Recombinant Neurospora crassa Glutamyl-tRNA (Gln) amidotransferase subunit B, mitochondrial (NCU09025)

What is the function of Glutamyl-tRNA(Gln) amidotransferase in Neurospora crassa?

Glutamyl-tRNA(Gln) amidotransferase in Neurospora crassa functions as part of the indirect pathway for aminoacylation of tRNA^Gln, particularly in mitochondria. This enzyme plays a critical role in the transamidation pathway, where it converts mischarged Glu-tRNA^Gln to the correctly charged Gln-tRNA^Gln. The enzyme participates in a two-step process: first, a non-discriminating glutamyl-tRNA synthetase mischarges tRNA^Gln with glutamate, then glutamyl-tRNA(Gln) amidotransferase converts this intermediate to the correctly charged Gln-tRNA^Gln by amidating the glutamate . Evidence for the participation of this pathway in the utilization of glutamine in Neurospora crassa has been experimentally obtained through various biochemical approaches . The NCU09025 gene specifically encodes the B subunit of this enzyme complex, which is localized to the mitochondria and contributes to mitochondrial protein synthesis .

How does the indirect aminoacylation pathway differ from direct aminoacylation?

The indirect aminoacylation pathway represents an evolutionarily distinct mechanism for tRNA charging compared to direct aminoacylation. In direct aminoacylation, a specific glutaminyl-tRNA synthetase (GlnRS) directly attaches glutamine to tRNA^Gln in a single step—a method primarily used in the cytoplasm of most eukaryotes . In contrast, the indirect pathway involves two sequential steps: first, a non-discriminating glutamyl-tRNA synthetase attaches glutamate to tRNA^Gln, creating mischarged Glu-tRNA^Gln; second, glutamyl-tRNA(Gln) amidotransferase converts this intermediate to the correctly charged Gln-tRNA^Gln through amidation .

This indirect pathway is particularly significant in organelles like mitochondria and chloroplasts, reflecting their bacterial evolutionary origins . In fact, most bacteria and archaea use this indirect route for Gln-tRNA synthesis rather than employing dedicated GlnRS enzymes. Evidence from studies in Saccharomyces cerevisiae has shown that yeast mitochondria, unlike what might be expected from their bacterial ancestry, may actually import components of the cytosolic pathway for glutaminyl-tRNA synthesis rather than relying exclusively on the transamidation pathway .

What are the recommended methods for expressing and purifying recombinant NCU09025?

Expressing and purifying functional recombinant NCU09025 requires careful optimization of several experimental parameters. The following methodological approach is recommended:

Expression System Selection:

• Bacterial systems: E. coli BL21(DE3) or Rosetta strains can accommodate codon bias issues common with fungal proteins

• Yeast systems: Consider S. cerevisiae or P. pastoris for expression of eukaryotic proteins with proper folding

• Insect cell systems: Baculovirus expression provides excellent folding environment for complex eukaryotic proteins

Construct Design:

• Remove the mitochondrial targeting sequence to improve expression

• Test multiple fusion tags (6xHis, GST, MBP, SUMO) to enhance solubility

• Consider co-expression with other subunits of the complex for stability

• Include TEV or PreScission protease sites for tag removal

Expression Optimization:

• Test induction at reduced temperatures (16-20°C) to improve folding

• Optimize inducer concentration and induction time

• Consider autoinduction media for gradual protein expression

• Include osmolytes or chaperone co-expression to enhance solubility

Purification Strategy:

• Implement a multi-step purification protocol:

  • Affinity chromatography (IMAC, GST, etc.)

  • Ion exchange chromatography

  • Size exclusion chromatography for final polishing

• Include reducing agents (DTT, β-mercaptoethanol) in buffers

• Add glycerol (5-10%) to prevent aggregation

• Consider including glutamine or substrate analogs to stabilize the protein

Quality Assessment:

• Verify protein identity by mass spectrometry

• Check homogeneity by dynamic light scattering

• Assess proper folding using circular dichroism

• Confirm activity using appropriate enzymatic assays

If expression yields inclusion bodies, consider on-column refolding strategies or solubilization with mild detergents rather than harsh denaturants. For structural studies, thermal shift assays can help identify optimal buffer conditions that maximize protein stability.

