Recombinant Aspergillus niger Exportin-T (los1), partial

What is Exportin-T (los1) and what is its biological function?

Exportin-T (Xpo-t) is a nuclear export receptor responsible for the translocation of tRNAs from the nucleus to the cytoplasm. It functions by binding mature tRNAs cooperatively with GTP-loaded Ran protein to form export-competent complexes. Xpo-t interacts extensively with the backbone of the TΨC and acceptor arms of tRNA molecules, recognizing primarily the three-dimensional structure rather than the sequence specificity of the tRNA . In Saccharomyces cerevisiae, the homolog Los1p serves a similar function, although it is not essential, suggesting redundant pathways for tRNA export in yeast . The primary biological function of Exportin-T/Los1 is to facilitate the selective transport of properly processed tRNAs to the cytoplasm where they participate in protein synthesis.

How does Exportin-T (los1) differ between yeast and filamentous fungi like Aspergillus niger?

While the basic function of tRNA nuclear export is conserved, there are notable differences in the exportin system between yeasts and filamentous fungi like A. niger:

• In S. cerevisiae, three distinct exporters (Los1, Mex67-Mtr2, and Crm1) have been documented to participate in tRNA nuclear export, each with different preferences for tRNA families .

• Los1 in S. cerevisiae is non-essential, unlike in some other organisms, indicating redundant export pathways .

• Filamentous fungi like A. niger have more complex cellular organization than yeast, which may affect the localization and regulation of nuclear export machinery.

• A. niger's increased capacity for protein secretion likely correlates with differences in the post-transcriptional processing machinery, potentially including tRNA export proteins .

What experimental evidence demonstrates that Los1 functions as a tRNA exporter?

Multiple experimental approaches have confirmed Los1's role as a tRNA exporter:

• Biochemical evidence: Los1/Xpo-t forms specific complexes with tRNAs and RanGTP, but not with RanGDP or other RNA classes like U1 or U6 .

• Antibody inhibition: Antibodies against Xpo-t, when injected into Xenopus oocyte nuclei, specifically inhibit export of tRNA^Phe and tRNA^i^Met .

• Genetic evidence: LOS1 was discovered in genetic screens for mutations affecting tRNA processing and for mutations producing synthetic lethality with defective nucleoporins .

• In vivo co-purification: Los1 forms in vivo nuclear export complexes with intron-containing tRNAs as demonstrated by immunoprecipitation followed by RNA analysis .

• Functional complementation: The vertebrate Xpo-t can functionally replace Los1 in yeast systems, confirming evolutionary conservation of function .

What are the most effective expression systems for recombinant Exportin-T production in A. niger?

For optimal recombinant Exportin-T production in A. niger, several expression systems can be utilized:

• Strong constitutive promoters: The gpdA (glyceraldehyde-3-phosphate dehydrogenase) promoter from Aspergillus nidulans provides strong, constitutive expression.

• Inducible promoters: For controlled expression, the glucoamylase (glaA) promoter can be used, which is induced by maltose or starch and repressed by glucose.

• Signal peptides: For secretion, the native glucoamylase signal sequence facilitates efficient protein transport through the secretory pathway.

• Selection markers: The pyrG gene (encoding orotidine-5'-phosphate decarboxylase) allows for selection of transformants on uridine/uracil-deficient media.

When selecting an expression system, consider the following methodology:

• Generate multiple transformants and screen for high expressors, as expression levels can vary widely between transformants.

• Verify genetic stability through single conidium isolation and analysis of protein production in subsequent generations .

• Optimize culture conditions (pH, temperature, media composition) to minimize proteolysis of the recombinant protein .

How can researchers optimize media conditions for recombinant Exportin-T expression in A. niger?

Optimization of media conditions is crucial for maximum yield and stability of recombinant Exportin-T in A. niger:

• Carbon source selection: Complex carbon sources like dairy whey can be used, with 10% whey providing good results for recombinant protein production .

• Nitrogen supplementation: Addition of 0.6% NaNO₃ to whey-based media can significantly enhance recombinant protein expression (demonstrated to increase activity up to 46 U/mL for other recombinant proteins) .

• pH control: Maintain culture pH between 4.5-6.0 to balance optimal growth with protein stability. For recombinant protein production, pH adjustment can dramatically affect stability regardless of media complexity .

• Protease inhibition strategy:

  • Include casamino acids (0.5-1.0%) to act as preferential substrates for proteases

  • Add PMSF (phenylmethylsulfonyl fluoride) at 1mM in late cultivation phases

  • Consider using protease-deficient A. niger strains like D15 for improved recombinant protein stability

• Culture mode: Fed-batch cultivation with controlled carbon source feeding can prevent metabolic overflow and improve protein yields.

