Recombinant Drosophila mojavensis Eukaryotic translation initiation factor 3 subunit L (GI12903), partial

What is eIF3 subunit L and what is its role in translation initiation?

eIF3 subunit L (eIF3l) is a component of the eukaryotic translation initiation factor 3 (eIF3) complex, which is the largest initiation factor complex involved in protein synthesis. The eIF3 complex in metazoans consists of 13 subunits (named a through m) and plays critical roles in multiple steps of translation initiation.

The eIF3 complex is involved in:

• Formation of the 43S pre-initiation complex (PIC)

• Binding of the PIC to the mRNA

• Scanning along the 5' UTR to locate the start codon

• Regulating the stringency of start codon selection

Why is studying D. mojavensis eIF3 subunit L important in evolutionary biology?

Studying eIF3l in D. mojavensis offers unique insights into how translation machinery evolves during ecological adaptation. D. mojavensis represents an excellent model for evolutionary biology research for several key reasons:

• It is endemic to the northwestern deserts of North America and has adapted to extreme desert environments with challenging thermal and desiccation stresses

• The species comprises four genetically isolated populations (host races), each specialized to different cactus species with distinct chemical profiles

• Each cactus host provides the resident D. mojavensis population with a unique chemical environment requiring specialized detoxification mechanisms

Translation regulation is crucial during stress responses, and components of the translation machinery may have evolved to optimize protein synthesis under these challenging conditions. By examining eIF3l across the four D. mojavensis populations, researchers can investigate whether translation factors show signatures of adaptive evolution similar to those observed in detoxification genes like GstD1 .

Furthermore, D. mojavensis exhibits elevated sleep need and stress resilience compared to D. melanogaster , suggesting potential adaptations in protein synthesis regulation that could involve translation initiation factors like eIF3l.

What are the optimal handling conditions for recombinant D. mojavensis eIF3 subunit L in laboratory settings?

Proper handling of recombinant D. mojavensis eIF3l is critical for maintaining its structural integrity and functional activity. Based on manufacturer recommendations, the following conditions should be observed:

Storage conditions:

• Store at -20°C for standard storage

• For extended storage, conserve at -20°C or -80°C

• Avoid repeated freezing and thawing cycles which can lead to protein denaturation

• Working aliquots can be stored at 4°C for up to one week

Reconstitution protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% for long-term storage (50% is recommended)

• Aliquot for long-term storage at -20°C/-80°C

Shelf life considerations:

• Liquid form: approximately 6 months at -20°C/-80°C

• Lyophilized form: approximately 12 months at -20°C/-80°C

• Note that shelf life depends on multiple factors including storage state, buffer ingredients, temperature, and the intrinsic stability of the protein

For experimental use, buffer conditions should be optimized based on the specific application. For interaction studies with other translation factors, buffers containing 50 mM Tris-HCl pH 7.5, 150 mM KCl/NaCl, 0.05-0.1% NP40, and 1 mM EDTA have been used successfully with translation factors in Drosophila .

How can researchers assess the functional activity of recombinant D. mojavensis eIF3 subunit L?

Several complementary approaches can be employed to evaluate the functional activity of recombinant D. mojavensis eIF3l:

1. Protein-Protein Interaction Assays:

• Co-immunoprecipitation experiments using antibodies against tagged eIF3l to pull down interacting partners

• Pull-down assays using recombinant His-tagged or HA-tagged eIF3l

• Size exclusion chromatography to analyze complex formation with other eIF3 subunits

2. In vitro Translation Assays:

• Cell-free translation systems reconstituted with purified components

• Measure translation efficiency of reporter mRNAs in the presence or absence of eIF3l

• Luciferase-based reporter assays similar to those used for eIF3d in D. melanogaster

3. RNA-Binding Assays:

• RNA electrophoretic mobility shift assays (EMSA) to test direct binding to mRNAs

• RNA chromatography using biotinylated RNA oligomers bound to streptavidin beads

• UV cross-linking assays to identify RNA-binding regions

4. Functional Complementation:

• Test if D. mojavensis eIF3l can rescue translation defects in systems where endogenous eIF3l has been depleted

• Compare activity to eIF3l from other Drosophila species

A typical experimental workflow might involve:

• Expressing and purifying the recombinant protein (>85% purity by SDS-PAGE)

• Verifying structural integrity using circular dichroism or thermal shift assays

• Testing incorporation into the eIF3 complex using co-immunoprecipitation

• Evaluating effects on translation using reporter assays

What expression systems are used to produce recombinant D. mojavensis eIF3 subunit L, and how do they affect protein functionality?

