Recombinant Mouse Solute carrier family 25 member 41 (Slc25a41), partial

What is the basic structure and function of Slc25a41?

Slc25a41 is a member of the mitochondrial carrier family (SLC25), which consists of proteins that transport various substrates across the inner mitochondrial membrane. The mouse Slc25a41 protein consists of 312 amino acids with a molecular weight of approximately 32 kDa. The protein sequence includes characteristic transmembrane domains typical of mitochondrial carriers .

Unlike some SLC25 family members with calcium-binding N-terminal extensions, Slc25a41 (also known as SCaMC-3L) lacks these calcium-binding domains. This structural difference is significant as it results in calcium-independent transport activity, representing a novel mechanism for adenine nucleotide transport across the inner mitochondrial membrane .

How does Slc25a41 differ from other SLC25 family members?

Slc25a41 differs from other SLC25 family members in several key aspects:

• Evolutionary origin: Slc25a41 orthologues are found exclusively in mammals, showing approximately 60% identity to the C-terminal half of SCaMC-3 (its closest paralogue). In mammalian genomes, SCaMC-3 and SCaMC-3L genes are adjacent on the same chromosome, forming a head-to-tail tandem array with identical exon-intron boundaries. This suggests that Slc25a41 arose from an SCaMC-3 ancestor through a partial duplication event that occurred before mammalian radiation .

• Expression pattern: Unlike the broadly expressed longer SCaMCs, mouse Slc25a41 shows a limited expression pattern, being preferentially expressed in testis and, to a lesser extent, in brain .

• Transport mechanism: While most mitochondrial carriers are obligatory exchangers, Slc25a41 performs ATP-Mg/Pi exchange in a calcium-independent manner, unlike other ATP-Mg/Pi carriers .

What expression systems are suitable for producing recombinant Slc25a41 protein?

Multiple expression systems have been successfully employed to produce recombinant Slc25a41 protein:

For functional characterization studies, both bacterial and yeast expression systems have proven effective when the purified protein is reconstituted into liposomes for transport assays .

What methods are most effective for characterizing Slc25a41 transport activity?

The most reliable approach for characterizing Slc25a41 transport activity involves:

• Reconstitution into liposomes: Purified Slc25a41 protein should be incorporated into phospholipid vesicles to create a controlled environment for transport studies.

• Homoexchange experiments: Testing transport activities using radiolabeled substrates (e.g., ATP) with the same substrate inside and outside the liposomes.

• Controls: Including inhibition controls (e.g., with mercurial compounds like HgCl₂) and negative controls (boiled protein or empty liposomes).

• Kinetic measurements: Determining transport parameters such as Km and Vmax values using varying substrate concentrations.

These methods, as demonstrated with other SLC25 family members, allow for definitive characterization of transport specificity, mechanism (exchange vs. uniport), and regulation .

What role does Slc25a41 play in cancer biology?

Research indicates that Slc25a41 expression is altered in several cancer types, suggesting a potential role in tumor progression:

• In lung adenocarcinoma, SLC25A41 shows elevated expression compared to normal tissue, contrasting with the decreased expression of SLC25A7 and SLC25A25 in the same cancer type .

• Comprehensive analysis of the SLC25 family in cancer reveals that various members, including SLC25A41, demonstrate strong correlations with immune cell infiltration, such as macrophages M2 and CD8+ T cells .

• The SLC25 family's involvement in cellular metabolism, particularly mitochondrial functions, positions these transporters as potential contributors to the metabolic reprogramming seen in cancer cells .

For experimental investigation of Slc25a41's role in cancer, researchers should consider:

• Performing comparative expression analyses across multiple cancer types

• Examining correlations with clinical outcomes

• Conducting knockdown/overexpression studies in cancer cell lines to assess effects on proliferation, invasion, and metabolism

How do alterations in Slc25a41 expression affect mitochondrial function?

Changes in Slc25a41 expression can significantly impact mitochondrial function through several mechanisms:

• ATP/ADP exchange: As a potential adenine nucleotide transporter, alterations in Slc25a41 expression may affect the exchange of ATP and ADP across the inner mitochondrial membrane, impacting energy production.

