Recombinant Macaca fascicularis ADP/ATP translocase 4 (SLC25A31)

What is the structure and function of Macaca fascicularis SLC25A31?

SLC25A31, also known as ADP/ATP translocase 4, belongs to the mitochondrial carrier family that facilitates the exchange of ADP and ATP across the mitochondrial inner membrane. In Macaca fascicularis (cynomolgus macaque), SLC25A31 plays a critical role in cellular energy metabolism by exporting ATP from the mitochondrial matrix while importing ADP from the cytosol.

The protein contains approximately 320 amino acids organized into multiple transmembrane domains that form a channel for adenine nucleotide transport . Like other members of the adenine nucleotide translocase (ANT) family, SLC25A31 is essential for oxidative phosphorylation, serving as the gatekeeper of energy flux between the mitochondria and cytosol . The protein shares significant structural homology with other ANT family members but has distinct tissue distribution patterns, being predominantly expressed in testicular tissue in mammals.

How does SLC25A31 differ from other ADP/ATP translocases in Macaca fascicularis?

While all ANT isoforms in Macaca fascicularis share the fundamental function of exchanging ADP for ATP, SLC25A31 (ANT4) exhibits several distinguishing characteristics:

• Tissue distribution: SLC25A31 is primarily expressed in testicular tissue and plays a specific role in spermatogenesis, whereas other ANT isoforms show different tissue expression patterns (ANT1 in heart and skeletal muscle, ANT2 in proliferating cells, and ANT3 with ubiquitous expression).

• Regulatory mechanisms: SLC25A31 is subject to distinct regulatory controls compared to other ANTs, particularly during germ cell development.

• Inhibitor sensitivity: SLC25A31 may have different responses to classical ANT inhibitors such as bongkrekic acid and carboxyatractyloside compared to other isoforms .

• Amino acid sequence: While maintaining the core structural elements necessary for transport function, SLC25A31 has unique sequence features that may influence its substrate binding affinity and interaction with other proteins.

What is the known role of SLC25A31 in mitochondrial function within experimental models?

In experimental models, SLC25A31 has been shown to:

• Physically and functionally interact with respirasomes (supercomplexes of the electron transport chain) , highlighting its integration into the broader oxidative phosphorylation machinery.

• Contribute to maintaining mitochondrial membrane potential by facilitating the exchange of negatively charged ATP⁴⁻ for negatively charged ADP³⁻.

• Support spermatogenesis and male fertility through its specialized expression in testicular tissue.

• Potentially participate in the regulation of the mitochondrial permeability transition, a process involved in cell death pathways.

Research in cynomolgus macaques has particular relevance as these primates are increasingly selected as experimental models due to their phylogenetic proximity to humans . The MHC polymorphism in Macaca fascicularis has been shown to influence immune responses against pathogens and vaccines , suggesting that genetic variability in key proteins like SLC25A31 may similarly impact individual responses in experimental medicine.

What expression systems are optimal for producing recombinant Macaca fascicularis SLC25A31?

Based on available research, several expression systems have proven effective for recombinant SLC25A31 production:

Table 1: Comparison of Expression Systems for Recombinant SLC25A31

For functional studies requiring properly folded and active SLC25A31, mammalian expression systems (particularly HEK-293 cells) are recommended despite their higher cost and lower yield . When selecting an expression system, researchers should consider their downstream applications and whether post-translational modifications are critical for their specific experiments.

What purification strategies yield highest activity for recombinant Macaca fascicularis SLC25A31?

