Recombinant Saccharomyces cerevisiae UPF0479 membrane protein YFL068W (YFL068W)

What is UPF0479 membrane protein YFL068W and what is known about its function?

UPF0479 membrane protein YFL068W is a membrane protein encoded by the YFL068W gene in Saccharomyces cerevisiae (baker's yeast). It belongs to the UPF0479 protein family, which is designated as a protein of unknown function. The protein consists of 160 amino acids and is predicted to be integrated into cellular membranes. While its precise biological function remains uncharacterized, structural analysis suggests potential roles in membrane transport or signaling. The "UPF" designation (Uncharacterized Protein Family) indicates that this protein's function has not been experimentally determined, making it a target for fundamental research in protein characterization studies .

What expression systems can be used to produce recombinant YFL068W protein?

Saccharomyces cerevisiae serves as an excellent expression system for the recombinant production of YFL068W, offering several advantages over other expression platforms. S. cerevisiae is particularly suitable because it provides a eukaryotic environment for protein expression while being relatively quick, easy, and economical to culture. The native cellular machinery in yeast can facilitate proper folding and post-translational modifications of the membrane protein, which are often critical for structural and functional studies .

For expression in S. cerevisiae, researchers commonly use galactose-inducible promoters (P GAL) to control expression levels. These promoters allow for tightly regulated induction of protein expression after the culture reaches the desired density. Alternative expression systems such as Pichia pastoris might be considered for higher yields, but S. cerevisiae remains preferred for studying proteins in their native context. Bacterial expression systems (E. coli) typically struggle with proper folding of eukaryotic membrane proteins, making yeast the preferred choice for YFL068W expression .

How is YFL068W protein typically stored and what are the best practices for maintaining stability?

Recombinant YFL068W protein requires specific storage conditions to maintain stability and functional integrity. Based on standard protocols for similar membrane proteins, it is recommended to store the purified protein at -20°C for short-term storage, and at -80°C for extended preservation. The optimal storage buffer typically consists of a Tris-based buffer supplemented with 50% glycerol, which has been optimized specifically for this protein .

To minimize protein degradation and denaturation, researchers should avoid repeated freeze-thaw cycles, as these can significantly compromise protein integrity. For working applications requiring frequent access to the protein, it is advisable to prepare small aliquots at the time of purification and keep working aliquots at 4°C for up to one week. When thawing frozen stocks, gradual thawing on ice is preferred over rapid warming to room temperature, as the latter may induce protein aggregation and functional loss .

What are the challenges in resolving the crystal structure of YFL068W and how can they be addressed?

Crystallizing membrane proteins like YFL068W presents several significant challenges that must be addressed with specialized methodologies. Membrane proteins are notoriously difficult to crystallize due to their hydrophobic transmembrane domains, which can lead to aggregation or misfolding when removed from the lipid bilayer. For YFL068W specifically, researchers must overcome several obstacles:

First, obtaining sufficient quantities of properly folded protein requires optimization of expression conditions in S. cerevisiae. The yeast expression system offers advantages for eukaryotic membrane proteins since it can perform necessary post-translational modifications while being relatively easy and inexpensive to culture at scale. Recent successes with other membrane proteins expressed in S. cerevisiae, such as the Arabidopsis thaliana NRT1.1 nitrate transporter and fungal TMEM16 lipid scramblase, provide methodological frameworks that can be adapted for YFL068W .

Researchers should implement detergent screening to identify optimal solubilization conditions that maintain protein stability while removing it from the membrane. Techniques such as lipidic cubic phase (LCP) crystallization have proven successful for recalcitrant membrane proteins and should be considered. Additionally, protein engineering approaches—such as creating fusion constructs with crystallization chaperones or truncating flexible regions—may enhance crystallization propensity while preserving the core structural features of YFL068W.

How can researchers distinguish between functional and non-functional recombinant YFL068W protein preparations?

Distinguishing functional from non-functional recombinant YFL068W presents a complex challenge due to the protein's uncharacterized function. Researchers must employ multiple complementary approaches to assess protein quality and functionality.