What purification strategies are most suitable for recombinant A. niger Exportin-T?

Purification of recombinant Exportin-T from A. niger can be achieved through a sequential approach:

• Initial capture:

  • If secreted: Ammonium sulfate precipitation (40-60% saturation) followed by dialysis

  • If intracellular: Cell disruption using glass beads or mechanical homogenization in buffer containing 20mM HEPES (pH 7.5), 150mM NaCl, 10% glycerol, and protease inhibitors

• Intermediate purification:

  • Ion exchange chromatography (IEX): Use SP-Sepharose for cation exchange or Q-Sepharose for anion exchange depending on the protein's pI

  • Hydrophobic interaction chromatography (HIC): Particularly useful after ammonium sulfate precipitation

• Affinity purification:

  • If tagged: Ni-NTA for His-tagged proteins, glutathione-Sepharose for GST-tagged proteins

  • Untagged: Consider using RanGTP-coupled resins to exploit the natural affinity of Exportin-T for RanGTP

• Polishing step:

  • Size exclusion chromatography to remove aggregates and achieve high purity

For quality control, assess:

• Purity by SDS-PAGE (target >95%)

• Identity by Western blotting with anti-Exportin-T antibodies

• Functional activity through tRNA binding assays in the presence of RanGTP

How do mutations in the Exportin-T binding site affect tRNA export efficiency and specificity?

The impact of mutations in Exportin-T binding sites on tRNA export has revealed several critical insights:

Research methodology for studying binding site mutations:

• Generate tRNA variants with specific mutations using in vitro transcription

• Assess binding affinity using gel mobility shift assays with recombinant Exportin-T and RanGTP

• Monitor export efficiency through microinjection experiments in Xenopus oocytes

• Perform chemical and enzymatic footprinting to map interaction sites

What is the mechanism of tRNA selectivity among different nuclear exporters (Los1, Mex67-Mtr2, and Crm1) in fungi?

The three known tRNA nuclear exporters in S. cerevisiae (Los1, Mex67-Mtr2, and Crm1) demonstrate distinct tRNA preferences and quality control mechanisms:

• Family-specific preferences:

  • Los1 can export all 10 families of intron-containing pre-tRNAs but shows lower efficiency for tRNA^Phe^GAA and tRNA^Ser^CGA .

  • Mex67-Mtr2 shows preference for tRNA^Ile^UAU, tRNA^Pro^UGG, tRNA^Trp^CCA and tRNA^Tyr^GUA .

  • Crm1 has been confirmed as a bona fide tRNA nuclear exporter with its own set of tRNA preferences, though less thoroughly characterized .

• Quality control differences:

  • Los1 and Crm1 maintain high fidelity, preferentially exporting tRNAs with mature 5′ termini .

  • Mex67-Mtr2 is more error-prone, capable of binding and exporting pre-tRNAs with unprocessed 5′ leaders .

• Error correction mechanism:

  • tRNA retrograde nuclear import serves as a quality control mechanism for aberrantly exported tRNAs, returning them to the nucleus where 3′ to 5′ exonucleases facilitate their degradation .

Methodological approach for studying exporter selectivity:

• In vivo co-purification of tRNAs with endogenously expressed nuclear exporters

• Northern blot analysis to detect specific tRNA families

• Mutational analysis of exporters to identify tRNA recognition domains

• Competitive binding assays to determine relative affinities for different tRNAs

How does the intron status of tRNAs affect their interaction with Exportin-T in A. niger?

The relationship between tRNA intron status and Exportin-T interaction reveals a complex interplay between processing and export:

• Binding of intron-containing tRNAs: Intron-containing, end-processed pre-tRNAs can bind to Xpo-t–RanGTP, particularly when Xpo-t is present in excess .

• Normal export hierarchy:

  • Mature, intron-removed tRNAs are the preferred substrate for Exportin-T

  • End-processed, intron-containing pre-tRNAs have intermediate binding affinity

  • Unprocessed pre-tRNAs with 5' leaders or 3' trailers show minimal binding

• Correlation between binding and export: For most tRNAs tested, those that bound tightly to Xpo-t–RanGTP in vitro were efficiently exported from the nucleus in vivo, with intron-containing pre-tRNA being the notable exception .

• Proofreading mechanism: The retention of intron-containing pre-tRNAs in the nucleus despite their ability to bind Exportin-T suggests additional proofreading mechanisms beyond simple binding affinity .

