Recombinant Saccharomyces cerevisiae Vesicle-associated membrane protein-associated protein SCS22 (SCS22), partial

What is the primary function of SCS22 in Saccharomyces cerevisiae?

SCS22 is a vesicle-associated membrane protein (VAP) that primarily functions as an endoplasmic reticulum-plasma membrane (ER-PM) tethering protein. It works in conjunction with SCS2, another VAP family protein, to establish and maintain contact sites between the ER and PM. These contact sites are crucial for various cellular processes including lipid transfer, calcium signaling, and cell polarity establishment. Research indicates that SCS22 plays a secondary role compared to SCS2, which is the dominant ER-PM tethering protein in yeast cells. The functional differences between these two proteins appear to be determined by their intrinsic structural properties, particularly within their MSP (major sperm protein) domains .

How does the expression level of SCS22 compare to SCS2 in wild-type yeast?

SCS22 is expressed at significantly lower levels compared to SCS2 in wild-type yeast cells. This differential expression contributes to their distinct roles in ER-PM tethering, with SCS2 playing the dominant role. Studies have demonstrated that when the open reading frames of SCS2 and SCS22 are swapped at their genomic loci, ER-PM contact formation is compromised, suggesting that the natural expression levels of these proteins are optimized for their respective functions . This differential expression is part of the regulatory mechanism that ensures appropriate ER-PM contact site formation under normal physiological conditions.

What expression systems are most suitable for producing recombinant SCS22 in yeast?

For recombinant expression of SCS22 in Saccharomyces cerevisiae, several vector systems can be employed:

For fundamental characterization studies of SCS22, YCp vectors might be suitable due to their stability and expression levels that more closely approximate physiological conditions. For studies requiring higher protein yields, YEp vectors would be more appropriate. If stable, long-term expression is needed, YIp vectors that integrate multiple copies (up to 15-20) have been developed for overexpressing specific genes . The choice of promoter is also critical, with options including inducible (e.g., ADH2, SUC2) or constitutive (e.g., GAPDH) promoters depending on experimental requirements .

What reference genes should be used when analyzing SCS22 expression by RT-qPCR?

When analyzing SCS22 expression by RT-qPCR in Saccharomyces cerevisiae, it is essential to select appropriate reference genes for accurate normalization. Recent research has identified several stable reference genes suitable for dynamic gene expression studies in yeast:

• For experiments involving glucose availability perturbations: TPI1, FBA1, CDC19, and ACT1 have been identified as the most stable reference genes .

• For nitrogen/ammonium availability studies: FBA1, TDH3, CCW12, and ACT1 are recommended .

These newly identified reference genes significantly outperform the traditional sole use of common reference genes in determining dynamic transcriptional responses. Using multiple reference genes from these sets is strongly recommended for robust normalization. When designing an experiment to analyze SCS22 expression, researchers should select at least three reference genes from the appropriate set based on their experimental conditions, and confirm their stability under the specific experimental parameters being used .

How do the structural differences between SCS22 and SCS2 MSP domains affect their functions?

The MSP (major sperm protein) domains of SCS22 and SCS2 exhibit critical structural differences that directly influence their function as ER-PM tethers. Research has revealed that specific lysine residues (K36/K38/K43) in the VAP consensus sequence (VCS) within the MSP domain are essential for ER-PM tethering capacity. Mutations of these residues (K36/38/43A) in either protein abolish their ability to form ER-PM contacts, regardless of their expression levels .

Comparative functional analyses demonstrate that SCS2 consistently displays higher plasma membrane affinity than SCS22 when expressed at similar levels. This is evidenced by:

• Higher cortex-to-nuclear envelope (cortex-to-NE) ratios for SCS2 compared to SCS22

• Slower fluorescence recovery after photobleaching (FRAP) for SCS2, indicating stronger PM association

• More efficient restoration of ER-PM contacts in scs2Δscs22Δ cells when SCS2 is expressed

When chimeric variants were constructed by swapping MSP domains between SCS2 and SCS22, the PM binding properties correlated with the origin of the MSP domain, confirming that the intrinsic structural features of these domains are the primary determinants of their differential PM affinity . These findings provide critical insights for researchers designing experiments to manipulate SCS22 function or to engineer synthetic ER-PM tethers with specific properties.

