Recombinant Saccharomyces cerevisiae Uncharacterized protein YFL042C (YFL042C)

What is YFL042C and what is its structural characteristics?

YFL042C, also known as LAM5 (Lipid transfer protein Anchored at Membrane contact sites 5) or LTC2 (Lipid Transfer at Contact site protein 2), is an uncharacterized protein from Saccharomyces cerevisiae. It is a membrane-anchored lipid-binding protein comprising 674 amino acids with a molecular function associated with lipid transfer at membrane contact sites . The full amino acid sequence begins with MSDVDNWEPVSDNEDSTDSVKQLGPPFEHAS and continues through to LHQLVKLQLVELKL at the C-terminal end . The protein contains multiple regions of interest, including potential membrane-anchoring domains in its C-terminal region. When produced recombinantly, the full-length protein (amino acids 1-674) can be fused with tags such as His-tag to facilitate purification and detection in experimental settings .

What expression systems are commonly used for recombinant YFL042C production?

E. coli is the predominant expression system used for the recombinant production of YFL042C. The protein can be successfully expressed as a full-length construct (amino acids 1-674) with an N-terminal His-tag fusion . The bacterial expression system offers advantages for protein production including high yield, cost-effectiveness, and established purification protocols. After expression, the recombinant protein is typically available in lyophilized powder form with purity greater than 90% as determined by SDS-PAGE . Alternative expression systems such as yeast (including S. cerevisiae itself) could potentially be used, particularly when post-translational modifications or proper folding might be concerns, though specific protocols for these alternatives are not detailed in the available literature.

What are the recommended storage and handling conditions for recombinant YFL042C?

Recombinant YFL042C protein requires specific handling protocols to maintain stability and functionality. The lyophilized protein should be stored at -20°C to -80°C upon receipt . For reconstitution, it is recommended to briefly centrifuge the vial prior to opening to ensure all contents are at the bottom. The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage, it is advisable to add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation) and to aliquot the solution to avoid repeated freeze-thaw cycles, which can degrade protein quality . Working aliquots can be stored at 4°C for up to one week . The reconstitution buffer typically consists of a Tris/PBS-based solution containing 6% trehalose at pH 8.0 .

How is the purity of recombinant YFL042C assessed?

The purity of recombinant YFL042C is primarily assessed using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), with commercial preparations typically achieving greater than 90% purity . This analytical technique separates proteins based on molecular weight, allowing researchers to verify both the purity and approximate molecular weight of the recombinant protein. Additional analytical methods that could be employed (though not specifically mentioned in the search results) include western blotting using antibodies against the His-tag or the protein itself, mass spectrometry for precise molecular weight determination, and size exclusion chromatography for assessing aggregation state and homogeneity. For researchers requiring higher purity standards for specific applications, additional purification steps such as ion exchange chromatography or gel filtration may be necessary following the initial immobilized metal affinity chromatography (IMAC) used for His-tagged proteins.

What approaches can be used to characterize the function of uncharacterized proteins like YFL042C?

Characterizing uncharacterized proteins like YFL042C requires multi-faceted approaches combining biochemical, genetic, and computational methods. Several effective strategies include:

• Protein-Protein Interaction Studies: Employing yeast two-hybrid assays, bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (CoIP), and GST-pulldown techniques to identify binding partners that may suggest functional roles .

• Subcellular Localization: Determining where the protein operates within the cell using fluorescent protein tagging or immunofluorescence microscopy, which can provide insights into functional context.

• Genetic Manipulation: Creating knockout or knockdown variants in S. cerevisiae to observe resultant phenotypes. Synthetic genetic arrays can also identify genetic interactions that suggest functional pathways.

• Expression Pattern Analysis: Using RNA-Seq or other transcriptomic approaches to determine under which conditions YFL042C is expressed, potentially revealing functional contexts .

• Computational Prediction: Employing bioinformatic tools to predict domains, structural motifs, and potential functions based on sequence similarity with other characterized proteins.

• Biochemical Assays: Developing specific assays to test predicted functions, such as lipid binding and transfer activities, which would be particularly relevant given YFL042C's annotation as a lipid transfer protein .

