Recombinant Human Protein FAM173A (FAM173A)

What is the primary function of FAM173A protein?

FAM173A functions as a mitochondrial protein-lysine N-methyltransferase that specifically trimethylates adenine nucleotide translocases (ANTs). This enzyme catalyzes the methylation of Lysine-52 in ANT2/SLC25A5 and ANT3/SLC25A6, a modification that appears to regulate mitochondrial respiration. FAM173A is also predicted to methylate ANT1/SLC25A4 at the same position . The identification of FAM173A as the enzyme responsible for ANT methylation represents a significant advancement in understanding post-translational modifications in mitochondrial proteins, particularly those involved in energy metabolism and ADP/ATP exchange across the inner mitochondrial membrane.

How is FAM173A targeted to mitochondria?

FAM173A contains a noncanonical mitochondrial targeting sequence (MTS) in its N-terminal region. Specifically, the preMT sequence (amino acids 43-77) is responsible for its mitochondrial localization. This has been demonstrated through fluorescence microscopy of live cells expressing FAM173A-GFP fusion proteins, where deletion mutants lacking this sequence failed to localize to mitochondria . The protein also contains a predicted transmembrane domain (TMD) that appears to anchor it to the mitochondrial inner membrane. Western blot analysis of mitoplast extracts has revealed the presence of both full-length FAM173A and a truncated version consisting of only the methyltransferase domain, suggesting that the protein undergoes partial processing in mitochondria while retaining its membrane association .

What experimental methods are suitable for detecting FAM173A in cell samples?

To detect FAM173A in cellular samples, researchers can employ several complementary approaches:

• Immunohistochemistry (IHC-P): Paraffin-embedded tissues can be probed with specific antibodies such as ab126340 at a recommended dilution of 1/20, which has successfully detected FAM173A in human cerebral cortex tissue .

• Immunocytochemistry/Immunofluorescence (ICC/IF): For cultured cells, immunofluorescent staining using antibodies at 1-4 μg/ml concentration shows cytoplasmic positivity, particularly in mitochondria. Cells should be treated with PFA/Triton X-100 for optimal results .

• Western Blotting: For protein expression quantification, Western blotting with specific antibodies against FAM173A can identify both the full-length protein (approximately 28.2 kDa) and its processed form (approximately 19.8 kDa) in mitochondrial fractions .

• Fluorescence Microscopy: Creating GFP fusion constructs allows for live-cell visualization of FAM173A localization using confocal microscopy, which can be co-stained with mitochondrial markers like MitoTracker Orange to confirm mitochondrial localization .

How can researchers effectively disrupt FAM173A function to study its physiological role?

Disrupting FAM173A function can be accomplished through several approaches:

• CRISPR/Cas9 Gene Editing: The most definitive approach involves creating knockout cell lines using CRISPR/Cas9 technology. Guide RNAs should target sequences upstream of motif "Post I," which contains the catalytically important Glu-105 residue. Successful disruption can be confirmed by sequencing genomic DNA to verify frameshift mutations that result in truncated, inactive proteins .

• Site-Directed Mutagenesis: Creating point mutations at catalytic residues, particularly the E105A mutation, produces enzymatically inactive FAM173A that can be used in complementation studies. This approach allows researchers to specifically attribute phenotypes to the methyltransferase activity rather than structural roles of the protein .

• Complementation Assays: Reintroducing wild-type or mutant FAM173A into knockout cells allows for rescue experiments that can confirm the specific function of the protein and separate enzymatic from non-enzymatic roles .

• RNA Interference: Though less complete than CRISPR-mediated knockout, siRNA or shRNA approaches can achieve temporary knockdown of FAM173A expression to study acute effects of its depletion.

The selection of methodology should consider the specific research question, with CRISPR-based approaches providing the most definitive results for long-term functional studies.

What methods are most effective for measuring the methyltransferase activity of FAM173A?

Measuring FAM173A methyltransferase activity requires specialized methods that can detect protein methylation:

For ANT methylation specifically, immunoblotting with methyllysine-specific antibodies has successfully identified Lys-52 methylation status. Mass spectrometry analysis provides the most definitive identification of methylation sites and can distinguish between mono-, di-, and trimethylation . Functional complementation of FAM173A knockout cells with wild-type versus E105A mutant FAM173A confirms that enzymatic activity is required for ANT methylation .

How does FAM173A deficiency affect mitochondrial respiration, and how can these changes be measured?