What protein-protein interactions does SCS22 engage in, and how do they compare to SCS2 interactions?

SCS22 engages in distinct protein-protein interactions that differ significantly from those of SCS2, despite their shared role as ER-PM tethers. Interactome analyses using both full-length and transmembrane domain-truncated variants (SCS22N and SCS2N) have revealed divergent binding preferences:

Notably, SCS22 shows interaction with SCS2 itself, as confirmed by Co-immunoprecipitation . This suggests potential heteromeric complex formation that may have functional significance in regulating ER-PM contact sites.

The differential binding affinities explain several observed phenotypes, including the reduced capacity of SCS22 to establish ER-PM contacts at cell ends compared to SCS2. This has important implications for experimental design when studying either protein, as manipulating one may have indirect effects through their interaction network. Researchers should consider these interaction profiles when designing experiments to investigate the specific functions of SCS22 or when using SCS22 as a tool to study ER-PM contacts .

What methodologies are most effective for visualizing and quantifying SCS22 localization and dynamics?

The visualization and quantification of SCS22 localization and dynamics require sophisticated imaging techniques and quantitative analysis approaches. Based on current research practices, the following methodologies have proven most effective:

• Fluorescence microscopy with quantitative metrics:

  • Cortex-to-Nuclear Envelope (cortex-to-NE) ratio analysis provides a quantitative measure of SCS22's ER-PM localization efficiency

  • This approach allows comparison of different mutants or conditions and enables statistical analysis of localization patterns

• Fluorescence Recovery After Photobleaching (FRAP):

  • FRAP analysis along the cell cortex provides crucial information about SCS22 mobility and its interaction strength with the PM

  • SCS22 typically shows faster recovery than SCS2, indicating weaker PM association

  • Protocol recommendation: Photobleach a small region of the cortical ER and monitor fluorescence recovery over 60-120 seconds with images captured every 2-5 seconds

• Chimeric protein analysis:

  • Construction of chimeric proteins by swapping domains between SCS22 and SCS2 enables precise determination of which protein regions are responsible for specific localization and dynamics properties

  • This approach requires careful design of fusion points to avoid disrupting protein folding

• Point mutation analysis:

  • Introduction of specific mutations (e.g., K36/38/43A in the MSP domain) to assess the contribution of individual amino acids to protein localization and function

  • This method is particularly valuable for structure-function analyses

When implementing these methodologies, it is crucial to use appropriate controls and to normalize expression levels when comparing different proteins or mutants, as expression level differences can significantly impact localization patterns and dynamics measurements.

How can CRISPR-Cas9 be optimized for generating SCS22 mutants in Saccharomyces cerevisiae?

Optimizing CRISPR-Cas9 for generating SCS22 mutants in Saccharomyces cerevisiae requires careful consideration of several factors to ensure high efficiency and specificity:

• Guide RNA (gRNA) design:

  • Select target sites with minimal off-target effects using algorithms specifically optimized for yeast genomes

  • Avoid regions with secondary structures that might impair Cas9 binding

  • Target the N-terminal region of SCS22 for full or partial knock-outs, or specific functional domains (e.g., MSP domain) for precise mutations

• Delivery system optimization:

  • For transient expression, use a two-plasmid system with Cas9 on one plasmid and gRNA on another

  • For stable expression, integrate Cas9 into the genome under an inducible promoter (e.g., GAL1) and deliver gRNA on a plasmid

  • Consider using YEp vectors for higher expression or YIp vectors for stable, consistent expression

• Repair template design:

