Recombinant Saccharomyces cerevisiae Myosin-4 (MYO4), partial

What is Myosin-4 (MYO4) in Saccharomyces cerevisiae and why is it important for research?

Myosin-4 (MYO4) is a class V myosin motor protein in S. cerevisiae that plays essential roles in mRNA transport and localization. Unlike conventional muscle myosins, MYO4 functions as a monomeric motor protein that associates with adapter proteins to transport mRNA to specific cellular locations, particularly during asymmetric cell division.

When working with recombinant MYO4, researchers should consider the following methodological approaches:

• Use fluorescence microscopy with GFP-tagged MYO4 to visualize transport dynamics

• Employ immunoprecipitation with specific antibodies to identify interaction partners

• Conduct in vitro motility assays to assess motor function

S. cerevisiae provides an excellent model system for studying MYO4 because it allows for straightforward genetic manipulation and has a well-characterized genome, making it valuable for extrapolating results to higher eukaryotes including humans .

How does partial recombinant MYO4 differ from full-length MYO4 in functional studies?

Partial recombinant MYO4 typically includes only specific domains of interest, most commonly the motor domain or cargo-binding tail region. When conducting research with partial constructs, consider these methodological distinctions:

To properly interpret results:

• Always compare activity parameters between partial and full-length constructs

• Include domain-specific controls in binding assays

• Consider how domain truncation might affect protein folding and stability

• Document the exact amino acid sequences included in your partial construct

The use of partial constructs often provides cleaner experimental systems for specific questions but requires validation with full-length protein for biological relevance .

What expression systems are optimal for recombinant S. cerevisiae MYO4?

When designing expression systems for recombinant MYO4, consider these methodological approaches:

Homologous expression in S. cerevisiae:

• Use GAL1 promoter-based vectors for inducible expression

• Consider integrating constructs at the native locus for physiological expression levels

• TAP-tag or His-tag fusions facilitate purification while minimizing interference with function

Heterologous expression:

• Baculovirus-insect cell system provides high yields for functional myosins

• E. coli expression typically limited to individual domains due to size and complexity

The experimental design should follow systematic optimization:

• Clone MYO4 coding sequence into appropriate vectors with suitable tags

• Transform into compatible host strains

• Test multiple induction conditions (temperature, time, inducer concentration)

• Evaluate protein solubility and functionality through pilot purifications

• Scale up optimal conditions for preparative expression

Similar to the approach used for recombinant yeast proteins in immunotherapy research, purification protocols must be optimized to maintain protein folding and activity .

How should I design experimental controls when working with recombinant MYO4?

Essential controls for MYO4 functional studies:

When designing a randomized block experimental design, consider:

• Grouping experiments by protein preparation batch

• Randomizing treatment conditions within blocks

• Including technical and biological replicates

• Blinding analysis where possible

This approach minimizes the impact of batch-to-batch variability and controls for confounding variables that might influence MYO4 activity measurements .

What techniques can reveal MYO4's role in mRNA transport and localization?

To investigate MYO4's function in mRNA transport, employ these methodological approaches:

In vivo visualization methods:

• MS2-GFP system for tracking specific mRNAs in real-time

• Dual-color imaging with fluorescently tagged MYO4 and mRNA

• FRAP (Fluorescence Recovery After Photobleaching) to measure transport kinetics

Biochemical approaches:

• RNA immunoprecipitation (RIP) to identify MYO4-associated transcripts

• Proximity labeling (BioID/TurboID) to map the MYO4 interaction network

• In vitro reconstitution of the transport complex with purified components

Genetic manipulation strategies:

• CRISPR-Cas9 genome editing to create specific MYO4 variants

• Anchor-away techniques to conditionally relocalize MYO4

• Auxin-inducible degron for rapid protein depletion

These approaches share similarities with the experimental design principles used in Rad52-dependent DNA repair studies, where specific protein functions are isolated through systematic manipulation of the system components .

How can recombinant MYO4 be used to study cargo specificity and regulatory mechanisms?

To investigate the molecular basis of MYO4's cargo selection and regulation, implement these methodological approaches:

Cargo binding characterization:

• Generate a library of truncated MYO4 constructs to map binding regions

• Perform pull-down assays with potential cargo adapters (She2p, She3p)

• Use fluorescence polarization to measure binding affinities

• Employ hydrogen-deuterium exchange mass spectrometry to identify interaction interfaces

Regulatory mechanism investigation:

• Site-directed mutagenesis of potential phosphorylation sites

• In vitro kinase assays to identify regulatory modifications

• Phosphomimetic mutations (S→D or S→E) to study constitutive activation

• Cryo-EM structural analysis of MYO4 in different nucleotide states

When studying protein-protein interactions, control for non-specific binding by including:

• GST-only controls for GST-fusion proteins

• Pre-blocked beads in pull-down experiments

• Competition assays with unlabeled proteins

This systematic approach allows for detailed characterization of MYO4's interaction network and regulatory mechanisms, similar to approaches used in studies of S. cerevisiae as a model organism for conserved cellular pathways .