Two main expression systems are employed for producing recombinant D. mojavensis eIF3l, each with distinct advantages and implications for protein functionality:

1. E. coli Expression System:

• Product code: CSB-EP007541DLY-B

• Advantages: High yield, cost-effective, rapid production

• Limitations: Lacks eukaryotic post-translational modifications, may form inclusion bodies requiring refolding

• Impact on functionality: May lack critical modifications needed for certain protein-protein or protein-RNA interactions

2. Baculovirus Expression System:

• Product code: CSB-BP007541DLY

• Advantages: Provides eukaryotic post-translational modifications, better protein folding, higher solubility

• Limitations: More expensive, longer production time, typically lower yield than E. coli

• Impact on functionality: Likely to retain more native-like activity due to proper folding and modifications

Comparative analysis of proteins from different expression systems:

When selecting an expression system, researchers should consider:

• The intended experimental application (structural vs. functional studies)

• Whether post-translational modifications are critical for function

• Required quantity and budget constraints

• The need for soluble, properly folded protein

For comprehensive functional characterization, comparing proteins from both expression systems can provide valuable insights into the role of eukaryotic modifications in eIF3l activity.

How does eIF3 subunit L contribute to translation regulation in D. mojavensis?

While the specific function of eIF3l in D. mojavensis has not been directly characterized, its likely roles can be inferred based on studies of eIF3 in other Drosophila species and organisms:

Assembly and structural roles:

• eIF3l likely contributes to the assembly and structural integrity of the eIF3 complex

• It may form specific interactions with other subunits, potentially as part of a sub-module within the larger complex

mRNA-specific regulation:

• Similar to eIF3d in D. melanogaster, which regulates msl-2 mRNA translation , eIF3l may be involved in message-specific translation control

• This specificity could be particularly relevant for D. mojavensis' adaptation to different cactus hosts, where specialized proteins may be required for detoxification

Stress adaptation mechanisms:

• Given D. mojavensis' adaptation to desert conditions, eIF3l might play a role in stress-responsive translation

• It could participate in translational reprogramming during heat shock, desiccation stress, or exposure to cactus-specific toxins

Developmental regulation:

• eIF3 components often show tissue-specific or developmental stage-specific functions

• eIF3l might regulate translation during specific developmental processes that are crucial for D. mojavensis' life cycle in desert environments

To confirm these hypothesized functions, experimental approaches such as RNAi knockdown in D. mojavensis cells, ribosome profiling to identify affected mRNAs, and expression of the protein in heterologous systems would be necessary.

Are there tissue-specific expression patterns of eIF3 subunit L in D. mojavensis?

Potential expression patterns:

• Based on the tissue-specific expression of other translation factors like eIF4E variants in Drosophila , eIF3l might show differential expression across tissues

• Tissues involved in detoxification (midgut, fat body) might show specialized expression patterns given D. mojavensis' adaptation to toxic cactus hosts

• The nervous system might show distinct expression patterns, considering D. mojavensis' unique sleep patterns and stress responses compared to D. melanogaster

• Expression might differ across the four D. mojavensis populations in response to their distinct cactus host adaptations

Experimental approaches to determine tissue-specific expression:

Understanding tissue-specific expression would provide insights into whether eIF3l has specialized functions in certain tissues relevant to D. mojavensis' unique ecological adaptations and would guide further functional studies.

How does eIF3 subunit L interact with other components of the translation initiation machinery in D. mojavensis?

Understanding the interaction network of eIF3l within the translation initiation machinery is crucial for elucidating its function. While specific interaction data for D. mojavensis eIF3l is not available, its likely interactions can be inferred from studies of eIF3 in other systems:

Interactions with other eIF3 subunits:

• eIF3l likely interacts directly with specific eIF3 subunits to form the complete eIF3 complex

• Based on studies in other systems, eIF3l might form a specific sub-module within the complex, similar to how eIF3d and eIF3e form a module in D. melanogaster

• These interactions are likely mediated by conserved protein-protein interaction domains

Interactions with the translation machinery:

• As part of the eIF3 complex, eIF3l contributes to interactions with:

  • The 40S ribosomal subunit

  • Other initiation factors including eIF1, eIF1A, eIF2, eIF4G, and eIF5

  • mRNA, potentially through direct RNA binding or via other eIF3 subunits

Experimental approaches to characterize interactions:

• Co-immunoprecipitation experiments:

  • Using antibodies against tagged D. mojavensis eIF3l

  • Mass spectrometry identification of co-precipitated proteins

  • RNase treatment to distinguish RNA-dependent interactions

• Protein crosslinking:

  • Chemical or UV crosslinking to capture transient interactions

  • Mass spectrometry analysis to identify crosslinked residues

  • Mapping interaction interfaces at amino acid resolution

• Yeast two-hybrid or mammalian two-hybrid assays:

  • Testing direct binary interactions with other translation factors

  • Identifying minimal interaction domains

• In vitro binding assays:

  • Pull-down assays with purified components

  • Surface plasmon resonance to measure binding affinities

  • Isothermal titration calorimetry for thermodynamic parameters

These studies would help construct a comprehensive interaction map for eIF3l in D. mojavensis and reveal how these interactions might contribute to the species' adaptation to its unique ecological niche.

