Recombinant Saccharomyces cerevisiae Elongation factor 2 (EFT1), partial

What is EFT1 and what is its role in Saccharomyces cerevisiae?

EFT1 is one of two genes (along with EFT2) that encode elongation factor 2 (EF-2) in the yeast Saccharomyces cerevisiae. EF-2 plays a critical role in the elongation phase of protein synthesis, specifically catalyzing the ribosomal translocation step. During this step, EF-2 facilitates the movement of the ribosome along the mRNA by one codon after peptide bond formation, moving the peptidyl-tRNA from the A-site to the P-site . This process is essential for cellular viability, as genetic deletion experiments have shown that at least one functional copy of either EFT1 or EFT2 is required for yeast cell survival .

The expression of EF-2 is tightly regulated, with messenger RNA levels paralleling cellular growth and reaching peak levels during mid-log phase cultures . This correlation between EF-2 expression and growth reflects its essential role in protein synthesis, which is particularly important during active cell division and proliferation.

How do EFT1 and EFT2 genes compare in S. cerevisiae?

Although EFT1 and EFT2 are located on separate chromosomes in S. cerevisiae, they exhibit remarkable similarity at the DNA level. The nucleotide sequences of these two genes differ at only four positions out of 2526 base pairs, representing an almost perfect duplication event . More significantly, despite these minor DNA sequence differences, the predicted protein sequences encoded by both genes are identical .

This high degree of conservation suggests strong evolutionary pressure to maintain the precise sequence of EF-2, highlighting its critical role in cellular function. The redundancy provided by having two identical copies likely ensures adequate EF-2 production under various growth conditions and provides a safety mechanism against mutational damage to either gene. At least one functional copy of either EFT gene must be present for cell viability, as demonstrated through genetic deletion studies .

What are the key functional domains of EFT1-encoded protein?

The EFT1-encoded elongation factor 2 (EF-2) contains several critical functional domains that enable its role in translation:

• GTP-binding domain (G-domain): This domain is responsible for binding and hydrolyzing GTP, providing the energy required for ribosomal translocation. The G-domain of EF-2 exhibits strong homology to the G-domain of Escherichia coli elongation factor Tu (EF-Tu) and other G-protein family members .

• Diphthamide-containing domain: EF-2 contains a domain with a unique post-translationally modified histidine residue that is converted to diphthamide. This modification is specifically targeted for ADP-ribosylation by diphtheria toxin, which inhibits protein synthesis by inactivating EF-2 .

• Phosphorylation site: Yeast and mammalian EF-2 share identical sequences at the threonine residue that is specifically phosphorylated in mammalian cells by calmodulin-dependent protein kinase III (also known as EF-2 kinase) .

• Effector sequence motif: The protein contains a conserved Glu-X-X-Arg-X-Ile-Thr-Ile "effector" sequence motif that is found among all known elongation factors and is involved in functional interactions during translation .

These domains work in concert to enable EF-2's role in catalyzing ribosomal translocation during protein synthesis, a process essential for cellular viability.

What are the optimal approaches for expressing recombinant EFT1 in heterologous systems?

Expressing recombinant EFT1 from S. cerevisiae in heterologous systems requires careful optimization to ensure high yields of functional protein. The choice of expression system depends on research objectives, particularly regarding post-translational modifications and functional studies.

For bacterial expression systems, E. coli BL21(DE3) or Rosetta strains are commonly used with pET-series vectors featuring the T7 promoter system. When expressing yeast proteins in E. coli, codon optimization is often necessary to account for codon usage bias. Induction conditions should be optimized, with lower temperatures (16-20°C) and moderate IPTG concentrations (0.1-0.5 mM) typically yielding better results for complex eukaryotic proteins .

For applications requiring authentic post-translational modifications, particularly the unique diphthamide modification found on EF-2, yeast expression systems may be preferable. S. cerevisiae or Pichia pastoris systems using vectors like pYES2 or pPICZ, respectively, can provide a more native environment for proper protein folding and modification .

The addition of affinity tags (His, GST, or MBP) can facilitate purification while potentially enhancing solubility. Position the tag carefully to avoid interference with functional domains, particularly the GTP-binding domain and diphthamide-containing region .

What assays can be used to evaluate the functionality of recombinant EFT1-encoded protein?

Several complementary assays can be employed to assess the functionality of recombinant EF-2 encoded by EFT1:

• GTPase activity assays: These measure the intrinsic GTP hydrolysis activity of EF-2. Methods include colorimetric phosphate detection assays (malachite green), radioactive assays using [γ-³²P]GTP, or fluorescent assays with labeled GTP analogs. This approach confirms the basic catalytic function of the protein .

