Recombinant Saccharomyces cerevisiae Uncharacterized mitochondrial outer membrane protein YPR098C (YPR098C)

What is YPR098C and where is it localized in the cell?

YPR098C is classified as a hypothetical protein identified in Saccharomyces cerevisiae that localizes to the mitochondrial outer membrane . Proteomic analysis has confirmed its presence in the mitochondrial outer membrane proteome with an MLR (mitochondrial localization of mRNA) value of 51.0, providing strong evidence for its mitochondrial association . The protein consists of 161 amino acids with a molecular weight of approximately 17.7 kDa and an isoelectric point (pI) of 9.7 .
For researchers studying this protein, localization confirmation can be performed using subcellular fractionation followed by Western blotting or fluorescent protein tagging approaches. Co-localization studies with known mitochondrial outer membrane markers would provide additional verification of its submitochondrial localization.

What is the complete amino acid sequence of YPR098C?

The full amino acid sequence of YPR098C (161 amino acids) is:
MCLVKTTAHLLFYSFVFGGTTFYSYVASPIAFKVLEKDQFSALQNKIFPYFFQMQAASPVILALTAPIALTTGPLSSLVVASVSGLTNLFWLLPWTHKVKEQRKNIAKKYTGSELEAKDAILRKEFGKSHGLSLLFNLSNVCGMLAYGVCLSGGLLRKIPK
Researchers should analyze this sequence using bioinformatics tools to identify potential transmembrane domains, functional motifs, and structural features relevant to its membrane association. Hydrophobicity analysis using Kyte-Doolittle plots or TMHMM would help identify potential membrane-spanning regions that might be critical for proper membrane insertion and function.

How does YPR098C compare to other mitochondrial outer membrane proteins?

Proteomic analysis data reveals that YPR098C is part of a larger set of mitochondrial outer membrane proteins, some of which remain uncharacterized . When compared to other hypothetical proteins in this compartment:

What expression systems are optimal for recombinant YPR098C production?

E. coli has been successfully used as an expression system for recombinant YPR098C production, specifically with an N-terminal His-tag . For researchers planning to express this protein:

• Bacterial expression systems (E. coli): The available data confirms successful expression in E. coli . Consider specialized strains designed for membrane protein expression such as C41(DE3) or C43(DE3) to minimize toxicity.

• Yeast expression systems: Though not explicitly mentioned in the search results, S. cerevisiae or P. pastoris expression systems might provide more native-like post-translational modifications and membrane insertion for this yeast protein.

• Optimization parameters: For membrane proteins like YPR098C, expression conditions should be carefully optimized, including induction temperature (typically lower temperatures of 16-25°C are preferable), inducer concentration, and duration to minimize aggregation.

• Fusion tags: The N-terminal His-tag approach has been validated , but researchers might consider alternative tags such as MBP or SUMO to potentially improve solubility while maintaining the option for tag removal.

What are the optimal conditions for storage and handling of recombinant YPR098C?

Based on the product information, recombinant YPR098C requires specific storage and handling conditions :

• Storage buffer: Tris/PBS-based buffer containing 6% Trehalose at pH 8.0 is recommended .

• Long-term storage: Store lyophilized protein at -20°C/-80°C upon receipt. After reconstitution, add glycerol to 5-50% (with 50% being the default recommendation) and store in aliquots at -20°C/-80°C .

• Working storage: Aliquots can be stored at 4°C for up to one week .

• Stability considerations: Repeated freeze-thaw cycles should be avoided as they can lead to protein degradation and loss of activity .

• Reconstitution: Reconstitute protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL . Brief centrifugation prior to opening is recommended to bring contents to the bottom of the vial.
  These handling recommendations reflect the challenges associated with maintaining membrane protein stability once removed from their native lipid environment.

What purification strategies are most effective for recombinant His-tagged YPR098C?

For researchers purifying recombinant His-tagged YPR098C:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins is the primary method, leveraging the N-terminal His-tag .

