Recombinant Saccharomyces cerevisiae Prefoldin subunit 3 (PAC10)

What is the primary function of Prefoldin subunit 3 (PAC10) in Saccharomyces cerevisiae?

Prefoldin subunit 3, also known as PAC10, Gim2, or Pfd3, is a component of the hexameric prefoldin complex that assists in the folding of newly synthesized polypeptides produced on ribosomes. In eukaryotes including S. cerevisiae, the primary canonical function of prefoldin is to transfer cytoskeletal proteins, particularly actin and tubulin, to the TRiC/CCT chaperonin complex . This process is ATP-independent, classifying prefoldin as a molecular "holdase" similar to stress-inducible small heat shock proteins .

To study this function experimentally, researchers typically employ deletion mutants (e.g., pfd3Δ) and observe the resulting phenotypes related to cytoskeletal organization, which often manifest as defects in microtubule assembly and function. Comparative analysis of wild-type and mutant strains using microscopy techniques and protein quantification assays allows for comprehensive assessment of PAC10's impact on protein folding pathways.

How is PAC10 structurally organized and what domains are critical for its function?

PAC10 (Pfd3) is one of the six subunits that form the prefoldin complex, which has a distinctive jellyfish-like structure with tentacle-like coiled coils that capture unfolded proteins. Though not explicitly detailed in the provided search results, standard structural analysis methods such as X-ray crystallography and cryo-electron microscopy have been employed to characterize prefoldin's architecture.

For experimental characterization of functional domains, researchers typically use site-directed mutagenesis to create truncated or modified versions of PAC10, followed by complementation assays in pfd3Δ strains to assess which regions are essential for function. Co-immunoprecipitation experiments are also valuable for determining interaction domains with other prefoldin subunits and client proteins.

How is the expression of PAC10 regulated in different growth conditions?

The expression of PAC10 in S. cerevisiae appears to be responsive to environmental conditions, particularly stress conditions. Research indicates that Pfd3/PAC10 is involved in oxidative and osmotic stress-activated transcription , suggesting that its expression might be upregulated during these stress conditions to help manage protein quality control.

To study PAC10 expression regulation experimentally, researchers typically employ:

• Quantitative PCR to measure transcript levels under various conditions

• Western blotting with anti-PAC10 antibodies to quantify protein levels

• Reporter gene constructs (e.g., PAC10 promoter driving GFP expression) to visualize expression dynamics in live cells

• Chromatin immunoprecipitation (ChIP) to identify transcription factors that bind to the PAC10 promoter

What phenotypes are associated with PAC10 deletion in yeast?

Deletion of PAC10 (pfd3Δ) in S. cerevisiae leads to several observable phenotypes that reflect its role in protein folding and quality control. Based on studies of prefoldin components and related proteins, these phenotypes likely include:

• Cytoskeletal defects: Improper folding and degradation of tubulin and actin, leading to abnormal cell morphology and division

• Stress sensitivity: Increased vulnerability to environmental stressors, particularly oxidative and osmotic stress

• Transcriptional alterations: Changes in gene expression patterns, especially for stress-responsive genes

How does PAC10 cooperate with other molecular chaperones in the protein quality control network?

PAC10, as part of the prefoldin complex, functions within a sophisticated network of molecular chaperones that collectively maintain protein homeostasis. This network includes the TRiC/CCT chaperonin complex, the Hsp70/Hsp40 system, and others. The interaction between these components is dynamic and context-dependent.

Research demonstrates that prefoldin interacts with nascent polypeptide chains in the initial phase of folding and subsequently transfers them to TRiC/CCT in later stages . This process involves physical interactions between prefoldin subunits (including PAC10) and TRiC/CCT subunits, which facilitate the release of nascent chains from prefoldin and their ATP-dependent folding by TRiC/CCT .

To experimentally investigate these interactions, researchers can employ:

• Co-immunoprecipitation assays with tagged PAC10 to identify interacting partners

• Fluorescence resonance energy transfer (FRET) to visualize interactions in live cells

• In vitro reconstitution experiments with purified components to dissect the mechanism of substrate transfer

• Genetic interaction screens to identify functional relationships with other chaperone systems

What role does PAC10 play in transcriptional regulation beyond its chaperone function?

Beyond its canonical role in protein folding, PAC10 has been implicated in transcriptional processes. Research has shown that all prefoldin subunits, including PAC10, exhibit nucleo-cytoplasmic localization in S. cerevisiae , suggesting potential nuclear functions.

Specifically, PAC10 (Pfd3/Gim2) has been demonstrated to be involved in oxidative and osmotic stress-activated transcription . This function appears to be independent of its cytoplasmic role in actin and tubulin folding, representing a distinct biological activity.

