Recombinant Vanderwaltozyma polyspora Mitochondrial escape protein 2 (YME2)

What is YME2 and what is its fundamental role in mitochondrial function?

YME2 (Yeast Mitochondrial Escape protein 2) was initially discovered in a genetic screen conducted in Saccharomyces cerevisiae, where loss of YME2 resulted in the escape of mitochondrial DNA (mtDNA) from mitochondria to the nucleus . In Vanderwaltozyma polyspora, YME2 is believed to perform similar functions related to mtDNA maintenance and protein biogenesis. The protein has been found to co-localize with mtDNA nucleoids and associate with MIOREX complexes, which are large expressosome-like assemblies comprising factors bound to mitoribosomes involved in mitochondrial gene expression . This association suggests that YME2 plays a critical role in maintaining mitochondrial genome stability and potentially in coordinating mitochondrial translation with membrane protein insertion.

What is the subcellular localization and topology of YME2?

YME2 is a single-spanning transmembrane protein embedded in the inner mitochondrial membrane. It has a distinctive topology with its N-terminus exposed to the mitochondrial matrix and its C-terminus facing the intermembrane space . This orientation is critical for its function, as it positions different functional domains in separate mitochondrial compartments. Specifically, the RNA recognition motif (RRM) faces the matrix where mitochondrial ribosomes and nucleoids reside, while its AAA+ domain is positioned in the intermembrane space . This arrangement allows YME2 to potentially interact with both matrix components (such as mtDNA, RNA, or ribosomes) and proteins in the intermembrane space.

What structural domains and motifs characterize YME2?

Structural analysis of YME2 using bioinformatics tools like AlphaFold has revealed distinct N-terminal and C-terminal domains separated by a transmembrane alpha helix. The protein contains:

• An RNA recognition motif (RRM) in the matrix-facing N-terminal domain

• A transmembrane domain between residues 287 and 305

• An AAA+ domain in the intermembrane space-facing C-terminal portion

• Putative Walker motifs that are important for YME2 function

The presence of an RRM suggests that YME2 can interact with RNA, potentially mitochondrial mRNAs or rRNAs, while the AAA+ domain (typically associated with ATP binding and hydrolysis) indicates that YME2 may have ATPase activity involved in protein complex assembly or disassembly functions.

How does YME2 interact with other mitochondrial proteins?

YME2 exhibits negative genetic interactions with several components of the mitochondrial protein biogenesis machinery. The strongest negative genetic interaction is observed between YME2 and MDM38, a protein that acts as a receptor to recruit mitochondrial ribosomes to the inner membrane . Additionally, YME2 genetically interacts with MBA1 and OXA1, which are involved in co-translational membrane insertion of mitochondrially-encoded proteins . These interactions suggest that YME2 functions in coordination with the mitochondrial translation and protein insertion machinery, potentially coupling these processes to prevent aberrant accumulation of unfolded proteins or improperly assembled complexes.

What approaches can researchers use to study the role of YME2 in mitochondrial protein biogenesis?

Researchers investigating YME2's role in mitochondrial protein biogenesis can employ several sophisticated approaches:

• Ribosome profiling analysis: To determine if YME2 affects translation efficiency of specific mitochondrial mRNAs, researchers can compare ribosome occupancy on mitochondrial transcripts in wild-type and YME2-deficient cells.

• Proximity labeling techniques: BioID or APEX2 fusions with YME2 can identify proteins in close proximity, revealing potential interaction partners involved in protein biogenesis.

• In organello translation assays: These can assess whether YME2 deletion affects the synthesis rate of mitochondrially-encoded proteins.

• Pulse-chase experiments: These experiments can determine if YME2 affects the stability or membrane insertion efficiency of newly synthesized mitochondrial proteins.

• Co-immunoprecipitation with components of the MIOREX complex: This approach can confirm direct interactions between YME2 and specific factors involved in mitochondrial gene expression .

