COX17-1 Antibody

What is COX17 and what is its biological significance?

COX17 (cytochrome c oxidase assembly homolog) is a small protein (7 kDa, 63 amino acids) that functions as a metal chaperone, delivering copper ions to Sco1 and Cox11 and ultimately to cytochrome c oxidase . It contains twin-CX9C motifs and requires the MIA pathway for its import and assembly into mitochondria . COX17 is essential for cellular respiration, as cox17Δ cells cannot grow on respiratory medium . Recent research has identified COX17 as an interaction partner of the MICOS complex, which is crucial for maintaining the architecture of the mitochondrial inner membrane . Additionally, COX17 acetylation via the MOF-KANSL complex appears to regulate energy metabolism and mitochondrial structure .

What are the technical specifications of the COX17 Antibody (11464-1-AP)?

The COX17 Antibody (11464-1-AP) is a rabbit polyclonal antibody that targets COX17 in multiple applications . Key specifications include:

What experimental applications has this antibody been validated for?

The COX17 antibody has been validated for multiple applications with specific recommended dilutions :

The antibody has been cited in numerous publications for these applications, with 14 publications specifically referencing its use in Western blotting, 4 in immunofluorescence, and 1 each in IHC, KD/KO studies, and CoIP experiments .

How does COX17 interact with the MICOS complex and what is the significance of this interaction?

Research has revealed that COX17 directly interacts with the MICOS complex, particularly with Mic60, a core component of MICOS . This interaction was demonstrated through affinity purification experiments using both COX17-FLAG as bait (pulling down Mic60) and Mic60-ProtA as bait (pulling down native COX17) . Interestingly, this interaction is promoted by copper ions .

Four models have been proposed for COX17's interaction network :

• COX17 interacts with Sco1 indirectly via Mic60 or the MICOS complex

• COX17 interacts with the MICOS complex via Sco1 protein

• COX17 mediates the interaction between MICOS and Sco1

• COX17 forms separate complexes with Sco1 and the MICOS complex

The functional significance of this interaction relates to the maintenance of mitochondrial inner membrane architecture. Loss of COX17 results in reduced cristae density, similar to the phenotype observed with depletion of MICOS components . This suggests that COX17, through its interaction with MICOS, plays a critical role in establishing and maintaining proper mitochondrial ultrastructure .

What role does COX17 acetylation play in mitochondrial function?

COX17 acetylation represents a critical regulatory mechanism for mitochondrial function . Research has identified two key acetylation sites on COX17: K18 (located in its disordered N-terminal region) and K30 (within the first twin-CX9C motif and exposed at the protein surface) . These sites are acetylated by the MOF-KANSL complex .

Experimental evidence using acetylation-mimicking (K18,30Q) and non-acetylated (K18,30R) variants of COX17 has demonstrated that acetylation at these sites is essential for :

• Maintaining normal mitochondrial morphology

• Ensuring proper cristae density

• Preserving mitochondrial membrane potential

• Supporting cytochrome c oxidase (Complex IV) activity

In cells with depleted MOF (the acetyltransferase), only the acetylation-mimicking COX17 variant could restore complex IV activity, indicating that MOF-mediated acetylation of COX17 is essential for its function in promoting cytochrome c oxidase activity .

How can I investigate COX17's role in mitochondrial cristae architecture?

To investigate COX17's role in mitochondrial cristae architecture, consider the following experimental approaches:

• Genetic manipulation: Generate COX17 knockdown or knockout cells using shRNA or CRISPR-Cas9 technology. The research indicates that Cox17 KD leads to fragmented mitochondrial morphology and reduced cristae density .

• Rescue experiments: Express wild-type or mutant COX17 variants in COX17-depleted cells. Particularly informative are acetylation mutants (K18,30Q as acetylation-mimicking and K18,30R as non-acetylated), as they differentially affect cristae architecture .

• Microscopy analysis:

  • Confocal microscopy to assess mitochondrial morphology (fragmentation vs. normal)

  • Transmission electron microscopy (TEM) for detailed analysis of cristae ultrastructure and density

• Functional assays:

  • Mitochondrial membrane potential measurements

  • Cytochrome c oxidase activity assays

• MICOS interaction studies: Investigate the interaction between COX17 and MICOS components (particularly Mic60) using co-immunoprecipitation, as this interaction appears crucial for maintaining cristae architecture .

What are the optimal protocols for immunoprecipitation of COX17 and its interaction partners?