• Membrane protein considerations: Prior to IMAC, careful solubilization using appropriate detergents is crucial. Test multiple detergents (DDM, LDAO, Triton X-100) at various concentrations to optimize extraction while maintaining protein structure.

• Polishing steps: Size exclusion chromatography is recommended after IMAC to remove aggregates and obtain homogeneous preparations.

• Quality control: SDS-PAGE analysis can verify purity, which should exceed 90% as reported in previous preparations .

• Detergent exchange: Consider exchanging harsh solubilization detergents for milder ones during purification if downstream structural or functional studies are planned.

What approaches can researchers use to investigate the function of uncharacterized YPR098C?

As YPR098C remains functionally uncharacterized, several complementary approaches can help elucidate its role:

• Genetic approaches:

  • Gene deletion/disruption studies in S. cerevisiae to observe phenotypic effects

  • CRISPR interference (CRISPRi) screening, which has been effective for studying mitochondrial membrane proteins

  • Synthetic genetic array (SGA) analysis to identify genetic interactions

• Protein interaction studies:

  • Affinity purification coupled with mass spectrometry to identify binding partners

  • Proximity labeling approaches (BioID, APEX) to identify neighboring proteins in the mitochondrial membrane

  • Crosslinking mass spectrometry to capture transient interactions

• Structural studies:

  • Recent advances in AlphaFold and similar AI-based structure prediction tools can provide initial structural insights

  • Experimental structure determination through X-ray crystallography or cryo-EM

  • Analysis for features like hydrophilic grooves that might suggest functions similar to MTCH2

• Localization dynamics:

  • Live-cell imaging with fluorescently tagged YPR098C to observe dynamics during cellular processes

  • Sub-mitochondrial localization using super-resolution microscopy

How might YPR098C relate to mitochondrial protein import pathways?

Given YPR098C's localization to the mitochondrial outer membrane, it may play a role in protein import or membrane organization:

• Potential import role: Other mitochondrial outer membrane proteins function as "doorways" that facilitate protein insertion into membranes . YPR098C might serve a similar role, possibly for a specific subset of mitochondrial proteins.

• Experimental approaches:

  • In vitro import assays with isolated mitochondria from wild-type and YPR098C-deletion strains

  • Analysis of potential physical interactions with known import machinery components

  • Examining if YPR098C has structural features similar to known import components like MTCH2, which contains a hydrophilic groove that functions as a "funnel" for protein insertion

• Comparative analysis:

  • Examine if YPR098C displays structural similarities to characterized outer membrane proteins involved in protein import

  • Test if YPR098C deletion affects the import of specific mitochondrial protein classes

What role might YPR098C play in mitochondrial membrane organization and dynamics?

Mitochondrial outer membrane proteins often participate in membrane organization and dynamics:

• Potential functions to investigate:

  • Involvement in mitochondrial fusion/fission processes

  • Role in establishing contact sites between mitochondria and other organelles

  • Participation in lipid transfer or membrane remodeling

• Experimental approaches:

  • Examine mitochondrial morphology in YPR098C deletion strains using fluorescence microscopy

  • Investigate localization of YPR098C during mitochondrial fusion/fission events

  • Analyze lipid composition of mitochondria lacking YPR098C

• Mutation studies:

  • Similar to studies with MTCH2 where "a single point mutation was enough to really change how the protein behaved" , targeted mutations in YPR098C could reveal functional domains

  • Creation of chimeric proteins or truncation variants to identify functional regions

How can researchers investigate potential binding partners of YPR098C?

Identifying interaction partners is crucial for understanding uncharacterized proteins like YPR098C:

• Affinity purification strategies:

  • Leverage the His-tagged construct for pull-down experiments followed by mass spectrometry

  • Consider crosslinking approaches to capture transient interactions

  • Employ stringent controls to distinguish specific from non-specific interactions

• Proximity labeling:

  • BioID or APEX2 fusion proteins can biotinylate proximal proteins in the native environment

  • TurboID offers faster labeling kinetics for capturing dynamic interactions

  • These methods are particularly valuable for membrane proteins where traditional co-immunoprecipitation may disrupt interactions

• Real-time interaction analysis:

  • Förster resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) for live-cell interaction studies

  • Split-GFP complementation assays to visualize interactions in intact cells

• Quantitative analysis:

  • Surface plasmon resonance (SPR) or microscale thermophoresis (MST) for quantitative binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters of interactions

What methodological approaches are suitable for analyzing YPR098C membrane integration?

