C6orf203 Antibody

What is C6orf203 and why is it important in mitochondrial research?

C6orf203 is a mitochondrial matrix protein that interacts weakly with the inner mitochondrial membrane (IMM) and plays dual roles in cellular function. It participates in mitochondrial transcription under stress conditions and has a critical role in OXPHOS (oxidative phosphorylation) biogenesis under normal culture conditions . Recently identified as part of the mitochondrial proteome, C6orf203 contains a conserved RNA-binding S4 domain and is evolutionarily conserved from insects and worms to mammals . The protein is particularly significant because knockout studies demonstrate it severely affects the translation of mitochondrial mRNAs, making it essential for proper mitochondrial function .

Research using C6orf203 antibodies has revealed its presence in high molecular weight complexes compatible with mitoribosomes, suggesting its involvement in mitochondrial protein synthesis . C6orf203 depletion leads to impaired oxygen consumption and reduced growth in galactose medium, both indicators of compromised OXPHOS function . Given these characteristics, C6orf203 is considered a potential candidate gene for mitochondrial diseases, particularly those presenting with mild phenotypes.

How should I validate the specificity of a C6orf203 antibody?

Validating antibody specificity for C6orf203 requires multiple complementary approaches:

• Knockout/knockdown controls: Generate CRISPR/Cas9 C6orf203 knockout cells as negative controls . The complete absence of signal in these cells provides strong evidence of antibody specificity.

• Overexpression controls: Express tagged versions of C6orf203 (such as FLAG-tagged or GFP-tagged constructs) and confirm co-detection with the antibody .

• Subcellular fractionation: Verify that the antibody detects C6orf203 primarily in mitochondrial fractions, consistent with its known localization . The protein should appear at approximately 19 kDa (the mature form after N-terminal cleavage of 84 amino acids) .

• Immunofluorescence co-localization: Confirm co-localization with established mitochondrial markers such as Mitotracker Red . The pattern should match the punctate distribution typical of mitochondrial proteins.

• Western blot analysis: Observe a single band at the expected molecular weight (19 kDa for the mature form) in wild-type cells that is absent in knockout cells .

What are optimal western blot conditions for detecting C6orf203?

Optimal western blot conditions for C6orf203 detection include:

Sample Preparation:

• Extract mitochondria using standard differential centrifugation protocols

• Solubilize samples in buffer containing digitonin (a relatively mild detergent suitable for preserving protein interactions)

• Include protease inhibitors to prevent degradation

Electrophoresis and Transfer:

• Use 12-15% SDS-PAGE gels to properly resolve the 19 kDa mature protein

• Transfer to PVDF membranes (preferred over nitrocellulose for small proteins)

• Use wet transfer systems at lower voltage for extended periods to ensure efficient transfer of small proteins

Detection Parameters:

• Primary antibody dilution: Start with manufacturer's recommendation (typically 1:1000 for commercial antibodies like Sigma's HPA049535)

• Secondary antibody: Anti-rabbit IgG coupled to HRP or fluorescent tags

• Blocking: 5% non-fat dry milk or BSA in TBS-T (1 hour at room temperature)

• Include positive controls (purified C6orf203 or lysates from cells overexpressing C6orf203)

• Include negative controls (lysates from C6orf203 knockout cells)

Special Considerations:

• The mature form of C6orf203 has a molecular weight of 19 kDa after cleavage of the mitochondrial targeting sequence

• For detection of interactions with high molecular weight complexes, consider using blue native PAGE (BN-PAGE) followed by second-dimension SDS-PAGE

How can I use C6orf203 antibodies to study its interaction with mitoribosomes?