What are common challenges in recombinant MYO4 expression and how can they be overcome?

Recombinant expression of large motor proteins like MYO4 presents several challenges. Apply these methodological solutions:

Challenge: Low expression yields

• Optimize codon usage for the host organism

• Test different promoter strengths and induction conditions

• Co-express molecular chaperones (Hsp90, Hsp70, GroEL/ES)

• Use specialized host strains with enhanced protein expression capabilities

Challenge: Protein aggregation

• Reduce expression temperature (16-20°C)

• Include stabilizing additives (glycerol, low concentrations of non-ionic detergents)

• Express as fusion with solubility tags (MBP, SUMO)

• Test different buffer compositions during purification

Challenge: Proteolytic degradation

• Add protease inhibitor cocktails during all purification steps

• Use host strains deficient in specific proteases

• Identify and remove flexible regions prone to proteolysis

• Optimize purification speed and maintain low temperatures

Similar challenges are encountered when expressing recombinant yeast proteins for immunotherapy applications, where maintaining protein integrity is critical for proper function .

How should contradictory data regarding MYO4 function be analyzed and reconciled?

When faced with contradictory results in MYO4 research, apply these methodological approaches:

• Systematic analysis of experimental variables:

  • Compare buffer compositions and reaction conditions

  • Evaluate protein preparation methods (tags, purification strategies)

  • Assess expression systems and potential post-translational modifications

  • Consider isoform-specific differences

• Statistical approaches:

  • Perform meta-analysis of multiple independent studies

  • Use larger sample sizes to increase statistical power

  • Apply appropriate statistical tests for your experimental design

  • Calculate effect sizes rather than relying solely on p-values

• Validation through orthogonal methods:

  • Confirm key findings using different experimental techniques

  • Combine in vitro biochemical assays with in vivo functional studies

  • Employ both gain-of-function and loss-of-function approaches

  • Use CRISPR-Cas9 to create specific mutations at the endogenous locus

This approach to data reconciliation mirrors principles used in experimental design for controlled studies, where systematic variation of parameters helps identify the factors responsible for differing results .

How can single-molecule techniques advance our understanding of MYO4 mechanochemistry?

Single-molecule approaches provide unique insights into MYO4 function. Implement these methodological strategies:

Optical tweezers studies:

• Attach single MYO4 molecules to polystyrene beads

• Measure force generation during interaction with surface-immobilized actin filaments

• Determine step size, stall force, and force-velocity relationships

• Compare mechanical properties with other myosin classes

Single-molecule TIRF microscopy:

• Label MYO4 and actin with different fluorophores

• Track movement of individual MYO4 molecules along actin filaments

• Measure processivity, run length, and velocity

• Analyze the effect of load, nucleotide concentration, and binding partners

Data analysis considerations:

• Apply hidden Markov modeling to identify discrete states

• Use bootstrapping methods for statistical confidence intervals

• Implement drift correction algorithms for long-duration experiments

• Analyze dwell times to extract kinetic parameters

These advanced biophysical approaches provide quantitative insights into MYO4 function similar to techniques used to characterize other motor proteins in model organisms .

What genome editing approaches can be used to study MYO4 function in vivo?

CRISPR-Cas9 and other genome editing techniques offer powerful tools for studying MYO4. Implement these methodological strategies:

CRISPR-Cas9 editing for MYO4 modification:

• Design sgRNAs targeting specific regions of the MYO4 gene

• Prepare repair templates containing desired mutations with homology arms

• Transform cells with Cas9, sgRNA, and repair template

• Screen transformants for successful editing using sequencing

• Validate mutant phenotypes through functional assays

Endogenous tagging strategies:

• C-terminal tagging preserves native regulation but may affect cargo binding

• N-terminal tagging can interfere with motor function

• Internal tagging at flexible loops minimizes functional disruption

• Split fluorescent protein approaches for studying protein interactions

Inducible systems for temporal control:

• Auxin-inducible degron for rapid protein depletion

• Anchor-away system for conditional relocalization

• Temperature-sensitive alleles for conditional inactivation

When designing genome editing experiments, consider:

• Off-target effects by validating multiple independent clones

• Potential impacts on neighboring genes

• The need for complementation controls

• Phenotypic analysis at both cellular and molecular levels

This approach shares similarities with experimental design principles used in DNA repair studies, where specific mutations are introduced to study protein function in relevant biological contexts .