cerevisiae (budding yeast), indicating evolutionary conservation of its function .

How was SPAC26A3.14c identified as Atg44?

SPAC26A3.14c was discovered through a screening of a fission yeast (S. pombe) genome-wide deletion library for mitophagy-deficient mutants. According to the unified nomenclature for autophagy-related genes in yeast, researchers named this gene atg44+. The gene's role in mitophagy was confirmed through experiments monitoring the vacuolar processing of chimeric mitochondrial proteins like Tuf1-RFP or Sdh2-GFP, where deletion of atg44 prevented the normal progression of mitophagy . Its mitochondrial localization was confirmed through cell fractionation studies and proteinase K protection assays, which demonstrated that the protein specifically resides in the mitochondrial intermembrane space .

What is the relationship between SPAC26A3.14c (Atg44) and mitophagy?

Atg44 is specifically required for mitophagy in yeast. Deletion of the atg44+ gene results in mitophagy deficiency while not significantly affecting non-selective macroautophagy, the Cvt pathway, reticulophagy, or pexophagy . This specificity makes Atg44 a valuable marker for studying selective mitochondrial degradation. Studies have shown that Atg44 localizes in the intermembrane space of mitochondria and is not a transmembrane protein. While Atg44 is not involved in the initial cargo selection process of mitophagy in S. cerevisiae (which involves Atg32 phosphorylation and interaction with Atg11), it plays a critical role in promoting mitochondrial fission, which is a prerequisite step for mitophagy .

What types of SPAC26A3.14c antibodies are available for research purposes?

Several commercial antibodies against SPAC26A3.14c are available from vendors. For example, Cusabio offers a polyclonal antibody (catalog number CSB-PA608750XA01SXV) specific to Schizosaccharomyces pombe (strain 972/ATCC 24843) SPAC26A3.14c protein (UniProt accession number Q10167) . MyBioSource also offers a rabbit polyclonal antibody (MBS7188925) against this target . When selecting an antibody, researchers should consider the specific application needs (Western blot, immunoprecipitation, immunofluorescence) and the validation data available for each antibody in those applications.

How should researchers validate SPAC26A3.14c antibodies before experimental use?

Antibody validation is essential before use in critical experiments. A comprehensive validation strategy should include:

• Specificity testing: Using wild-type vs. atg44Δ yeast strains to confirm antibody specificity. The antibody should detect a band of appropriate molecular weight (~8 kDa) in wild-type but not in knockout samples .

• Cross-reactivity assessment: Testing the antibody against recombinant Atg44 protein and cell lysates from different organisms to evaluate potential cross-reactivity .

• Application-specific validation: For each intended application (Western blot, immunoprecipitation, immunofluorescence), perform validation experiments with appropriate positive and negative controls .

• Reproducibility testing: Ensure consistent results across different batches of the antibody and experimental conditions .

Validation should follow standards similar to those described for other antibodies in the literature, including testing appropriate controls, comparison with known expression patterns, and evaluation of non-specific binding .

What controls should be included when using SPAC26A3.14c antibodies in experiments?

When using SPAC26A3.14c antibodies, the following controls should be incorporated:

• Positive control: Wild-type S. pombe lysate or recombinant Atg44 protein.

• Negative control: Lysate from atg44Δ strain.

• Loading control: Antibody against a constitutively expressed protein (e.g., actin or a ribosomal protein like Rps2) .

• Isotype control: For immunoprecipitation or immunofluorescence, include an isotype control antibody to assess non-specific binding.

• Secondary antibody control: Samples processed with secondary antibody only to evaluate background.

• RNase treatment control: For co-immunoprecipitation experiments, include RNase treatment to determine if protein-protein interactions are RNA-dependent .

What are the recommended protocols for using SPAC26A3.14c antibodies in Western blotting?

For Western blotting with SPAC26A3.14c antibodies, follow these methodological steps:

• Sample preparation: Prepare total cell lysates from S. pombe using a method that preserves protein integrity. For mitochondrial proteins like Atg44, consider isolating mitochondrial fractions to enrich the target protein.

• Protein separation: Use 12-15% SDS-PAGE gels that are optimal for resolving low molecular weight proteins (~8 kDa).

• Transfer conditions: Transfer proteins to PVDF membrane using 10 mM CAPS buffer (pH 11) with 1% methanol to ensure efficient transfer of small proteins .

• Blocking: Block membrane with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute SPAC26A3.14c antibody (typical starting dilution 1:500-1:1000) in blocking buffer and incubate overnight at 4°C.

• Secondary antibody: Use IRdye 800 or Alexa Fluor 680-conjugated secondary antibodies for fluorescent detection, or HRP-conjugated secondary antibodies for chemiluminescent detection .

• Controls: Include both positive control (wild-type) and negative control (atg44Δ) samples.