For comprehensive functional characterization, a combination of these approaches is typically necessary, with experimental design iteratively refined based on preliminary findings.

How can RNA-Seq be utilized to study expression patterns and regulation of YFL042C?

RNA-Seq provides powerful insights into the expression patterns and regulation of genes like YFL042C. A comprehensive RNA-Seq approach would include:

• Experimental Design Considerations:

  • Sample multiple physiological conditions (nutrient limitation, stress conditions, growth phases)

  • Include biological replicates (minimum of 3) to ensure statistical validity

  • Select appropriate sequencing depth (10-30 million reads per sample for S. cerevisiae)

• Data Analysis Pipeline:

  • Quality control and preprocessing of raw sequencing data

  • Alignment to the S. cerevisiae reference genome

  • Quantification of YFL042C expression levels

  • Differential expression analysis across conditions

  • Co-expression network analysis to identify functionally related genes

• Validation Strategies:

  • RT-qPCR to confirm expression patterns

  • Reporter gene assays to study promoter activity

  • ChIP-Seq to identify transcription factors binding to YFL042C regulatory regions

RNA-Seq data can reveal condition-specific expression patterns that suggest functional contexts for YFL042C and identify co-regulated genes that may participate in the same biological processes . Additionally, RNA-Seq can uncover alternative splicing events or non-coding RNAs that might regulate YFL042C expression, though alternative splicing is less common in S. cerevisiae than in higher eukaryotes.

What synthetic biology approaches can be employed to investigate YFL042C function?

Synthetic biology offers innovative approaches for investigating the function of uncharacterized proteins like YFL042C:

• Domain Swapping and Protein Engineering:

  • Creating chimeric proteins by swapping domains with related characterized proteins

  • Introducing specific mutations to test hypotheses about functional residues

  • Engineering protein variants with modified regulatory elements or localization signals

• Synthetic Genetic Circuits:

  • Developing reporter systems linked to YFL042C expression or activity

  • Creating conditional expression systems to control protein levels

  • Implementing feedback loops to study regulatory dynamics

• Recombinant Population Construction:

  • Generating synthetic recombinant populations of S. cerevisiae strains with varying YFL042C alleles

  • Tracking phenotypic changes across generations in these populations

• CRISPR-Cas9 Applications:

  • Precise genome editing to introduce tagged versions of YFL042C

  • Creating regulated expression systems

  • Implementing CRISPRi for targeted gene repression

• Heterologous Expression Systems:

  • Expressing YFL042C in alternative hosts to study function outside its native context

  • Using complementation assays in mutant strains to test functional hypotheses

These synthetic biology approaches allow researchers to systematically manipulate YFL042C in ways that can reveal its functional properties and biological significance.

What protein-protein interaction methods are most suitable for studying YFL042C?

Several protein-protein interaction methods are particularly valuable for studying uncharacterized proteins like YFL042C:

• Yeast Two-Hybrid (Y2H) System:

  • Advantage: Native environment for a yeast protein, high-throughput capability

  • Implementation: Clone YFL042C as bait in fusion with a DNA-binding domain

  • Analysis: Screen against a S. cerevisiae genomic library or focused candidate proteins

  • Consideration: May detect interactions that occur in the nucleus

• Bimolecular Fluorescence Complementation (BiFC):

  • Advantage: Visualizes interactions in their native cellular context

  • Implementation: Fuse YFL042C and potential interactors with complementary fragments of a fluorescent protein

  • Analysis: Fluorescence microscopy to detect and localize interactions

  • Consideration: Provides spatial information about interactions

• Co-Immunoprecipitation (CoIP):

  • Advantage: Detects native complexes under physiological conditions

  • Implementation: Use antibodies against YFL042C or its tag to pull down interacting partners

  • Analysis: Mass spectrometry to identify co-precipitated proteins

  • Consideration: Requires efficient antibodies or well-expressing tagged constructs

• Proximity-Dependent Biotin Identification (BioID):

  • Advantage: Captures transient and weak interactions

  • Implementation: Fuse YFL042C with a biotin ligase to biotinylate proximal proteins

  • Analysis: Affinity purification of biotinylated proteins followed by mass spectrometry

  • Consideration: Maps the protein's "neighborhood" rather than direct interactions

• GST-Pulldown Assays:

  • Advantage: Direct biochemical validation of specific interactions

  • Implementation: Express YFL042C as a GST fusion protein for pulldown experiments

  • Analysis: SDS-PAGE and western blotting or mass spectrometry

  • Consideration: Useful for confirming interactions identified by other methods

A comprehensive characterization would employ multiple complementary methods to build confidence in the identified interactions and provide insights into YFL042C's functional network.