Purification of functional SLC25A31 typically involves a multi-step approach:

• Affinity Chromatography: Using protein tags for selective capture:

  • His-tagged SLC25A31 can be purified using nickel or cobalt affinity resins

  • Strep-tagged SLC25A31 can be isolated using Strep-Tactin columns

• Size Exclusion Chromatography (SEC):

  • Critical for separating correctly folded protein from aggregates

  • Analytical SEC (HPLC) can evaluate protein homogeneity with >90% purity achievable

• Detergent Selection and Exchange:

  • Mild detergents (e.g., n-dodecyl-β-D-maltoside or digitonin) preserve protein activity

  • Detergent concentration must be optimized to prevent aggregation while maintaining solubility

• Reconstitution into Liposomes:

  • Essential for functional studies of membrane proteins

  • Enables measurement of transport activity in a controlled membrane environment

  • Liposome composition should include cardiolipin to mimic the mitochondrial inner membrane

The protein-to-lipid ratio during reconstitution significantly affects activity measurements and should be carefully optimized. Purity assessment using SDS-PAGE, Western blotting, and analytical SEC should be performed at each purification stage to ensure high-quality protein preparation.

How can researchers verify the activity of purified recombinant Macaca fascicularis SLC25A31?

Multiple complementary approaches should be employed to confirm SLC25A31 activity:

• Fluorescence-based ADP/ATP Exchange Assay:

  • Using Magnesium Green (MgGr™) to detect changes in Mg²⁺ concentration during nucleotide exchange

  • This method leverages different binding affinities of Mg²⁺ for ATP versus ADP

  • Enables real-time, non-radioactive measurement with comparable sensitivity to traditional methods

  • Expected exchange rates of approximately 3-4 mmol/min/g protein based on studies with related ANT proteins

• Inhibitor Sensitivity Testing:

  • Bongkrekic acid and carboxyatractyloside specifically inhibit ANT-mediated transport

  • Functional SLC25A31 should show dose-dependent inhibition by these compounds

• Liposome-based Transport Assays:

  • After reconstitution, measure uptake/exchange of radiolabeled substrates

  • Can determine kinetic parameters (Km, Vmax) for transport activity

• Structural Integrity Assessment:

  • Circular dichroism spectroscopy to evaluate secondary structure content

  • Thermal stability assays to assess protein folding and stability

For robust verification, researchers should demonstrate both binding of known inhibitors and substrate transport activity, as functional deficiencies may not be apparent from structural assessments alone.

What fluorescence-based methods are most effective for studying SLC25A31 transport kinetics?

The fluorescence-based Magnesium Green (MgGr™) assay has emerged as a preferred non-radioactive method for studying ANT transport kinetics and can be effectively applied to SLC25A31 research:

Methodological approach:

• Principle: The assay exploits differential binding of Mg²⁺ to ATP (high affinity) versus ADP (lower affinity). When ATP is exchanged for ADP by SLC25A31, free Mg²⁺ concentration increases, enhancing MgGr™ fluorescence .

• Protocol outline:

  • Reconstitute purified SLC25A31 into unilamellar liposomes

  • Load liposomes with ATP and MgGr™ dye

  • Add external ADP to initiate exchange

  • Record fluorescence changes in real-time (excitation ~490 nm, emission ~520 nm)

  • Calculate transport rates from the initial velocity of fluorescence increase

• Quantification: Exchange rates can be determined using a calibration curve with known concentrations of free Mg²⁺. For adenine nucleotide translocases, rates of 3-4 mmol/min/g protein have been documented in proteoliposome systems .

• Advantages over radioactive assays:

  • Real-time continuous monitoring

  • No radioactive waste or safety concerns

  • Compatible with high-throughput screening applications

  • Comparable sensitivity to traditional methods when optimized

• Kinetic parameter determination:

  • Km and Vmax can be calculated by varying external ADP concentrations

  • Inhibition constants (Ki) can be determined with ANT inhibitors

This method has successfully measured ADP/ATP exchange rates of 3.49 ± 0.41 mmol/min/g for recombinant ANT1 reconstituted into unilamellar liposomes , providing a validated benchmark for similar studies with SLC25A31.

How can researchers investigate SLC25A31 interactions with respirasomes?