A comprehensive quality assessment begins with biophysical characterization techniques. Circular dichroism (CD) spectroscopy can verify proper secondary structure formation, while size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) can determine if the protein exists in the expected oligomeric state rather than as misfolded aggregates. Thermal shift assays provide valuable information about protein stability under various buffer conditions, allowing optimization of purification and storage protocols.

Since the precise function of YFL068W remains unknown, researchers should develop activity assays based on predicted functions of similar membrane proteins. These might include testing for potential transport activity using reconstituted proteoliposomes with fluorescent substrates, or investigating protein-lipid interactions through lipid binding assays. Additionally, complementation studies in S. cerevisiae strains with YFL068W gene deletions can reveal whether the recombinant protein restores any observable phenotypes.

Another approach involves structural integrity assessment through limited proteolysis, which can distinguish between properly folded proteins (which show resistance to digestion in transmembrane regions) and misfolded variants. Combined with mass spectrometry analysis, this technique provides insights into the protein's structural organization and stability.

What approaches can be used to identify potential interaction partners of YFL068W in yeast cells?

Identifying interaction partners of YFL068W requires sophisticated protein interaction detection methods adapted for membrane proteins. A multi-tiered strategy combining in vivo and in vitro approaches offers the most comprehensive results.

Affinity purification coupled with mass spectrometry (AP-MS) represents a powerful approach when adapted for membrane proteins. By creating strains expressing epitope-tagged YFL068W (e.g., with FLAG, HA, or TAP tags), researchers can isolate the protein along with its interacting partners using mild detergent conditions that preserve physiologically relevant interactions. Critical to this approach is the careful selection of detergents that solubilize membrane complexes while maintaining interaction integrity. Crosslinking prior to purification can stabilize transient interactions that might otherwise be lost during purification.

Proximity-based labeling methods such as BioID or APEX2 are particularly valuable for studying membrane protein interactions. By fusing a biotin ligase or peroxidase enzyme to YFL068W, researchers can biotinylate proteins in close proximity in living cells. This approach captures both stable and transient interactions in their native cellular environment, providing a more comprehensive interaction network.

Genetic interaction screens, including synthetic genetic array (SGA) analysis, can identify functional relationships between YFL068W and other genes. By systematically creating double mutants and assessing phenotypic consequences, researchers can infer biological pathways in which YFL068W participates. These genetic interactions often suggest physical interaction partners for further investigation.

Split-ubiquitin yeast two-hybrid systems, specifically designed for membrane proteins, offer another valuable approach. Unlike traditional yeast two-hybrid assays, this modified system allows for detection of interactions involving membrane-bound proteins, making it suitable for screening potential YFL068W interactors.

What are the optimal conditions for expressing recombinant YFL068W in S. cerevisiae?

Optimizing expression of recombinant YFL068W in S. cerevisiae requires careful consideration of multiple parameters to maximize protein yield while ensuring proper folding and functionality. The GAL promoter system represents an excellent choice for controlled induction, allowing separation of the growth phase from the protein expression phase .

A standardized expression protocol begins with transformation of S. cerevisiae with a suitable expression vector containing the YFL068W sequence. Select a strain optimized for recombinant protein expression, such as BJ5460, which lacks specific proteases that could degrade the target protein. Initially, grow cells in glucose-containing medium (e.g., YPAD) at 30°C until reaching mid-log phase (OD600 of approximately 0.8-1.0). At this point, harvest cells by centrifugation and resuspend in medium containing galactose (typically 2%) to induce protein expression .

The temperature during the induction phase critically impacts protein folding. While standard induction occurs at 30°C, lower temperatures (18-24°C) often improve proper folding of membrane proteins, albeit with slower expression kinetics. Conducting expression trials at different temperatures (18°C, 24°C, and 30°C) for varying durations (12-72 hours) helps identify optimal conditions.