How can computational approaches predict the function of uncharacterized proteins like YFL042C?

Computational approaches provide valuable predictions for uncharacterized proteins like YFL042C through several methodologies:

• Sequence-Based Analysis:

  • Homology detection using PSI-BLAST, HHpred, or HMMER

  • Domain identification through InterPro, Pfam, or SMART databases

  • Prediction of transmembrane regions and signal peptides

  • Assessment of evolutionary conservation patterns

• Structural Prediction and Analysis:

  • Ab initio modeling using Rosetta or AlphaFold2

  • Template-based modeling when partial homology exists

  • Identification of potential binding pockets or active sites

  • Molecular dynamics simulations to understand flexibility and potential interactions

• Systems Biology Integration:

  • Analysis of gene co-expression networks

  • Examination of genetic interaction profiles

  • Integration of multiple -omics datasets (transcriptomics, proteomics, metabolomics)

  • Pathway enrichment analysis for associated genes/proteins

• Functional Prediction Algorithms:

  • Gene Ontology term prediction

  • Enzyme commission number prediction for potential enzymatic functions

  • Interaction partner prediction through network analysis

  • Phenotype prediction based on knockout/mutation data

• Comparative Genomics:

  • Analysis of gene neighborhood conservation

  • Phylogenetic profiling to identify co-evolving genes

  • Examination of selective pressure through dN/dS ratios

These computational approaches can provide testable hypotheses about YFL042C's function that guide experimental design and prioritization. Predictions should be validated through experimental approaches discussed in previous sections, as computational methods provide probabilities rather than certainties.

What are the key considerations for designing knockout/knockdown experiments for YFL042C?

Designing effective knockout/knockdown experiments for YFL042C requires careful planning and consideration of several factors:

• Knockout Strategy Selection:

  Method	Advantages	Limitations	Considerations for YFL042C
  CRISPR-Cas9	Precise, efficient	Potential off-targets	Design guide RNAs with minimal off-target effects
  Homologous Recombination	Well-established in yeast	Labor intensive	Include selectable markers for screening
  Transposon Mutagenesis	Random insertion library	Less specific	Requires extensive screening

• Conditional Expression Systems:

  • Tetracycline-regulatable promoters for titratable expression

  • Auxin-inducible degron system for post-translational control

  • Temperature-sensitive alleles if complete knockout is lethal

• Phenotypic Analysis Plan:

  • Growth assays under various conditions (carbon sources, stress)

  • Microscopy for subcellular structure examination

  • Lipidomics analysis, particularly relevant given YFL042C's annotation as a lipid transfer protein

  • Stress response profiling (oxidative, osmotic, temperature)

• Controls and Validation:

  • Include wild-type controls processed identically

  • Complement knockout with wild-type YFL042C to confirm phenotype specificity

  • Verify knockout at DNA, RNA, and protein levels

  • Consider double knockouts with related genes to address redundancy

• Data Analysis Framework:

  • Statistical methods appropriate for expected effect sizes

  • Consideration of growth phase-dependent phenotypes

  • Plan for unexpected or subtle phenotypes

When designing these experiments, it's critical to consider that YFL042C may have subtle phenotypes or be conditionally essential, requiring examination under specialized conditions relevant to lipid metabolism or membrane contact site functions.

What methods can be used to study the potential lipid transfer function of YFL042C?