Investigation of SLC25A31 interactions with respirasomes requires specialized techniques that preserve both the structural integrity of membrane protein complexes and their functional associations:

• Co-immunoprecipitation approaches:

  • Use antibodies against SLC25A31 or respirasome components

  • Mild detergents (digitonin) preserve supercomplexes during solubilization

  • Mass spectrometry identification of co-precipitated proteins

  • Western blot confirmation of specific interactions

• Blue Native PAGE analysis:

  • Enables separation of intact protein complexes

  • Can reveal SLC25A31 migration with respirasome components

  • Second-dimension SDS-PAGE separates individual proteins within complexes

• Proximity labeling techniques:

  • Fusion of SLC25A31 with BioID or APEX2 enzymes

  • Allows biotinylation of proteins in close proximity in living cells

  • Mass spectrometry identification of biotinylated proteins

  • Has advantages over traditional pull-down methods for transient interactions

• Functional coupling analysis:

  • Measure respiration rates with purified components

  • Assess how SLC25A31 addition affects respirasome activity

  • Examine effects of inhibitors on coupled functions

• Cryo-electron microscopy:

  • Can resolve structures of membrane protein supercomplexes

  • May reveal physical contacts between SLC25A31 and respirasome components

Human adenine nucleotide translocases have been demonstrated to physically and functionally interact with respirasomes , suggesting that Macaca fascicularis SLC25A31 likely exhibits similar interactions that can be investigated using these approaches.

What techniques are available for studying SLC25A31 in mitochondrial disease models?

Several complementary approaches can be employed to investigate SLC25A31 in mitochondrial disease contexts:

• CRISPR/Cas9 gene editing:

  • Introduce disease-associated mutations into SLC25A31 in cell lines

  • Create isogenic cell lines differing only in SLC25A31 sequence

  • Analyze phenotypic consequences of mutations on mitochondrial function

• Patient-derived iPSC models:

  • Reprogram patient cells carrying mitochondrial disease mutations

  • Differentiate into relevant cell types (e.g., neurons, cardiomyocytes)

  • Compare with isogenic corrected controls

  • Allows study of mutations in appropriate genetic background

• Proteoliposome reconstitution with mutant proteins:

  • Express and purify disease-associated SLC25A31 variants

  • Reconstitute into liposomes for transport assays

  • Directly measure impact of mutations on transport kinetics

  • The fluorescence-based method in search result provides a sensitive approach

• Mitochondrial respiration analysis:

  • High-resolution respirometry to measure oxygen consumption

  • Seahorse XF analyzer for real-time cellular bioenergetics

  • Assess impact of SLC25A31 mutations on integrated respiratory function

• Mitochondrial membrane potential measurements:

  • Fluorescent probes (TMRM, JC-1) to monitor Δψm

  • Flow cytometry or microscopy-based readouts

  • Quantify effects of SLC25A31 dysfunction on bioenergetic parameters

Mutations in or dysregulation of adenine nucleotide translocases have been associated with mitochondrial diseases including progressive external ophthalmoplegia, cardiomyopathy, and nonsyndromic intellectual disabilities . These techniques allow researchers to establish mechanistic links between SLC25A31 dysfunction and disease phenotypes.

How does Macaca fascicularis SLC25A31 compare structurally and functionally to human SLC25A31?

Comparative analysis reveals important similarities and differences between Macaca fascicularis and human SLC25A31:

Structural comparison:

• Sequence homology: High sequence conservation exists between macaque and human SLC25A31, with expected identity >90%, reflecting their close evolutionary relationship and conserved function.

• Protein architecture: Both proteins contain approximately 320 amino acids organized into six transmembrane domains arranged in three similar repeats, forming a characteristic mitochondrial carrier family structure .

• Critical domains: Nucleotide binding sites and channel-forming regions show particularly high conservation, while surface-exposed loops may exhibit greater species-specific variation.

Functional comparison:

• Transport kinetics: The fundamental ADP/ATP exchange mechanism is conserved, though subtle species-specific differences in transport rates may exist.

• Inhibitor sensitivity: Both proteins are expected to be inhibited by classical ANT inhibitors (bongkrekic acid and carboxyatractyloside), though potentially with different affinities .