To study this transcriptional role experimentally:

• Perform ChIP-seq to identify genomic regions associated with PAC10

• Use RNA-seq to compare transcriptional profiles between wild-type and pfd3Δ strains under various stress conditions

• Employ nuclear-cytoplasmic fractionation followed by western blotting to quantify nuclear PAC10 levels in response to different stimuli

• Create separation-of-function mutants that retain either the chaperone or transcriptional activities to dissect these dual roles

How do post-translational modifications affect PAC10 function and interactions?

While the provided search results don't specifically mention post-translational modifications (PTMs) of PAC10, this represents an important area for investigation. Based on research on related proteins, PAC10 may be subject to modifications such as phosphorylation, ubiquitination, or acetylation that regulate its activity, localization, or interactions.

Experimental approaches to investigate PAC10 PTMs include:

• Mass spectrometry analysis of purified PAC10 to identify modification sites

• Generation of site-specific mutants (e.g., phospho-mimetic or phospho-deficient) to assess functional consequences

• In vitro kinase or acetylation assays to identify modifying enzymes

• Proteomic analysis under different stress conditions to detect dynamic changes in modification patterns

What are the evolutionary implications of PAC10 conservation across species?

PAC10 shows significant conservation across species, indicating its fundamental importance in cellular function. For example, Mgr (a Drosophila protein) shows 37% amino acid identity with yeast PAC10 and 57% identity with human VBP1/Pfdn3/Gim2 . This conservation suggests that the basic mechanism of prefoldin-mediated protein folding has been maintained throughout eukaryotic evolution.

To study evolutionary aspects experimentally:

• Perform phylogenetic analysis of PAC10 sequences across diverse species

• Test cross-species complementation (e.g., can human PFDN3 rescue pfd3Δ phenotypes in yeast?)

• Compare interaction networks of prefoldin complexes in different organisms

• Identify species-specific modifications or adaptations that might reflect specialized functions

What are the optimal conditions for expressing recombinant PAC10 in S. cerevisiae?

Expression of recombinant PAC10 in S. cerevisiae requires consideration of several factors to ensure proper protein production and function. Based on general principles of recombinant protein expression in yeast and the specific characteristics of prefoldin subunits:

• Vector selection: Plasmids carrying auxotrophic markers (e.g., LEU2 for leucine auxotrophy complementation) are commonly used for selection . Both integrative and episomal vectors can be employed depending on the experimental requirements.

• Promoter choice:

  • Constitutive promoters (e.g., TEF1, GPD) for stable expression

  • Inducible promoters (e.g., GAL1/10) for controlled expression

• Growth conditions:

  • Temperature: 30°C is standard, but lower temperatures (20-25°C) may improve folding of complex proteins

  • Media composition: Defined media for precise control or complex media for higher biomass

• Tags and fusion proteins:

  • C-terminal tags are generally preferred to minimize interference with N-terminal targeting sequences

  • Common tags include His6, FLAG, or fluorescent proteins for visualization

For optimal expression while maintaining plasmid stability, a fed-batch cultivation strategy that maintains low concentrations of yeast extract (below 0.05 g/L) can provide plasmid-containing cells with a competitive advantage over plasmid-free cells .

How can researchers assess the functional integrity of recombinant PAC10?

Verifying that recombinantly expressed PAC10 is functionally active is crucial for experimental validity. Several complementary approaches can be employed:

• Complementation assays:

  • Transform pfd3Δ strains with plasmids expressing recombinant PAC10

  • Assess restoration of wild-type phenotypes (growth rate, stress resistance, cytoskeletal organization)

• Protein interaction assays:

  • Co-immunoprecipitation to verify interaction with other prefoldin subunits and client proteins

  • Size exclusion chromatography to confirm incorporation into the prefoldin complex

• Subcellular localization:

  • Fluorescence microscopy of tagged PAC10 to verify proper nucleo-cytoplasmic distribution

  • Fractionation studies to quantify distribution between cellular compartments

• Functional assays:

  • In vitro protein folding assays using purified components

  • Measurement of cytoskeletal protein stability and folding efficiency

What strategies can resolve contradictory data regarding PAC10 function?

Researchers may encounter contradictory results when studying PAC10, particularly given its dual roles in protein folding and transcriptional regulation. Strategies to resolve such contradictions include:

• Genetic background considerations:

  • Use isogenic strains for all comparisons

  • Validate key findings in multiple strain backgrounds

  • Consider the impact of suppressor mutations that might arise in deletion strains

• Separation of functions:

  • Design domain-specific mutations that disrupt specific functions

  • Use condition-specific assays that isolate individual functions

• Temporal resolution:

  • Employ time-course experiments to distinguish primary from secondary effects

  • Use rapid-induction or depletion systems (e.g., auxin-inducible degron) to observe immediate consequences

• Systematic validation:

  • Apply multiple orthogonal techniques to measure the same parameter

  • Use both in vivo and in vitro approaches to validate findings

  • Collaborate with laboratories using different methodologies

How can high-throughput approaches advance our understanding of PAC10?