Supplementing the medium with specific additives can significantly enhance membrane protein expression. Consider adding glycerol (0.5-1%) to stabilize membrane proteins, and specific ions or cofactors if they are known to be required for YFL068W folding or function. Optimal induction times typically range from 16-24 hours, but this parameter should be empirically determined for YFL068W through time-course experiments with samples analyzed by Western blotting.

What purification strategy yields the highest purity and stability for YFL068W protein?

Purifying membrane proteins like YFL068W demands specialized strategies to maintain protein stability throughout the purification process while achieving high purity. A systematic approach begins with efficient cell disruption followed by careful membrane isolation and protein extraction.

For cell disruption, mechanical methods such as glass bead homogenization or high-pressure homogenization (using a French press or microfluidizer) are effective for yeast cells. After disruption, differential centrifugation isolates the membrane fraction, typically through a low-speed centrifugation (3,000-5,000 × g) to remove unbroken cells and debris, followed by ultracentrifugation (100,000 × g for 1-2 hours) to collect membrane fractions.

Solubilization represents a critical step requiring careful detergent selection. Screen multiple detergents including mild options like n-dodecyl-β-D-maltoside (DDM), n-decyl-β-D-maltoside (DM), or digitonin, which often preserve membrane protein structure and function. Typically, solubilization occurs at 4°C for 1-2 hours with detergent concentrations ranging from 0.5-2%, depending on the specific detergent's critical micelle concentration.

For affinity purification, incorporate a purification tag (His6, FLAG, or Strep-tag) at either the N- or C-terminus of YFL068W, avoiding disruption of transmembrane domains. Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is commonly employed for His-tagged proteins. Throughout purification, maintain detergent concentrations above their critical micelle concentration to prevent protein aggregation.

Size exclusion chromatography serves as a final polishing step, separating different oligomeric states and removing aggregates. Consider replacing harsh detergents with milder alternatives or amphipols during this step to enhance long-term stability.

The purification buffer composition significantly impacts stability. A typical buffer contains 20-50 mM Tris or HEPES (pH 7.0-8.0), 100-300 mM NaCl, glycerol (10-20%), and detergent at concentrations just above CMC. Addition of specific lipids (0.01-0.1 mg/ml) that mimic the native membrane environment can further stabilize the protein.

How can researchers effectively introduce site-specific mutations in YFL068W for structure-function studies?

Site-directed mutagenesis of YFL068W enables researchers to probe structure-function relationships by selectively modifying specific amino acid residues. For S. cerevisiae genes like YFL068W, several specialized approaches have been developed that accommodate the unique characteristics of yeast molecular biology.

The restriction enzyme-mediated integration approach represents an efficient method for introducing mutations into yeast genes. This technique utilizes the yeast transformation protocol with a BamHI restriction enzyme to facilitate integration of DNA fragments. The process begins with preparation of high molecular weight yeast DNA following established rapid procedures. Cells from an overnight culture are resuspended in appropriate media, grown to optimal density, and harvested by centrifugation .

For site-specific mutations, design primers that incorporate your desired mutation flanked by 15-25 nucleotides of perfectly matched sequence on each side. PCR amplification using these primers generates a fragment containing the mutation. Following PCR, digest the product with DpnI to eliminate template DNA, then transform the PCR product directly into competent S. cerevisiae cells. The transformation mixture should include salmon sperm DNA as a carrier and polyethylene glycol to enhance transformation efficiency .

Alternatively, the CRISPR-Cas9 system offers superior precision for introducing mutations. This approach requires designing a guide RNA targeting the YFL068W region of interest, along with a repair template containing your desired mutation. The repair template should include 50-60 bp homology arms flanking the mutation site. When co-transformed into yeast cells expressing Cas9, this system creates a specific double-strand break that is repaired using the template, thereby introducing the desired mutation.

Verification of successful mutagenesis is essential. Perform colony PCR followed by sequencing to confirm the presence of the intended mutation. Additionally, Western blotting can verify that the mutant protein maintains appropriate expression levels. For functional mutations, develop assays specific to YFL068W's predicted function or use complementation tests to assess the impact of mutations.

How should researchers analyze membrane topology data for YFL068W?

