Recombinant Saccharomyces cerevisiae Vacuolar protein sorting-associated protein 62 (VPS62)

What is the function of Vacuolar protein sorting-associated protein 62 (VPS62) in Saccharomyces cerevisiae?

VPS62 (also known as Vps62p) is one of the novel proteins identified in genome-wide screens for components involved in the vacuolar protein sorting pathway in Saccharomyces cerevisiae. This protein participates in the carboxypeptidase Y (CPY) trafficking pathway, which is one of the main routes for hydrolase transport to the yeast vacuole. Research indicates that VPS62 plays a specific role in the proper targeting of proteins to the vacuole, with deletion mutants exhibiting missorting of CPY to the extracellular space rather than to the vacuole .

Methodologically, the function has been determined through reverse genetic approaches, particularly using homozygous diploid deletion strains and monitoring the secretion of CPY as a reporter. The CPY colony blot assay, as described by Roberts et al. (1991), is a primary technique used to identify the functional role of VPS genes including VPS62 .

How is VPS62 related to other VPS proteins in the vacuolar protein sorting pathway?

VPS62 belongs to a group of novel vacuolar protein sorting (VPS) genes designated as VPS61p-VPS75p. Within the broader classification of VPS proteins, VPS62 appears to function primarily in the carboxypeptidase Y pathway, unlike some other VPS proteins (such as MON1, MON2, VPS61, and VPS67) that function in both the CPY and alkaline phosphatase pathways .

To investigate these pathway relationships experimentally, researchers typically employ pulse-chase experiments tracking the processing of enzyme precursors to mature forms. The data from such experiments reveals the following relationships among newer VPS proteins:

Note: + indicates degree of involvement; - indicates minimal involvement .

What are the basic experimental techniques for studying VPS62 expression and localization?

Several fundamental techniques can be employed to study VPS62 expression and localization:

• Recombinant protein expression systems: VPS62 can be expressed in various host systems including E. coli, yeast, baculovirus, or mammalian cells, with the choice depending on research requirements for post-translational modifications and protein folding .

• Colony blot assays: The CPY colony blot assay is particularly useful for screening VPS gene function. This involves transferring yeast colonies to nitrocellulose membranes and performing immunoblotting with anti-CPY antibodies to detect CPY secretion .

• Fluorescence microscopy: GFP-tagging of VPS62 allows for visualization of its subcellular localization. This approach can confirm vacuolar membrane association or reveal other unexpected localizations.

• Subcellular fractionation: Differential centrifugation followed by western blotting can be used to determine the subcellular compartments where VPS62 is enriched.

For experimental protocols, the following parameters have proven effective:

• Expression conditions: 30°C for yeast hosts

• Purification yield: >90% purity achievable with appropriate chromatography steps

• Storage stability: Maintain in liquid form containing glycerol at -20°C for short-term or -80°C for long-term storage .

What methodological approaches can be used to investigate the specific role of VPS62 in the CPY pathway?

To investigate the specific role of VPS62 in the CPY pathway, several advanced methodological approaches can be employed:

• Reciprocal hemizygosity assay: This technique is particularly valuable for determining allele-specific contributions to phenotype in diploid organisms. The method involves creating hybrid strains where only one functional copy of VPS62 remains (either from one strain or another), allowing researchers to evaluate the contribution of different allelic variants to the sorting phenotype .

• Pulse-chase analysis: To track the kinetics of CPY transport:

  • Pulse cells with 35S-methionine for 5-10 minutes

  • Chase with excess unlabeled methionine

  • Collect timepoints (0, 5, 15, 30 minutes)

  • Immunoprecipitate CPY from both intracellular and extracellular fractions

  • Analyze by SDS-PAGE and autoradiography to visualize p1, p2, and mature forms of CPY

• Quantitative assessment of sorting defects: Using densitometry to measure the ratio of intracellular to extracellular CPY forms:

• Yeast two-hybrid analysis: To identify protein-protein interactions of VPS62 with other components of the sorting machinery. This can reveal functional complexes and position VPS62 within the hierarchical organization of the pathway .

How does VPS62 interact with the actin cytoskeleton during vacuolar protein sorting?