• Tissue expression pattern: Both human and Macaca fascicularis SLC25A31 show predominant expression in testicular tissue, suggesting conserved specialization for reproductive functions.

• Respirasome interactions: Based on studies of human adenine nucleotide translocases, Macaca fascicularis SLC25A31 likely physically and functionally interacts with respirasomes in a similar manner .

These similarities make Macaca fascicularis SLC25A31 a valuable model for human mitochondrial carrier function, particularly for studies relevant to reproductive biology and mitochondrial disease modeling.

What genetic variations exist in SLC25A31 across Macaca fascicularis populations?

While specific information about SLC25A31 genetic variation across Macaca fascicularis populations is not explicitly detailed in the search results, we can infer potential patterns based on broader genomic studies of this species:

• Population structure: Cynomolgus macaques show distinct genetic differentiation between continental and island populations , suggesting potential population-specific variants in genes including SLC25A31.

• Regulatory elements: Genomic analysis of Macaca fascicularis has identified differences in transcription factor binding sites that could affect gene regulation , potentially including differential expression of SLC25A31 across populations.

• MHC comparison as proxy: Studies of MHC polymorphism in Macaca fascicularis reveal significant variation, with many novel alleles being regularly identified in Vietnamese and Cambodian populations . Similar patterns of genetic diversity might exist for SLC25A31.

• Methodological approaches: Population genetic analyses using techniques such as "window scans of various population genetic statistics" and analysis of "minor allele frequencies" could be applied to characterize SLC25A31 variation across macaque populations.

• Functional implications: Any identified variants would need functional characterization using methods like the fluorescence-based ADP/ATP exchange assay to determine their impact on protein function.

Understanding population-specific variation in SLC25A31 could provide insights into adaptive evolution of mitochondrial function in different environmental contexts and help researchers select appropriate macaque populations for specific research applications.

How has SLC25A31 evolved in primates and what are the implications for using Macaca fascicularis as a model organism?

Evolutionary analysis of SLC25A31 provides important context for its use in biomedical research:

The genetic differentiation observed between continental and island populations of Macaca fascicularis suggests researchers should carefully consider the geographical origin of animals when designing studies, as population-specific genetic variants might influence experimental outcomes.

What are the most common challenges in producing active recombinant Macaca fascicularis SLC25A31 and how can they be addressed?

Researchers frequently encounter several challenges when producing active recombinant SLC25A31:

• Protein aggregation issues:

  • Challenge: As a membrane protein, SLC25A31 tends to aggregate when removed from lipid environments

  • Solution: Use mild detergents (DDM, digitonin); maintain critical micelle concentration throughout purification; add stabilizing agents like glycerol (10-15%); consider adding specific lipids during purification

• Low expression levels:

  • Challenge: Membrane proteins often express poorly in heterologous systems

  • Solution: Optimize codon usage for expression host; use strong inducible promoters; lower induction temperature (16-18°C for E. coli, 30°C for mammalian cells); consider fusion tags that enhance folding (SUMO, MBP)

• Incorrect folding:

  • Challenge: SLC25A31 may not adopt native conformation in heterologous systems

  • Solution: Express in mammalian cells (HEK-293) for complex membrane proteins ; co-express with chaperones; use GFP fusion to monitor proper folding

• Protein instability:

  • Challenge: Purified SLC25A31 may rapidly lose activity

  • Solution: Minimize freeze-thaw cycles; add protease inhibitors; maintain constant low temperature during purification; consider protein engineering to increase stability

• Inefficient reconstitution:

  • Challenge: Poor incorporation into liposomes for functional assays

  • Solution: Optimize protein:lipid ratios; include cardiolipin in liposome composition; try different reconstitution methods (detergent dialysis, Bio-Beads, dilution)

• Activity verification difficulties:

  • Challenge: Distinguishing specific transport activity from background

  • Solution: Include proper controls (protein-free liposomes, inhibitor-treated samples); use the fluorescence-based MgGr™ assay which offers improved signal-to-noise ratio over traditional methods

Achieving ADP/ATP exchange rates of 3-4 mmol/min/g protein (similar to those reported for ANT1 ) indicates successful production of active recombinant SLC25A31.