Analyzing membrane topology data for YFL068W requires integration of computational predictions with experimental validation techniques to generate an accurate topological model. Begin with computational analysis using multiple topology prediction algorithms such as TMHMM, Phobius, and TOPCONS, which analyze the amino acid sequence to predict transmembrane segments based on hydrophobicity patterns and other sequence features.

The 160-amino acid sequence of YFL068W (MMPAKLQLDVLRTLQSSARHGTQTLKNSNFLERFHKDRIVFCLPFFPALFLVPVQKVLQHLCLRFTQVAPYFIIQLFDLPSRHAENLAPLLASCRIQYTNCFSSSSNGQVPSIISLYLRVDLSPFYAKKFQIPYRVPMIWLDVFQVFFVFLVISQHSLHS) contains several hydrophobic regions that likely form transmembrane domains . Create a consensus prediction by comparing results from multiple algorithms, particularly focusing on areas of agreement.

Experimental validation is crucial for confirming computational predictions. Design a systematic approach using reporter fusion techniques, where reporters such as alkaline phosphatase (PhoA) or green fluorescent protein (GFP) are fused at different positions throughout the YFL068W sequence. PhoA shows activity when located in the periplasmic space, while GFP fluorescence is detectable when positioned in the cytoplasm, allowing determination of segment orientation.

Cysteine accessibility methods offer another powerful approach. By introducing single cysteine residues at various positions and then testing their accessibility to membrane-impermeable sulfhydryl reagents, researchers can determine which regions are exposed to which side of the membrane. Combine these experimental data with the computational predictions to generate a refined topological model.

When analyzing experimental topology data, researchers should assess:

• Consistency between different experimental approaches

• Agreement with computational predictions

• Biological plausibility of the resulting model

• Comparison with known topologies of related proteins

Create a comprehensive topology map that integrates all data sources, clearly indicating confidence levels for different regions based on the consistency of evidence. This map should identify the number and orientation of transmembrane domains, along with the cellular localization of loop regions.

What statistical approaches are most appropriate for analyzing YFL068W protein-protein interaction data?

Protein-protein interaction (PPI) data for YFL068W requires robust statistical analysis to distinguish genuine interactions from false positives, particularly challenging for membrane proteins due to their hydrophobic nature and potential for non-specific interactions. A multi-layered statistical approach is essential.

For mass spectrometry-based interaction data, implement SAINTexpress (Significance Analysis of INTeractome) or similar algorithms specifically designed for analyzing AP-MS data. These tools compare prey proteins identified in experimental samples against control purifications, calculating the probability that each interaction is genuine rather than background contamination. Set a stringent threshold (typically SAINT score ≥ 0.7) to minimize false positives while capturing true interactions.

When analyzing data from multiple biological replicates, employ reproducibility-based filtering. Calculate the coefficient of variation (CV) for spectral counts or intensity values across replicates, removing interactions with high variability (CV > 40%). Additionally, implement fold-change cutoffs by comparing spectral counts or intensity values between bait and control samples, typically requiring at least a 2-fold enrichment.

Network analysis provides valuable context for interpreting YFL068W interactions. Calculate network parameters including node degree (number of interactions per protein), betweenness centrality, and clustering coefficients to identify central players in the interaction network. Visualization tools such as Cytoscape enable identification of functional modules within the interaction network through algorithms like MCODE or ClusterONE.

Comparative analysis across different interaction detection methods strengthens confidence in identified interactions. Interactions detected by multiple orthogonal techniques (e.g., both AP-MS and split-ubiquitin assays) receive higher confidence scores. Implement a scoring system that rewards interactions with multiple lines of evidence.

How can researchers effectively compare structural data of YFL068W with homologous proteins from other species?

Comparative structural analysis of YFL068W with homologous proteins requires systematic integration of sequence and structural information to identify conserved features and species-specific variations. The process begins with comprehensive homology identification.