  • For precise mutations (e.g., K36/38/43A in the MSP domain), design homology-directed repair (HDR) templates with:

    • At least 50 bp homology arms on each side of the cut site

    • Silent mutations in the PAM site to prevent re-cutting

    • Additional silent mutations to introduce restriction sites for screening

• Transformation and selection protocol:

  • Optimize transformation efficiency using lithium acetate/PEG method with heat shock

  • For selection, utilize auxotrophic markers (URA3, TRP1, HIS3, or LEU2) rather than antibiotic resistance when possible

  • Consider using the CRISPR-mediated marker-free approach for seamless editing

• Verification strategies:

  • Design PCR primers for initial screening

  • Confirm mutations by sequencing

  • Verify protein expression and localization using fluorescent tagging and microscopy

  • Validate functional changes using cortex-to-NE ratio analysis and FRAP

This optimized CRISPR-Cas9 approach enables the creation of precise SCS22 mutants for detailed structure-function studies, facilitating deeper understanding of its role in ER-PM tethering and interactions with partner proteins.

What role does SCS22 play in PI4P homeostasis and how does this compare to SCS2?

SCS22 contributes to phosphatidylinositol 4-phosphate (PI4P) homeostasis at the plasma membrane, though its role appears to be secondary to that of SCS2. Current research reveals several key aspects of this function:

• Restoration of PM PI4P levels:

  • In scs2Δscs22Δ double deletion cells, PI4P levels at the plasma membrane are compromised

  • Re-expression of either SCS2 or SCS22 can restore these levels, though SCS2 does so more efficiently

  • This restoration requires a functional MSP domain, as mutations of key lysine residues (K36/38/43A) abolish this ability

• Interaction with PI4P regulatory proteins:

  • Both SCS22 and SCS2 interact with Sec14 family proteins (Csr102 and Pdr16) which contribute to PI4P homeostasis

  • Interestingly, SCS22 shows higher affinity for Csr102 compared to SCS2 when expressed at comparable levels

  • This suggests that while SCS22 may bind regulatory proteins more effectively, SCS2's greater abundance and stronger PM association make it the dominant player in PI4P regulation

• Mechanism of action:

  • Evidence suggests that direct binding of anionic phospholipids by the MSP domain may be a primary driver of VAP-mediated ER-PM contact formation, rather than protein-protein interactions

  • This is supported by experiments with an SCS2 variant anchored directly to the PM (SCS2-PM), which could restore PI4P levels in a manner dependent on its functional MSP domain

The data indicates a model where SCS22 and SCS2 both contribute to establishing ER-PM contacts that facilitate PI4P homeostasis, potentially by creating microdomains where lipid transport or modification enzymes can function optimally. For researchers studying phosphoinositide signaling or membrane contact sites, understanding these distinct but complementary roles is essential for experimental design and interpretation.

What are the optimal vector and promoter combinations for differential expression studies of SCS22?

When designing differential expression studies for SCS22, selecting the appropriate vector and promoter combination is crucial for achieving the desired expression levels and patterns. Based on the characteristics of various expression systems in Saccharomyces cerevisiae, the following recommendations can be made:

For studies comparing SCS22 with SCS2, it is particularly important to normalize expression levels, as previous research has shown that expression levels significantly impact their functional differences. The PIL1 promoter has been successfully used to achieve high expression levels for comparative studies .

When selecting auxotrophic markers for these vectors, consider using URA3, TRP1, HIS3, or LEU2 based on your strain background. For experiments requiring multiple plasmids, ensure different markers are used for each plasmid to maintain selection pressure .

For N- or C-terminal tagging of SCS22 (e.g., with fluorescent proteins), careful design is necessary to avoid disrupting the transmembrane domain or functional MSP domain. Flexible linkers (e.g., GGGGS repeats) should be incorporated between SCS22 and any tags to minimize interference with protein function.

How should researchers design experiments to investigate SCS22-mediated ER-PM contact sites?