How can researchers optimize liposome-based functional assays for SLC25A31?

Optimizing liposome-based functional assays for SLC25A31 requires careful attention to multiple parameters:

Liposome preparation optimization:

• Lipid composition:

  • Include cardiolipin (15-20%) to mimic mitochondrial inner membrane

  • Optimize PC:PE ratio (typically 3:1 to 4:1)

  • Test different lipid sources (synthetic vs. extracted)

  • Document impact of lipid composition on baseline activity measurements

• Liposome size and homogeneity:

  • Use extrusion through defined pore size filters (100-200 nm)

  • Verify size distribution by dynamic light scattering

  • Ensure unilamellar vesicle formation (critical for consistent results)

• Protein:lipid ratio:

  • Test multiple ratios (typically 1:50 to 1:200 w/w)

  • Too high protein concentration leads to aggregation

  • Too low concentration yields insufficient signal

Assay conditions optimization:

• Buffer composition:

  • Test different pH values (typically 6.8-7.4)

  • Optimize ionic strength (100-150 mM is typical)

  • Include divalent cations (Mg²⁺, 1-5 mM)

• Temperature control:

  • Maintain consistent temperature (typically 25°C or 30°C)

  • Account for temperature effects on fluorescence measurements

  • Document temperature dependence of transport activity

• Substrate concentrations:

  • Internal ATP typically 5-10 mM

  • External ADP titration to determine Km (typically 10 μM-2 mM range)

• For fluorescence-based assays:

  • Optimize MgGr™ concentration to avoid inner filter effects

  • Establish calibration curve with known Mg²⁺ concentrations

  • Control for potential artifacts (photobleaching, dye leakage)

Applying these optimizations can yield reliable measurements of SLC25A31 transport activity with kinetic parameters comparable to those observed for other adenine nucleotide translocases (exchange rates of approximately 3-4 mmol/min/g protein) .

What are the best approaches for investigating SLC25A31 post-translational modifications and their impact on function?

Investigating post-translational modifications (PTMs) of SLC25A31 requires an integrated approach combining identification and functional characterization:

Identification strategies:

• Mass spectrometry-based approaches:

  • Enrichment methods for specific modifications (phosphopeptides, acetylated peptides)

  • Top-down proteomics to analyze intact protein preserving PTM combinations

  • Fragmentation techniques optimized for PTM identification (ETD, HCD)

  • Site-specific quantification using labeled standards

• Antibody-based detection:

  • Western blotting with modification-specific antibodies

  • Immunoprecipitation to enrich modified forms

  • Requires careful validation of antibody specificity

• In silico prediction:

  • Computational algorithms to predict potential modification sites

  • Cross-species comparison to identify conserved modification motifs

  • Structural modeling to assess accessibility of potential sites

Functional characterization:

• Site-directed mutagenesis:

  • Generate non-modifiable variants (e.g., S→A for phosphorylation sites)

  • Create phosphomimetic mutations (S→D/E) to mimic constitutive phosphorylation

  • Assess impact on transport activity using fluorescence-based assays

• In vitro modification:

  • Treat purified SLC25A31 with specific kinases or acetyltransferases

  • Compare activity before and after modification

  • Identify enzymes responsible for specific modifications

• Interaction studies:

  • Determine if modifications alter SLC25A31 interactions with respirasomes

  • Pull-down assays comparing modified and unmodified protein

  • Proximity labeling to detect modification-dependent interactions

• Physiological triggers:

  • Identify conditions that alter SLC25A31 modification status

  • Link modifications to specific cellular states or stresses

  • Establish temporal dynamics of modifications

This integrated approach can reveal how PTMs regulate SLC25A31 function in different physiological contexts and potentially identify targets for therapeutic intervention in mitochondrial diseases associated with adenine nucleotide translocase dysfunction .