Based on published research, optimal immunoprecipitation of COX17 and its interaction partners can be achieved using the following protocol :

For cell extract preparation:

• Culture cells expressing tagged COX17 (e.g., COX17-FLAG) or interaction partners (e.g., Mic60-ProtA)

• For inducible expression systems, induce expression (e.g., with 0.5% galactose for 12 hours in yeast systems)

• Lyse cells or isolate mitochondria

• Solubilize in digitonin-containing buffer:

  • 1% (w/v) digitonin

  • 10% (w/v) glycerol

  • 20 mM Tris-HCl (pH 7.4)

  • 300 mM NaCl

  • 50 mM iodoacetamide

  • 2 mM PMSF

• Incubate on ice for 20 minutes

• Perform affinity purification using appropriate beads for the tag system

This approach has successfully identified interactions between COX17 and multiple partners, including components of the MICOS complex (particularly Mic60), the MIA machinery (Mia40), and copper transport partners (Sco1) .

How do I optimize the COX17 Antibody for immunohistochemistry applications?

For optimal immunohistochemistry results with the COX17 Antibody (11464-1-AP), follow these guidelines :

• Tissue preparation:

  • Use appropriate fixation (typically formalin-fixed, paraffin-embedded sections)

  • Positive control: Human liver cancer tissue has been validated for this antibody

• Antigen retrieval:

  • Primary recommendation: TE buffer at pH 9.0

  • Alternative method: Citrate buffer at pH 6.0

• Antibody dilution:

  • Starting range: 1:50-1:500

  • Perform titration experiments to determine optimal dilution for your specific tissue

• Detection system:

  • Use detection systems compatible with rabbit IgG antibodies

  • Follow the manufacturer's specific IHC protocol available for download

• Controls:

  • Include positive controls (human liver cancer tissue)

  • Include negative controls (omitting primary antibody)

  • Consider using tissues from COX17 knockout models if available

What troubleshooting approaches should I consider when working with the COX17 Antibody in Western blot?

When troubleshooting Western blot applications with the COX17 Antibody, consider these technical aspects:

• Sample preparation for a small protein (7 kDa):

  • Use high percentage gels (15-20% acrylamide) for proper resolution

  • Consider specialized gel systems designed for low molecular weight proteins

  • Ensure complete transfer to membrane (adjust transfer conditions for small proteins)

• Antibody parameters:

  • Optimize within the recommended dilution range (1:500-1:3000)

  • Test different blocking agents to reduce background

  • Verify with positive controls (MCF-7 cells, rat heart tissue, or THP-1 cells)

• Special considerations for COX17:

  • As a cysteine-rich protein involved in copper transport, ensure samples contain appropriate reducing agents

  • Include protease inhibitors during sample preparation

  • For mitochondrial preparations, ensure proper isolation and solubilization (digitonin buffer as described in )

• Signal detection:

  • For this small protein, use highly sensitive detection methods

  • Adjust exposure times accordingly

  • Consider enhanced chemiluminescence systems for optimal detection

How can I design experiments to study the impact of COX17 acetylation on mitochondrial function?

To effectively study COX17 acetylation and its impact on mitochondrial function, consider this experimental framework :

What are the considerations for interpreting complex IV activity data in relation to COX17 function?

When interpreting cytochrome c oxidase (complex IV) activity data in relation to COX17 function, several factors should be considered:

How does the relationship between COX17, copper metabolism, and mitochondrial architecture inform experimental design?

The intricate relationship between COX17, copper metabolism, and mitochondrial architecture offers several experimental considerations:

• Copper manipulation experiments:

  • The interaction between COX17 and the MICOS complex is promoted by copper ions

  • Consider designing experiments with copper chelation or supplementation

  • Measure how copper availability affects COX17's interactions with MICOS components

• Structural and functional readouts:

  • Include both structural assessments (cristae morphology, mitochondrial fragmentation)

  • And functional measurements (complex IV activity, membrane potential)

  • This approach helps connect copper delivery functions with architectural roles

• Protein domain analysis:

  • K30 acetylation site is located within the first twin-CX9C motif

  • This domain is critical for copper binding

  • Consider domain-specific mutations to dissect copper-binding functions from structural roles

• Temporal dynamics:

  • Design time-course experiments to determine whether COX17's role in copper delivery precedes its role in maintaining mitochondrial architecture

  • Or whether these functions occur simultaneously or independently

• Systems biology approach:

  • Integrate data on COX17 acetylation, copper binding, MICOS interaction, and functional outcomes

  • This comprehensive approach can reveal whether these pathways operate in parallel or are interdependent

By carefully considering these experimental designs and interpretations, researchers can gain deeper insights into the multifaceted roles of COX17 in mitochondrial function and architecture.