Experimental approaches to study intron effects:

• Compare export rates of intron-containing and intronless tRNA variants

• Analyze the structural consequences of introns on the tRNA three-dimensional architecture

• Perform competition assays between intron-containing and mature tRNAs for Exportin-T binding

• Use in vivo RNA labeling and subcellular fractionation to track tRNA movement

What are the primary causes of genetic instability in recombinant A. niger strains expressing Exportin-T?

Genetic instability represents a significant challenge in maintaining consistent Exportin-T expression in recombinant A. niger strains. Key factors contributing to this instability include:

• Heterokaryosis: The presence of multiple genetically different nuclei within the mycelium can lead to segregation of the recombinant and wild-type nuclei during subculturing .

• Genomic modifications: Several types of alterations can occur:

  • Excision or modification of the recombinant DNA

  • Point mutations affecting promoter or coding regions

  • Chromosomal rearrangements altering gene expression

• Selection pressure: Absence of continuous selection pressure may lead to loss of expression cassettes, particularly if the recombinant protein imposes metabolic burden.

• Proteolytic degradation: Internal or external proteolysis can reduce apparent production levels, making it difficult to distinguish from genetic instability .

This genetic instability is demonstrated in single conidium isolates derived from transformants, which can show dramatic variations in recombinant protein production. For example, single conidium isolates of A. niger producing recombinant hen egg white lysozyme showed highly variable expression levels regardless of culture conditions .

Methodology to assess and mitigate genetic instability:

• Generate single conidium isolates from transformants and compare protein production levels

• Regularly verify the presence and integrity of the expression cassette by PCR

• Maintain selective pressure throughout cultivation

• Identify and preserve high-producing isolates at low temperatures (-80°C)

How can researchers differentiate between true Los1 homologs and related exportins in fungal genomic studies?

Distinguishing true Los1 homologs from related exportins in fungal genomics requires a multi-faceted approach:

• Sequence-based identification:

  • Primary sequence identity: True Los1 homologs typically share >30% amino acid identity with the S. cerevisiae Los1 protein

  • Conserved domain architecture: Look for the characteristic importin-β fold consisting of HEAT repeats

  • Motif analysis: Los1 contains specific Ran-binding domains and cargo-binding regions

• Phylogenetic analysis:

  • Construct phylogenetic trees including known exportins from model organisms

  • True Los1 homologs will cluster together separate from other exportin family members like Crm1, Cse1, etc.

• Functional verification:

  • Test for interaction with RanGTP using pull-down assays

  • Assess ability to bind tRNAs in the presence of RanGTP

  • Perform complementation assays in Los1-deficient yeast strains

• Structural prediction:

  • Use homology modeling and structural prediction tools to verify the characteristic solenoid structure

  • Compare predicted structures with solved structures of known exportins

Notably, Los1 belongs to the importin-β family but shares limited sequence similarity with other members, making structural and functional characteristics more reliable for identification than sequence alone .

What approaches can mitigate proteolysis of recombinant Exportin-T in A. niger expression systems?

Proteolysis represents a significant challenge in recombinant protein production using A. niger. Several strategies can be employed to minimize proteolytic degradation of Exportin-T:

• Strain engineering:

  • Use protease-deficient strains (e.g., A. niger D15)

  • Further modify strains through deletion of specific protease genes (e.g., pepA, pepB, pepD)

• Media optimization:

  • Control pH to reduce activity of acid proteases (maintain above pH 5.0)

  • Add casein or casamino acids as competitive substrates for proteases

  • Supplement media with defined carbon sources rather than complex ingredients

• Cultivation strategies:

  • Harvest at optimal time points before extensive proteolysis occurs

  • Consider reduced temperature cultivation (25-28°C instead of 30°C)

  • Implement fed-batch strategies to maintain controlled growth

• Protein engineering:

  • Identify and modify protease-sensitive sites in Exportin-T

  • Add stabilizing domains or fusion partners

  • Consider intracellular retention rather than secretion if appropriate

• Downstream processing:

  • Add protease inhibitors (PMSF, EDTA, pepstatin A) immediately upon harvesting

  • Perform rapid initial capture steps to separate the target protein from proteases

  • Maintain low temperature throughout processing

Effectiveness of these approaches can be monitored through pulse-chase experiments and proteome analysis to identify specific proteases responsible for degradation.

How can structural studies of recombinant Exportin-T inform drug design targeting nuclear transport in pathogenic fungi?