• Ribosomal translocation assays: These more directly assess the biological function of EF-2 in promoting translocation. They typically involve reconstituted translation systems with purified ribosomes, mRNA, tRNAs, and other translation factors. The movement of tRNAs through the ribosome upon addition of EF-2 and GTP can be measured using fluorescently labeled components and FRET-based detection systems .

• Diphtheria toxin ADP-ribosylation assay: This test evaluates whether the recombinant EF-2 contains the unique diphthamide modification. The assay measures the incorporation of ADP-ribose from NAD⁺ into EF-2 catalyzed by diphtheria toxin. This modification only occurs at the diphthamide residue, making this a specific test for this post-translational modification .

• Binding assays: Techniques such as surface plasmon resonance (SPR) or microscale thermophoresis can measure the binding of EF-2 to ribosomes or nucleotides, providing information about interaction affinities and kinetics .

A comprehensive evaluation should include multiple assays to ensure both structural integrity and functional activity of the recombinant protein.

How can researchers effectively purify recombinant EFT1-encoded protein while maintaining its functionality?

Purification of functional recombinant EF-2 encoded by EFT1 requires strategies that preserve the protein's complex structure and activity. The following approach has proven effective:

Buffer optimization: Use buffers containing 20-50 mM Tris or HEPES (pH 7.5-8.0), 100-300 mM NaCl, 5-10% glycerol, and 1-5 mM MgCl₂, which is critical for nucleotide binding. Include reducing agents (1-5 mM DTT or β-mercaptoethanol) to maintain thiol groups, and protease inhibitors to prevent degradation .

Purification strategy:

• Affinity chromatography: If using a His-tagged construct, employ Ni-NTA or TALON resins with careful optimization of imidazole concentrations in wash and elution buffers. For GST-tagged proteins, use Glutathione Sepharose.

• Ion exchange chromatography: As an intermediate purification step, particularly useful for removing nucleic acid contaminants.

• Size exclusion chromatography: As a final polishing step to obtain homogeneous protein and remove aggregates.

Activity preservation: Throughout purification, maintain samples at 4°C and include stabilizing agents such as glycerol (10-20%) and MgCl₂ (1-5 mM). The addition of GTP or non-hydrolyzable analogs (0.1-0.5 mM) may stabilize the protein. Avoid multiple freeze-thaw cycles by dividing the purified protein into single-use aliquots .

Quality control: Evaluate protein purity by SDS-PAGE and confirm identity by Western blotting or mass spectrometry. Assess functionality using GTPase activity assays to ensure the purification process has not compromised the protein's activity .

This systematic approach helps ensure that the purified recombinant EF-2 maintains its structural integrity and functional capabilities.

How is EFT1 evolutionarily conserved and what does this tell us about its function?

The extraordinary conservation of EF-2 across evolutionarily diverse species highlights its fundamental importance in protein synthesis. Yeast EF-2 shares 66% identity and over 85% homology with human EF-2, despite the vast evolutionary distance between these organisms . This high degree of conservation is particularly remarkable given the rapid evolutionary divergence typically observed between yeast and human proteins.

The conservation extends to critical functional sites, including the domain containing the histidine residue that is modified to diphthamide and the threonine residue that is specifically phosphorylated in mammalian cells . Additionally, yeast EF-2 contains the Glu-X-X-Arg-X-Ile-Thr-Ile "effector" sequence motif that is conserved among all known elongation factors .

This exceptional conservation suggests that EF-2 represents a core component of the eukaryotic translation machinery that has been under strong selective pressure throughout evolutionary history. The maintenance of identical amino acid sequences at critical functional sites indicates that the precise molecular mechanisms of ribosomal translocation have remained largely unchanged for billions of years.

Comparative analysis of EF-2 sequences across species can identify invariant residues that are likely essential for function, providing insights into structure-function relationships and potentially guiding the development of species-specific inhibitors .

How do sordarin derivatives specifically target fungal EF-2 without affecting mammalian EF-2?

Sordarin derivatives represent a family of antifungal compounds that selectively target fungal EF-2 without significantly affecting mammalian EF-2, despite the high conservation of this protein. This selectivity makes them valuable both as research tools and potential therapeutic agents.

Research has shown that sordarin derivatives bind to a specific region in fungal EF-2, likely a pocket formed by multiple domains. Resistance studies have identified mutations in EFT2 that confer resistance to these compounds, and these mutations cluster in the three-dimensional structure, helping to define the probable binding site . When mapped onto structural models of EF-2, these mutations form a potential drug binding pocket .