How can researchers design assays to measure NCU09025 enzymatic activity?

Designing robust assays to measure the enzymatic activity of NCU09025 requires careful consideration of substrate preparation, reaction conditions, and detection methods:

Substrate Preparation:

• Prepare mischarged Glu-tRNA^Gln using:

  • Recombinant non-discriminating GluRS

  • In vitro transcribed tRNA^Gln

  • ATP and glutamate

• Purify the mischarged tRNA via ethanol precipitation or HPLC

• Verify aminoacylation status using acid urea gel electrophoresis

Reaction Setup:

• Buffer components:

  • HEPES or Tris-HCl (pH 7.2-7.8)

  • Mg²⁺ (2-5 mM)

  • KCl (50-100 mM)

  • ATP (1-5 mM)

  • Glutamine (1-10 mM)

  • DTT (1-2 mM)

• Temperature: 25-30°C (typical for Neurospora proteins)

• Time: Establish a time course to determine linear reaction range

Detection Methods:

• Thin-layer chromatography (TLC):

  • Release amino acids from tRNA by alkaline hydrolysis

  • Separate glutamate from glutamine on TLC plates

  • Visualize using ninhydrin or autoradiography (if using ¹⁴C-labeled amino acids)

• HPLC analysis:

  • Analyze amino acid content after tRNA hydrolysis

  • Compare against glutamate and glutamine standards

• Mass spectrometry:

  • Analyze intact charged tRNAs by LC-MS

  • Determine mass shift indicative of Glu→Gln conversion

• Coupled enzyme assays:

  • Monitor ADP production using pyruvate kinase and lactate dehydrogenase

  • Follow NADH oxidation spectrophotometrically

Kinetic Analysis:

• Vary substrate concentrations to determine K_m and V_max

• Test specificity using different tRNA isoacceptors

• Examine pH dependence to identify optimal conditions

• Assess the effects of divalent cations and ionic strength

To properly characterize the full enzyme complex, it's crucial to co-express and purify all subunits (A, B, and C) since the B subunit alone may show limited or no activity. Including positive and negative controls in all assays is essential for proper interpretation of results.

What approaches can be used to localize NCU09025 within mitochondria?

Confirming the mitochondrial localization and precise submitochondrial distribution of NCU09025 requires a multi-faceted approach:

In vivo fluorescent protein fusions:

• Generate C-terminal or N-terminal GFP fusions with NCU09025

• Transform constructs into N. crassa using standard protocols

• Co-stain with mitochondrial markers (MitoTracker, TOM20 antibodies)

• Visualize using confocal microscopy with appropriate controls

Immunolocalization techniques:

• Generate specific antibodies against NCU09025 or use epitope tags

• Perform immunofluorescence microscopy on fixed cells

• For higher resolution, use immunogold electron microscopy

• Counter-stain with established mitochondrial markers

Subcellular fractionation:

• Isolate mitochondria using differential centrifugation

• Further fractionate mitochondria into:

  • Outer membrane

  • Intermembrane space

  • Inner membrane

  • Matrix

• Analyze fractions by Western blotting using anti-NCU09025 antibodies

• Include markers for each compartment (e.g., TOM20 for outer membrane, cytochrome c for intermembrane space)

Protease protection assays:

• Treat isolated mitochondria with proteases (trypsin, proteinase K)

• Compare protection in intact mitochondria versus detergent-permeabilized organelles

• Analyze by Western blotting to determine accessibility to proteases

Import assays:

• In vitro translate radiolabeled NCU09025

• Incubate with isolated mitochondria under appropriate conditions

• Analyze import by SDS-PAGE and autoradiography

• Test dependence on membrane potential and import machinery

Proximity labeling approaches:

• Fuse NCU09025 with proximity labeling enzymes (APEX2, BioID)

• Express in N. crassa and activate labeling

• Identify labeled proteins by mass spectrometry

• Map the proximal interactome to infer submitochondrial localization

Each approach has specific advantages and limitations, so combining multiple methods provides the most reliable determination of NCU09025's precise mitochondrial localization. Confirming localization is essential for understanding the protein's function in the context of mitochondrial translation and glutamine incorporation.