Modern high-throughput technologies offer powerful tools to investigate PAC10 function comprehensively:

• Genome-wide genetic interaction screens:

  • Synthetic genetic array (SGA) analysis to identify genes that interact with PAC10

  • Quantitative assessment of genetic interactions to build functional networks

• Proteomics approaches:

  • Proximity labeling (BioID or APEX) to identify proteins in close physical proximity to PAC10

  • SILAC or TMT-based quantitative proteomics to assess global protein changes in pfd3Δ strains

• Transcriptomics:

  • RNA-seq analysis of wild-type vs. pfd3Δ strains under various conditions

  • NET-seq or similar approaches to assess direct impacts on transcription elongation

• High-content microscopy:

  • Automated imaging of cellular phenotypes in large-scale genetic or chemical perturbation experiments

  • Machine learning analysis of complex cellular phenotypes

How can insights from PAC10 research inform understanding of human disease?

While avoiding commercial applications as requested, the fundamental research on yeast PAC10 has significant implications for understanding human pathologies:

• Protein misfolding disorders:

  • The human homolog of PAC10 (PFDN3/VBP1) may play roles in preventing protein aggregation relevant to neurodegenerative diseases

  • Comparative studies between yeast and human systems can reveal conserved protective mechanisms

• Cytoskeletal disorders:

  • Given PAC10's role in tubulin and actin folding, insights may be relevant to human diseases involving cytoskeletal dysfunction

  • The phenotypes of mgr mutants in Drosophila, which show improper folding and degradation of tubulin in the absence of the Prefoldin complex , suggest potential disease mechanisms

• Stress response deficiencies:

  • PAC10's involvement in stress-activated transcription points to potential roles of human PFDN3 in stress adaptation

  • This may be relevant to conditions involving impaired cellular stress responses

What methodological advances in PAC10 research could be applied to other protein studies?

The experimental approaches developed for studying PAC10 can be valuable for investigating other proteins:

• Co-chaperone interaction networks:

  • Methods to dissect the cooperation between PAC10 and other chaperones can be applied to study other co-chaperone systems

  • This includes techniques for monitoring substrate transfer between different chaperone complexes

• Dual-function protein analysis:

  • Approaches to separate PAC10's chaperone and transcriptional functions can be adapted for other proteins with multiple cellular roles

  • This includes development of separation-of-function mutants and condition-specific assays

• Protein quality control assessment:

  • Assays developed to measure PAC10's impact on proteostasis can be used to investigate other components of protein quality control networks

  • This facilitates comprehensive understanding of cellular proteostasis mechanisms

What are the unexplored aspects of PAC10 function that warrant investigation?

Several aspects of PAC10 biology remain incompletely understood and represent promising directions for future research:

• Client specificity:

  • While PAC10 is known to be involved in actin and tubulin folding, the full spectrum of its client proteins remains undefined

  • Comprehensive identification of client proteins through proteome-wide approaches could reveal additional functional roles

• Regulatory mechanisms:

  • The factors controlling PAC10 expression, localization, and activity under different conditions require further characterization

  • This includes investigation of transcriptional, post-transcriptional, and post-translational regulatory mechanisms

• Structure-function relationships:

  • Detailed structural analysis of PAC10 within the prefoldin complex could provide insights into its mechanism of action

  • This could be achieved through advanced structural biology techniques such as cryo-EM

• Non-canonical functions:

  • Beyond its roles in protein folding and transcription, PAC10 may have additional functions that remain to be discovered

  • Unbiased approaches such as synthetic genetic interaction screens could reveal unexpected functional connections

How might emerging technologies advance PAC10 research?

New technologies are continuously expanding the experimental possibilities for studying proteins like PAC10:

• CRISPR-based approaches:

  • Base editing or prime editing for precise modification of endogenous PAC10

  • CRISPRi/CRISPRa for temporal control of PAC10 expression

• Single-molecule techniques:

  • Single-molecule FRET to directly observe PAC10 interactions with client proteins

  • Super-resolution microscopy to visualize PAC10 distribution and dynamics in unprecedented detail

• In situ structural biology:

  • Cryo-electron tomography to visualize PAC10-containing complexes in their native cellular environment

  • Integrative structural biology approaches combining multiple data types

• Computational approaches:

  • Molecular dynamics simulations to understand conformational changes in PAC10

  • Network analysis to position PAC10 within the broader cellular proteostasis system