Several VPS proteins, including VPS62, have been implicated in actin cytoskeleton interactions. To investigate this relationship methodologically:

• Actin staining in vps62Δ mutants: Use rhodamine-phalloidin staining to visualize F-actin structures. Compare wild-type and vps62Δ strains at various temperatures (25°C, 30°C, and 37°C) to detect temperature-dependent defects.

• Co-immunoprecipitation studies: Use epitope-tagged VPS62 to identify physical interactions with actin or actin-related proteins such as Arp5p and Arp6p, which have been shown to affect vacuolar protein sorting .

• Latrunculin treatment: Disrupt the actin cytoskeleton using latrunculin and assess the effect on VPS62 localization and function. This can determine whether an intact actin cytoskeleton is required for VPS62 function.

Research findings suggest that some novel VPS proteins like VPS61p, VPS64p, and VPS67p display defects in the actin cytoskeleton at 30°C, suggesting an interplay between actin organization and vacuolar protein sorting . VPS62 may have similar connections that can be investigated using these approaches.

How do you design experiments to differentiate between direct and indirect effects of VPS62 deletion on protein sorting?

Differentiating between direct and indirect effects of VPS62 deletion requires carefully designed experimental approaches:

• Conditional expression systems: Use temperature-sensitive or galactose-inducible VPS62 alleles to allow for rapid inactivation or depletion of the protein. Monitor immediate versus delayed effects on sorting to distinguish direct from indirect consequences.

• Bypass suppression analysis:

  • Transform vps62Δ cells with a library of genes on high-copy plasmids

  • Screen for restoration of normal CPY sorting

  • Identify genes that can bypass the requirement for VPS62

  • This helps establish pathway relationships and order of action

• Cargo-specific analysis: Compare the trafficking of multiple vacuolar cargoes (CPY, ALP, PrA, CPS) in vps62Δ mutants to determine specificity:

• Epistasis analysis: Construct double mutants combining vps62Δ with deletions of genes functioning in defined steps of vacuolar transport (e.g., vps21Δ, pep12Δ, vps4Δ). Compare phenotypes to establish pathway relationships .

What statistical approaches are recommended for analyzing experimental data from VPS62 functional studies?

Robust statistical approaches are essential for proper interpretation of VPS62 functional studies:

How can population variability be addressed in yeast VPS62 research?

• Strain background considerations:

  • Use isogenic strains for all comparisons (BY4743, BY4739, BY4742)

  • When comparing results across studies, account for strain background differences

  • For genetic screens, consider using Diversity Outbred yeast strains to capture genetic diversity

• Phenotypic variation quantification:

  • Measure distribution of phenotypes across single cells rather than population averages

  • Apply flow cytometry to quantify cell-to-cell variation in protein sorting

  • Implement single-cell tracking to detect heterogeneity in response

• Experimental design strategies:

  • Implement minimax temporal designs to account for time-varying factors

  • Use adaptive experimental designs to optimize detection of variability

  • Characterize phenotypic distributions rather than simple means

• Statistical approaches for heterogeneity:

  • Apply variance component analysis to distinguish technical from biological variation

  • Implement mixed-effect models to account for batch and experimental variation

  • Consider Bayesian hierarchical modeling for integrating variable data sources

What are the key quality control parameters when working with recombinant VPS62?

When working with recombinant VPS62, several quality control parameters should be monitored to ensure experimental reliability:

• Protein purity assessment:

  • SDS-PAGE analysis: Should demonstrate >90% purity

  • Mass spectrometry validation of intact protein mass

  • N-terminal sequencing to confirm proper processing

• Functional validation:

  • Binding assays to known interaction partners

  • Complementation of vps62Δ phenotypes when reintroduced

  • In vitro assays relevant to hypothesized molecular function

• Stability monitoring:

  • Differential scanning fluorimetry to assess thermal stability

  • Size-exclusion chromatography to detect aggregation

  • Appropriate storage conditions: Liquid containing glycerol at -20°C or -80°C for extended storage

  • Avoid repeated freeze-thaw cycles

• Expression system considerations:

  • Expression host selection (E. coli, yeast, baculovirus, mammalian cells) based on experimental requirements

  • Post-translational modification analysis if expressed in eukaryotic systems

  • Codon optimization for the expression host

How should researchers design experiments to investigate VPS62 interactome?