Understanding how YPR098C integrates into the mitochondrial outer membrane requires specialized techniques:

• Topology determination:

  • Protease protection assays to determine which regions are exposed to the cytosol versus intermembrane space

  • Fluorescence quenching approaches with environment-sensitive dyes

  • Substituted cysteine accessibility method (SCAM) to map membrane-spanning segments

• Membrane insertion mechanisms:

  • In vitro reconstitution into liposomes to study autonomous insertion capabilities

  • Analysis of potential dependencies on known insertion machinery components

  • Investigation of sequence features that direct membrane targeting and insertion

• Structural studies in membrane environments:

  • Solid-state NMR to analyze protein structure in lipid bilayers

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map membrane-interacting regions

  • Electron paramagnetic resonance (EPR) spectroscopy with site-directed spin labeling

How should researchers approach mutational analysis of YPR098C?

Strategic mutational analysis can provide valuable insights into structure-function relationships:

• Targeted mutation strategies:

  • Site-directed mutagenesis of conserved residues

  • Alanine-scanning mutagenesis of predicted functional domains

  • Charge-reversal mutations to probe electrostatic interactions

• Functional readouts:

  • Growth phenotypes in various conditions

  • Mitochondrial morphology and function

  • Protein-protein interaction profiles

  • Membrane integration efficiency

• Experimental design considerations:

  • Create a panel of mutations rather than single mutants to comprehensively map functional regions

  • Include both conservative and non-conservative substitutions

  • Consider temperature-sensitive mutations that might reveal conditional phenotypes

How should researchers interpret phenotypic data from YPR098C deletion studies?

Interpreting phenotypic data from deletion studies requires careful consideration:

• Primary vs. secondary effects:

  • Distinguish direct consequences of YPR098C absence from compensatory responses

  • Consider acute depletion (e.g., using degron tags) versus chronic deletion

  • Examine time-course of phenotypic manifestations

• Condition-dependent phenotypes:

  • Test multiple growth conditions (carbon sources, stress conditions)

  • Examine mitochondrial function under different metabolic states

  • Consider genetic background effects that might mask or enhance phenotypes

• Quantitative analysis approaches:

  • Measure growth rates rather than endpoint measurements

  • Quantify mitochondrial morphology parameters

  • Assess mitochondrial membrane potential and respiratory capacity

What computational approaches can help predict YPR098C function?

Computational methods offer valuable insights for uncharacterized proteins:

• Sequence-based predictions:

  • Conserved domain analysis

  • Motif identification

  • Phylogenetic profiling to identify co-evolving genes

• Structure-based approaches:

  • AlphaFold or RoseTTAFold for structure prediction

  • Structure comparison with characterized proteins

  • Molecular docking to predict potential binding partners

• Network-based methods:

  • Gene co-expression analysis

  • Protein-protein interaction network integration

  • Functional enrichment analysis of genetic interactors

• Data integration strategies:

  • Bayesian integration of multiple data types

  • Machine learning approaches trained on characterized proteins

  • Literature-based discovery methods

How can researchers design rigorous controls for YPR098C functional studies?

• Genetic controls:

  • Complementation with wild-type YPR098C to confirm phenotype specificity

  • Use of known mitochondrial outer membrane protein mutants as benchmarks

  • Inclusion of unrelated mitochondrial protein controls

• Protein interaction controls:

  • Non-specific binding controls using unrelated proteins with similar physicochemical properties

  • Competition assays to verify binding specificity

  • Reciprocal tagging approaches to confirm interactions

• Localization study controls:

  • Co-localization with established mitochondrial markers

  • Controls for potential tag-induced mislocalization

  • Comparison with other mitochondrial compartment markers