Designing robust experiments to investigate SCS22-mediated ER-PM contact sites requires a multifaceted approach that combines genetic manipulation, advanced imaging techniques, and functional assays. A comprehensive experimental design should include:

• Genetic manipulation strategies:

  • Generate single and double deletion strains (scs22Δ, scs2Δ, scs2Δscs22Δ) using CRISPR-Cas9 or traditional homologous recombination

  • Create complementation strains expressing wild-type or mutant versions of SCS22 (e.g., K36/38/43A) under native or controlled promoters

  • Develop chimeric constructs swapping domains between SCS22 and SCS2 to identify functional determinants

  • Consider using the swapping of genomic loci technique to investigate the impact of expression levels versus protein properties

• Imaging and quantification approaches:

  • Implement fluorescent protein tagging of SCS22 (ensuring tags don't disrupt function)

  • Establish quantitative metrics for ER-PM contacts:

    • Cortex-to-nuclear envelope (cortex-to-NE) ratio measurements

    • Percentage of PM with adjacent ER

    • Distribution pattern analysis (e.g., cell tips vs. lateral cortex)

  • Apply FRAP analysis to assess SCS22 mobility at the cortex

  • Consider super-resolution microscopy to characterize contact site morphology

• Functional assays:

  • Measure PI4P levels using specific biosensors (e.g., GFP-2xPHOsh2)

  • Assess the cortical recruitment of known interacting proteins (e.g., Csr102)

  • Examine the localization of other ER-PM tethers in SCS22 mutant backgrounds

  • Investigate phenotypic consequences of SCS22 manipulation (e.g., stress resistance, lipid homeostasis)

• Controls and validation:

  • Include artificial ER-PM tethers (e.g., TM-GFP-CSS Ist2) as positive controls

  • Use non-tether ER proteins (e.g., mCherry-ADEL, TM-GFP) as negative controls

  • Validate key findings using orthogonal methods (e.g., biochemical fractionation, electron microscopy)

  • Confirm expression levels of all constructs by Western blotting or qPCR

This comprehensive experimental approach will enable researchers to dissect the specific contributions of SCS22 to ER-PM contact site formation and function, while distinguishing its role from that of the more abundant SCS2 protein.

What are the best approaches for analyzing potential post-translational modifications of SCS22?

Analyzing post-translational modifications (PTMs) of SCS22 requires a systematic approach combining multiple complementary techniques. The following methods represent the current best practices for comprehensive PTM characterization:

• Mass Spectrometry-Based Approaches:

  • Tandem mass spectrometry (MS/MS) following immunoprecipitation of tagged SCS22

  • Multiple fragmentation methods (CID, ETD, HCD) to improve PTM identification

  • Targeted analysis using parallel reaction monitoring (PRM) for specific modifications

  • Quantitative proteomics to compare modification states under different conditions

  Protocol Note: For optimal SCS22 purification, use YEp vectors with strong promoters like ADH1 or GAL1 to achieve high expression yields . Consider both N- and C-terminal tags to ensure complete coverage of the protein.

• Site-Directed Mutagenesis Strategies:

  • Systematic mutation of potential modification sites (Ser, Thr, Tyr for phosphorylation; Lys for ubiquitination/acetylation)

  • Creation of phosphomimetic (S/T→D/E) and phospho-deficient (S/T→A) mutations

  • Evaluation of mutant phenotypes using cortex-to-NE ratio and FRAP analysis

• PTM-Specific Detection Methods:

  • Phosphorylation: Phos-tag SDS-PAGE followed by Western blotting

  • Ubiquitination: Immunoprecipitation under denaturing conditions with ubiquitin-specific antibodies

  • Glycosylation: Mobility shift assays with glycosidase treatments

  • General PTM screening: 2D gel electrophoresis to separate modified forms

• Dynamics and Regulation Analysis:

  • Time-course studies following stimuli that affect ER-PM contacts

  • Inhibitor studies targeting specific kinases, phosphatases, or other PTM enzymes

  • Analysis of PTM changes in response to cell stress or membrane perturbations

  Experimental Design Note: For dynamic studies, select appropriate reference genes for qPCR validation based on the specific experimental conditions, such as TPI1, FBA1, CDC19, and ACT1 for glucose-related studies .