Interestingly, not all resistance mechanisms involve mutations in EFT1/EFT2, suggesting that the functional target for sordarins may involve additional factors or complex interactions with other components of the translational machinery . This complexity in the mechanism of action presents both challenges and opportunities for developing improved antifungal agents targeting this essential pathway.

What are the implications of EFT1/EFT2 redundancy for genetic studies in yeast?

The presence of two nearly identical genes (EFT1 and EFT2) encoding the same elongation factor 2 (EF-2) protein creates unique considerations for genetic studies in S. cerevisiae. This redundancy has several important implications:

Functional redundancy: Single deletion mutants (either eft1Δ or eft2Δ) remain viable due to compensation by the remaining gene, while deletion of both genes is lethal . This necessitates creating conditional alleles or employing sophisticated genetic approaches to study EF-2 function.

Genetic stability: The maintenance of two nearly identical genes with only four nucleotide differences across 2526 base pairs suggests either ongoing selection pressure or a relatively recent duplication event . This provides an interesting system for studying gene duplication and evolution.

Expression regulation: While the proteins are identical, differences in promoter strength, mRNA stability, or translation efficiency between EFT1 and EFT2 may exist. mRNA levels of EF-2 parallel cellular growth and peak in mid-log phase cultures, suggesting coordinated regulation .

Evolutionary insights: The EFT1/EFT2 pair represents an excellent model for studying the fate of duplicated genes and the evolutionary mechanisms that maintain functional redundancy .

Understanding these implications allows researchers to design more effective experiments and accurately interpret results when studying EF-2 function in S. cerevisiae.

How does the GTP-binding domain of EFT1-encoded protein compare to other G-proteins?

The GTP-binding domain (G-domain) of EFT1-encoded EF-2 belongs to the large superfamily of G-proteins that share common structural and functional features. Based on modeling studies comparing EF-2 to the crystallographic structure of E. coli elongation factor Tu (EF-Tu), several key features have been identified .

Like other G-proteins, EF-2 cycles between a GTP-bound "active" state and a GDP-bound "inactive" state. The GTP hydrolysis provides the energy for ribosomal translocation, coupling a chemical reaction to mechanical movement during protein synthesis .

A distinctive feature of EF-2's G-domain is its interaction with the ribosome, particularly with the sarcin-ricin loop of ribosomal RNA, which stimulates GTP hydrolysis. This interaction is mediated in part by the conserved "effector" sequence motif (Glu-X-X-Arg-X-Ile-Thr-Ile) found in elongation factors .

The homology between the G-domains of elongation factors from different species suggests a common evolutionary origin and similar basic mechanisms, despite their specialized functions in translation .

What is the significance of the diphthamide modification in EFT1-encoded protein?

Diphthamide is a unique post-translational modification found exclusively on a specific histidine residue of elongation factor 2 (EF-2) in all eukaryotes and archaebacteria. This modification has several significant aspects with implications for both basic research and potential therapeutic applications:

First, diphthamide is the specific target for ADP-ribosylation by bacterial toxins such as diphtheria toxin from Corynebacterium diphtheriae. This ADP-ribosylation inactivates EF-2, blocking protein synthesis and leading to cell death, which explains the pathogenicity of these toxins .

The diphthamide modification is highly conserved across species, with the histidine residue that undergoes this modification being identical in yeast and human EF-2 . This extraordinary conservation suggests an important functional role, potentially in ensuring translational fidelity or preventing frameshifting during translation.

The biosynthesis of diphthamide requires multiple enzymatic steps and several genes, making it a complex and energetically expensive modification. This further supports its functional importance, as cells would not maintain such a costly modification without significant selective advantage .

In research contexts, the unique nature of the diphthamide modification has made it a valuable tool for studying EF-2 function and for developing methods to specifically label or target this protein. The modification can be used as a marker for authentic, functionally active recombinant EF-2 .

How does EFT1-encoded protein interact with the ribosome during translocation?

EF-2 encoded by EFT1/EFT2 interacts with multiple components of the ribosome to facilitate translocation, the process by which the ribosome moves along the mRNA by one codon after peptide bond formation. These interactions involve a complex network of contacts with both ribosomal subunits and the tRNAs.

When GTP-bound EF-2 binds to the pre-translocation ribosome, it interacts with the small subunit (40S) at the decoding center and mRNA-binding channel. A critical interaction occurs with the large subunit (60S), particularly with the sarcin-ricin loop (SRL) in the 25S rRNA, which activates GTP hydrolysis .

The binding of EF-2 disrupts bridges between the ribosomal subunits, facilitating the ratcheting motion required for translocation. Domain IV of EF-2 reaches into the decoding center, disrupting codon-anticodon interactions and allowing the movement of the tRNAs and mRNA .