Structural studies of recombinant Exportin-T provide valuable insights for antifungal drug development targeting nuclear transport:

• Identifying unique binding pockets:

  • Structural characterization can reveal fungal-specific features in the tRNA binding domain

  • Differences in the RanGTP binding interface between human and fungal Exportin-T

  • Species-specific regions that could be targeted with minimal cross-reactivity

• Structure-based drug design approaches:

  • Virtual screening against identified binding pockets

  • Fragment-based drug discovery focusing on key interaction sites

  • Rational design of competitive inhibitors that mimic tRNA or RanGTP binding

• Allosteric inhibition opportunities:

  • Identification of allosteric sites that can modulate Exportin-T function

  • Design of molecules that lock the protein in inactive conformations

• Potential drug targets in the export pathway:

  • Exportin-T/Los1 itself as a direct target

  • The Exportin-T-RanGTP interface

  • The Exportin-T-tRNA interaction surface

  • The interaction between Exportin-T and nucleoporins

Methodology for structural studies:

• X-ray crystallography of Exportin-T alone and in complex with RanGTP and/or tRNA

• Cryo-electron microscopy for larger complexes

• NMR spectroscopy for dynamic interaction studies

• Hydrogen-deuterium exchange mass spectrometry to map binding interfaces

What are the implications of redundant tRNA export pathways for genetic manipulation of A. niger?

The presence of redundant tRNA export pathways has significant implications for genetic engineering and industrial applications of A. niger:

• Genetic engineering considerations:

  • Single knockouts of export factors may show minimal phenotypic effects due to redundancy

  • Multiple gene deletions may be required to significantly impair tRNA export

  • The specific preferences of each exporter for different tRNA families must be considered when targeting specific cellular processes

• Strain development strategies:

  • Partial inhibition of tRNA export might create strains with altered translation rates beneficial for certain recombinant proteins

  • Overexpression of specific exporters could potentially enhance expression of certain recombinant proteins by increasing availability of specific tRNAs in the cytoplasm

  • Fine-tuning tRNA export could optimize codon usage for heterologous gene expression

• Metabolic engineering applications:

  • Manipulating tRNA availability through export modulation could affect translation efficiency of enzymes with rare codons

  • Engineering strain-specific tRNA export regulation might create conditional expression systems

• Synthetic biology potential:

  • The redundant export system provides multiple intervention points for synthetic regulatory circuits

  • tRNA export could potentially be engineered as a sensing mechanism for metabolic states

Methodological approach:

• Generate single and combinatorial knockouts of Los1, Mex67-Mtr2, and Crm1 homologs

• Analyze effects on growth, protein production, and stress response

• Perform transcriptomics and proteomics to assess global impacts

• Measure tRNA subcellular distribution using fluorescence in situ hybridization

How might comparative analysis of Exportin-T across fungal species inform evolutionary understanding of nuclear transport?

Comparative analysis of Exportin-T/Los1 across fungal species offers valuable insights into the evolution of nuclear transport systems:

• Evolutionary conservation and divergence:

  • Core functional domains show high conservation, reflecting essential transport functions

  • Species-specific variations may reveal adaptations to different cellular environments and metabolic needs

  • Comparison between yeast (S. cerevisiae) and filamentous fungi (A. niger) can highlight adaptations related to morphological differences

• Functional redundancy patterns:

  • The essentiality of Los1 varies between species, with S. cerevisiae able to survive without it

  • Comparative genomics can reveal if redundant exporters evolved through gene duplication or repurposing of existing transport factors

  • Analysis may indicate if redundant systems evolved as backup mechanisms or for specialized functions

• Co-evolution with tRNA processing machinery:

  • Correlation between Exportin-T structure and species-specific aspects of tRNA processing

  • Potential co-evolution with splicing machinery, particularly in species with different intron frequencies

  • Relationships between export factors and tRNA modification enzymes

• Implications for horizontal gene transfer:

  • Potential instances of horizontal gene transfer of tRNA export factors

  • Impact on species adaptation and specialization

Methodological approach:

• Phylogenetic analysis of Los1/Exportin-T sequences across fungal kingdom

• Structural prediction and comparison of binding sites

• Functional complementation experiments across species

• Correlation analysis between Los1 features and tRNA gene architecture in different genomes

What are the best assays to measure the functional activity of recombinant Exportin-T?