• Metabolite transport: Based on studies of other SLC25 family members, Slc25a41 may transport specific metabolites crucial for mitochondrial metabolism.

• Membrane potential: Studies on related SLC25 transporters show that their deficiency can affect mitochondrial membrane potential, which is critical for oxidative phosphorylation .

In experimental models using SLC25A51 (another family member), knockdown resulted in reduced mitochondrial NAD+ levels and respiratory capacity without affecting membrane potential . Similar experimental approaches could be applied to study Slc25a41's specific effects on mitochondrial function.

How can one determine the precise substrates of Slc25a41?

Determining the precise substrates of Slc25a41 requires a multi-faceted approach:

• Liposome reconstitution assays with potential substrates: This approach, successfully used with SLC25A29, involves testing radiolabeled potential substrates in liposome exchange experiments. For Slc25a41, candidates may include adenine nucleotides, phosphate, and other metabolites .

• Substrate competition assays: Testing whether non-labeled potential substrates can compete with the transport of a known radiolabeled substrate.

• Structural modeling and site-directed mutagenesis: Identifying and mutating potential substrate-binding residues based on homology modeling with other characterized SLC25 family members.

• Metabolomic analysis: Comparing metabolite profiles in mitochondria with normal versus altered Slc25a41 expression.

As demonstrated with SLC25A29, these approaches can definitively establish substrate specificity, showing that despite prior suggestions, it transports basic amino acids rather than carnitine/acylcarnitines .

What are the molecular mechanisms regulating Slc25a41 expression and activity?

Understanding the regulation of Slc25a41 requires investigation at multiple levels:

• Transcriptional regulation: Analysis of the promoter region and identification of transcription factors controlling tissue-specific expression, particularly in testis and brain tissues where Slc25a41 is preferentially expressed .

• Post-translational modifications: Phosphorylation, acetylation, or other modifications may regulate Slc25a41 activity, as seen with other mitochondrial carriers.

• Protein-protein interactions: Identification of binding partners that may modulate Slc25a41 transport activity or localization.

• Substrate regulation: Investigation of whether substrate availability affects Slc25a41 expression or activity through feedback mechanisms.

Studies of other SLC25 family members have shown that their activity can be regulated by Ca²⁺ binding, redox state, and interaction with regulatory proteins . While Slc25a41 lacks the calcium-binding domain present in longer SCaMCs, it may still be subject to other regulatory mechanisms that warrant investigation .

How can CRISPR-Cas9 gene editing be optimized for studying Slc25a41 function?

Optimizing CRISPR-Cas9 approaches for Slc25a41 research requires careful consideration of several factors:

• Guide RNA design: Multiple sgRNAs targeting different exons should be designed and validated. The highly conserved nature of SLC25 family members necessitates careful sgRNA selection to avoid off-target effects on related transporters.

• Functional readouts: Since mitochondrial carriers like Slc25a41 can affect metabolism, appropriate functional assays should include:

  • Respirometry (oxygen consumption rate measurements)

  • Metabolite profiling

  • Mitochondrial membrane potential assessment

  • Cell growth and viability under different metabolic conditions

• Complementation controls: Re-expression of wild-type Slc25a41 in knockout cells should rescue the phenotype, confirming specificity.

• Inducible systems: For essential genes, inducible CRISPR systems or degradation tag approaches may be necessary to avoid cell lethality while studying acute effects of Slc25a41 loss.

Studies using similar approaches with SLC25A51 demonstrated that its knockout affected mitochondrial NAD+ levels and respiratory capacity, establishing its role as a mitochondrial NAD+ transporter .

How does Slc25a41 interact with other mitochondrial transporters and metabolic pathways?