The genetic interactions between YME2 and components like MDM38, MBA1, and OXA1 suggest that combining these approaches with strains lacking these factors could provide particularly insightful results regarding YME2's specific contribution to mitochondrial protein biogenesis.

How can researchers investigate the structure-function relationship of YME2's domains?

To explore the structure-function relationships of YME2's domains, researchers can implement the following methodological approaches:

• Site-directed mutagenesis: Introducing specific mutations in the RRM, Walker motifs, or AAA+ domain can help determine which residues are critical for function. For instance, mutations in the Walker A motif (typically GxxGxGKT/S) would disrupt ATP binding and hydrolysis, potentially affecting YME2 function .

• Domain swapping experiments: Replacing domains with homologous regions from related proteins can reveal which domains confer specificity for particular functions.

• Truncation analysis: Creating truncated versions of YME2 lacking specific domains can determine which regions are necessary and sufficient for different functions.

• Structural studies: X-ray crystallography or cryo-EM of purified recombinant YME2 can provide detailed structural information about domain organization and potential conformational changes upon substrate binding.

• Crosslinking mass spectrometry: This technique can identify residues that are in close proximity in the native protein structure and map interaction interfaces between YME2 and binding partners.

A combination of these approaches could reveal how the unusual arrangement of domains (with the RRM in the matrix and AAA+ domain in the intermembrane space) contributes to YME2's function in mitochondrial DNA maintenance and protein biogenesis.

What explains the assembly of YME2 into high molecular weight complexes?

YME2 has been shown to assemble into high molecular weight complexes , raising important questions about the functional significance of this multimerization. Researchers can explore this phenomenon through:

• Blue native PAGE analysis: To determine the precise molecular weight and composition of YME2-containing complexes under different conditions.

• Size exclusion chromatography combined with multi-angle light scattering (SEC-MALS): To determine the absolute molecular weight and stoichiometry of purified YME2 complexes.

• Negative stain or cryo-electron microscopy: To visualize the architecture of YME2 complexes and determine if they form specific geometric arrangements.

• Crosslinking studies: To identify which regions of YME2 are involved in self-association.

• Mutational analysis targeting predicted oligomerization interfaces: To determine which residues are critical for complex formation and whether complex formation is essential for function.

The AAA+ domain present in YME2 provides a potential mechanism for oligomerization, as many AAA+ proteins form hexameric rings. Researchers should investigate whether ATP binding or hydrolysis regulates YME2 complex assembly and if complex formation is linked to specific physiological conditions or stresses.

How does YME2 prevent mitochondrial DNA escape to the nucleus?

The original identification of YME2 came from its role in preventing mitochondrial DNA escape to the nucleus . Advanced research approaches to investigate this function include:

• Quantitative PCR assays: To measure the rate of mtDNA transfer to the nucleus in wild-type versus YME2-deficient strains.

• Fluorescence microscopy with DNA-specific dyes: To visualize mtDNA localization and potential escape events in real-time.

• ChIP-seq with YME2 antibodies: To determine if YME2 directly binds to specific regions of mtDNA.

• Electron microscopy: To examine whether YME2 affects mitochondrial membrane integrity or nucleoid structure.

• Genetic screens for suppressors of the mtDNA escape phenotype: To identify other factors that work in concert with YME2.

Understanding the mechanism by which YME2 prevents mtDNA escape could provide insights into mitochondrial genome stability and the evolutionary significance of horizontal gene transfer from mitochondria to the nucleus.

What expression systems are optimal for producing recombinant Vanderwaltozyma polyspora YME2?

For successful expression and purification of recombinant V. polyspora YME2, researchers should consider the following expression systems and methodological approaches:

When expressing membrane proteins like YME2, it's crucial to consider:

• Expression of individual domains (RRM or AAA+ domain) separately may yield better results than full-length protein

• Lower induction temperatures (16-20°C) often improve proper folding

• Inclusion of detergents like DDM, LMNG, or GDN during extraction is essential for maintaining native structure

• Screening multiple constructs with varying N- and C-terminal boundaries can identify optimal expression constructs

The RRM domain alone may be more amenable to structural studies and RNA-binding assays, while the AAA+ domain could be studied for its ATPase activity independently of the full-length protein.