• Functional Impact Assessment:

  • Comparison of wild-type and PTM-mutant SCS22 for:

    • Protein localization and dynamics (using fluorescence microscopy)

    • Interaction partner binding (using Co-IP or proximity labeling)

    • Ability to complement phenotypes in scs22Δ or scs2Δscs22Δ cells

    • Impact on PI4P levels and distribution

This comprehensive approach will not only identify the PTMs present on SCS22 but also provide insights into their functional significance in regulating ER-PM contact sites and membrane tethering activities.

What are common pitfalls when expressing and purifying recombinant SCS22, and how can they be addressed?

Expression and purification of recombinant SCS22 present several challenges due to its transmembrane domain and specific structural features. The following table outlines common pitfalls and their solutions:

For functional studies, consider expressing SCS22 using the same strong promoters used in previous studies (e.g., PIL1 promoter) to achieve comparable expression levels to published work . For structural studies, the MSP domain can be expressed separately from the transmembrane region, similar to approaches used with other VAP proteins.

When expressing full-length SCS22 for membrane studies, integration vectors (YIp) provide the most stable expression, while episomal vectors (YEp) with inducible promoters offer higher yields for biochemical studies . If SCS22 toxicity is observed, tightly controlled inducible systems with short induction periods are recommended.

How can researchers effectively analyze contradictory data regarding SCS22 function and interactions?

Analyzing contradictory data regarding SCS22 function and interactions requires a systematic approach to identify sources of variability and reconcile disparate findings. Researchers should implement the following structured methodology:

• Experimental Design Comparison:

  • Create a detailed comparison table of contradictory studies examining:

    • Strain backgrounds and genotypes

    • Expression systems and levels (vector types, promoters)

    • Tags and their positions (N- vs. C-terminal)

    • Experimental conditions (temperature, media, growth phase)

  • Identify key variables that differ between studies and systematically test their impact

• Expression Level Analysis:

  • Quantify SCS22 expression levels across different experimental systems

  • Test the hypothesis that functional observations are dose-dependent

  • Consider that SCS22 is naturally expressed at lower levels than SCS2, and artificial overexpression may create non-physiological effects

• Interaction Context Evaluation:

  • Assess whether contradictory protein interactions might be context-dependent

  • Implement proximity-dependent labeling approaches (BioID, APEX) to capture transient or weak interactions

  • Compare interaction profiles under different cellular conditions

  • Consider that SCS22 shows divergent binding preferences compared to SCS2, which may explain functional differences

• Functional Redundancy Assessment:

  • Systematically compare phenotypes of single (scs22Δ) versus double (scs2Δscs22Δ) mutants

  • Test whether SCS22 function becomes more apparent in the absence of SCS2

  • Analyze whether interactions unique to SCS22 (e.g., stronger binding to Csr102) reveal specialized functions

• Quantitative Validation Approach:

  • Implement quantitative metrics for ER-PM contacts (cortex-to-NE ratios)

  • Use FRAP analysis to compare protein dynamics between studies

  • Apply appropriate statistical tests to determine significance of observed differences

  • Ensure proper normalization using validated reference genes for expression studies

• Reconciliation Strategies:

  • Develop a unified model that accommodates apparently contradictory data

  • Consider that SCS22 may have condition-specific functions or serve as a regulatory partner to SCS2

  • Test whether differential binding partners of SCS22 (regulatory trafficking proteins) versus SCS2 (coatomers and SNARE/NSF factors) suggest specialized roles in different cellular processes

This methodical approach will help researchers distinguish genuine biological complexity from experimental artifacts, leading to a more comprehensive understanding of SCS22 function in the context of ER-PM tethering and membrane contact site biology.