Investigating the protein interaction network (interactome) of VPS62 requires multiple complementary approaches:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Express epitope-tagged VPS62 (e.g., TAP-tag, FLAG-tag) in S. cerevisiae

  • Perform single-step or tandem affinity purifications

  • Analyze co-purifying proteins by mass spectrometry

  • Use SILAC or TMT labeling for quantitative comparison

  • Include appropriate controls (untagged strains, irrelevant tagged proteins)

• Proximity-based labeling approaches:

  • Fuse VPS62 to BioID or APEX2 enzymes

  • Allow in vivo biotinylation of proximal proteins

  • Purify biotinylated proteins and identify by mass spectrometry

  • This detects transient and stable interactions in the native cellular environment

• Yeast two-hybrid screening:

  • Use VPS62 as bait to screen against genomic or cDNA prey libraries

  • Validate positive interactions through secondary assays

  • Map interaction domains through deletion constructs

• Co-localization studies:

  • Employ fluorescently tagged VPS62 and candidate interactors

  • Use high-resolution microscopy to assess spatial overlap

  • Apply FRET or BiFC techniques to confirm direct interactions

From existing studies, potential VPS62 interactions should be investigated with:

• Other VPS pathway components (especially VPS61-VPS75)

• Actin-related proteins (Arp5p, Arp6p)

• GTP-binding proteins involved in vesicle trafficking (Arl1p, Arl3p)

What experimental design is optimal for studying contradictions in VPS62 phenotypic data?

Contradictions in phenotypic data are common in complex biological systems. To systematically address contradictions in VPS62 research:

• Integrated analysis approach:

  • Employ meta-analysis techniques for quantitative phenotypes

  • Use Bayesian network modeling to reconcile conflicting data

  • Implement decision tree frameworks to guide interpretation of inconsistent results

How can advanced imaging techniques be applied to study VPS62 dynamics and localization?

Advanced imaging techniques offer powerful approaches to understand VPS62 dynamics and localization:

• Live-cell imaging methodologies:

  • Express VPS62-GFP/mCherry fusion under native promoter

  • Use spinning disk confocal microscopy for fast acquisition

  • Apply TIRF microscopy to visualize events near the plasma membrane

  • Implement photoactivatable or photoconvertible fluorophores (PA-GFP, mEos) to track protein subpopulations

• Super-resolution microscopy approaches:

  • STED microscopy: Achieve 50-70 nm resolution in yeast cells

  • PALM/STORM: Single-molecule localization for 20-30 nm resolution

  • SIM: Structured illumination for 100 nm resolution with less photodamage

  • These techniques can resolve subdomains within organelles that may be specifically enriched for VPS62

• Correlative light and electron microscopy (CLEM):

  • Locate VPS62-tagged structures by fluorescence

  • Examine the same structures at ultrastructural resolution by EM

  • This is particularly valuable for characterizing novel compartments

• Advanced analysis techniques:

  • Particle tracking for dynamic studies

  • Fluorescence recovery after photobleaching (FRAP) for mobility assessment

  • Single-particle tracking for diffusion coefficient calculation

  • Object-based colocalization for interaction studies with other proteins

When implementing these techniques, consider:

• Minimizing phototoxicity through reduced laser power or oxygen scavengers

• Appropriate controls for fusion protein functionality

• Statistical analysis of dynamic data (MSD plots, diffusion coefficients)

• Integration with complementary biochemical approaches

How can understanding VPS62 function contribute to broader research on vesicular trafficking systems?

Understanding VPS62 function has several implications for broader research on vesicular trafficking:

• Comparative analysis across species:

  • Identify VPS62 homologs in other organisms to establish evolutionarily conserved functions

  • Compare phenotypes of VPS62 disruption across model systems

  • Investigate whether higher organisms employ similar mechanisms for specialized trafficking

• Integration with systems biology approaches:

  • Position VPS62 within the larger network of trafficking pathways

  • Apply computational modeling to predict system-level consequences of VPS62 dysfunction

  • Use network analysis to identify critical nodes in vesicular trafficking systems

• Extension to disease-relevant models:

  • Several human diseases are linked to defects in vacuolar/lysosomal sorting

  • Determining whether VPS62 homologs are implicated in human disease

  • Development of yeast-based models for trafficking-related disorders

• Application to biotechnology:

  • Engineering improved secretion systems through manipulation of VPS pathways

  • Development of yeast strains with modified trafficking for enhanced recombinant protein production

  • The understanding gained from VPS62 research could enhance recombinant protein expression systems

What methodological approaches are needed to resolve contradictory findings about VPS62 function?

