Recombinant Saccharomyces cerevisiae Eukaryotic translation initiation factor 3 subunit I (TIF34)

What is TIF34 and what is its role in Saccharomyces cerevisiae?

TIF34 is a 39 kDa protein component (also referred to as eIF3-p39) of the eukaryotic initiation factor-3 (eIF3) complex in the yeast Saccharomyces cerevisiae . It plays a central role in the initiation of translation, which is the process of assembling ribosomes on mRNA to begin protein synthesis. TIF34 is an essential gene in yeast, as demonstrated by the lethal phenotype of a tif34 null mutation .

To study the function of this essential gene, temperature-sensitive (ts) alleles provide the most effective approach. When using tif34-ts mutants, researchers can observe normal growth under permissive conditions (24°C) while inducing functional inactivation under restrictive conditions (37°C), enabling observation of immediate consequences of TIF34 loss without complications from long-term absence .

How is TIF34 structurally characterized and what functional domains does it contain?

TIF34 contains five WD-40 repeats, which are structural domains that typically facilitate protein-protein interactions . These WD-40 repeats likely form a propeller-like configuration similar to the structure observed in other WD-40 containing proteins such as Gβ, providing a structural platform for interactions with other components of the eIF3 complex .

To investigate structure-function relationships in TIF34, researchers should:

• Generate site-directed mutations targeting conserved residues within individual WD-40 repeats

• Create truncation mutants that remove specific repeats

• Test mutant proteins for their ability to interact with known partners (PRT1 and p33)

• Assess functional complementation of tif34-ts or tif34Δ mutants

• Examine effects on translation initiation through polysome profile analysis

What experimental systems are available for studying TIF34 function?

Several complementary experimental systems can be employed to study TIF34 function:

• Temperature-sensitive mutants: The tif34-ts alleles (such as tif34-ts6 and tif34-ts31) allow for conditional inactivation of TIF34, enabling the study of immediate consequences of TIF34 loss . These mutants show different degrees of severity, with tif34-ts6 conferring no growth at 30°C while tif34-ts31 grows normally at 30°C but not at 37°C .

• Epitope-tagged versions: Myc-tagged TIF34 or His-tagged TIF34 can be expressed from plasmids for detection, purification, and interaction studies . These constructs should be validated by testing their ability to complement tif34Δ strains.

• Heterologous expression systems: TIF34 can be expressed in E. coli using vectors like pTrcHis for biochemical and structural studies . The recombinant protein can be purified using affinity chromatography (e.g., Co3+-agarose for His-tagged proteins).

• Yeast two-hybrid system: This approach can identify proteins that interact directly with TIF34 and map interaction domains . When implementing this system, researchers should include appropriate controls to confirm specificity of interactions.

How does TIF34 contribute to translation initiation?

TIF34 plays an essential role in translation initiation in Saccharomyces cerevisiae. When TIF34 function is disrupted in temperature-sensitive mutants, several key observations can be made:

• Polysome profile analysis reveals a strong reduction in the polysome to monosome ratio within 30 minutes of shifting to restrictive temperature, indicating a rapid defect in translation initiation .

• The immediate effect on translation initiation occurs while other eIF3 components are still present, allowing researchers to assign an essential role specifically to TIF34 .

• Depletion of TIF34 results in accelerated degradation of all eIF3 subunits, suggesting a role in assembly and maintenance of the complex .

To quantitatively assess the effects of TIF34 on global protein synthesis rates, researchers should conduct pulse-labeling experiments with radioactive amino acids followed by measurement of incorporation into proteins at various times after temperature shift .

What is the relationship between TIF34 function and cell cycle regulation?

TIF34 plays a critical role in cell cycle regulation, particularly at the G1/S and G2/M transitions . Experimental evidence supporting this relationship includes:

• Following temperature shift, tif34-ts mutants accumulate primarily (60%) as unbudded cells or cells with small buds (characteristic of G1), with an additional 35% arresting with larger buds (characteristic of G2/M) .

• Cell synchronization experiments demonstrate specific requirements:

  • When synchronized with α-factor (G1 arrest), tif34-ts cells cannot exit G1 at restrictive temperature

  • When synchronized with nocodazole (G2/M arrest), only a small proportion of tif34-ts cells can exit G2/M

  • When synchronized with hydroxyurea (early S phase), tif34-ts cells can complete DNA replication

These findings indicate that TIF34's role in translation is particularly critical at these cell cycle transitions, likely due to the requirement for synthesis of specific cell cycle regulatory proteins .