C6orf203 has been found in high molecular weight complexes compatible with mitoribosomes and may play a role in mitochondrial translation . To study these interactions:

Co-immunoprecipitation approach:

• Isolate mitochondria from cells expressing tagged C6orf203 (e.g., TAP-tagged or FLAG-tagged)

• Solubilize mitochondria with digitonin (0.5-1%) to preserve protein-protein interactions

• Perform immunoprecipitation using anti-C6orf203 antibodies

• Analyze co-precipitated proteins by mass spectrometry or western blot using antibodies against mitoribosomal proteins (e.g., MRPS22, MRLP49)

Density gradient analysis:

• Separate mitochondrial lysates on isokinetic sucrose gradients (10-30%)

• Collect fractions and analyze by western blot using antibodies against C6orf203 and mitoribosomal markers

• Compare distribution profiles to determine co-sedimentation patterns

2D BN/SDS-PAGE analysis:

• Solubilize mitochondria with digitonin

• Separate protein complexes by blue native PAGE

• Cut out lanes and run second-dimension SDS-PAGE

• Perform western blot to detect C6orf203 and mitoribosomal components

Research has shown that C6orf203 forms complexes ranging from monomeric forms to very high molecular weight assemblies (~1,500 kDa), being particularly enriched in fractions corresponding to the 39S mitochondrial ribosome large subunit (mtLSU) .

What techniques can distinguish between C6orf203's roles in transcription versus translation?

Distinguishing between C6orf203's transcriptional and translational roles requires targeted methodological approaches:

For transcriptional role assessment:

• RNA pulse labeling: Use 4-thiouridine incorporation followed by biotinylation and pull-down to measure newly synthesized mitochondrial RNA

• RT-qPCR: Quantify steady-state levels of mitochondrial transcripts in wild-type versus C6orf203 knockout cells

• RNA processing analysis: Examine the processing of polycistronic mitochondrial transcripts using northern blotting

• Stress condition experiments: Measure transcriptional changes under mtDNA depletion stress (e.g., ethidium bromide treatment) compared to normal conditions

For translational role assessment:

• In vivo metabolic labeling: Pulse cells with 35S-methionine/cysteine in the presence of cytosolic translation inhibitors (e.g., emetine) to measure newly synthesized mitochondrial proteins

• Mitoribosome assembly analysis: Use sucrose gradients to examine the distribution of mitoribosomal subunits in the presence/absence of C6orf203

• Polysome profiling: Analyze the distribution of mitochondrial mRNAs across polysome fractions to assess translation efficiency

• Proteomics: Quantify mitochondrially-encoded proteins using mass spectrometry

The research indicates that C6orf203 knockout cells show normal mtDNA content and transcript levels but significantly reduced mitochondrial protein synthesis under normal growing conditions . This suggests a primary role in translation under basal conditions, while its role in transcription may become more prominent under stress.

How can I study the phosphorylation state of C6orf203?

C6orf203 is known to be phosphorylated at Ser-106/Ser-110 positions . To study its phosphorylation:

Detection methods:

• Phospho-specific antibodies: Generate or obtain antibodies that specifically recognize phosphorylated Ser-106/Ser-110

• Phos-tag SDS-PAGE: Use Phos-tag containing gels that cause mobility shifts in phosphorylated proteins

• Mass spectrometry: Perform LC-MS/MS analysis on immunoprecipitated C6orf203 to identify phosphorylation sites and quantify phosphorylation levels

Functional analysis:

• Site-directed mutagenesis: Generate phosphomimetic (S→D/E) and phospho-deficient (S→A) mutants of C6orf203

• Rescue experiments: Express phospho-mutants in C6orf203 knockout cells and assess their ability to rescue phenotypes

• Kinase inhibitor studies: Treat cells with various kinase inhibitors to identify the responsible kinase

Regulation studies:

• Stress response: Examine changes in C6orf203 phosphorylation under various stress conditions (oxidative stress, nutrient deprivation, etc.)

• Cell cycle analysis: Synchronize cells and analyze phosphorylation status throughout the cell cycle

• Respiration correlation: Correlate phosphorylation levels with measures of mitochondrial respiration

These approaches can help determine whether phosphorylation affects C6orf203's localization, stability, or functional interactions with mitoribosomal components or the inner mitochondrial membrane.