Following GTP hydrolysis, EF-2 undergoes conformational changes that drive the translocation process, moving the peptidyl-tRNA from the A-site to the P-site. The GDP-bound EF-2 then adopts a "compact" conformation with lower affinity for the ribosome, facilitating its dissociation .

These interactions are highly conserved but contain subtle species-specific differences. The specific interactions between fungal EF-2 and fungal ribosomes explain why sordarin derivatives can selectively target fungal translation, as they may act at the interface between EF-2 and the ribosome .

What mechanisms of resistance to EF-2 targeting antifungals have been identified?

Resistance to EF-2 targeting antifungals, particularly sordarin derivatives, has been identified through laboratory studies and provides valuable insights into both drug action mechanisms and potential challenges for therapeutic development.

The primary resistance mechanism involves target-site mutations in the EFT2 gene encoding EF-2. In studies with the semisynthetic sordarin derivative GM193663, the major group of resistant isolates (21 members) had mutations in EFT2 . These mutations clustered in a three-dimensional region that likely forms the drug binding pocket when mapped onto structural models of EF-2, providing valuable information about the binding site of these compounds .

Interestingly, a minor complementation group of resistant isolates (four members) did not have mutations in EFT1, suggesting alternative resistance mechanisms . This indicates that the functional target for sordarin derivatives may be more complex than initially thought, potentially involving additional factors or protein complexes beyond EF-2 itself.

Cell extracts from resistant mutants lost the capacity to bind to the inhibitors, confirming that the resistance mechanism involves altered drug binding rather than drug metabolism or efflux . This provides strong evidence that EF-2 is indeed the primary target of these compounds.

Understanding these resistance mechanisms is crucial for the development of new derivatives that can overcome resistance, as well as for designing combination therapies that might prevent or delay the emergence of resistant strains.

How can structural studies of EFT1 contribute to antifungal drug development?

Structural studies of EFT1-encoded elongation factor 2 (EF-2) provide valuable insights that can significantly advance antifungal drug development. The approach combines modeling, experimental data, and comparative analysis to identify targetable features specific to fungal EF-2.

Modeling the G-domain of yeast EF-2 based on the crystallographic structure of E. coli EF-Tu has provided insights into GTP binding and hydrolysis mechanisms . This structural information, combined with data from resistance mutations, has helped define the binding pocket for sordarin derivatives . These compounds represent a promising family of antifungals that specifically target fungal EF-2, demonstrating the potential of this protein as a drug target .

The clustering of resistance mutations to sordarin derivatives has been particularly valuable in mapping the drug binding site on EF-2 . When these mutations are mapped onto three-dimensional models of EF-2, they form a potential drug binding pocket, providing a structural basis for understanding drug-target interactions and guiding the design of improved compounds .

Despite the high conservation of EF-2 across species (66% identity between yeast and human), subtle structural differences exist that can be exploited for selective targeting . Detailed comparison of fungal and mammalian EF-2 structures can identify regions that differ sufficiently to allow selective binding of antifungal compounds without affecting the host protein.

This structure-based approach to drug design holds promise for developing new antifungal agents with improved specificity, potency, and resistance profiles.

What are the future research directions for studying recombinant EFT1?

Future research on recombinant EFT1 from S. cerevisiae will likely focus on several promising directions that build upon our current understanding of this essential translation factor.

The development of improved expression and purification protocols for obtaining functional recombinant EF-2 with authentic post-translational modifications, particularly the unique diphthamide modification, remains an important technical challenge . Advanced structural studies, including cryo-electron microscopy of EF-2-ribosome complexes at different stages of translocation, will provide deeper insights into the molecular mechanisms of protein synthesis .

The redundancy between EFT1 and EFT2 genes offers an interesting system for studying gene duplication and evolution . Investigating potential differences in regulation and expression patterns between these nearly identical genes under various growth conditions could reveal subtle functional specialization.

The continued exploration of EF-2 as a target for antifungal development holds particular promise. Structure-based drug design approaches, informed by the binding mode of sordarin derivatives and the mapping of resistance mutations, could lead to improved compounds with enhanced specificity and reduced resistance potential .

Finally, investigating the broader roles of EF-2 beyond its canonical function in translocation, such as potential regulatory roles in specific mRNA translation or interactions with other cellular components, represents an exciting frontier. The evolution of such a highly conserved protein suggests it may participate in networks beyond our current understanding .

These research directions will continue to advance our understanding of this fundamental component of the translation machinery while potentially yielding practical applications in medicine and biotechnology.