Recombinant Aspergillus niger Eukaryotic translation initiation factor 3 subunit K (An16g02940)

What is the structural organization of eIF3k within the eIF3 complex?

Eukaryotic translation initiation factor 3 subunit K (eIF3k) is part of the structural scaffold known as the PCI/MPN octamer within the larger eIF3 complex. This octamer consists of eight eIF3 subunits (a, c, e, f, h, k, l, and m) that form an essential structural foundation for the complex. The eIF3k subunit integrates with this octamer, which is positioned near the mRNA exit site on the 40S ribosomal subunit during the translation process . When studying An16g02940 (the Aspergillus niger homolog), it's important to note that the protein functions within this complex structural arrangement, which influences its functional properties and experimental handling requirements.

What expression systems are optimal for producing Recombinant An16g02940?

Recombinant An16g02940 can be produced using multiple expression systems, each with distinct advantages depending on your experimental requirements:

All these systems typically yield protein with >85% purity as determined by SDS-PAGE. For structural studies requiring authentic post-translational modifications, yeast or mammalian systems are preferable. For biochemical assays where higher yields are prioritized, E. coli expression may be more suitable. The protein is typically obtained as a lyophilized powder regardless of expression system.

How does the eIF3 complex contribute to translation initiation in eukaryotes?

The eIF3 complex plays multiple critical roles in eukaryotic translation initiation through the following mechanisms:

• Binds to the 40S ribosomal subunit and stimulates recruitment of other initiation factors

• Facilitates the formation of the 43S pre-initiation complex by promoting binding of the eIF2-GTP-Met-tRNAiMet ternary complex

• Prevents premature attachment of 60S ribosomal subunits prior to mRNA binding

• Promotes ribosomal recycling by inhibiting 60S-43S interaction after recycling

• Facilitates loading of charged 40S onto capped mRNAs by forming a complex with eIF4F

• Influences both scanning and recognition of the start codon on mRNA

The eIF3 complex extends its regulatory influence across multiple stages of translation initiation, making it a crucial component for proper protein synthesis in eukaryotic cells. Experimental designs targeting An16g02940 should consider these diverse functional roles within the broader translation machinery.

What techniques can researchers employ to study the interaction between An16g02940 and other translation machinery components?

To investigate interactions between An16g02940 and other translation components, researchers should consider these methodological approaches:

• Co-immunoprecipitation (Co-IP) with GFP-trap beads: This approach has successfully identified 531 MoOeIF3K-GFP co-immunoprecipitation protein complexes in related systems. Implement by:

  • Creating An16g02940-GFP fusion constructs

  • Extracting proteins under non-denaturing conditions

  • Using GFP-Trap beads for immunoprecipitation

  • Analyzing precipitated complexes via mass spectrometry

• Gene Ontology (GO) and pathway enrichment analyses: Apply these to Co-IP results to categorize interacting proteins. Previous studies with eIF3k homologs have identified interactions with ribonucleoprotein complexes (171 proteins), rRNA processing machinery (93 proteins), and gene expression regulators (215 proteins) .

• Proximity-dependent biotin identification (BioID): This technique can identify transient or weak interactions that may be missed by Co-IP:

  • Fuse An16g02940 to a promiscuous biotin ligase

  • Express in Aspergillus niger

  • Identify biotinylated proteins using streptavidin pulldown followed by mass spectrometry

• Cryo-electron microscopy: For structural analysis of An16g02940 within the intact eIF3 complex bound to ribosomes, revealing its spatial positioning relative to the 40S subunit (typically positioned near the mRNA exit site) .

How does An16g02940 potentially affect fungal stress responses?

To investigate An16g02940's role in stress responses, implement these methodological approaches:

• Targeted gene disruption: Generate ΔAn16g02940 knockout strains using CRISPR-Cas9 or homologous recombination. Based on studies of homologous proteins, expect potential impacts on:

  • Vegetative growth under normal and starvation conditions

  • Hyphal morphogenesis

  • Responses to ER stress

• Comparative growth analysis: Assess growth of wild-type versus ΔAn16g02940 strains under:

  • Nutrient-rich versus nutrient-deficient media

  • Various stress conditions (oxidative, osmotic, temperature)

  • Monitor growth rates, colony morphology, and stress tolerance

• Biochemical assessment of energy metabolism: Studies of eIF3k homologs suggest involvement in regulating the utilization of stored cellular nutrients under starvation:

  • Measure glycogen mobilization and degradation

  • Assess ATP production under stress conditions

  • Quantify protein synthesis rates using puromycin incorporation assays

• Transcriptomic and proteomic profiling: Comparative analysis of wild-type versus ΔAn16g02940 strains under stress:

  • RNA-seq to identify differentially expressed genes

  • Ribosome profiling to detect translation efficiency changes

  • Quantitative proteomics to measure protein abundance alterations

Research in related fungi has shown that eIF3k homologs can support survival by regulating vegetative growth and energy reserve utilization under starvation conditions .

What methodologies are effective for analyzing the impact of An16g02940 on ribosomal RNA generation and protein synthesis?

To investigate An16g02940's impact on rRNA generation and protein synthesis, implement these approaches:

Studies of eIF3k homologs have shown that deletion can accelerate rRNA generation with a corresponding increase in total protein output . These methodologies will help determine if An16g02940 functions similarly in Aspergillus niger.

How can researchers investigate the potential role of An16g02940 in autophagy regulation?