Given YFL042C's annotation as a membrane-anchored lipid-binding protein (LAM5) involved in lipid transfer at membrane contact sites , several specialized methods can be employed to investigate this function:

• In Vitro Lipid Binding Assays:

  • Lipid overlay assays using recombinant YFL042C protein

  • Liposome flotation assays to assess membrane binding

  • Isothermal titration calorimetry (ITC) for binding affinity measurement

  • Surface plasmon resonance (SPR) for kinetic analysis of lipid interactions

• Lipid Transfer Activity Assessment:

  • FRET-based assays using fluorescently labeled lipids

  • Radiolabeled lipid transfer assays between distinct membrane fractions

  • Reconstituted systems with artificial membrane compartments

• Membrane Contact Site (MCS) Visualization:

  • Super-resolution microscopy (STED, PALM, STORM) to localize YFL042C at MCSs

  • Electron microscopy to examine ultrastructural changes in knockout strains

  • Split-GFP approaches to detect YFL042C proximity to MCS marker proteins

• Lipidomic Analysis:

  • Quantitative lipidomics comparing wild-type and YFL042C knockout strains

  • Analysis of lipid distribution between organelles using subcellular fractionation

  • Pulse-chase experiments with labeled lipid precursors to track lipid movement

• Genetic Interaction Mapping:

  • Synthetic genetic arrays (SGA) focusing on genes involved in lipid metabolism

  • Chemical-genetic profiling using lipid metabolism modulators

  • Suppressor screens to identify functional relationships

These methodologies can be combined to build a comprehensive understanding of YFL042C's role in lipid transfer, with particular attention to which specific lipid species might be transported and between which membrane compartments this transfer occurs.

How should researchers interpret conflicting results when characterizing YFL042C?

When characterizing uncharacterized proteins like YFL042C, researchers often encounter conflicting results that require careful interpretation and resolution strategies:

• Systematic Error Assessment:

  • Evaluate experimental conditions for each conflicting result

  • Consider differences in strain backgrounds, expression levels, and tags

  • Assess whether protein localization or function may be affected by experimental manipulations

  • Examine differences in assay sensitivity or specificity

• Biological Context Considerations:

  • YFL042C may have context-dependent functions that vary with conditions

  • Consider growth phase, nutrient availability, and stress responses

  • Evaluate whether post-translational modifications might alter function

  • Assess whether protein interactors present in one system might be absent in another

• Resolution Strategies:

  • Perform orthogonal experiments using complementary methods

  • Standardize experimental conditions across different approaches

  • Use native expression levels where possible to avoid artifacts

  • Apply quantitative rather than qualitative measurements

  • Conduct time-course experiments to capture dynamic behaviors

• Integrated Data Analysis Framework:

  • Develop weighted assessment of evidence based on methodology robustness

  • Apply Bayesian approaches to update hypotheses as new data emerges

  • Use computational modeling to reconcile seemingly contradictory observations

  • Consider evolutionary context and conservation patterns

• Community Standards:

  • Follow established guidelines for validation (multiple replicates, statistical analysis)

  • Consider independent validation in different laboratories

  • Transparently report all results, including negative or contradictory findings

When facing conflicting results specifically for YFL042C's lipid transfer function, researchers should consider whether the protein might transfer different lipid species under different conditions or whether it might have additional functions beyond lipid transfer.

What are the best practices for validating protein-protein interactions involving YFL042C?

Validating protein-protein interactions for an uncharacterized protein like YFL042C requires rigorous methodology and multiple lines of evidence:

• Multi-Method Validation Approach:

  Method	Strengths	Considerations
  Yeast Two-Hybrid	High-throughput screening	Validate with complementary methods
  Co-Immunoprecipitation	Detects native complexes	Use appropriate controls for specificity
  BiFC	Visualizes interactions in situ	Confirm with biochemical methods
  GST-Pulldown	Direct biochemical validation	Use physiological conditions

• Controls and Specificity Assessment:

  • Include non-interacting protein pairs as negative controls

  • Use structure-guided mutagenesis to disrupt specific interaction surfaces

  • Perform competition assays with unlabeled proteins

  • Test interaction dependency on relevant cofactors or lipids

• Quantitative Interaction Characterization:

  • Determine binding affinities using methods like SPR or ITC

  • Assess stoichiometry through analytical ultracentrifugation

  • Measure interaction kinetics when possible

  • Evaluate concentration dependence of interactions

• Functional Validation:

  • Demonstrate phenotypic consequences of disrupting the interaction

  • Show co-localization under physiologically relevant conditions

  • Establish correlation between interaction and known functions

  • Perform genetic epistasis analysis between interaction partners

• Structural Characterization:

  • Identify interaction interfaces through techniques like hydrogen-deuterium exchange

  • Use cross-linking mass spectrometry to map interaction regions

  • When possible, obtain structural data through X-ray crystallography, cryo-EM, or NMR

Following these validation practices builds confidence in protein interactions identified for YFL042C and provides a foundation for understanding its functional network within the cell.

