Recombinant Kluyveromyces lactis Mitochondrial escape protein 2 (YME2)

What is the Kluyveromyces lactis Mitochondrial escape protein 2 (YME2) and what is its cellular localization?

YME2 (Yeast Mitochondrial Escape protein 2) was first identified in Saccharomyces cerevisiae through genetic screening where its deletion led to escape of mitochondrial DNA into the nucleus . The K. lactis ortholog is a single-spanning transmembrane protein localized to the inner mitochondrial membrane, with a specific topology where:

• N-terminus faces the mitochondrial matrix

• Transmembrane domain spans the inner mitochondrial membrane

• C-terminus extends into the intermembrane space

Experimental studies have shown that YME2 co-localizes with mitochondrial DNA nucleoids and associates with MIOREX complexes, which are large expressosome-like assemblies comprising factors bound to mitoribosomes involved in mitochondrial gene expression .

What expression systems are available for recombinant K. lactis YME2 production?

While native expression in K. lactis is possible, heterologous E. coli expression systems have proven effective for recombinant YME2 production. For example, the commercially available recombinant full-length K. lactis YME2 protein (covering amino acids 31-808) is expressed in E. coli with an N-terminal His tag for purification purposes .

For researchers working directly with K. lactis, several promoter systems can be employed:

• The traditional galactose/lactose-inducible and glucose-repressible LAC4 promoter, though it lacks tight regulation

• The engineered hybrid promoter P350, which combines segments of the strong constitutive GAP1 promoter with carbon source-sensitive elements, providing tight regulation and strong expression

The P350 promoter system is particularly advantageous as it:

• Maintains tight repression in the presence of glucose or glycerol

• Strongly induces expression once carbon sources are depleted

• Permits "autoinduction" without requiring addition of inducer molecules

What methodologies are most effective for studying YME2 complex formation?

Based on experimental data, K. lactis YME2 forms high molecular weight complexes (~1250 kDa) that contain multiple copies of the protein. Several complementary approaches have proven effective for investigating these complexes:

Blue Native PAGE Analysis:
This technique enables visualization of native protein complexes and has revealed that YME2 migrates at a size similar to dimeric complex V (~1250 kDa) .

Co-immunoprecipitation with Differentially Tagged Variants:
Studies employing strains expressing YME2 with different epitope tags (e.g., YME2-9Myc and YME2-6HA) have demonstrated:

• A visible size shift in Blue Native PAGE when both tagged variants are present

• Successful co-purification of YME2-6HA with YME2-9Myc (and vice versa)

• No co-purification in control strains with only one tagged variant

Mutational Analysis of AAA+ Domain:
Strategic mutations in the Walker A (K393A) and Walker B (D522A) motifs provide insights into complex assembly:

• Single K393A mutations do not disrupt complex formation

• Single D522A mutations partially impair complex formation

• Double K393A/D522A mutations severely compromise complex formation

What genetic interactions exist between YME2 and the mitochondrial protein biogenesis machinery?

YME2 exhibits important genetic interactions with components of the mitochondrial protein export machinery. Key findings include:

Notably, despite genetic interactions, Blue Native PAGE analysis revealed that YME2 complex formation is not compromised in either Δmdm38 or Δmba1 mutants, suggesting that neither protein is an integral component of the YME2 complex .

How can CRISPR-Cas9 technology be optimized for YME2 modification in K. lactis?

While K. lactis historically showed variable and generally low gene targeting efficiency compared to S. cerevisiae, several strategies have been developed to enhance targeted genetic modifications:

Optimization of Homologous Recombination:

• Wild-type K. lactis requires substantial homologous flanking sequences (up to 600 bp) to achieve 88% targeting efficiency

• Targeting efficiency drops dramatically with shorter flanking sequences (0% with 50 bp flanks)

NHEJ Pathway Disruption:

• Deletion of KlKU80 (component of non-homologous end joining pathway) dramatically improves targeting efficiency

• KlKU80 deletion strains show >97% targeting efficiency regardless of homologous flank length

Competition Strategy:

• Transformation in the presence of excess small DNA fragments increases targeting efficiency

• Enables efficient gene replacement using PCR-generated constructs with only 50 bp homologous flanking sequences

When designing CRISPR-Cas9 experiments for YME2 modification, researchers should consider:

• Using KlKU80 deletion strains if available

• Including at least 600 bp homologous flanking sequences in wild-type backgrounds

• Employing excess competitor DNA fragments during transformation

How does environmental light stress affect YME2 function in K. lactis?

While not directly focusing on YME2, research has revealed that K. lactis is responsive to light, which acts as an environmental stressor. This is particularly relevant when studying mitochondrial proteins like YME2:

• Light exposure induces oxidative stress in yeasts, similar to what has been observed in S. cerevisiae

• Light stress affects pathways related to mitochondrial function:

  • Growth rate

  • Respiration rate

  • Mitochondrial morphology

  • Expression of detoxifying enzymes (catalase and SOD)

• Membrane composition plays a role in light stress response:

  • Phenotypes related to mitochondrial function are partially or completely suppressed by darkness or unsaturated fatty acids

  • This suggests a link between light stress, membrane functionality, and mitochondrial proteins

When designing experiments to study YME2 function, researchers should control for light exposure and consider how this environmental variable might influence experimental outcomes, particularly when examining mitochondrial phenotypes.

What are the recommended experimental conditions for analyzing YME2-dependent phenotypes?

Based on available research, the following conditions are recommended for investigating YME2-dependent phenotypes:

Growth Media Considerations:

• Non-fermentable carbon sources should be used to accentuate respiratory phenotypes

• Control experiments with both respiratory (glycerol) and fermentative (glucose) carbon sources are advisable

Environmental Controls:

• Light/dark conditions should be carefully controlled and documented

• Growth at different temperatures may reveal conditional phenotypes

Genetic Background:

• Wild-type and YME2 deletion strains should be compared under identical conditions

• Consider creating double mutants with known genetic interactors (e.g., MDM38, MBA1)

Phenotypic Assays:

• Mitochondrial morphology: Fluorescence microscopy with mitochondrial-targeted fluorophores

• Respiration rate: Oxygen consumption measurements

• mtDNA stability: qPCR analysis of mitochondrial to nuclear DNA ratio

• Complex formation: Blue Native PAGE followed by Western blotting

How should recombinant K. lactis YME2 protein be handled and stored for optimal stability?

For researchers working with purified recombinant K. lactis YME2 protein, the following handling and storage conditions are recommended:

Storage Buffer:

• Tris/PBS-based buffer with 6% Trehalose at pH 8.0

Reconstitution Protocol:

• Briefly centrifuge the vial prior to opening

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (recommended: 50%)

• Aliquot for long-term storage

Storage Conditions:

• Store at -20°C/-80°C upon receipt

• Working aliquots can be stored at 4°C for up to one week

• Avoid repeated freeze-thaw cycles

This handling protocol maximizes protein stability and prevents activity loss during storage, ensuring reliable experimental results.