Recombinant Ashbya gossypii 37S ribosomal protein S10, mitochondrial (RSM10)

What is RSM10 and what is its functional role in Ashbya gossypii mitochondria?

RSM10 (gene name AGOS_AGR035C) in Ashbya gossypii is a component of the mitochondrial ribosomal small subunit. It plays a critical role in the translation of mitochondrially encoded proteins essential for respiratory function. As a mitochondrial 37S ribosomal protein, RSM10 contributes to the assembly and stability of the small ribosomal subunit within mitochondria, ultimately affecting mitochondrial protein synthesis and cellular respiration.

To experimentally determine RSM10's function, researchers typically employ gene deletion studies followed by mitochondrial functional assays. The approach involves creating RSM10 knockout strains and measuring oxygen consumption rates, ATP production, and mitochondrial translation efficiency. Additionally, blue native PAGE analysis can be used to assess mitochondrial ribosome assembly, providing insights into how RSM10 deficiency affects ribosomal structure .

What expression systems are most effective for producing Recombinant Ashbya gossypii RSM10?

For optimal expression of Recombinant Ashbya gossypii RSM10, multiple expression systems have been tested with varying efficiency. While prokaryotic and eukaryotic expression systems can be utilized, each offers distinct advantages depending on research requirements.

For standard biochemical studies, E. coli expression systems typically provide sufficient quantity and quality of the recombinant protein. When designing expression constructs, include an appropriate affinity tag (His, GST, or MBP) for purification, and consider codon optimization for the expression host to enhance yield .

How can researchers verify the structural integrity and functionality of purified Recombinant RSM10?

Verifying the structural integrity and functionality of purified Recombinant RSM10 requires a multi-faceted approach combining biophysical and functional assays. This comprehensive validation is crucial before proceeding to complex experiments.

The methodological workflow should include:

• Primary Structure Verification: Perform mass spectrometry analysis (MALDI-TOF or ESI-MS) to confirm the correct molecular weight and sequence coverage through peptide mapping.

• Secondary Structure Assessment: Utilize circular dichroism (CD) spectroscopy to analyze protein folding patterns. Compare the CD spectrum with predicted secondary structure elements based on homology modeling.

• Tertiary Structure Evaluation: Employ thermal shift assays to determine protein stability and proper folding. Dynamic light scattering can assess homogeneity and detect aggregation.

• Functional Validation: Conduct RNA binding assays using filter binding experiments or isothermal titration calorimetry to measure the interaction between RSM10 and mitochondrial rRNA targets.

• Assembly Competence: Perform reconstitution experiments with other mitochondrial ribosomal components to verify the ability of RSM10 to incorporate into ribosomal subassemblies .

These validation steps ensure that the recombinant protein maintains native-like properties and functionality for downstream experiments.

What are the common challenges in working with mitochondrial ribosomal proteins like RSM10?

Mitochondrial ribosomal proteins present several technical challenges that researchers must address through methodological adaptations. When working with RSM10 specifically, consider the following approaches to overcome common obstacles:

Challenge 1: Protein Solubility Issues

• Implement solubility screening with different buffer compositions (varying pH, salt concentration, and additives)

• Utilize fusion partners (SUMO, MBP, TRX) to enhance solubility

• Develop refolding protocols from inclusion bodies when necessary

• Consider expression at lower temperatures (16-20°C) to promote proper folding

Challenge 2: Maintaining Structural Integrity

• Add stabilizing agents such as glycerol (5-10%), reducing agents (DTT or β-mercaptoethanol), and appropriate metal ions

• Minimize freeze-thaw cycles by preparing single-use aliquots

• Perform activity assays immediately after purification when possible

Challenge 3: Reconstitution of Functional Complexes

• Develop stepwise reconstitution protocols for assembling mitochondrial ribosomal subunits

• Use gradient ultracentrifugation to isolate intact ribosomal subcomplexes

• Validate assembly through cryo-EM or biochemical crosslinking approaches

The integration of these methodological adaptations significantly improves the success rate when working with challenging mitochondrial ribosomal proteins like RSM10 .

How can researchers investigate the relationship between RSM10 function and mitochondrial morphology in Ashbya gossypii?

Investigating the relationship between RSM10 function and mitochondrial morphology in Ashbya gossypii requires a multidisciplinary approach leveraging advanced imaging techniques and genetic manipulations. A. gossypii presents a unique experimental system due to its multinucleate nature and asynchronous nuclear division within a common cytoplasm.

A comprehensive experimental design should include:

• Conditional RSM10 Expression System: Develop a tetracycline-inducible or repressible system to modulate RSM10 expression levels, allowing temporal control over protein abundance.

• Live-Cell Mitochondrial Imaging: Implement time-lapse confocal microscopy using mitochondria-targeted fluorescent proteins (e.g., mito-GFP) to visualize morphological changes. Advanced super-resolution techniques (STED or SIM) can provide nanoscale resolution of structural alterations.

• Quantitative Morphological Analysis: Apply computational image analysis using tools like MitoGraph or MitoAnalyzer to quantify parameters including mitochondrial network connectivity, branching frequency, and organelle dimensions.

• Correlative Approach: Combine fluorescence microscopy with electron microscopy through correlative light and electron microscopy (CLEM) to bridge ultrastructural information with functional data.