How can researchers investigate the role of NCU09025 in mitochondrial translation?

Investigating the role of NCU09025 in mitochondrial translation requires a comprehensive experimental approach:

Genetic Manipulation Strategies:

• Generate NCU09025 knockout strains using CRISPR-Cas9 or homologous recombination

• Create conditional knockdown systems if complete deletion is lethal

• Develop point mutations targeting key functional residues

• Generate reporter strains expressing mitochondrially-targeted fluorescent proteins

Mitochondrial Translation Analysis:

• In vivo labeling:

  • Pulse cells with ³⁵S-methionine in the presence of cycloheximide (to inhibit cytosolic translation)

  • Chase with cold methionine to follow protein turnover

  • Analyze mitochondrial translation products by SDS-PAGE and autoradiography

  • Quantify differences between wild-type and NCU09025 mutant strains

• Ribosome profiling:

  • Isolate mitochondrial ribosomes and extract ribosome-protected fragments

  • Prepare libraries for next-generation sequencing

  • Analyze ribosome occupancy across mitochondrial transcripts

  • Identify specific translational defects at glutamine codons

• Polysome analysis:

  • Fractionate mitochondrial extracts on sucrose gradients

  • Analyze distribution of mitochondrial mRNAs in polysome fractions

  • Compare polysome profiles between wild-type and mutant strains

Functional Assessment:

• Measure oxygen consumption rates in intact cells and isolated mitochondria

• Analyze activities of respiratory chain complexes containing mitochondrially-encoded subunits

• Assess mitochondrial membrane potential using fluorescent indicators (TMRM, JC-1)

• Quantify ATP production in wild-type versus mutant strains

tRNA Analysis:

• Northern blotting to detect mitochondrial tRNA^Gln levels

• Acid urea gel electrophoresis to assess aminoacylation status

• Mass spectrometry to directly analyze amino acids attached to tRNAs

• In vitro charging assays to examine tRNA^Gln aminoacylation

This methodological framework allows researchers to comprehensively assess how disruption of NCU09025 affects mitochondrial translation, with particular focus on glutamine incorporation and its downstream effects on mitochondrial function. Correlating molecular defects with physiological outcomes provides important insights into the biological significance of this enzyme.

What approaches can be used to identify the interactome of NCU09025?

Identifying the interactome of NCU09025 requires multiple complementary approaches to capture both stable and transient protein-protein interactions:

Affinity Purification-Mass Spectrometry (AP-MS):

• Generate strains expressing tagged NCU09025 (e.g., FLAG, HA, His)

• Optimize extraction conditions to preserve native interactions

• Perform affinity purification under various stringency conditions

• Identify co-purifying proteins by mass spectrometry

• Use SILAC or TMT labeling for quantitative comparison

• Apply appropriate statistical analysis to distinguish true interactors from background

Proximity-based Labeling:

• Fuse NCU09025 with BioID or APEX2 proximity labeling enzymes

• Express in N. crassa and activate labeling (biotin incubation for BioID, H₂O₂ for APEX2)

• Purify biotinylated proteins using streptavidin beads

• Identify labeled proteins by mass spectrometry

• Create spatial interaction maps based on labeling patterns

Crosslinking Mass Spectrometry (XL-MS):

• Apply chemical crosslinkers to intact mitochondria or purified complexes

• Digest crosslinked proteins and enrich crosslinked peptides

• Analyze by LC-MS/MS with specialized search algorithms

• Identify interaction interfaces at amino acid resolution

• Integrate with structural modeling approaches

Yeast Two-Hybrid Screens:

• Use NCU09025 as bait against N. crassa cDNA library

• Focus on mitochondrial proteins to reduce false positives

• Validate interactions using complementary methods

• Consider membrane yeast two-hybrid for membrane-associated interactions

Co-immunoprecipitation Validation:

• Generate antibodies against predicted interaction partners

• Perform reciprocal co-immunoprecipitation experiments

• Test interaction dependency on different conditions (ATP, substrate presence)

• Examine the effect of mutations on interaction patterns

Functional Validation:

• Assess genetic interactions through double-mutant analysis

• Test enzymatic activities of reconstituted complexes

• Examine localization dependencies using fluorescence microscopy

• Analyze the effect of partner depletion on NCU09025 stability and function

Combining these approaches provides a comprehensive view of NCU09025's interaction network, including both the core amidotransferase complex components and potentially novel interaction partners involved in regulation or assembly.