The expected band size for Atg44 is approximately 8 kDa based on the 73-amino acid sequence of the protein.

How can SPAC26A3.14c antibodies be used to study mitochondrial dynamics and mitophagy?

SPAC26A3.14c antibodies can be employed in several experimental approaches to study mitochondrial dynamics and mitophagy:

• Co-localization studies: Use immunofluorescence with SPAC26A3.14c antibodies alongside mitochondrial markers to examine Atg44 localization during different cellular conditions.

• Proximity ligation assays: Detect in situ protein-protein interactions between Atg44 and other mitochondrial or autophagy-related proteins.

• Co-immunoprecipitation: Identify protein interaction partners of Atg44 during normal growth and mitophagy-inducing conditions.

• Chromatin immunoprecipitation (ChIP): If using tagged versions of Atg44, investigate potential DNA binding or transcriptional regulation roles.

• Proteinase K protection assays: Confirm mitochondrial intermembrane space localization by treating isolated mitochondria with proteinase K under different membrane permeabilization conditions .

• Subcellular fractionation: Monitor Atg44 distribution between mitochondrial and cytosolic fractions during mitophagy induction.

These methodologies can help elucidate the precise role of Atg44 in the mitophagy pathway and its contribution to mitochondrial fission processes.

What experimental design considerations should be made when studying mitophagy using SPAC26A3.14c antibodies?

When designing experiments to study mitophagy using SPAC26A3.14c antibodies, consider these methodological factors:

• Mitophagy induction: Use established methods for inducing mitophagy in yeast, such as nitrogen starvation, and monitor the process over appropriate time points .

• Reporter systems: Incorporate mitophagy reporter systems like Tuf1-RFP or Sdh2-GFP to correlate antibody-detected changes with functional mitophagy .

• Appropriate controls: Include both positive controls (wild-type cells) and negative controls (strains with deletions of known mitophagy factors like atg32Δ).

• Experimental replications: Follow robust experimental design principles with appropriate biological replicates (n≥3) and technical replicates to ensure statistical validity .

• Randomization and blinding: When possible, randomize sample processing and blind analysis to minimize bias .

• Quantification methods: Use standardized methods to quantify Western blot band intensities or fluorescence signals, with appropriate normalization to loading controls.

• Validation with complementary approaches: Confirm antibody-based results with orthogonal methods such as live-cell imaging of tagged proteins or electron microscopy of mitochondrial morphology .

How do SPAC26A3.14c knockout and overexpression models affect mitochondrial morphology and function?

Research indicates that deletion of atg44 in both S. pombe and S. cerevisiae results in abnormal mitochondrial morphology, specifically causing spherically enlarged mitochondria similar to those observed in mitochondrial fission defects (e.g., dnm1Δ mutants) . This suggests Atg44 plays a positive role in mitochondrial fission.

For conducting such studies:

• Morphological analysis: Use fluorescence microscopy with mitochondrial markers (e.g., MitoTracker or mitochondria-targeted fluorescent proteins) to visualize changes in mitochondrial network structure.

• Electron microscopy: Analyze ultrastructural changes in mitochondrial morphology, particularly the cristae structure which appears unaffected in atg44Δ cells despite the enlarged morphology .

• Functional assays: Measure oxygen consumption rate, membrane potential, and ATP production to assess mitochondrial functional changes.

• Mitochondrial dynamics: Analyze rates of mitochondrial fusion and fission events using time-lapse microscopy of fluorescently-labeled mitochondria.

• Stress response: Examine how atg44 deletion or overexpression affects cell survival under conditions that promote mitochondrial stress.

Overexpression studies can provide complementary information about Atg44's role in mitochondrial dynamics and whether its function is dose-dependent.

What is the relationship between SPAC26A3.14c and other proteins involved in mitophagy and mitochondrial dynamics?

The relationship between Atg44 and other mitophagy and mitochondrial dynamics proteins can be studied through:

• Epistasis analysis: Create double mutants of atg44Δ with other autophagy or mitochondrial genes to determine their functional relationships. For example, research shows that while Atg44 is essential for mitophagy, it does not affect Atg32 phosphorylation or interaction with Atg11 in S. cerevisiae, suggesting it functions downstream of or parallel to initial cargo recognition .

• Protein-protein interaction studies: Use co-immunoprecipitation with SPAC26A3.14c antibodies followed by mass spectrometry to identify interaction partners. Validate specific interactions using targeted co-IP or yeast two-hybrid assays.

• Localization studies: Examine co-localization patterns between Atg44 and other proteins using dual-label immunofluorescence or tagged proteins.

• Temporal analysis: Monitor the recruitment timing of Atg44 and other proteins during mitophagy to establish their sequential relationships.