Slc25a41 likely participates in complex interactions with other mitochondrial components:

• Metabolic pathway integration: As a proposed ATP-Mg/Pi carrier, Slc25a41 would interface with energy metabolism pathways, potentially affecting:

  • Oxidative phosphorylation

  • Tricarboxylic acid (TCA) cycle

  • Amino acid metabolism

  • Nucleotide metabolism

• Transporter networks: Mitochondrial carriers often function in coordinated networks. For example, the oxodicarboxylate carrier (ODC) encoded by SLC25A21 regulates the efflux of α-ketoglutarate, affecting glutamine metabolism in cancer cells . Similar functional interactions may exist for Slc25a41.

• Protein complexes: Some SLC25 proteins interact with other mitochondrial components. For instance, SLC25A51 reportedly interacts with C7orf55 (an assembly factor for respiratory complex V) and Bola1 (involved in iron-sulfur metabolism) .

Experimental approaches to study these interactions include co-immunoprecipitation, proximity labeling techniques (BioID, APEX), and metabolic flux analysis using isotope-labeled substrates.

What evolutionary insights can be gained from comparative analysis of Slc25a41 across species?

Evolutionary analysis of Slc25a41 provides valuable insights into its functional significance:

• Mammalian-specific adaptation: Slc25a41 orthologues are found exclusively in mammals, suggesting it evolved to meet specific metabolic demands in mammalian physiology .

• Evolutionary origin: The genomic arrangement shows that SCaMC-3 and Slc25a41 genes form a head-to-tail tandem array with identical exon-intron boundaries, indicating that Slc25a41 arose through a partial gene duplication of SCaMC-3 prior to mammalian radiation .

• Functional divergence: Following duplication, Slc25a41 acquired more restrictive functions and expression patterns compared to SCaMC-3, demonstrating subfunctionalization .

• Structure-function relationships: Comparison of conserved residues across species can identify essential functional domains for substrate binding and transport.

This evolutionary scenario suggests that Slc25a41 may fulfill specialized roles in mammalian-specific physiological processes, particularly in testis and brain tissues where it shows predominant expression .

What are the most common challenges in working with recombinant Slc25a41, and how can they be addressed?

Researchers face several technical challenges when working with recombinant Slc25a41:

• Protein solubility and stability:

  • Challenge: Membrane proteins like Slc25a41 often aggregate during expression and purification.

  • Solution: Optimize detergent selection for solubilization; consider using mild detergents like dodecylmaltoside or digitonin. Expression as inclusion bodies followed by refolding has proven successful for other SLC25 family members .

• Functional reconstitution:

  • Challenge: Ensuring proper insertion and orientation in liposomes for functional studies.

  • Solution: Optimize lipid composition and reconstitution protocols; verify protein incorporation using proteoliposome flotation assays.

• Transport assay sensitivity:

  • Challenge: Detecting potentially low transport activities.

  • Solution: Use radiolabeled substrates for maximum sensitivity; optimize internal substrate concentrations to enhance exchange rates.

• Substrate identification:

  • Challenge: Identifying the physiological substrate from numerous possibilities.

  • Solution: Perform broad screening of potential substrates using the liposome reconstitution system; prioritize testing based on homology to characterized family members.

For SLC25A29, expression in E. coli as inclusion bodies, purification, and reconstitution into liposomes yielded functionally active protein suitable for comprehensive transport characterization . Similar approaches may be applicable to Slc25a41.

How can contradicting research findings about Slc25a41 function be reconciled?

When faced with contradictory findings regarding Slc25a41 function, researchers should:

• Examine experimental systems:

  • Different expression systems may yield proteins with varying post-translational modifications or folding properties

  • Cell-based versus reconstitution assays may provide different results due to the presence/absence of regulatory factors

• Consider methodological differences:

  • Transport assay conditions (pH, temperature, membrane composition)

  • Detection methods and their sensitivity limits

  • Time course of measurements (initial rates versus equilibrium)

• Evaluate genetic background effects:

  • Compensatory mechanisms in knockout models

  • Strain or cell line-specific differences in metabolic dependencies

• Perform comprehensive validation studies:

  • Use multiple complementary approaches to confirm substrate specificity

  • Validate in vitro findings with in vivo models

  • Apply CRISPR-based methods to confirm specificity of observed phenotypes