Recent research has revealed unexpected roles for eIF3k in selective autophagy pathways. To investigate whether An16g02940 shares these functions:

• Co-localization studies with autophagy markers:

  • Generate fluorescently tagged An16g02940 and autophagy proteins (ATG5, ATG8)

  • Perform fluorescence microscopy under autophagy-inducing conditions

  • Quantify co-localization coefficients between An16g02940 and autophagy markers

• Protein-protein interaction analysis:

  • Use yeast two-hybrid or split-GFP assays to test direct interaction with ATG proteins

  • Perform co-immunoprecipitation followed by western blotting for autophagy components

  • Investigate whether An16g02940 interacts with ubiquitinated proteins

• Autophagic flux measurement:

  • Compare autophagosome formation in wild-type and ΔAn16g02940 strains

  • Use GFP-ATG8 processing assays to measure autophagic flux

  • Apply autophagy inhibitors to determine pathway specificity

• Selective substrate degradation analysis:

  • Identify potential targets of An16g02940-mediated selective autophagy

  • Track their degradation kinetics in wild-type versus ΔAn16g02940 strains

  • Perform ubiquitination assays to determine if An16g02940 recognizes ubiquitin-tagged proteins

Research with other eIF3k homologs has demonstrated that eIF3k can function as a selective autophagic receptor, acting as a bridge linking ubiquitin-tagged proteins and ATG5 to promote selective autophagy . This suggests a mechanism by which translation regulators may directly interface with protein degradation pathways.

What experimental approaches can determine if An16g02940 impacts pathogenicity in Aspergillus niger?

Studies of eIF3k homologs in plant fungal pathogens suggest roles in pathogenicity. To investigate potential pathogenicity functions:

• Host infection assays:

  • Compare virulence of wild-type and ΔAn16g02940 strains in appropriate host models

  • Measure fungal burden, spread, and host tissue damage

  • Assess appressorium formation and function if applicable

• Host immune response analysis:

  • Measure host cytokine production (IL-6, IL-8, TNF-α) in response to wild-type versus ΔAn16g02940 strains

  • Evaluate NF-κB pathway activation using reporter assays

  • Compare MyD88 expression and degradation rates

• Adhesion and invasion quantification:

  • Perform adhesion assays measuring colony forming units adhering to host cells

  • Conduct microscopy to visualize fungal invasion processes

  • Compare host cell proliferation in response to infection

• Analysis of stored energy mobilization:

  • Measure glycogen mobilization during infection

  • Assess appressorium integrity

  • Evaluate host penetration and colonization efficiency

Research in Magnaporthe oryzae has shown that eIF3k domain-containing proteins can promote disease by regulating glycogen mobilization and degradation, appressorium integrity, and host colonization . Other studies suggest eIF3k can inhibit host immune responses by targeting MyD88 for autophagy-mediated degradation .

How can researchers distinguish between direct translational effects of An16g02940 and its influence on transcriptional regulation?

Distinguishing between translational and transcriptional effects requires careful experimental design:

• Integrated transcriptomic and translatomic approach:

  • Perform parallel RNA-seq and ribosome profiling on the same samples

  • Calculate translation efficiency (TE) for each transcript by normalizing ribosome footprints to mRNA abundance

  • Identify differentially translated genes (DTEGs) using statistical tools like DESeq2

  • Compare changes in mRNA levels versus translation efficiency to separate effects

• Reporter assay systems:

  • Design dual-reporter constructs with varying 5' UTRs and coding sequences

  • Measure reporter activity in wild-type versus ΔAn16g02940 backgrounds

  • Include transcriptional versus translational inhibitors to parse mechanisms

• In vitro translation systems:

  • Develop a reconstituted translation system with purified components

  • Test translation with and without recombinant An16g02940

  • Measure effects on initiation, elongation, and termination rates

• Analysis of eIF3k-interacting transcription factors:

  • Identify transcription factors that interact with An16g02940 using Co-IP

  • Perform ChIP-seq to detect changes in transcription factor binding upon An16g02940 deletion

  • Correlate with transcriptional changes

Studies of eIF3k homologs have identified interactions with 34 transcription factors, suggesting potential transcriptional regulatory functions beyond its canonical translation role . These approaches will help delineate the direct translational effects from indirect transcriptional influence.

What methodological considerations should be made when investigating stoichiometric changes in eIF3 complex assembly?

To study how changes in An16g02940 levels affect eIF3 complex assembly:

• Quantitative proteomics of eIF3 complexes:

  • Immunoprecipitate eIF3 complexes from cells with varying An16g02940 expression levels

  • Use TMT or SILAC quantitative proteomics to measure stoichiometric changes in subunits

  • Develop a mathematical model of complex assembly based on quantitative data

• Sucrose gradient fractionation:

  • Separate native eIF3 complexes based on size and composition

  • Compare profiles between wild-type and An16g02940-altered strains

  • Identify subcomplex formation or altered assembly states

• Blue native PAGE analysis:

  • Preserve native protein complexes during electrophoresis

  • Visualize intact eIF3 complexes and subcomplexes

  • Compare complex integrity across different An16g02940 expression conditions

• Integrated structural approaches:

  • Combine cryo-EM with crosslinking mass spectrometry

  • Map subunit interactions within the eIF3 complex

  • Identify structural changes resulting from An16g02940 alteration

Recent research has shown that perturbations in eIF3 subunit stoichiometry can alter expression of specific mRNAs, indicating mRNA-specific regulation of translation by individual subunits . These methodologies will help determine whether An16g02940 exerts similar regulatory effects through stoichiometric changes in the eIF3 complex.