Perform iterative sequence searches using PSI-BLAST or HHpred against protein databases to identify remote homologs across diverse species. The UPF0479 protein family likely contains members across various fungi and possibly other eukaryotes. Create a multiple sequence alignment (MSA) using tools like MUSCLE or MAFFT, carefully adjusting parameters to accommodate the challenges of aligning membrane protein sequences with their hydrophobic regions.

For structural comparison, use existing structural data for homologs as templates for modeling YFL068W. If experimental structures exist for homologs, employ comparative modeling tools such as MODELLER or SWISS-MODEL to generate structural models of YFL068W. Assess model quality using validation tools like MolProbity or PROCHECK, paying particular attention to transmembrane regions where traditional validation metrics may not apply effectively.

Analyze conservation patterns within the context of the structural model. Map sequence conservation scores (calculated from the MSA using methods like Jensen-Shannon divergence) onto the three-dimensional structure to identify functionally important regions. Highly conserved surface patches often indicate binding sites or functional interfaces, while conserved transmembrane regions may be involved in transport functions or structural integrity.

Compare predicted or experimentally determined membrane topologies across homologs to identify conserved structural features. Create topology diagrams that highlight conserved transmembrane segments, loop regions, and potential functional motifs across species. Analyze variations in loop regions, which often evolve more rapidly than transmembrane domains but may contain species-specific functional elements.

Molecular dynamics simulations can provide insights into conserved dynamic properties across homologs. By simulating the behavior of YFL068W and its homologs in membrane environments, researchers can identify conserved conformational changes or interaction patterns that suggest functional mechanisms.

How can researchers address low expression yields of recombinant YFL068W in yeast?

Low expression yields of YFL068W in yeast represent a common challenge that requires systematic optimization strategies. The approach should address multiple aspects of the expression system, beginning with genetic construct optimization.

Codon optimization significantly impacts expression levels. Analyze the YFL068W sequence for rare codons in S. cerevisiae and optimize accordingly. Online tools such as JCat or Codon Optimization Tool can generate sequences with codons preferred by S. cerevisiae, potentially increasing translation efficiency. Additionally, optimize the 5' and 3' untranslated regions (UTRs) to enhance mRNA stability and translation initiation.

Expression vector selection is critical. Test different promoter systems beyond the standard GAL promoter, including constitutive promoters (TEF1, TDH3) or alternative inducible systems (CUP1, MET25). For each promoter, evaluate expression levels through Western blotting. Consider utilizing centromeric (low-copy) versus 2μ (high-copy) plasmids, as membrane proteins often express better from low-copy vectors due to reduced cellular stress.

Host strain optimization can address proteolytic degradation issues. Utilize protease-deficient strains such as BJ5460 or BJ5464, which lack specific vacuolar proteases. Alternatively, consider specialized strains developed for membrane protein expression that contain modifications to the secretory pathway or chaperone systems.

For expression conditions, implement a factorial design experiment testing multiple variables:

• Induction temperature (18°C, 24°C, 30°C)

• Induction duration (12, 24, 48, 72 hours)

• Inducer concentration (0.5%, 1%, 2% galactose)

• Media composition (standard vs. enriched)

• Cell density at induction (OD600 of 0.6, 1.0, 1.5)

Addition of specific chemical chaperones or membrane-stabilizing compounds can increase functional expression. Test supplementation with compounds such as DMSO (2-5%), glycerol (5-10%), or specific lipids that might stabilize the membrane protein during expression.

What approaches help resolve difficulties in solubilizing and purifying YFL068W while maintaining its functional integrity?

Solubilization and purification challenges for YFL068W require specialized strategies to extract the protein from membranes while preserving its native structure and function. Begin with systematic detergent screening, as detergent selection critically impacts both extraction efficiency and protein stability.

Conduct a comprehensive detergent screen testing:

• Maltosides (DDM, DM, UDM) - generally mild and effective for many membrane proteins

• Glucosides (OG, NG) - more harsh but efficient for extraction

• Neopentyl glycols (LMNG, DMNG) - enhanced stability properties

• Fos-cholines - highly efficient but potentially denaturing

• Digitonin or GDN - very mild natural or synthetic detergents

For each detergent, test multiple concentrations (typically 0.5-2%) and solubilization conditions (time: 1-4 hours; temperature: 4°C vs. room temperature). Assess extraction efficiency by Western blotting and functional integrity through activity assays appropriate for YFL068W.