Resolving contradictory findings requires systematic approaches:

• Standardization of experimental conditions:

  • Develop consensus protocols for VPS62 functional assays

  • Create reference strain collections available to the research community

  • Establish minimal reporting requirements for experimental details

• Multi-laboratory validation studies:

  • Implement collaborative projects to test key findings across different laboratories

  • Use identical reagents and protocols to eliminate technical variability

  • Apply statistical meta-analysis techniques to integrate results

• Integration of diverse experimental approaches:

  • Combine genetic, biochemical, and imaging approaches

  • Develop computational frameworks for integrating heterogeneous datasets

  • Apply Bayesian techniques to weight evidence based on methodological strength

• Formal contradiction detection frameworks:

  • Implement structured approaches as outlined in source :

    • Systematic documentation of evidence

    • Classification of contradiction types (local vs. global)

    • Analysis of contradiction scope and dependencies

• Advanced statistical methods:

  • Apply methods developed for adaptive experiments as described in source

  • Use sample splitting techniques to validate findings

  • Implement sensitivity analyses to identify factors driving contradictory results

How can genomic screening approaches be optimized to discover new aspects of VPS62 function?

Building on the successful genomic screening approaches that originally identified VPS62 , several advanced methodologies can be employed:

• Synthetic genetic array (SGA) analysis:

  • Cross vps62Δ with the entire yeast deletion collection

  • Identify synthetic lethal/sick interactions

  • Discover genetic suppressors

  • Map the genetic interaction network surrounding VPS62

• CRISPR-based screening approaches:

  • Apply CRISPR interference (CRISPRi) for partial loss-of-function phenotypes

  • Use CRISPR activation (CRISPRa) to identify genes that, when overexpressed, modify vps62Δ phenotypes

  • Implement multiplexed CRISPR screening for combinatorial genetic perturbations

• Chemical-genetic profiling:

  • Screen vps62Δ against libraries of small molecules

  • Identify compounds that specifically enhance or suppress vps62Δ phenotypes

  • Use these compounds as chemical probes to dissect VPS62 function

• Quantitative phenotyping platforms:

  • Implement high-content microscopy screening

  • Apply automated image analysis to quantify multiple phenotypic parameters

  • Use the PHENotyping On Solid media (PHENOS) platform for growth-based screens

• Integration with genome-wide datasets:

  • Correlate genetic interactions with protein-protein interaction data

  • Analyze transcriptome responses to VPS62 deletion

  • Examine proteome-wide changes in protein localization or abundance

For optimal results, these approaches should be combined with appropriate statistical methods and experimental designs as outlined in source .

How can researchers leverage recombinant VPS62 for structural studies?

Structural studies of VPS62 would provide valuable insights into its molecular function. Methodological approaches include:

• Optimized protein expression and purification:

  • Test multiple expression systems (E. coli, yeast, insect cells)

  • Implement multi-step purification protocols including affinity, ion exchange, and size exclusion chromatography

  • Achieve >95% purity and monodispersity for structural studies

  • Consider expression of domains separately if full-length protein proves challenging

• Protein crystallization approaches:

  • Screen extensive crystallization conditions

  • Apply surface entropy reduction through targeted mutations

  • Consider co-crystallization with binding partners or ligands

  • Use crystallization chaperones such as antibody fragments

• Cryo-electron microscopy (cryo-EM):

  • Particularly valuable if VPS62 forms part of a larger complex

  • Optimize sample preparation to achieve uniform particle distribution

  • Consider GraFix method for stabilizing complexes

  • Implement computational approaches for heterogeneity analysis

• Hybrid structural approaches:

  • Combine X-ray crystallography of domains with cryo-EM of full complexes

  • Integrate small-angle X-ray scattering (SAXS) for solution structure

  • Use NMR for dynamic regions and interaction interfaces

• Computational structure prediction:

  • Apply AlphaFold2 or RoseTTAFold for initial structural models

  • Validate predictions through targeted experimental approaches

  • Use molecular dynamics simulations to explore conformational states

Understanding the structural features of VPS62 would provide mechanistic insights into its role in vacuolar protein sorting and potentially reveal unexpected functions or interaction capabilities.