How does TIF34 function affect mating and pheromone response in yeast?

Temperature-sensitive TIF34 mutants show defects in mating and α-factor response at restrictive temperature, establishing a link between translation initiation and mating signaling pathways . To investigate this phenotype, researchers can:

• Quantify mating efficiency using standard quantitative mating assays with tif34-ts mutants and wild-type strains at permissive and restrictive temperatures .

• Assess α-factor response by:

  • Examining morphological changes (shmoo formation) by microscopy

  • Measuring induction of pheromone-responsive genes using reporter constructs like FUS1-lacZ

  • Analyzing cell cycle arrest in response to α-factor

• Determine the timing of the defect by shifting cultures to restrictive temperature at different times relative to α-factor addition .

The requirement for TIF34 in mating links general protein synthesis to the ability of yeast cells to respond to mating pheromones and successfully mate .

Which proteins directly interact with TIF34 in the eIF3 complex?

TIF34 directly interacts with at least two other components of the eIF3 complex:

• PRT1: A previously characterized eIF3 subunit that was identified as a TIF34 interactor through yeast two-hybrid screening .

• A novel protein of 33 kDa (eIF3-p33/TIF35): This protein contains an RNA binding domain and was also identified through yeast two-hybrid screening . It was subsequently demonstrated to be part of the eIF3 complex.

The direct interaction between TIF34 and p33 was further confirmed by co-immunoprecipitation experiments using epitope-tagged versions of both proteins . Interestingly, deletion of the RNA binding domain in p33 did not affect its association with TIF34, indicating that this domain is not required for the protein-protein interaction .

The following table summarizes key features of known TIF34 interacting proteins:

What methodological approaches can be used to identify and characterize TIF34 interactions?

Several complementary approaches are effective for studying TIF34 interactions:

• Yeast two-hybrid system: This method identified direct binary interactions between TIF34 and other proteins . When implementing this system:

  • Use full-length TIF34 as bait to screen cDNA libraries

  • Include appropriate controls to rule out auto-activation

  • Confirm interactions by independent methods

• Co-immunoprecipitation: This technique confirms interactions in vivo . Researchers should:

  • Construct expression plasmids for epitope-tagged proteins (e.g., myc-tagged TIF34)

  • Express proteins in yeast under appropriate promoters (e.g., GAL1)

  • Immunoprecipitate with antibodies against the tag

  • Detect co-precipitating proteins by Western blotting

• Domain mapping: Creating deletion mutants helps determine which protein domains are essential for interactions . For example, p33ΔC (lacking the RNA binding domain) still interacts with TIF34 .

• Functional complementation: Testing whether overexpression of an interacting protein can rescue defects caused by mutations in TIF34 provides functional evidence for the interaction's biological significance . The study showed that overexpression of p33 complements the growth defect of tif34-ts mutants .

How can suppressor screens be used to identify functional partners of TIF34?

Genetic suppressor screens can identify genes that, when overexpressed, can compensate for defects in TIF34 function. The methodology involves:

• Transforming tif34-ts mutant strains with a high-copy yeast genomic library (e.g., YEp13-based)

• Selecting transformants that grow at semi-restrictive temperature (e.g., 35°C for tif34-ts31)

• Isolating plasmids from suppressor colonies and retransforming to confirm the suppression phenotype

• Sequencing the inserts to identify the suppressor genes

In the study, this approach identified 41 suppressor colonies from approximately 40,000 transformants . While most (36/41) contained the TIF34 gene itself, other suppressors were identified, including p33/TIF35 . The finding that overexpression of p33 suppresses tif34-ts defects provides functional evidence for their interaction and suggests that increased levels of p33 can compensate for reduced TIF34 function .

How can temperature-sensitive TIF34 mutants be generated and characterized?

Temperature-sensitive (ts) TIF34 mutants can be generated through the following protocol:

• PCR amplification of the TIF34 coding region under mutagenic conditions:

  • Use manganese chloride (0.5 mM MnCl2) to increase error rate

  • Use unbalanced dNTP concentrations (1 mM each dGTP, dTTP, dCTP; 0.2 mM dATP)

  • Use appropriate primers containing restriction sites (e.g., BamHI)

• Clone the mutagenized PCR products into a yeast expression vector (e.g., pTCA)

• Transform the plasmid library into a strain with the endogenous TIF34 gene deleted (tif34Δ) but kept viable by a wild-type TIF34 plasmid with a URA3 marker

• Screen for transformants that grow at permissive temperature (24°C) but not at restrictive temperature (37°C)

• Counter-select on 5-FOA plates to remove the wild-type TIF34 URA3 plasmid

Characterization of ts mutants should include:

• Growth assays at different temperatures (24°C, 30°C, 37°C) to determine the severity of the phenotype

• Cell cycle analysis using FACS after synchronization with α-factor, hydroxyurea, or nocodazole

• Polysome profile analysis to assess effects on translation initiation

• Protein synthesis measurements using radioactive pulse labeling

• Mating efficiency and pheromone response assays

What methods can be used to analyze polysome profiles in TIF34 mutants?