• Structural studies: If possible, investigate the structural basis of Atg44 interactions with other proteins through techniques like X-ray crystallography or cryo-electron microscopy.

Understanding these relationships will help place Atg44 within the larger context of mitochondrial quality control pathways.

How does SPAC26A3.14c function compare between fission yeast and other organisms with orthologs?

Comparative analysis of Atg44 function across species provides evolutionary insights:

• Complementation experiments: Research has shown that expression of budding yeast Atg44 in fission yeast atg44Δ cells can rescue the mitophagy deficiency, and vice versa, indicating functional conservation . This approach can be extended to test orthologs from other species.

• Sequence comparison: Perform detailed sequence alignments to identify conserved domains or motifs across different species' orthologs, which might indicate functional regions.

• Localization conservation: Determine if orthologs share the same subcellular localization in the mitochondrial intermembrane space.

• Expression pattern analysis: Compare expression patterns and regulation of orthologs under various conditions, particularly during mitophagy induction.

• Interaction partner conservation: Identify whether protein-protein interactions are conserved across species using techniques like co-immunoprecipitation with species-specific antibodies.

• Phenotypic analysis: Compare phenotypes resulting from deletion of orthologous genes in different model organisms to assess functional conservation.

These comparative approaches can help identify evolutionarily conserved aspects of Atg44 function that represent core mechanisms in mitophagy.

What are common challenges when using SPAC26A3.14c antibodies and how can they be addressed?

Researchers might encounter several challenges when working with SPAC26A3.14c antibodies:

• Low signal intensity: Atg44 is a small protein (73 amino acids) that may be expressed at low levels, making detection challenging.

  • Solution: Use enrichment strategies like mitochondrial isolation or immunoprecipitation before Western blotting. Consider enhanced chemiluminescence detection systems or more sensitive fluorescent secondary antibodies .

• Non-specific binding: Antibodies may detect cross-reactive bands.

  • Solution: Optimize antibody dilution and incubation conditions. Always include atg44Δ negative controls. Use more stringent washing conditions and consider pre-adsorption of antibodies with non-specific proteins .

• Inconsistent results: Variation between experiments.

  • Solution: Standardize protein extraction protocols, use fresh reagents, and include positive controls in each experiment. Document lot numbers of antibodies used .

• Poor reproducibility across different applications: An antibody working for Western blotting may not work for immunofluorescence.

  • Solution: Validate the antibody separately for each application. Optimize fixation and permeabilization conditions for immunofluorescence or immunohistochemistry .

• Difficulty detecting protein-protein interactions: Low-abundance interactions may be challenging to capture.

  • Solution: Use crosslinking approaches before immunoprecipitation. Consider proximity ligation assays as an alternative for detecting in situ interactions.

How should researchers design experiments to quantitatively analyze SPAC26A3.14c expression and localization?

For quantitative analysis of SPAC26A3.14c expression and localization:

• Expression quantification by Western blot:

  • Use loading controls appropriate for the cell fraction being analyzed (whole cell vs. mitochondrial)

  • Include a concentration gradient of recombinant Atg44 protein for calibration

  • Apply digital image analysis software for densitometry with proper background subtraction

  • Report results as normalized relative units across multiple biological replicates (n≥3)

• Quantitative immunofluorescence:

  • Use standardized image acquisition settings

  • Include co-staining with mitochondrial markers

  • Apply automated image analysis to measure colocalization coefficients (Manders, Pearson)

  • Analyze sufficient cell numbers (>30 per condition) from multiple independent experiments

  • Consider advanced techniques like FRET or FLIM for protein-protein interaction studies

• Flow cytometry:

  • If using tagged versions of Atg44, develop flow cytometry protocols for rapid quantification in large cell populations

  • Include appropriate compensation controls and gating strategies

• Experimental design considerations:

  • Employ factorial or randomized complete block designs to analyze multiple variables simultaneously

  • Calculate appropriate sample sizes based on power analysis

  • Include time-course analyses to capture dynamic changes in expression or localization

How can researchers differentiate between specific and non-specific binding when using SPAC26A3.14c antibodies?

To differentiate between specific and non-specific binding:

• Genetic controls: The most definitive approach is comparing antibody reactivity in wild-type versus atg44Δ samples. Specific signals should be absent in the deletion strain .

• Peptide competition assays: Pre-incubate the antibody with excess antigenic peptide before application to samples. Specific signals should be blocked while non-specific signals remain.

• Multiple antibodies approach: Use two or more antibodies raised against different epitopes of Atg44. Signals detected by all antibodies are more likely to be specific.

• Signal correlation with known biology: Specific signals should change predictably under conditions that affect Atg44 (e.g., mitophagy induction).

• Recombinant protein controls: Include lanes with purified recombinant Atg44 as a positive control to confirm the expected molecular weight.

