Recombinant Chaetomium globosum Mitochondrial escape protein 2 (YME2)

What is Chaetomium globosum YME2 and what is its primary function?

YME2 (Mitochondrial escape protein 2) is a protein found in the inner mitochondrial membrane of Chaetomium globosum, a filamentous fungus. The protein plays a critical role in mitochondrial protein biogenesis, specifically in the co-translational integration of mitochondrial DNA-encoded proteins into the inner membrane. Functionally, YME2 appears to be linked to the mitochondrial protein export machinery and interacts genetically with components such as Mdm38, Mba1, and Oxa1 . The protein consists of 831 amino acids (with the mature form spanning residues 49-831) and contains specific motifs including an RNA recognition motif (RRM) that faces the mitochondrial matrix and an AAA+ domain located in the intermembrane space .

What structural domains characterize YME2 protein?

YME2 contains two primary structural domains that are essential for its function:

• RNA Recognition Motif (RRM): Located on the matrix-facing side of the protein, this domain likely facilitates interaction with RNA molecules, potentially mitochondrial RNA .

• AAA+ Domain: Positioned in the intermembrane space, this domain typically functions in ATP binding and hydrolysis. In YME2, this domain contains the characteristic Walker A and Walker B motifs, though with an unusual substitution in the Walker B motif (an Arginine replaces the typical Glutamate) .

How is recombinant YME2 typically expressed and purified?

Recombinant full-length Chaetomium globosum YME2 protein is commonly expressed in E. coli expression systems with an N-terminal His tag to facilitate purification . The expression construct typically includes residues 49-831, representing the mature form of the protein after processing of the mitochondrial targeting sequence. Following expression, the protein is purified using affinity chromatography, leveraging the His tag, and is generally supplied as a lyophilized powder . For research purposes, the protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the addition of 5-50% glycerol for long-term storage at -20°C/-80°C .

How do the Walker A and Walker B motifs affect YME2 complex formation?

The Walker A and B motifs in YME2's AAA+ domain are crucial for complex formation and function. Research has shown that YME2 assembles into a high molecular weight complex of approximately 1250 kDa, similar in size to dimeric complex V . Mutational studies have revealed the following:

• Walker A Motif (K393): Mutation of the conserved lysine (K393A) does not significantly impair complex formation, suggesting this residue may not be critical for assembly .

• Walker B Motif (D522, R523): The Walker B motif in YME2 is unusual, with an arginine residue replacing the typical glutamate. Mutation of the aspartate residue (D522A) partially impairs complex formation .

• Double Mutation (K393A/D522A): When both Walker A and B motifs are mutated simultaneously, complex formation is severely compromised, indicating their collective importance in maintaining the structural integrity of the YME2 complex .

These findings suggest that the AAA+ domain plays a crucial role in YME2 oligomerization and function, with the Walker B motif being particularly important for proper assembly.

What is known about YME2 oligomerization and complex formation?

Blue Native PAGE and Western Blot analysis have revealed that YME2 forms a distinct high molecular weight complex of approximately 1250 kDa . This suggests that multiple copies of YME2 assemble together, possibly forming a homooligomeric complex. Interestingly, the complex formation appears to be independent of other mitochondrial proteins such as Mdm38 and Mba1, as deletion of these proteins does not affect YME2 complex formation .

The complex likely contains multiple copies of YME2, and its assembly depends on the integrity of the AAA+ domain, particularly the Walker B motif. The exact stoichiometry and three-dimensional structure of the complex remain areas for further investigation.

What is the role of YME2 in mitochondrial protein biogenesis?

YME2 plays a significant role in mitochondrial protein biogenesis, particularly in the co-translational integration of mitochondrial DNA-encoded proteins into the inner membrane. Research has established genetic interactions between YME2 and other components of the mitochondrial protein export machinery, including Mdm38, Mba1, and Oxa1 .

The functional model suggests that:

• Mdm38 and Mba1 act as ribosome receptors, recruiting mitochondrial ribosomes to the inner membrane

• Oxa1 functions as an insertase, facilitating membrane integration of client proteins

• YME2 likely works in concert with these components, potentially assisting in the proper positioning or processing of nascent mitochondrial peptides

The RNA recognition motif (RRM) facing the matrix suggests that YME2 may interact with mitochondrial RNA, potentially playing a role in coordinating translation with membrane insertion .

How do genetic interactions between YME2 and other mitochondrial proteins inform its function?

Genetic interaction studies have revealed important functional relationships between YME2 and other mitochondrial proteins:

• YME2 and MDM38: Previous large-scale screens have identified a negative genetic interaction between YME2 and MDM38 . This suggests that these proteins may have partially redundant functions or work in parallel pathways.

• YME2, MBA1, and OXA1: Genetic interactions with these components of the mitochondrial protein export machinery link YME2 to the process of integrating mitochondrial-encoded proteins into the inner membrane .

These genetic interactions provide valuable insights into YME2's functional network within mitochondria and suggest its involvement in the coordinated process of mitochondrial protein synthesis and membrane integration.

What are the optimal conditions for expressing and purifying recombinant YME2?