FAM173A deficiency significantly impacts mitochondrial respiration, with knockout cells displaying approximately 50% increase in both State II (basal) and State III (ADP-stimulated) respiration compared to FAM173A-proficient cells . These changes can be accurately measured using:

• Seahorse XF Analyzer: This platform allows measurement of oxygen consumption rate (OCR) under various conditions. The recommended protocol involves:

  • Isolating mitochondria from FAM173A-proficient and FAM173A-deficient cells

  • Incubating with succinate (Complex II substrate) and rotenone (Complex I inhibitor)

  • Measuring OCR under basal conditions and after sequential addition of:

    • ADP (stimulates ATP synthesis)

    • Oligomycin (inhibits ATP synthase)

    • FCCP (uncoupling agent)

    • Antimycin A (Complex III inhibitor)

• Respiration States Analysis: Calculate different respiration states:

  • State II (basal respiration) = OCR basal − OCR AntA

  • State III (ATP synthesis-stimulated) = OCR ADP − OCR AntA

  • State IVo (proton leak) = OCR oligomycin − OCR AntA

  • State IIIu (maximal uncoupled) = OCR FCCP − OCR AntA

• Protein Expression Control: Always verify that the expression levels of key mitochondrial proteins (COX IV, ATPSc, ATP5A, and ANT2) remain unchanged to ensure observed differences are due to methylation status rather than protein abundance .

The significant increase in mitochondrial respiration in FAM173A-deficient cells suggests that methylation of ANT at Lys-52 normally functions to reduce translocase activity, potentially limiting ADP/ATP exchange across the inner mitochondrial membrane and decreasing respiration rates .

What is the structural significance of Lys-52 in ANT proteins, and how does its methylation potentially affect function?

Lysine-52 occupies a critical position in the structure of adenine nucleotide translocase (ANT) proteins:

• Structural Location: Lys-52 is located in a large solvent-exposed segment that connects the first and second transmembrane α-helices of the ANT pore and faces the mitochondrial matrix .

• Functional Implications: The position of Lys-52 in this matrix-facing loop suggests its methylation could affect:

  • Protein conformation and stability

  • Interaction with other matrix proteins

  • Electrostatic properties influencing substrate binding or transport kinetics

  • Allosteric regulation of the transport mechanism

• Methylation Status In Vivo: Analysis of rat tissues has shown that Lys-52 is exclusively trimethylated rather than existing in various methylation states, suggesting this is a constitutive rather than dynamic regulatory modification .

• Functional Effects: Experimental evidence indicates that loss of Lys-52 methylation in FAM173A knockout cells results in increased mitochondrial respiration, suggesting methylation normally restricts ANT translocase activity and thereby limits ADP/ATP exchange and respiratory rates .

The model supported by current research suggests that FAM173A is inserted into the inner mitochondrial membrane via its transmembrane domain, positioning its catalytic domain to access and methylate the matrix-exposed Lys-52 residue on ANT proteins. This methylation appears to fine-tune ANT activity and consequently mitochondrial respiration rates .

What are the key considerations when using recombinant FAM173A protein or antibodies in experimental settings?

When working with recombinant FAM173A protein or antibodies, researchers should consider several critical factors:

• Antibody Selection and Validation:

  • Commercial antibodies like ab126340 have been validated for IHC-P and ICC/IF applications in human samples .

  • Researchers should verify specificity using FAM173A knockout cells as negative controls.

  • Appropriate dilutions (e.g., 1/20 for IHC-P, 1-4 μg/ml for ICC/IF) are essential for optimal results .

• Expression Constructs Design:

  • Include the complete coding sequence with the N-terminal targeting sequence for proper localization.

  • Consider using epitope tags (FLAG, GFP) positioned at the C-terminus to avoid interfering with mitochondrial targeting .

  • When creating mutants, the E105A mutation provides a catalytically inactive control .

• Experimental Controls:

  • Include wild-type cells, knockout cells, and knockout cells complemented with wild-type or mutant FAM173A for comprehensive analysis .

  • Verify expression levels of ANT proteins and other mitochondrial components to ensure phenotypes are not due to altered protein abundance.

• Species Considerations:

  • FAM173A is present only in vertebrates, while its paralogue ATPSc-KMT is ubiquitously found in all metazoans .

  • Cross-species reactivity of antibodies should be verified experimentally.

• Storage and Handling:

  • Recombinant proteins and antibodies should be stored according to manufacturer recommendations to preserve activity.