Polysome profile analysis is a powerful technique to assess the role of TIF34 in translation initiation. The method involves:

• Preparation:

  • Grow yeast cultures (wild-type and tif34-ts mutants) at permissive temperature

  • Transfer half of each culture to restrictive temperature for a specific time (e.g., 30 minutes)

  • Add cycloheximide to freeze ribosomes on mRNAs

  • Prepare cell extracts under conditions that preserve polyribosomes

• Sucrose gradient centrifugation:

  • Layer extracts onto 7-47% sucrose gradients

  • Centrifuge at high speed (e.g., 39,000 rpm) for 2.5 hours

  • Collect fractions while monitoring absorbance at 254 nm

• Analysis:

  • Compare the polysome to monosome (P/M) ratio between wild-type and mutant strains

  • A decreased P/M ratio indicates defective translation initiation

The study demonstrated that tif34-ts mutants show a strong reduction in the P/M ratio within 30 minutes of shifting to 37°C, indicating a rapid defect in translation initiation . This technique provides direct evidence for TIF34's role in translation initiation rather than elongation or termination.

How can the effects of TIF34 mutations on cell cycle progression be analyzed?

To analyze the effects of TIF34 mutations on cell cycle progression, researchers can employ these methodological approaches:

• Cell synchronization followed by FACS analysis:

  • Synchronize cells in G1 using α-factor (5-10 μg/ml for 2-3 hours)

  • Synchronize cells in early S phase using hydroxyurea (200 mM)

  • Synchronize cells in G2/M using nocodazole (15 μg/ml)

  After synchronization:

  • Release cells at permissive or restrictive temperature

  • Collect samples at different time points

  • Fix cells, stain DNA with propidium iodide

  • Analyze DNA content by flow cytometry

• Morphological analysis:

  • Fix cells with formaldehyde

  • Count the proportion of unbudded, small-budded, and large-budded cells using microscopy

  • A predominance of unbudded or small-budded cells indicates G1 arrest

  • An accumulation of large-budded cells suggests G2/M arrest

• Cell cycle marker analysis:

  • Western blotting for cyclins or other cell cycle-regulated proteins

  • Assays for CDK activity

  • Monitoring specific phosphorylation events that mark cell cycle transitions

The study found that tif34-ts cells primarily accumulate in G1 (60% as unbudded or small-budded cells) with an additional 35% arresting with larger buds (G2/M) after 3 hours at restrictive temperature .

What is the significance of the WD-40 repeats in TIF34?

The five WD-40 repeats in TIF34 are likely crucial for its protein-protein interactions within the eIF3 complex . These structural features deserve special consideration:

• Structural implications:

  • WD-40 repeats typically form a β-propeller structure that provides multiple interaction surfaces

  • Crystallographic analysis of similar domains in other proteins (like Gβ) shows that these repeats create a propeller-like configuration that forms a platform for protein interactions

• Functional significance:

  • The WD-40 repeats likely mediate the direct interactions between TIF34 and other eIF3 components (PRT1 and p33/TIF35)

  • These interactions appear essential for assembly and stability of the eIF3 complex

• Experimental approaches to study WD-40 functions:

  • Site-directed mutagenesis targeting conserved residues within individual repeats

  • Creation of chimeric proteins where WD-40 repeats are exchanged with those from other proteins

  • Deletion or insertion analysis to determine which repeats are essential

  • Structural studies (X-ray crystallography or cryo-EM) of TIF34 alone or in complex with interacting partners

Different tif34-ts alleles likely contain mutations affecting different aspects of WD-40 repeat function, explaining their varying severity in phenotypic assays .

How does TIF34 contribute to eIF3 complex assembly and maintenance?

Evidence suggests TIF34 plays a crucial role in the assembly and maintenance of the eIF3 complex:

• Previous research has shown that depletion of TIF34 results in degradation of all eIF3 subunits at a rate much faster than their normal turnover, suggesting TIF34 is important for complex stability .