What are the most reliable methods for quantifying SCS22-SCS2 interactions in vivo?

Quantifying SCS22-SCS2 interactions in vivo requires methods that can detect protein associations within their native membrane environment. The following techniques represent the most reliable approaches, listed in order of increasing spatial resolution and sensitivity:

• Co-Immunoprecipitation (Co-IP) with Quantitative Analysis:

  • Use differentially tagged versions of SCS22 and SCS2 (confirmed functional by localization studies)

  • Implement gentle solubilization conditions to preserve membrane protein interactions

  • Quantify interaction strength using Western blot band intensity ratios

  • Include appropriate controls (e.g., non-interacting membrane proteins, MSP domain mutants)

  This approach has successfully confirmed SCS22-SCS2 interaction in previous studies but requires careful optimization of detergent conditions to maintain the integrity of membrane protein complexes.

• Bimolecular Fluorescence Complementation (BiFC):

  • Fuse complementary fragments of a fluorescent protein (e.g., Venus) to SCS22 and SCS2

  • Quantify interaction by measuring reconstituted fluorescence intensity at ER-PM contact sites

  • Include proper controls for fragment self-association and expression levels

  • Analyze spatial distribution of interactions along the cell periphery

  This method offers spatial information about where interactions occur but may stabilize transient interactions.

• Förster Resonance Energy Transfer (FRET):

  • Tag SCS22 and SCS2 with appropriate FRET pairs (e.g., mTurquoise2/mVenus)

  • Measure FRET efficiency using acceptor photobleaching or fluorescence lifetime imaging (FLIM)

  • Quantify interaction proximity (1-10 nm resolution)

  • Analyze interaction dynamics in living cells under different conditions

  FRET provides higher spatial resolution and allows dynamic measurements but requires careful controls for expression levels and fluorophore orientation.

• Proximity Ligation Assay (PLA):

  • Use antibodies against epitope tags on SCS22 and SCS2

  • Detect proximity (<40 nm) through rolling circle amplification and fluorescent probe hybridization

  • Quantify discrete interaction puncta throughout the cell

  • Correlate interaction sites with ER-PM contact sites

  PLA offers high sensitivity for detecting endogenous protein levels but requires fixed samples.

• Split-Ubiquitin Membrane Yeast Two-Hybrid:

  • Fuse ubiquitin halves to SCS22 and SCS2

  • Measure interaction through reconstitution of ubiquitin and reporter gene activation

  • Quantify interaction strength by reporter signal intensity

  • Test mutant variants to map interaction domains

  This method is specifically designed for membrane protein interactions and can be used for screening interaction determinants.

For optimal results, researchers should implement at least two complementary methods and validate that tagged protein versions maintain their functional properties by confirming their cortex-to-NE ratios and mobility characteristics using established microscopy techniques . Quantitative analysis should include statistical testing of replicate experiments to ensure reproducibility.

What emerging technologies could advance our understanding of SCS22 function in membrane contact sites?

Emerging technologies offer unprecedented opportunities to elucidate SCS22 function at membrane contact sites with increased precision, resolution, and throughput. The following cutting-edge approaches hold particular promise:

• Cryo-Electron Tomography (Cryo-ET):

  • Enables visualization of ER-PM contact sites at nanometer resolution in their native state

  • Can reveal the detailed organization of SCS22 and SCS2 within contact sites

  • When combined with gold-labeled antibodies, can precisely localize SCS22 relative to other tethering components

  • Could resolve structural changes in contact sites upon mutation or deletion of SCS22

• Engineered Proximity Sensors:

  • Split fluorescent proteins optimized for membrane contact sites

  • FRET-based biosensors that report on contact site formation/dissolution

  • Chemically induced dimerization systems to artificially manipulate contact sites

  • These tools would allow real-time monitoring of SCS22 function during dynamic cellular processes