Assessing the functional activity of recombinant Exportin-T requires assays that reflect its biological role in tRNA nuclear export:

• In vitro binding assays:

  • Electrophoretic mobility shift assay (EMSA): Measures the formation of Exportin-T-RanGTP-tRNA complexes

  • Filter binding assay: Quantifies the interaction between radiolabeled tRNAs and Exportin-T

  • Surface plasmon resonance (SPR): Determines binding kinetics and affinity constants

  • Fluorescence anisotropy: Measures binding using fluorescently labeled tRNAs

• Nuclear export assays:

  • Microinjection of labeled tRNAs into Xenopus oocyte nuclei with subsequent measurement of nuclear/cytoplasmic distribution

  • Permeabilized cell assay: Uses digitonin-permeabilized cells to measure transport of labeled tRNAs

  • Cell-based reporter systems: Utilizing split fluorescent or luminescent proteins fused to nuclear export-dependent constructs

• Protection assays:

  • Chemical and enzymatic footprinting to map interaction sites between Exportin-T and tRNAs

  • Phosphate modification interference to identify critical contacts within the tRNA structure

  • Hydrogen-deuterium exchange mass spectrometry to map binding interfaces

• Competition assays:

  • Testing substrate specificity by competing various tRNA species for binding

  • Assessing the effect of tRNA modifications on binding affinity

  • Determining the preference for mature versus precursor tRNAs

For quantitative assessment, prepare a standard curve with known quantities of purified active Exportin-T and normalize results to positive controls.

How do post-translational modifications affect Exportin-T function, and how can these be analyzed in recombinant systems?

Post-translational modifications (PTMs) can significantly impact Exportin-T function, and their analysis in recombinant systems requires specialized approaches:

• Common PTMs affecting Exportin-T function:

  • Phosphorylation: May regulate binding affinity to tRNAs or RanGTP

  • Ubiquitination: Can affect protein stability and turnover

  • Acetylation: May influence nuclear localization or protein-protein interactions

  • Glycosylation: Could affect secretion and stability of recombinant protein in A. niger

• Detection and characterization methods:

  • Mass spectrometry-based proteomics for comprehensive PTM mapping

  • Phospho-specific antibodies for detecting phosphorylation events

  • Pro-Q Diamond staining for phosphoprotein detection in gels

  • Periodic acid-Schiff staining for glycoprotein detection

• Functional impact assessment:

  • Site-directed mutagenesis of modified residues to mimic or prevent modifications

  • In vitro modification using purified kinases, acetyltransferases, etc.

  • Comparison of protein from different growth conditions or cellular compartments

• A. niger-specific considerations:

  • Fungal PTM patterns may differ from those in native host organisms

  • A. niger may lack certain modification enzymes present in the original host

  • The secretory pathway in A. niger may introduce non-native glycosylation patterns

Methodological approach:

• Express Exportin-T with epitope tags that don't interfere with potential modification sites

• Purify protein under conditions that preserve PTMs (phosphatase inhibitors, etc.)

• Compare PTM patterns between recombinant and native proteins

• Correlate PTM profiles with functional activity measurements

What approaches can be used to study the interaction between Exportin-T and the nuclear pore complex in fungal systems?

Understanding the interactions between Exportin-T and nuclear pore complexes (NPCs) in fungi requires specialized techniques spanning biochemical, genetic, and imaging approaches:

• Biochemical interaction studies:

  • Affinity purification followed by mass spectrometry to identify interacting nucleoporins

  • In vitro binding assays using recombinant nucleoporin fragments

  • Chemical cross-linking followed by mass spectrometry to map interaction interfaces

  • Surface plasmon resonance to measure binding kinetics to specific nucleoporins

• Genetic interaction mapping:

  • Synthetic lethality screening with nucleoporin mutants (as identified for Los1 in S. cerevisiae)

  • Suppressor screens to identify compensatory mutations

  • CRISPR-based genetic screens to identify functional interactions

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize co-localization at the nuclear envelope

  • Single-molecule tracking to follow Exportin-T movement through NPCs

  • Fluorescence recovery after photobleaching (FRAP) to measure transport kinetics

  • Förster resonance energy transfer (FRET) to detect direct interactions

• In silico approaches:

  • Molecular docking between Exportin-T and nucleoporin models

  • Molecular dynamics simulations of transport complexes

  • Integration of structural and interaction data into transport models

• Cargo-dependent interaction studies:

  • Compare interactions in the presence and absence of cargo (tRNAs)

  • Assess how RanGTP binding affects interactions with nucleoporins

  • Examine the effect of tRNA mutations on NPC passage efficiency

Methodological consideration: When studying A. niger, the cell wall can present challenges for imaging approaches, requiring optimized protocols for spheroplast preparation while maintaining nuclear envelope integrity.