What are the optimal fixation methods for immunofluorescence studies of C6orf203?

For optimal immunofluorescence detection of C6orf203 in mitochondria, consider the following protocol:

Fixation options:

Permeabilization considerations:

• After PFA fixation, permeabilize with 0.1-0.2% Triton X-100 (10 minutes at room temperature)

• For methanol-fixed cells, additional permeabilization is often unnecessary

Mitochondrial counterstaining:

• Live-cell MitoTracker staining prior to fixation

• Immunostaining with antibodies against mitochondrial markers (TOM20, COX IV, etc.)

Special considerations:

• Include C6orf203 knockout cells as negative controls

• For overexpressed C6orf203-GFP constructs, ensure fixation doesn't quench GFP fluorescence

• Use confocal microscopy for optimal resolution of mitochondrial structures

Previous studies have successfully used C6orf203-GFP constructs in HEK293T and HeLa cells, confirming mitochondrial localization through co-localization with Mitotracker Red . Thorough washing steps and appropriate blocking (3% BSA in PBS) help reduce background and improve signal-to-noise ratio.

How should I design experiments to study C6orf203's association with the inner mitochondrial membrane?

C6orf203 is a mitochondrial matrix protein that weakly associates with the inner mitochondrial membrane (IMM) . To study this association:

Biochemical fractionation approaches:

• Carbonate extraction assay: Treat isolated mitochondria with sodium carbonate (pH 11.5) to distinguish between integral membrane, peripheral membrane, and soluble proteins

  • Include appropriate controls: MT-CO1 (integral IMM), SDHA (peripheral IMM), and MnSOD (soluble matrix)

• Proteinase K protection assay: Incubate mitochondria with proteinase K under iso-osmotic and hypo-osmotic conditions

  • Hypo-osmotic conditions cause mitochondrial swelling and outer membrane rupture

  • Matrix proteins remain protected from proteinase K after swelling

Microscopy approaches:

• Super-resolution microscopy: Use techniques like STED or STORM to visualize the submitochondrial localization of C6orf203

• Immunogold electron microscopy: Precisely locate C6orf203 relative to the IMM

Protein interaction studies:

• Cross-linking experiments: Use membrane-permeable crosslinkers to capture transient interactions with IMM proteins

• Proximity labeling: Express C6orf203 fused to BioID or APEX2 to identify neighboring proteins

Experimental design table:

Research has shown that C6orf203 behaves similarly to SDHA in carbonate extraction assays, suggesting it is a matrix protein with weak association to the IMM , similar to other proteins involved in mitochondrial translation such as Mba1 or PET111 .

How should I interpret OXPHOS changes in relation to C6orf203 expression levels?

When analyzing OXPHOS changes in relation to C6orf203 expression, consider these methodological approaches and interpretation guidelines:

Key measurements to include:

• Oxygen consumption rate (OCR): C6orf203 knockout cells show significantly reduced OCR, indicating OXPHOS dysfunction

• Growth in galactose medium: C6orf203-deficient cells exhibit impaired growth when forced to rely on OXPHOS (galactose as carbon source)

• Mitochondrial ATP synthesis: Measure ATP production capacity in isolated mitochondria or permeabilized cells

• OXPHOS complex activity assays: Individual enzymatic activities of respiratory chain complexes are reduced in C6orf203 knockout cells

Interpretation framework:

• Primary vs. secondary effects: Determine whether OXPHOS changes are directly caused by C6orf203 deficiency or are secondary consequences

  • Primary effect indicators: Immediate changes in mitochondrial protein synthesis

  • Secondary effect indicators: Later changes in nuclear-encoded OXPHOS subunits

• Partial vs. complete defect: C6orf203 knockout severely but not completely affects mitochondrial translation