• Genome-wide CRISPR Screens:

  • Systematic identification of genes that modify SCS22 function

  • Design screens using fluorescent reporters of ER-PM contacts as readouts

  • Could identify novel regulatory pathways and interacting partners

  • May reveal condition-specific functions of SCS22 distinct from SCS2

• Advanced Optogenetic Approaches:

  • Light-controlled recruitment or dissociation of SCS22 from contact sites

  • Spatiotemporally precise manipulation of SCS22 activity in specific subcellular regions

  • Could reveal the acute consequences of SCS22 recruitment to or removal from contact sites

  • May help distinguish primary from secondary effects of SCS22 manipulation

• Single-Molecule Tracking:

  • Super-resolution microscopy to track individual SCS22 molecules

  • Quantification of dwell times at contact sites versus free diffusion

  • Comparison of SCS22 versus SCS2 molecular behavior at the single-molecule level

  • Could provide mechanistic insights into how SCS22 establishes and maintains contact sites

• Proximity Labeling Proteomics with Improved Spatial Resolution:

  • TurboID or miniTurbo systems for faster labeling kinetics

  • Split-BioID approaches to specifically label proteins at contact sites

  • Quantitative proteomics to compare SCS22-proximal versus SCS2-proximal proteins

  • May identify unique SCS22 interactors missed by conventional approaches

These emerging technologies, particularly when used in combination, could address fundamental questions about SCS22's specific contributions to ER-PM contact site biology and reveal how it complements or differs from SCS2 in ways that conventional approaches have been unable to resolve.

How might systems biology approaches be applied to understand the broader impact of SCS22 in cellular homeostasis?

Systems biology approaches offer powerful frameworks for understanding how SCS22 functions within broader cellular networks and contributes to homeostasis. Implementing these approaches can reveal emergent properties and system-level impacts that may not be apparent from reductionist studies:

• Multi-omics Integration:

  • Combine transcriptomics, proteomics, lipidomics, and metabolomics data from scs22Δ versus wild-type cells

  • Identify pathway-level changes rather than individual molecular alterations

  • Use time-series experiments to capture dynamic responses following perturbation

  • Apply appropriate reference genes such as TPI1, FBA1, CDC19, and ACT1 for accurate transcriptomic normalization

• Network Analysis and Modeling:

  • Construct protein-protein interaction networks centered on SCS22 and SCS2

  • Apply differential network analysis to identify condition-specific interactions

  • Develop mathematical models of ER-PM contact dynamics incorporating both VAPs

  • Simulate the system-level consequences of SCS22 perturbation under various conditions

• Flux Analysis of Lipid Transport:

  • Implement pulse-chase experiments with fluorescent or isotope-labeled lipids

  • Quantify transport rates at ER-PM contacts in the presence/absence of SCS22

  • Develop computational models of lipid flux that incorporate SCS22-mediated contacts

  • This approach could reveal why SCS22 influences PI4P homeostasis despite having lower PM affinity than SCS2

• Synthetic Biology Approaches:

  • Design minimal synthetic tethers based on SCS22 domains

  • Systematically vary tether properties (strength, number, distribution)

  • Measure cellular responses to synthetic tethering perturbations

  • Identify emergent properties of tethering systems that might explain the evolutionary conservation of multiple VAPs

• Comparative Systems Analysis:

  • Apply comparative genomics and functional analysis across fungal species

  • Identify conserved versus species-specific aspects of SCS22 function

  • Correlate evolutionary changes in SCS22 with alterations in cellular systems

  • This could explain why Scs2 plays a dominant role over Scs22 in ER-PM tethering in fission yeast

• Multi-scale Modeling:

  • Integrate molecular-level simulations of SCS22 structure and interactions

  • Scale up to membrane-level models of contact site formation

  • Further scale to whole-cell models incorporating multiple membrane contact sites

  • This approach could reveal how local molecular properties of SCS22 translate to global cellular phenotypes