For optimal expression and purification of recombinant Chaetomium globosum YME2:

Expression System:

• E. coli is the preferred expression system for full-length YME2 protein (residues 49-831)

• N-terminal His-tagging facilitates purification and detection

Purification Protocol:

• Express in E. coli with appropriate induction conditions

• Lyse cells under native conditions

• Purify using nickel affinity chromatography

• Consider additional purification steps (ion exchange, size exclusion) for higher purity

• Lyophilize the purified protein for stability

Storage Conditions:

• Store lyophilized powder at -20°C/-80°C

• After reconstitution, add 5-50% glycerol for long-term storage

• Avoid repeated freeze-thaw cycles

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

What techniques are most effective for analyzing YME2 complex formation?

Several techniques have proven effective for studying YME2 complex formation:

• Blue Native PAGE: This technique has successfully revealed the high molecular weight YME2 complex (~1250 kDa) and is useful for comparing wild-type and mutant forms of the protein .

• Size Exclusion Chromatography: Useful for determining the native molecular weight of the complex in solution and confirming oligomerization state.

• Crosslinking Approaches: Chemical crosslinking followed by mass spectrometry can help identify interaction interfaces within the complex.

• Mutational Analysis: Systematic mutation of conserved residues, particularly in the AAA+ domain, can provide insights into regions critical for complex formation .

• Electron Microscopy: Negative staining or cryo-EM can be employed for structural characterization of the YME2 complex.

For reliable results, researchers should consider using multiple complementary techniques to validate their findings regarding YME2 complex formation.

How can researchers generate and characterize YME2 mutants?

To generate and characterize YME2 mutants:

• Mutation Design:

  • Target conserved residues in functional domains

  • Key residues to consider include K393 (Walker A motif), D522 (Walker B motif), R523 (unusual Walker B residue), and R565 (potential arginine finger)

• Mutagenesis Approach:

  • Site-directed mutagenesis using PCR-based methods

  • Gibson assembly or other seamless cloning methods for introducing mutations

• Expression Systems:

  • Express in E. coli with appropriate tags for purification and detection

  • Consider TAP-tagging for complex isolation studies

• Functional Characterization:

  • Assess protein stability through western blotting

  • Evaluate complex formation using Blue Native PAGE

  • Test genetic interactions with known partners (Mdm38, Mba1, Oxa1)

  • Measure ATPase activity to assess the impact of mutations on enzymatic function

• In vivo Analysis:

  • Complement YME2 deletion strains with mutant variants

  • Assess growth phenotypes on different carbon sources

  • Monitor mitochondrial function and morphology

Previous studies have successfully characterized YME2 mutants using these approaches, particularly focusing on the AAA+ domain and its role in complex formation .

How does YME2 relate to other characterized proteins from Chaetomium globosum?

Chaetomium globosum produces various proteins with diverse functions, but the relationship between YME2 and other characterized proteins requires further investigation. Current research has identified:

Understanding the broader proteome of C. globosum and potential functional relationships between YME2 and other proteins would require additional research, possibly including interactome studies and comparative genomics approaches.

What approaches can be used to investigate YME2's role in mitochondrial protein biogenesis?

Investigating YME2's role in mitochondrial protein biogenesis requires a multi-faceted approach:

• Genetic Interaction Studies:

  • Generate double mutants with components of the mitochondrial protein export machinery (Mdm38, Mba1, Oxa1)

  • Perform growth analyses under different conditions to assess functional relationships

  • Conduct synthetic genetic array (SGA) analysis to identify novel genetic interactions

• Biochemical Approaches:

  • Co-immunoprecipitation to identify physical interaction partners

  • Proximity labeling techniques (BioID, APEX) to map the local protein environment

  • Ribosome profiling to assess the impact on mitochondrial translation

• Structural Biology:

  • Cryo-electron microscopy of the YME2 complex

  • Crosslinking mass spectrometry to identify interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry to identify dynamic regions

• In vitro Reconstitution:

  • Reconstitute YME2 into liposomes to study membrane-associated functions

  • In vitro translation assays with purified components to assess the direct role in protein synthesis/insertion

• Microscopy Techniques:

  • Super-resolution microscopy to visualize YME2 localization relative to ribosomes and other components

  • Live-cell imaging with fluorescently tagged YME2 to monitor dynamics

These complementary approaches would provide a comprehensive understanding of YME2's functional role in mitochondrial protein biogenesis.

How can researchers assess the RNA-binding capabilities of YME2's RRM domain?

To characterize the RNA-binding function of YME2's RNA Recognition Motif (RRM):

• RNA Electrophoretic Mobility Shift Assay (EMSA):

  • Express and purify the isolated RRM domain

  • Test binding to various RNA substrates (mitochondrial mRNAs, rRNAs, tRNAs)

  • Determine binding affinity and specificity

• UV Crosslinking and Immunoprecipitation (CLIP):

  • Perform CLIP analysis to identify in vivo RNA targets

  • Sequence bound RNAs to determine binding motifs or preferences

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI):

  • Quantitatively measure binding kinetics and affinity

  • Compare wild-type and mutant RRM domains

• RNA Interactome Capture:

  • Use RNA baits to capture YME2 and confirm RNA-binding capability

  • Identify specific RNA sequences or structures that preferentially bind to YME2

• Structural Analysis:

  • Solve the structure of the RRM domain in complex with RNA using X-ray crystallography or NMR

  • Identify key residues involved in RNA recognition

• Functional Validation:

  • Generate RRM domain mutants and assess their impact on YME2 function

  • Perform in vitro translation assays to determine if RNA binding affects mitochondrial translation

These approaches would establish whether the RRM domain is functional and identify its specific RNA targets, providing insights into YME2's role in coordinating RNA processing or translation in mitochondria.