How might studying YFL042C contribute to broader understanding of membrane contact sites?

Investigating YFL042C (LAM5) offers significant potential to advance our understanding of membrane contact sites (MCSs) in several ways:

• Molecular Architecture of MCSs:

  • YFL042C's role as a membrane-anchored lipid transfer protein could reveal mechanisms of tethering between organelles

  • Structural characterization may uncover novel domains involved in MCS formation

  • Interaction mapping could identify new proteins involved in MCS establishment and regulation

• Lipid Transfer Mechanisms:

  • Studying YFL042C's lipid binding and transfer activities may reveal fundamental principles of non-vesicular lipid transport

  • Characterization could establish whether transfer occurs through tunnel-like structures or shuttle mechanisms

  • Research might determine specificity for particular lipid species and directional preferences

• Regulatory Networks:

  • Investigation of YFL042C regulation may uncover how cells control lipid distribution in response to environmental changes

  • Studies could reveal how MCS formation and function are coordinated with metabolic state

  • Research might identify signaling pathways that modulate MCS activity

• Evolutionary Conservation:

  • Comparative studies between YFL042C and related proteins in other organisms could reveal conserved principles of MCS function

  • Research might identify specialized adaptations in different biological systems

  • Findings could bridge understanding between yeast models and more complex eukaryotes

• Disease Relevance:

  • Insights from YFL042C may inform understanding of human diseases linked to MCS dysfunction

  • Findings could be relevant to disorders of lipid metabolism or membrane homeostasis

  • Research might suggest new therapeutic approaches targeting MCS proteins

Studying this uncharacterized protein has the potential to fill critical knowledge gaps in how organelles communicate and coordinate lipid distribution, with implications extending beyond yeast biology to fundamental eukaryotic cell biology principles.

What emerging technologies might accelerate characterization of proteins like YFL042C?

Several cutting-edge technologies show promise for accelerating the functional characterization of uncharacterized proteins like YFL042C:

• Cryo-Electron Tomography:

  • Enables visualization of protein complexes in their native cellular environment

  • Could reveal YFL042C's arrangement at membrane contact sites

  • Provides structural insights without protein isolation or crystallization

• Proximity Labeling Proteomics:

  • Advanced methods like TurboID and APEX2 offer improved spatial and temporal resolution

  • Can map the protein neighborhood of YFL042C under various conditions

  • Helps identify weak or transient interactions missed by traditional methods

• Single-Molecule Techniques:

  • Single-molecule FRET to observe conformational changes during lipid binding/transfer

  • Optical tweezers to measure forces involved in protein-membrane interactions

  • Super-resolution tracking to follow individual YFL042C molecules in living cells

• Mass Spectrometry Innovations:

  • Crosslinking MS to map interaction interfaces at amino acid resolution

  • Native MS to determine complex stoichiometry and stability

  • Targeted proteomics for absolute quantification of low-abundance proteins

• Genome Engineering Advances:

  • CRISPR base editing for precise amino acid substitutions

  • Multiplexed CRISPR screens to systematically test YFL042C variants

  • Prime editing for scarless genomic modifications

• Artificial Intelligence Applications:

  • Deep learning for improved structural prediction (beyond AlphaFold)

  • Network-based function prediction incorporating multi-omics data

  • Automated hypothesis generation and experimental design optimization

• Microfluidics and Lab-on-a-Chip:

  • High-throughput phenotypic screening of YFL042C variants

  • Single-cell analysis of heterogeneous responses to YFL042C manipulation

  • Reconstituted membrane systems for controlled lipid transfer assays

These technologies, particularly when used in combination, have the potential to dramatically accelerate our understanding of YFL042C and similar uncharacterized proteins by providing higher resolution, more physiologically relevant data, and enabling studies that were previously technically infeasible.