• Mitochondrial Membrane Potential Assessment: Utilize potential-sensitive dyes (TMRM, JC-1) to simultaneously monitor membrane potential and morphology, providing insight into functional consequences of structural changes.

In A. gossypii, heterogeneity in mitochondrial morphology and membrane potential exists within a single multinucleated cell, but this heterogeneity appears to be independent of nuclear division states. This suggests that RSM10's role in mitochondrial function might be regulated by factors beyond the nuclear cycle .

What approaches can be used to study how RSM10 interacts with other mitochondrial ribosomal proteins in A. gossypii?

Studying RSM10 interactions with other mitochondrial ribosomal proteins in A. gossypii requires integrating structural biology approaches with functional genomics. The multinucleate nature of A. gossypii adds complexity to these analyses but also provides unique insights into organelle-specific protein interactions.

A methodological framework should include:

• Proximity-Based Protein Interaction Mapping: Implement BioID or APEX2 proximity labeling by fusing these enzymes to RSM10. This approach identifies neighboring proteins in the native cellular environment through biotinylation followed by streptavidin pulldown and mass spectrometry.

• Quantitative Interaction Proteomics: Perform SILAC or TMT-based quantitative proteomics coupled with RSM10 immunoprecipitation to identify and quantify interaction partners under different physiological conditions.

• In vivo Crosslinking Mass Spectrometry (XL-MS): Apply protein crosslinking with MS-compatible reagents (DSS, EDC) followed by digestion and MS analysis to determine precise interaction interfaces between RSM10 and other ribosomal components.

• Structural Analysis of Mitochondrial Ribosomes: Utilize cryo-electron microscopy to determine the structure of A. gossypii mitochondrial ribosomes, with specific focus on RSM10's position and interacting partners within the assembled complex.

• Functional Validation Through Genetic Approaches: Create a panel of point mutations at predicted interaction interfaces and assess their impact on ribosome assembly and translation efficiency. This can be accomplished through complementation studies in RSM10-depleted strains.

These methodological approaches provide a comprehensive view of RSM10's interaction network within the mitochondrial ribosome structure, essential for understanding mitochondrial translation mechanisms in multinucleate fungi .

How might gene expression patterns of RSM10 correlate with mitochondrial function across different growth phases of A. gossypii?

Understanding RSM10 expression patterns and their correlation with mitochondrial function throughout A. gossypii growth phases requires integration of transcriptomics, proteomics, and functional analyses. This multilayered approach provides insight into regulatory mechanisms governing mitochondrial ribosome assembly during fungal development.

An integrated experimental approach should include:

• Time-Resolved Transcriptomics: Implement RNA-seq analysis across defined growth stages (spore germination, hyphal extension, branching, and sporulation) to quantify RSM10 mRNA expression patterns. Compare expression profiles against other mitochondrial ribosomal proteins to identify co-regulated gene clusters.

• Translational Regulation Analysis: Employ ribosome profiling (Ribo-seq) to assess translational efficiency of RSM10 mRNA across growth phases, revealing potential post-transcriptional regulatory mechanisms.

• Protein Abundance Quantification: Use targeted proteomics approaches (SRM/MRM) to precisely quantify RSM10 protein levels in mitochondrial fractions isolated from different growth phases. This provides direct correlation between protein abundance and functional output.

• Mitochondrial Activity Measurement: Simultaneously assess mitochondrial function through respirometry (oxygen consumption rate), membrane potential measurements, and ATP production assays across growth phases.

• Multi-omics Data Integration: Apply computational modeling to integrate transcriptomic, proteomic, and functional data, establishing causal relationships between RSM10 expression patterns and mitochondrial functional states.

What are the optimal experimental approaches for creating and characterizing RSM10 mutants to study mitochondrial function?

Creating and characterizing RSM10 mutants in A. gossypii requires specialized approaches that account for the multinucleate nature of this filamentous fungus. The following comprehensive methodology enables systematic analysis of RSM10's role in mitochondrial function:

Mutant Generation Strategy:

• CRISPR-Cas9 Genome Editing: Design guide RNAs targeting the RSM10 gene (AGOS_AGR035C) with homology-directed repair templates containing desired mutations. For multinucleate A. gossypii, optimize transformation protocols to achieve high editing efficiency across multiple nuclei.

• Structure-Guided Mutagenesis: Based on homology modeling or available structural data, create a mutation library targeting:

  • RNA binding residues

  • Interface residues for protein-protein interactions

  • Conserved domains across fungal species

• Conditional Expression Systems: Develop tetracycline-inducible/repressible constructs to control mutant expression temporally, allowing study of acute effects versus adaptive responses.

Characterization Framework:

• Phenotypic Analysis Matrix:

• Heterokaryon Analysis: Create heterokaryons containing mixtures of wild-type and mutant nuclei to assess dominant-negative effects and protein diffusion limitations. This approach is particularly valuable in A. gossypii, where heterokaryon mutants with deletions in mitochondrial fusion/fission genes show dominant effects, suggesting limited diffusion of gene products .

• Synthetic Genetic Interactions: Combine RSM10 mutations with mutations in mitochondrial dynamics genes (e.g., DNM1, FZO1) to uncover functional relationships between mitochondrial translation and morphology regulation.

These methodological approaches provide a comprehensive framework for elucidating RSM10's role in mitochondrial function within the unique context of multinucleate A. gossypii cells .