• The identification of direct interactions between TIF34 and two other eIF3 components (PRT1 and p33/TIF35) suggests TIF34 serves as a scaffold within the complex .

• The suppression of tif34-ts defects by overexpression of p33 suggests that increased levels of this interaction partner can stabilize partially functional TIF34 mutant proteins .

To further investigate this assembly role, researchers could:

• Use in vitro reconstitution experiments with purified components

• Perform order-of-addition experiments to determine the assembly pathway

• Use protein crosslinking approaches to capture intermediate complexes

• Apply quantitative proteomic approaches to monitor complex composition in different mutant backgrounds

What are potential future research directions for TIF34 studies?

Several promising research directions could enhance our understanding of TIF34:

• Structural biology approaches:

  • Determine the three-dimensional structure of TIF34 and its complexes with interacting partners

  • Map the binding interfaces between TIF34 and PRT1 or p33

  • Investigate conformational changes that might occur during translation initiation

• Systems biology approaches:

  • Identify the complete set of mRNAs whose translation is most affected by TIF34 inactivation using ribosome profiling

  • Integrate translational control by TIF34 with other cellular networks

  • Model the kinetics of translation initiation with varying levels of functional TIF34

• Regulatory mechanisms:

  • Investigate whether TIF34 is subject to post-translational modifications that regulate its function

  • Determine if TIF34 activity is regulated during stress responses or cell cycle transitions

  • Identify factors that interact transiently with TIF34 to modulate its function

• Comparative studies:

  • Analyze the function of TIF34 homologs in other organisms

  • Determine whether the roles in cell cycle progression and mating are conserved

  • Investigate whether the human homolog could complement yeast tif34 mutations

What are common challenges in generating temperature-sensitive TIF34 mutants?

Researchers frequently encounter these challenges when generating temperature-sensitive TIF34 mutants:

• Mutation efficiency:

  • Problem: Too few mutations lead to insufficient temperature-sensitive candidates

  • Solution: Optimize mutagenic PCR conditions by adjusting MnCl2 concentration (0.5-1.0 mM) and dNTP imbalance

• Screening efficiency:

  • Problem: Labor-intensive screening of many colonies

  • Solution: Use replica plating techniques with accurate temperature control; consider developing a high-throughput screening system

• Leaky phenotypes:

  • Problem: Some ts mutants show partial defects even at permissive temperature

  • Solution: Screen larger numbers of candidates to identify truly conditional mutants with wild-type behavior at permissive temperature

• Variable penetrance:

  • Problem: Inconsistent phenotypes between experiments

  • Solution: Integrate ts alleles into the genome at the native locus; ensure consistent growth conditions and precise temperature control

• Validation challenges:

  • Problem: Distinguishing direct from indirect effects of TIF34 inactivation

  • Solution: Analyze phenotypes at multiple time points after temperature shift; confirm protein levels by Western blotting; include appropriate controls in all experiments

What controls should be included in TIF34 protein interaction studies?

When studying TIF34 protein interactions, these essential controls should be included:

How can polysome profile analysis be optimized for TIF34 studies?

To obtain reliable and informative polysome profiles when studying TIF34 function:

• Sample preparation optimization:

  • Add cycloheximide (final concentration 100 μg/ml) directly to cultures before harvesting

  • Avoid temperature changes during cell collection and lysis

  • Use buffer conditions that preserve polysome integrity (include Mg2+ and avoid EDTA)

  • Perform lysis rapidly using methods that minimize ribonuclease contamination

• Gradient quality control:

  • Use a gradient maker to ensure reproducible gradients

  • Pre-chill gradients before use

  • Include control samples (wild-type at permissive and restrictive temperatures) in each experiment

  • Process all samples in parallel to ensure comparability

• Data analysis considerations:

  • Normalize traces to the 80S peak for comparison between samples

  • Calculate polysome to monosome (P/M) ratios as a quantitative measure

  • Analyze multiple biological replicates to ensure reproducibility

  • Consider collecting gradient fractions for subsequent RNA analysis to identify specific affected mRNAs

• Timing considerations:

  • Analyze profiles at multiple time points after temperature shift (e.g., 15, 30, 60 minutes)

  • This temporal analysis can distinguish direct effects of TIF34 inactivation from secondary consequences

The study demonstrated that tif34-ts mutants show a strong reduction in polysome to monosome ratio within 30 minutes of temperature shift, indicating a rapid and direct effect on translation initiation .