How can structure-function relationships of NCU09025 be investigated?

Investigating structure-function relationships of NCU09025 requires integrating structural biology approaches with functional assays:

Homology Modeling and Structural Prediction:

• Generate homology models based on crystal structures of related bacterial amidotransferases

• Use advanced prediction tools like AlphaFold2 for more accurate modeling

• Identify conserved catalytic residues, substrate binding sites, and subunit interfaces

• Predict the impact of mutations using computational tools (PROVEAN, PolyPhen-2)

Site-Directed Mutagenesis Strategy:

• Design mutations targeting:

  • Predicted catalytic residues

  • Substrate binding sites

  • Subunit interaction interfaces

  • Conserved motifs identified by sequence alignment

• Generate a panel of single-residue mutants using PCR-based methods

• Express and purify wild-type and mutant proteins under identical conditions

• Assess effects on activity, complex formation, and stability

Structural Analysis:

Functional Characterization:

• Develop quantitative activity assays for wild-type and mutant proteins

• Determine enzyme kinetics (K_m, k_cat, substrate specificity)

• Assess thermal stability using differential scanning fluorimetry

• Examine oligomeric state by size exclusion chromatography and analytical ultracentrifugation

Domain Analysis:

• Create truncation constructs to identify minimal functional domains

• Generate chimeric proteins by swapping domains with orthologs

• Test the function of isolated domains

• Examine domain interactions using FRET-based approaches

In vivo Validation:

• Introduce mutations into genomic NCU09025 using CRISPR-Cas9

• Assess phenotypic effects of mutations

• Perform complementation tests with mutant constructs

• Correlate biochemical defects with physiological outcomes

This systematic approach allows researchers to establish clear relationships between specific structural features of NCU09025 and their functional importance. The integration of in vitro biochemical data with in vivo phenotypic analysis provides a comprehensive understanding of how structure dictates function in this important enzyme.

What are common challenges in working with recombinant NCU09025 and how can they be overcome?

Researchers working with recombinant NCU09025 typically encounter several challenges that can be addressed through specific technical approaches:

Challenge 1: Poor expression in heterologous systems

• Solution: Optimize codon usage for the expression host to improve translation efficiency

• Solution: Try different expression vectors with various promoter strengths

• Solution: Test multiple fusion tags (MBP, GST, SUMO) to enhance solubility

• Solution: Lower induction temperature (16-20°C) and reduce inducer concentration

• Solution: Consider specialized expression strains designed for toxic or difficult proteins

Challenge 2: Protein aggregation and inclusion body formation

• Solution: Co-express with molecular chaperones (GroEL/ES, DnaK/J)

• Solution: Add solubilizing agents to growth media (sorbitol, glycine betaine)

• Solution: Include stabilizing additives in lysis buffer (glycerol, arginine, glutamine)

• Solution: Use mild detergents (CHAPS, n-Dodecyl β-D-maltoside) in extraction buffers

• Solution: If inclusion bodies form, develop gentle solubilization and refolding protocols

Challenge 3: Enzyme instability during purification

• Solution: Maintain constant cold temperature throughout purification

• Solution: Include protease inhibitor cocktails to prevent degradation

• Solution: Add reducing agents (DTT, TCEP) to prevent oxidation of cysteine residues

• Solution: Minimize purification time by optimizing each step

• Solution: Consider stabilizing ligands or substrates in purification buffers

Challenge 4: Lack of enzymatic activity in purified protein

• Solution: Co-express with other subunits of the amidotransferase complex

• Solution: Ensure proper assembly of the multi-subunit complex

• Solution: Verify protein folding using circular dichroism spectroscopy

• Solution: Test various buffer conditions for activity assays

• Solution: Confirm the presence of necessary cofactors and substrates

Challenge 5: Difficulties in substrate preparation

• Solution: Optimize in vitro transcription of tRNA^Gln

• Solution: Ensure proper folding of tRNA by denaturation and controlled refolding