What techniques can be used to analyze YME2's RNA recognition capabilities?

Given that YME2 contains an RNA recognition motif (RRM) facing the mitochondrial matrix , researchers can employ several techniques to characterize its RNA-binding properties:

• RNA Electrophoretic Mobility Shift Assays (EMSA): To determine if purified YME2 or its RRM domain binds to specific RNA sequences.

• RNA immunoprecipitation followed by sequencing (RIP-seq): To identify RNAs that associate with YME2 in vivo.

• Fluorescence anisotropy: To measure binding affinities between YME2 and fluorescently labeled RNA substrates.

• CRISPR-mediated RNA tracking: To visualize interactions between YME2 and specific RNAs in living cells.

• Systematic Evolution of Ligands by Exponential Enrichment (SELEX): To identify RNA motifs that bind with high affinity to the YME2 RRM domain.

• UV crosslinking mass spectrometry: To map the exact residues involved in RNA recognition.

Researchers should focus on testing both mitochondrial transcripts (mRNAs, rRNAs, tRNAs) and potential non-coding RNAs, as the specificity of the RRM domain could provide crucial insights into YME2's functional role in mitochondrial gene expression.

How can researchers experimentally validate the genetic interactions between YME2 and other mitochondrial proteins?

To validate and characterize the reported genetic interactions between YME2 and other mitochondrial proteins like MDM38, MBA1, and OXA1 , researchers can employ:

• Synthetic genetic array (SGA) analysis: To systematically quantify genetic interactions between YME2 and other genes on a genome-wide scale.

• Tetrad analysis: To analyze the viability of double mutants and quantify the strength of genetic interactions.

• Growth curve analysis: To precisely measure growth defects of single and double mutants under various conditions (temperature, carbon source, oxidative stress).

• Mitochondrial function assays: To determine if the genetic interactions correlate with specific mitochondrial defects:

  • Oxygen consumption measurements

  • Membrane potential assays

  • ATP production capacity

  • Cytochrome content analysis

• Biochemical isolation of protein complexes: To determine if the genetic interactions reflect physical interactions or shared functions in the same pathway.

• In vivo protein complementation assays: Split fluorescent proteins or split ubiquitin assays can reveal direct physical interactions.

The strong negative genetic interaction between YME2 and MDM38 suggests they function in parallel pathways with overlapping essential functions, potentially in coupling mitochondrial translation to membrane protein insertion.

What approaches can be used to investigate the ATPase activity of YME2's AAA+ domain?

The presence of a AAA+ domain and Walker motifs in YME2 suggests it may possess ATPase activity critical for its function. Researchers can characterize this activity using:

• Colorimetric ATPase assays: Malachite green assays can quantify inorganic phosphate release from ATP hydrolysis.

• Radioactive ATPase assays: Using [γ-32P]ATP to measure ATP hydrolysis with high sensitivity.

• Enzyme-coupled ATPase assays: NADH-coupled assays that link ATP hydrolysis to NADH oxidation for continuous monitoring.

• Structure-based mutagenesis: Targeting key residues in the Walker A (ATP binding) and Walker B (ATP hydrolysis) motifs to correlate ATPase activity with biological function.

• Nucleotide binding assays: Using fluorescent ATP analogs or isothermal titration calorimetry to measure ATP binding affinity and stoichiometry.

• ATP-dependent conformational change analysis: Using limited proteolysis or hydrogen-deuterium exchange mass spectrometry to detect ATP-induced structural changes.