• Immunodepletion: Perform sequential immunoprecipitations to deplete Atg44 from samples and confirm the disappearance of specific signals in subsequent Western blots.

• Mass spectrometry validation: For immunoprecipitation experiments, confirm the identity of pulled-down proteins using mass spectrometry .

• Use of tagged proteins: Compare antibody detection with epitope tag detection when working with tagged versions of Atg44.

By applying these methodologies systematically, researchers can establish high confidence in the specificity of their SPAC26A3.14c antibody signals.

What are potential applications of SPAC26A3.14c antibodies in studying human diseases related to mitochondrial dysfunction?

While SPAC26A3.14c (Atg44) is a yeast protein, studying its function and utilizing antibodies against it can inform research on human mitochondrial diseases through:

• Identification of human orthologs or functional equivalents: Though direct orthologs may not be obvious, functional screening could identify human proteins with similar roles in mitochondrial fission and mitophagy.

• Comparative pathway analysis: Use insights from yeast Atg44 studies to investigate corresponding mitophagy pathways in human cells, potentially identifying novel therapeutic targets.

• Model system validation: Test whether findings from Atg44 studies in yeast translate to mammalian systems using similar experimental approaches with mammalian-specific antibodies.

• Biomarker development: Knowledge gained about mitophagy mechanisms through Atg44 research might help identify potential biomarkers for mitochondrial dysfunction in human diseases.

• Therapeutic screening platforms: Develop yeast-based screening systems using Atg44 function as a readout to identify compounds that modulate mitophagy, which could have therapeutic potential for human mitochondrial disorders.

• Structure-function insights: Detailed understanding of how Atg44 contributes to mitophagy could inform the design of peptides or small molecules targeting equivalent human pathways.

What advanced imaging techniques can be combined with SPAC26A3.14c antibodies to gain new insights into mitochondrial dynamics?

Advanced imaging techniques that can be combined with SPAC26A3.14c antibodies include:

• Super-resolution microscopy: Techniques like STORM, PALM, or STED can resolve structures beyond the diffraction limit, allowing visualization of Atg44 localization within mitochondrial subcompartments.

• Live-cell imaging: While direct antibody use requires fixation, insights from antibody studies can inform the design of fluorescent protein fusions for real-time tracking of Atg44 dynamics.

• Correlative light and electron microscopy (CLEM): Combine immunofluorescence detection of Atg44 with electron microscopy to correlate protein localization with ultrastructural features.

• FRET/FLIM analysis: Detect protein-protein interactions involving Atg44 by measuring energy transfer between fluorophores attached to interaction partners.

• Single-molecule tracking: Apply techniques like single-particle tracking to follow individual molecules of fluorescently-labeled Atg44 to understand their dynamics.

• Expansion microscopy: Physically expand cellular structures to achieve super-resolution imaging with standard microscopes.

• Lattice light-sheet microscopy: Achieve fast, high-resolution imaging of 3D volumes to capture rapid mitochondrial dynamics events with minimal phototoxicity.

• APEX2 proximity labeling: Combined with Atg44 fusion proteins, this technique can identify proteins in close proximity to Atg44 within living cells.

These advanced imaging approaches can provide unprecedented spatial and temporal resolution of Atg44's role in mitochondrial dynamics and mitophagy.

How can multi-omics approaches be integrated with SPAC26A3.14c antibody studies to provide a systems-level understanding of mitophagy?

Integration of multi-omics approaches with SPAC26A3.14c antibody studies can provide comprehensive insights:

• Proteomics integration:

  • Use SPAC26A3.14c antibodies for immunoprecipitation followed by mass spectrometry (IP-MS) to identify protein interaction networks

  • Compare whole proteome changes between wild-type and atg44Δ strains during mitophagy induction

  • Apply phosphoproteomics to identify signaling events regulated by or regulating Atg44

• Transcriptomics correlation:

  • Correlate gene expression changes with Atg44 protein levels or localization changes

  • Identify transcriptional responses to Atg44 deletion or overexpression

• Metabolomics analysis:

  • Profile metabolic changes associated with Atg44 function or dysfunction

  • Focus particularly on mitochondrial metabolites to understand functional consequences

• Lipidomics:

  • Analyze changes in mitochondrial membrane lipid composition in relation to Atg44 function

  • Investigate lipid requirements for Atg44 localization and function

• Data integration strategies:

  • Apply network analysis to integrate multiple data types

  • Use machine learning approaches to identify patterns across multi-omics datasets

  • Develop predictive models of mitophagy regulation incorporating Atg44 function

• Temporal profiling:

  • Perform time-course analyses across multiple omics layers to understand the dynamics of mitophagy regulation

  • Correlate these changes with Atg44 localization and modification states as determined by antibody-based methods

These integrated approaches can place Atg44 function within the broader cellular context and identify unexpected connections to other cellular processes.