• Solution: Develop efficient methods for preparation of mischarged Glu-tRNA^Gln

• Solution: Verify aminoacylation status using acid gel electrophoresis

• Solution: Consider chemical aminoacylation methods as alternatives

Challenge 6: Crystallization difficulties for structural studies

• Solution: Screen extensive crystallization conditions (>1000 conditions)

• Solution: Try surface entropy reduction mutations to enhance crystallizability

• Solution: Consider crystallization with antibody fragments or nanobodies

• Solution: Test co-crystallization with substrates, inhibitors, or stabilizing ligands

• Solution: Explore alternative structural approaches (cryo-EM, SAXS) if crystallization fails

Implementing these solutions systematically can significantly improve success rates when working with recombinant NCU09025, allowing researchers to overcome the inherent challenges associated with this complex mitochondrial enzyme.

How can researchers differentiate between direct and indirect effects when studying NCU09025 mutations?

Differentiating between direct and indirect effects of NCU09025 mutations requires sophisticated experimental design and careful data interpretation:

Rescue Experiments:

• Complement NCU09025 mutants with wild-type gene expression

• Test whether phenotypes can be rescued by exogenous enzyme expression

• Examine whether restoration of enzymatic activity correlates with phenotypic rescue

• Use domain-specific complementation to identify critical functional regions

Temporal Analysis:

• Employ time-course experiments to establish cause-effect relationships

• Use inducible or repressible systems to control NCU09025 expression

• Monitor primary biochemical changes versus secondary adaptations

• Identify the earliest detectable alterations following NCU09025 disruption

Biochemical Parsing:

• Directly measure glutamyl-tRNA(Gln) amidotransferase activity in vitro

• Quantify levels of mischarged Glu-tRNA^Gln versus correctly charged Gln-tRNA^Gln

• Analyze aminoacylation status of specific tRNAs

• Compare these direct biochemical measurements with downstream phenotypes

Targeted Interventions:

• Supplement growth media with glutamine to bypass potential shortages

• Express alternative aminoacylation pathways (e.g., cytosolic GlnRS) in mitochondria

• Target specific downstream pathways with selective inhibitors

• Assess which phenotypes can be modified by these interventions

Genetic Interaction Analysis:

• Perform epistasis analysis with mutations in related pathways

• Create double mutants to test genetic interactions

• Use synthetic genetic array approaches for genome-wide interaction mapping

• Identify suppressor mutations that restore function in NCU09025 mutants

Multi-omics Approach:

• Integrate data from:

  • Transcriptomics (RNA-seq)

  • Proteomics (quantitative mass spectrometry)

  • Metabolomics (focusing on glutamine-related metabolites)

  • Ribosome profiling (for translation effects)

• Apply network analysis to distinguish primary from secondary effects

• Use mathematical modeling to predict causal relationships

Subcellular Specificity:

• Compare effects in mitochondria versus other compartments

• Analyze compartment-specific processes (e.g., mitochondrial versus cytosolic translation)

• Examine organelle-specific stress responses

• Determine whether effects propagate from mitochondria to other cellular compartments

By systematically applying these approaches, researchers can establish clear causal relationships between NCU09025 mutations, their immediate biochemical consequences, and broader physiological effects. This comprehensive strategy helps distinguish genuine functional roles from secondary adaptations or compensatory responses.

How might alternative open reading frames impact our understanding of mitochondrial genes like NCU09025?

Recent discoveries about alternative open reading frames (altORFs) in mitochondrial genes could significantly impact our understanding of genes like NCU09025, opening new research directions:

Potential for Hidden Coding Capacity:
Recent research has identified unexpected coding potential within mitochondrial genomes through alternative reading frames. For example, MTALTCO1, a 259 amino-acid protein encoded by an alternative open reading frame in the +3 reading frame of the cytochrome oxidase 1 gene, has been experimentally confirmed . This discovery suggests that similar alternative proteins might exist in other mitochondrial genes, including potentially in genes related to NCU09025 or its substrates. These alternative proteins could have undiscovered regulatory or functional roles in mitochondrial biology.