How conserved is eIF3 subunit L across Drosophila species?

While the search results don't provide direct sequence comparison data for eIF3l across Drosophila species, a comprehensive analysis would involve several approaches to assess conservation:

Sequence conservation analysis:

• Multiple sequence alignment of eIF3l from D. mojavensis, D. melanogaster, and other Drosophila species

• Calculation of percent identity and similarity scores

• Identification of conserved domains versus variable regions

• Phylogenetic analysis to understand evolutionary relationships

Expected conservation patterns:

• Core functional domains involved in essential functions (e.g., incorporation into the eIF3 complex) would likely show high conservation

• Regulatory regions or surfaces that interact with species-specific factors might show accelerated evolution

• Similar to what has been observed with GstD1 in D. mojavensis , eIF3l might show population-specific variations related to different cactus host adaptations

Evolutionary rate analysis:

• Calculation of Ka/Ks ratios to identify regions under purifying or positive selection

• Comparison with evolutionary rates of other translation factors

• Assessment of whether any accelerated evolution correlates with ecological adaptations in D. mojavensis

Does D. mojavensis eIF3 subunit L show adaptations related to the ecological niche of this species?

D. mojavensis has adapted to challenging desert environments and cactus hosts with distinct chemical profiles . While direct evidence of eIF3l adaptations is not available in the search results, several approaches can be used to investigate this question:

Potential ecological adaptations in eIF3l:

• Stress response regulation:

  • D. mojavensis exhibits elevated sleep need and stress resilience

  • eIF3l might show adaptations that optimize translation during heat shock, desiccation, or other stresses

  • These adaptations could include thermal stability enhancements or regulatory modifications

• Host plant adaptation:

  • Similar to GstD1, which shows evidence of adaptive evolution in response to cactus host chemistry

  • eIF3l might be involved in regulating translation of detoxification genes

  • Population-specific variants could correlate with different cactus hosts

• Metabolic adaptation:

  • Desert adaptation requires efficient resource utilization

  • eIF3l might show changes that optimize translation efficiency under resource-limited conditions

Methodological approaches:

The analysis of genomic variation in D. mojavensis has revealed that genes with elevated rates of molecular evolution tend to be involved in metabolism, detoxification, chemosensory reception, and stress response – all functions potentially regulated by translation factors like eIF3l.

How does D. mojavensis eIF3 subunit L differ from the homologous protein in D. melanogaster?

A comprehensive comparison between D. mojavensis and D. melanogaster eIF3l would provide insights into how translation machinery components evolve during species diversification and ecological adaptation. While the search results don't provide direct comparison data, such an analysis would include:

Sequence comparison:

• Alignment of amino acid sequences to calculate percent identity and similarity

• Identification of conserved domains versus variable regions

• Mapping differences onto predicted structural models

• Assessment of whether differences correlate with ecological adaptations

Functional comparison:

• Cross-species complementation experiments (can D. mojavensis eIF3l rescue D. melanogaster eIF3l mutants?)

• Comparison of biochemical properties like binding affinities to other initiation factors

• Assessment of incorporation into the respective eIF3 complexes

Expected differences:

• Core functional domains would likely be conserved

• Regulatory regions might show more variation

• D. mojavensis eIF3l might show adaptations related to desert stress resistance

• Temperature optima might differ, reflecting adaptation to different thermal environments

• Binding interfaces with species-specific partners might show accelerated evolution

This comparative analysis would be particularly valuable because D. mojavensis and D. melanogaster represent contrasting ecological adaptations – D. mojavensis to desert environments and toxic cactus hosts, and D. melanogaster to more temperate conditions. Differences in their translation machinery could reveal how core cellular processes adapt to different ecological niches.

What CRISPR-Cas9 approaches can be used to study eIF3 subunit L function in D. mojavensis?