Researchers should investigate whether the ATPase activity of YME2 is regulated by RNA binding, protein interactions, or specific physiological conditions. The coupling between ATP hydrolysis and biological function could provide mechanistic insights into how YME2 contributes to mitochondrial DNA maintenance and protein biogenesis.

How does V. polyspora YME2 compare to its homologs in other yeast species?

Comparing YME2 across different yeast species can provide evolutionary insights and functional clues. Researchers should consider:

• Sequence conservation analysis: Multiple sequence alignment of YME2 homologs can identify highly conserved residues likely critical for function, particularly within the RRM and AAA+ domains.

• Functional complementation experiments: Testing whether YME2 from V. polyspora can rescue phenotypes in S. cerevisiae yme2 deletion strains would determine functional conservation.

• Domain architecture comparison: Examining whether the unique arrangement of domains in YME2 is conserved across species or whether alternative domain organizations exist in some lineages.

• Phylogenetic analysis: Constructing a phylogenetic tree of YME2 homologs to track the evolutionary history of this protein and identify potential instances of functional divergence.

The genetic interaction pattern of YME2 with proteins like MDM38, MBA1, and OXA1 should be compared across species to determine if these functional relationships are evolutionarily conserved or represent species-specific adaptations in mitochondrial biogenesis pathways.

What can we learn from the comparison between V. polyspora YME2 and the alanyl-tRNA synthetase genes?

The search results provide information about alanyl-tRNA synthetase genes in V. polyspora , which presents an interesting comparative case with YME2:

• Both systems involve proteins with dual cellular localization patterns (cytoplasmic and mitochondrial).

• V. polyspora uniquely possesses two distinct AlaRS genes (VpALA1 and VpALA2) that arose from duplication of a dual-functional predecessor , which parallels questions about the evolutionary origin of YME2.

• The use of non-ATG initiation codons in VpALA2 represents a specialized translational control mechanism that might also be relevant for understanding YME2 expression regulation.

• Both systems highlight V. polyspora as a valuable model organism for studying the evolution of mitochondrial protein targeting and function.

Researchers investigating YME2 should consider whether similar evolutionary processes (gene duplication, subfunctionalization, alternative translation initiation) have shaped its current structure and function in V. polyspora compared to other yeast species.

What emerging technologies could advance our understanding of YME2 function?

Several cutting-edge approaches could significantly advance YME2 research:

• Cryo-electron tomography: To visualize YME2 complexes in their native membrane environment.

• Single-molecule FRET: To analyze conformational dynamics of YME2 during its functional cycle.

• Proximity-dependent biotinylation (BioID or TurboID): To map the dynamic interactome of YME2 under different conditions.

• AlphaFold-based structural modeling: To predict interactions between YME2 and its partners at atomic resolution.

• CRISPR base editing: For precise introduction of specific mutations without completely disrupting gene function.

• Microfluidics-based single-cell analysis: To examine cell-to-cell variability in YME2 function and mitochondrial phenotypes.

These approaches, combined with classical genetic and biochemical techniques, could resolve the molecular mechanism by which YME2 prevents mitochondrial DNA escape and participates in mitochondrial protein biogenesis.

What are the implications of YME2 research for understanding human mitochondrial diseases?

While YME2 has been primarily studied in yeast, its research has broader implications:

• Identification of human homologs: Bioinformatic searches for human proteins with similar domain architecture could identify functional equivalents.

• Conservation of mechanisms: The fundamental processes by which YME2 prevents mtDNA escape may be conserved in human cells, with implications for mitochondrial DNA stability.

• Therapeutic targets: Understanding how YME2 maintains mitochondrial integrity could reveal new approaches for treating mitochondrial disorders.

• Evolutionary insights: The study of YME2 contributes to our understanding of how mitochondrial genomes have evolved and how nuclear-mitochondrial communication is maintained.

Future research should explore whether mechanisms discovered in YME2 studies could help explain pathogenic mitochondrial DNA deletions or nuclear insertions of mitochondrial sequences (NUMTs) observed in human diseases.