Consider alternative solubilization strategies beyond conventional detergents. Native nanodiscs, formed by membrane scaffold proteins and phospholipids, provide a more native-like environment. Styrene maleic acid (SMA) copolymers extract membrane proteins with their surrounding lipids as SMA lipid particles (SMALPs), preserving the native lipid environment.

Optimize purification buffers to enhance stability. Include glycerol (10-20%) to prevent aggregation, and test various salt concentrations (150-500 mM) to mitigate non-specific interactions. Consider adding specific lipids (0.01-0.1 mg/ml) that might stabilize the protein structure. For proteins with multiple domains, evaluate the impact of adding osmolytes such as trehalose or sucrose.

Implement a rational purification strategy designed specifically for membrane proteins:

• Solubilize membranes with the optimal detergent identified through screening

• Perform initial capture using affinity chromatography (typically IMAC for His-tagged proteins)

• Consider an ion exchange chromatography step if necessary to remove contaminants

• Finalize with size exclusion chromatography to separate different oligomeric states and aggregates

Throughout purification, monitor protein quality using techniques such as fluorescence-detection size exclusion chromatography (FSEC) or analytical ultracentrifugation to assess homogeneity and stability. For long-term stability, explore detergent exchange into more stabilizing detergents or consider transferring the protein into amphipols or nanodiscs after initial purification.

What emerging technologies could advance the structural and functional characterization of YFL068W?

The structural and functional characterization of YFL068W can be significantly advanced through emerging technologies that address the unique challenges posed by membrane proteins. Integration of these cutting-edge approaches offers new avenues for understanding this uncharacterized protein.

Cryo-electron microscopy (cryo-EM) has revolutionized membrane protein structural biology by enabling structure determination without crystallization. Recent technological advances in direct electron detectors and computational methods have pushed resolution limits below 2 Å for membrane proteins. For YFL068W, cryo-EM offers advantages over X-ray crystallography, particularly if the protein resists crystallization. Single-particle cryo-EM analysis could reveal the protein's structure in detergent micelles, while cryo-electron tomography could visualize it in its native membrane environment, providing context for its cellular organization.

Integrative structural biology approaches combine multiple experimental techniques with computational modeling. For YFL068W, researchers could integrate data from:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map solvent-accessible regions

• Cross-linking mass spectrometry (XL-MS) to identify spatial proximity between protein regions

• Small-angle X-ray scattering (SAXS) for low-resolution envelope determination

• Nuclear magnetic resonance (NMR) for dynamic information and ligand binding studies

These diverse datasets can be integrated using computational platforms like IMP (Integrative Modeling Platform) to generate comprehensive structural models even with limited high-resolution data.

Artificial intelligence approaches, particularly AlphaFold2 and RoseTTAFold, have demonstrated remarkable accuracy in predicting protein structures. For membrane proteins like YFL068W, these tools can provide initial structural models to guide experimental design. The integration of predicted models with sparse experimental data represents a powerful strategy for accelerating structure determination. As these AI methods continue to improve in modeling membrane protein-specific features and protein-lipid interactions, their utility for characterizing proteins like YFL068W will increase.

Single-molecule techniques offer unique insights into protein dynamics and heterogeneity. Single-molecule FRET can reveal conformational dynamics of YFL068W by measuring distance changes between strategically placed fluorophores. Single-molecule force spectroscopy can characterize mechanical properties and folding landscapes. These approaches are particularly valuable for membrane proteins, which often adopt multiple conformational states difficult to capture with ensemble methods.

Mass photometry represents an emerging technique for studying membrane proteins in solution at the single-molecule level. This label-free approach can determine oligomeric states and heterogeneity of membrane protein samples with minimal material requirements. For YFL068W, mass photometry could rapidly assess sample quality and characterize interactions with potential binding partners under near-native conditions.