While CRISPR-Cas9 has not been extensively applied to D. mojavensis based on the search results, several approaches could be employed to study eIF3l function:

CRISPR-Cas9 strategies for D. mojavensis eIF3l:

• Gene knockout:

  • Design guide RNAs (gRNAs) targeting coding regions of eIF3l

  • Create frameshift mutations that eliminate functional protein

  • Assess phenotypic consequences, with special attention to:

    • Viability and development

    • Stress resistance (given D. mojavensis' desert adaptation)

    • Population-specific effects related to cactus host use

• Domain-specific mutations:

  • Design precise edits to modify specific functional domains

  • Create point mutations in predicted binding interfaces

  • Test how these mutations affect function in vivo

• Fluorescent protein tagging:

  • Use homology-directed repair to insert fluorescent protein tags

  • Create C-terminal or N-terminal fusions

  • Visualize subcellular localization and tissue distribution

  • Track dynamics during stress responses

• Conditional alleles:

  • Create temperature-sensitive or drug-inducible alleles

  • Enable temporal control of eIF3l function

  • Study acute effects of eIF3l loss

Technical considerations specific to D. mojavensis:

• Embryo microinjection protocols may need optimization compared to D. melanogaster

• Efficiency of homology-directed repair might differ

• Appropriate genetic markers for screening would need to be established

• Rearing conditions for desert-adapted flies might require special consideration

Similar CRISPR-based approaches have been successfully used to study other translation factors in Drosophila, such as eIF4E-5, which was found to be essential for male fertility in D. melanogaster . These techniques could reveal the in vivo function of eIF3l in D. mojavensis, particularly in relation to this species' unique ecological adaptations.

How can mass spectrometry be used to study post-translational modifications of eIF3 subunit L in D. mojavensis?

Mass spectrometry (MS) offers powerful approaches for characterizing post-translational modifications (PTMs) of eIF3l, which could be crucial for its function in D. mojavensis:

Mass spectrometry workflow for PTM analysis:

• Sample preparation:

  • Express and purify recombinant D. mojavensis eIF3l

  • Alternatively, immunoprecipitate native eIF3l from D. mojavensis tissues

  • Perform in-gel or in-solution digestion with proteases (trypsin, chymotrypsin)

  • Enrichment for modified peptides if necessary (e.g., phosphopeptide enrichment)

• LC-MS/MS analysis:

  • High-resolution liquid chromatography-tandem mass spectrometry

  • Data-dependent acquisition to identify peptides and modifications

  • Search for common modifications:

    • Phosphorylation (Ser, Thr, Tyr)

    • Acetylation (Lys)

    • Methylation (Lys, Arg)

    • Ubiquitination (Lys)

• Comparative analysis under different conditions:

  • Normal vs. stress conditions (heat shock, desiccation)

  • Different developmental stages

  • Comparison across the four D. mojavensis populations

  • Label-free quantification or stable isotope labeling (SILAC)

• Functional characterization of identified PTMs:

  • Creation of site-directed mutants that mimic or prevent specific modifications

  • Testing how these mutations affect eIF3l function

  • Identification of enzymes responsible for adding or removing modifications

Potential significance for D. mojavensis biology:

• PTMs might regulate eIF3l in response to desert stress conditions

• Population-specific PTM patterns might contribute to cactus host adaptation

• Dynamic PTM changes could coordinate translation regulation during development

• Comparison with D. melanogaster could reveal species-specific regulatory mechanisms

This approach would provide insights into how post-translational regulation of eIF3l contributes to D. mojavensis biology and adaptation to its unique ecological niche.

What are the challenges in crystallizing D. mojavensis eIF3 subunit L for structural studies?

Determining the three-dimensional structure of D. mojavensis eIF3l presents several challenges typical of translation factors, along with potential species-specific considerations:

Key challenges in crystallizing eIF3l:

• Protein purity and homogeneity:

  • High-purity protein (>95%) would be needed, beyond the >85% mentioned in product specifications

  • Conformational heterogeneity could hinder crystal formation

  • Post-translational modifications might create multiple species

  • The "partial" nature of the available recombinant protein suggests potential expression challenges

• Protein stability and flexibility:

  • Translation factors often contain flexible regions that impede crystallization

  • Buffer optimization would be critical for long-term stability during crystallization trials

  • Temperature considerations may be important given D. mojavensis' adaptation to desert conditions

• Protein-protein interactions:

  • eIF3l may require other eIF3 subunits for proper folding or stability

  • Co-crystallization with binding partners might be necessary

  • The large size of complexes would create additional challenges

Strategies to overcome these challenges:

These approaches would help overcome the inherent challenges in determining the structure of this important translation factor. Given the evolutionary adaptations of D. mojavensis to desert environments, structural studies might reveal unique features that contribute to protein stability under extreme conditions.