  • Remember that all commercial products typically state they are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES" .

How might researchers investigate the broader physiological significance of FAM173A-mediated ANT methylation?

Investigating the broader physiological significance of FAM173A-mediated ANT methylation presents several promising research directions:

• Tissue-Specific Knockout Models:

  • Generate tissue-specific FAM173A knockout mice to investigate organ-specific effects.

  • Focus on tissues with high metabolic demands such as brain, heart, and skeletal muscle where ANT function is critical.

  • Analyze metabolic parameters, exercise capacity, and adaptation to metabolic stress.

• Pathological Conditions Investigation:

  • Examine FAM173A expression and ANT methylation status in models of:

    • Mitochondrial disorders

    • Neurodegenerative diseases

    • Metabolic syndrome and diabetes

    • Ischemia-reperfusion injury

    • Aging

• Interactome Analysis:

  • Identify protein interaction partners of FAM173A using techniques such as BioID, proximity labeling, or co-immunoprecipitation.

  • Investigate whether FAM173A functions in larger regulatory complexes that coordinate mitochondrial function.

• Regulatory Mechanisms:

  • Investigate how FAM173A expression and activity are regulated under different physiological and pathological conditions.

  • Determine whether post-translational modifications affect FAM173A activity.

• Therapeutic Potential:

  • Develop small molecule inhibitors or activators of FAM173A to modulate mitochondrial respiration.

  • Evaluate whether modulating FAM173A activity could provide therapeutic benefits in conditions characterized by mitochondrial dysfunction.

Given that FAM173A deficiency increases mitochondrial respiration, this enzyme represents a potential target for therapeutic approaches aimed at enhancing mitochondrial function in conditions where energy metabolism is compromised .

How can researchers address potential discrepancies between in vitro and in vivo findings regarding FAM173A function?

Researchers may encounter discrepancies between in vitro experiments and in vivo observations regarding FAM173A function. These can be addressed through systematic approaches:

• Methodological Reconciliation:

  • Compare cell culture conditions with physiological environments, particularly oxygen tension, substrate availability, and metabolic demands.

  • Determine whether observed differences are due to technical limitations or true biological variations.

  • Design experiments that bridge the gap between simplified in vitro systems and complex in vivo environments, such as organoid cultures or ex vivo tissue preparations.

• Context-Dependent Function Analysis:

  • Investigate whether FAM173A activity and its effects on ANT methylation vary across:

    • Different cell types and tissues

    • Developmental stages

    • Metabolic states (fed vs. fasted, rest vs. exercise)

    • Stress conditions

• Compensatory Mechanisms Identification:

  • Acute vs. chronic loss-of-function studies to identify adaptive responses

  • Combined knockout of FAM173A with related enzymes to identify redundant systems

  • Temporal analysis of phenotypes following FAM173A inactivation

• Technical Validation Across Platforms:

  • Confirm ANT methylation status using complementary techniques:

    • Antibody-based detection

    • Mass spectrometry

    • Metabolic flux analysis

    • Structural studies

• Translational Relevance Assessment:

  • Correlate experimental findings with human genetic data on FAM173A variants

  • Analyze ANT methylation in human biospecimens from relevant pathological conditions

  • Develop humanized models to better predict clinical relevance

The observation that Lys-52 is exclusively trimethylated in rat tissues suggests this modification is constitutive rather than dynamically regulated , which should be considered when interpreting experimental manipulations of FAM173A activity.

What technical challenges exist in the experimental study of membrane-associated methyltransferases like FAM173A?

Studying membrane-associated methyltransferases like FAM173A presents unique technical challenges that researchers must overcome:

FAM173A presents additional specific challenges:

• Dual Localization: FAM173A exists in both full-length membrane-anchored and processed soluble forms, complicating analysis of which form is enzymatically active in different contexts .

• Noncanonical Targeting: Its unusual mitochondrial targeting mechanism may result in variable mitochondrial import efficiency across different experimental systems .

• Substrate Accessibility: The methylation target (Lys-52) is located in a loop region of ANT that faces the mitochondrial matrix, requiring FAM173A to be correctly oriented in the inner membrane for catalysis .

• Functional Readouts: Changes in methylation status need to be linked to functional outcomes like transport activity, which requires specialized assays for ADP/ATP exchange and respiratory measurements .

Overcoming these challenges requires interdisciplinary approaches combining biochemistry, cell biology, structural biology, and advanced imaging techniques.