  • Quantify the degree of translation impairment

  • Compare with complete translation inhibition controls

• Complementation analysis: Rescue experiments with wild-type C6orf203 confirm the specificity of OXPHOS defects

Data normalization considerations:

• Normalize OXPHOS measurements to mitochondrial mass (using markers like citrate synthase)

• Compare results to both wild-type controls and other translation defect models

Research shows that C6orf203 knockout leads to global OXPHOS alterations, including decreased oxygen consumption, mitochondrial ATP synthesis, steady-state levels and activity of respiratory chain complexes, and steady-state levels of mitochondrially encoded OXPHOS subunits . These phenotypes are consistent with defects in mitochondrial protein synthesis rather than mtDNA maintenance or transcription under normal conditions .

What approaches should I use to analyze C6orf203's high molecular weight complexes?

Analyzing C6orf203's high molecular weight complexes requires specialized techniques and careful data interpretation:

Recommended analytical techniques:

• Blue Native PAGE (BN-PAGE): Separates intact protein complexes based on size

  • Follow with second-dimension SDS-PAGE (2D BN/SDS-PAGE) for component analysis

  • Western blot using anti-C6orf203 antibodies to detect complex distribution

• Isokinetic sucrose gradients (10-30%): Separate complexes based on sedimentation coefficient

  • Collect fractions and analyze by western blot

  • Compare C6orf203 distribution with known markers (e.g., MRPS22 for mt-SSU, MRLP49 for mt-LSU)

• Co-immunoprecipitation with mass spectrometry:

  • Immunoprecipitate C6orf203 using specific antibodies

  • Identify interacting proteins by mass spectrometry

  • Validate key interactions by reciprocal co-IP

Data analysis framework:

Interpretation guidelines:

• Compare complex distribution in different cell types and conditions

• Correlate complex formation with mitochondrial translation efficiency

• Analyze changes in complex composition after genetic manipulations

Research has shown that C6orf203 forms part of several complexes ranging from monomeric C6orf203 to high molecular weight complexes of approximately 1,500 kDa . It is particularly enriched in fractions corresponding to the 39S mitochondrial ribosome large subunit (mtLSU) . Bioinformatic analyses further support this association, with 23.8% to 45.9% of C6orf203's predicted interactors being mitoribosomal proteins .

How should C6orf203 be evaluated as a candidate gene for mitochondrial diseases?

C6orf203 has been identified as a potential candidate gene for mitochondrial diseases, particularly those with milder phenotypes . When evaluating it in a clinical context:

Patient selection criteria:

• Patients with suspected mitochondrial disease showing:

  • Mild to moderate OXPHOS deficiency

  • Combined respiratory chain complex deficiencies (suggesting translation defects)

  • Normal mtDNA maintenance

Genetic evaluation approach:

• Targeted sequencing: Include C6orf203 in gene panels for mitochondrial translation defects

• Whole exome/genome sequencing: Analyze for variants in C6orf203

• Variant classification: Evaluate missense variants for potential impact on:

  • Protein stability

  • RNA binding capacity (S4 domain)

  • Interaction with mitoribosomes

  • Phosphorylation at Ser-106/Ser-110

Functional validation methodologies:

• Patient fibroblast studies:

  • Measure C6orf203 protein levels

  • Assess mitochondrial translation using 35S-methionine/cysteine labeling

  • Evaluate OXPHOS complex assembly and activity

• Complementation studies:

  • Express wild-type C6orf203 in patient cells

  • Assess rescue of translation and OXPHOS defects

Clinical correlation considerations:

• The double function of C6orf203 (transcription protection under stress and translation regulation under normal conditions) may result in different clinical manifestations depending on which function is affected

• Synergistic effects with other gene mutations may exacerbate the phenotype

Research suggests that patients with C6orf203 mutations would likely present with a mild rather than severe phenotype, unless there are synergistic effects with other genetic defects . Its involvement in both transcription and translation processes makes it an important candidate to include in clinical gene panels for mitochondrial disease diagnosis.