Methodological Approaches for altORF Discovery:

• Bioinformatic screening of NCU09025 and related genes for potential altORFs

• Ribosome profiling with focus on non-canonical translation initiation sites

• Mass spectrometry-based proteomics with custom databases including predicted altORF products

• Generation of epitope-tagged constructs to detect potential alternative proteins

• CRISPR-based manipulation of altORFs without disrupting the main reading frame

Evolutionary Significance:
Research on mitochondrial altORFs has revealed interesting evolutionary patterns, with some showing "fusion-fission dynamics at the interspecies level" while maintaining full-length proteins throughout lineages . Similar analysis of NCU09025 and related genes across fungal species could reveal whether such evolutionary dynamics apply to the glutamyl-tRNA(Gln) amidotransferase system, potentially indicating selective pressures on both the canonical and alternative reading frames.

Functional Interplay:

• Investigate potential regulatory relationships between canonical proteins and products of altORFs

• Examine whether altORF products might modulate aminoacylation pathways

• Assess coexpression patterns of main ORFs and altORFs under different conditions

• Determine if altORF products interact with the canonical protein complexes

Implications for Mitochondrial Translation:
The discovery of additional coding capacity in mitochondrial genomes could have significant implications for our understanding of mitochondrial translation and its regulation. For NCU09025 specifically, researchers should consider whether alternative reading frames within this gene or its substrates might produce proteins that influence the aminoacylation process or mitochondrial translation more broadly.

Technical Considerations:

• Develop specific antibodies against predicted altORF products

• Create reporter constructs to monitor altORF translation in vivo

• Employ ribosome profiling with drugs that stall ribosomes at initiation codons

• Utilize nanopore direct RNA sequencing to identify potential alternative transcripts

This emerging area represents a significant shift in our understanding of mitochondrial gene expression and could reveal previously unrecognized layers of regulation and function in the mitochondrial translational machinery, including potential impacts on NCU09025 function and regulation.

How can comparative genomics inform our understanding of NCU09025 evolution and function?

Comparative genomics provides powerful insights into the evolution and function of NCU09025, revealing conservation patterns that highlight functional constraints and adaptations across species:

Evolutionary Trajectory Analysis:

• Conduct phylogenetic analysis of glutamyl-tRNA(Gln) amidotransferase B subunits across:

  • Different fungal lineages

  • Other eukaryotic groups

  • Bacterial and archaeal domains

• Identify key evolutionary transitions in the aminoacylation pathway

• Map the presence/absence of direct versus indirect glutaminyl-tRNA synthesis pathways

• Correlate evolutionary patterns with mitochondrial genome features

Sequence Conservation Mapping:

• Align NCU09025 orthologs to identify universally conserved residues

• Map conservation scores onto structural models to reveal functional hotspots

• Distinguish between conservation in catalytic domains versus structural regions

• Identify lineage-specific insertions or deletions that might confer specialized functions

Coevolution with Partner Proteins:

• Examine coordinated evolution between NCU09025 and other amidotransferase subunits

• Analyze coevolution with tRNA^Gln genes and aminoacyl-tRNA synthetases

• Identify potential compensatory mutations between interacting partners

• Apply statistical coupling analysis to detect coevolving residue networks

Patterns of Selection:

• Calculate dN/dS ratios to identify regions under purifying or positive selection

• Perform branch-site tests to detect episodic selection in specific lineages

• Correlate selection patterns with functional domains and protein interfaces

• Examine selection pressure differences between fungal lineages with different ecological niches

Horizontal Gene Transfer Assessment:

• Investigate potential horizontal gene transfer events involving aminoacylation pathways

• Compare bacterial-like features of fungal glutamyl-tRNA(Gln) amidotransferases

• Assess whether mitochondrial targeting represents recent or ancient adaptation

• Evaluate the role of endosymbiotic gene transfer in shaping current NCU09025 properties

Synteny and Genomic Context:

• Analyze gene neighborhood conservation around NCU09025 across fungal genomes

• Identify conserved regulatory elements in promoter regions

• Examine whether genomic location correlates with expression patterns

• Detect potential operon-like structures or co-regulated gene clusters

This comparative genomics approach provides an evolutionary framework for understanding NCU09025 function, revealing which features represent ancestral traits versus lineage-specific adaptations. Such insights can guide experimental approaches by highlighting the most functionally significant regions and suggesting how the indirect aminoacylation pathway has been adapted to specific organismal needs across evolutionary time.