Recombinant Clavispora lusitaniae Genetic interactor of prohibitin 7, mitochondrial (GEP7)

What is the GEP7 gene in Clavispora lusitaniae and how is it related to its homolog in Saccharomyces cerevisiae?

GEP7 (Genetic interactor of prohibitin 7, mitochondrial) in Clavispora lusitaniae is a homolog of the S. cerevisiae GEP7 gene, which encodes a protein of unknown function that interacts genetically with prohibitins. In S. cerevisiae, GEP7 belongs to the GEP7 family and null mutants exhibit respiratory growth defects and synthetic interactions with prohibitin (phb1) and gem1 . The protein is detected in highly purified mitochondria and is believed to play a role in mitochondrial function. While the exact function remains unclear in both species, comparative genomic analyses suggest conservation of the mitochondrial localization and potential interactions with prohibitins across fungal species.

What are the most reliable methods for isolating and identifying C. lusitaniae strains for GEP7 research?

For reliable isolation and identification of C. lusitaniae strains, researchers should employ a multi-faceted approach:

• Molecular identification: PCR amplification and sequencing of ribosomal DNA internal transcribed spacer (ITS) regions for species confirmation

• Genomic fingerprinting: Use of restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) analysis to determine strain relatedness and verify isolate identity

• Phenotypic confirmation: Assessment of drug susceptibility profiles using standardized protocols

• Morphological verification: Microscopic examination of cellular and colony characteristics

When working with clinical isolates specifically, researchers should document the treatment history as this can significantly impact genetic profiles, particularly regarding drug resistance genes . For example, in published studies, researchers have successfully used RFLP and RAPD analysis to confirm that multiple C. lusitaniae isolates from a single patient were genetically related despite exhibiting different antifungal susceptibility profiles .

How can GEP7 be distinguished from other mitochondrial proteins in C. lusitaniae?

Distinguishing GEP7 from other mitochondrial proteins in C. lusitaniae requires several complementary approaches:

• Epitope tagging: Generate recombinant strains expressing GEP7 with C-terminal or N-terminal epitope tags (e.g., HA, FLAG, GFP) that can be detected with specific antibodies

• Subcellular fractionation: Isolate highly purified mitochondria using differential centrifugation and density gradient separation techniques

• Proteomic analysis: Perform liquid chromatography-mass spectrometry (LC-MS/MS) on mitochondrial fractions to identify GEP7 and distinguish it from other mitochondrial proteins

• Immunoprecipitation: Use tagged versions of GEP7 to pull down the protein complex and identify interacting partners through mass spectrometry

• Bioinformatic prediction: Analyze protein sequences for mitochondrial targeting sequences and transmembrane domains using tools specific for fungal proteins

Notably, in S. cerevisiae, GEP7 has been confirmed in highly purified mitochondrial fractions through high-throughput proteomic studies , suggesting similar approaches would be effective in C. lusitaniae.

What is the most efficient CRISPR-Cas9 system for targeting GEP7 in C. lusitaniae?

The most efficient CRISPR-Cas9 system for targeting GEP7 in C. lusitaniae employs a transient expression approach optimized specifically for this species, which has historically been recalcitrant to genomic manipulation . The system should include:

• Codon-optimized Cas9 expression driven by a strong constitutive promoter (such as TEF1 or ENO1)

• sgRNA design targeting unique sequences within the GEP7 open reading frame with minimal off-target potential

• Homology-directed repair templates with at least 50 bp homology arms flanking the cut site

• Selection markers appropriate for C. lusitaniae (such as nourseothricin resistance)

• Efficient transformation protocol optimized for C. lusitaniae (typically lithium acetate/PEG method with heat shock)

The transient CRISPR system has been successfully used for efficient genetic manipulation of C. lusitaniae, as reported in recent literature . For GEP7 specifically, researchers should design at least three different sgRNAs targeting conserved regions of the gene to increase the likelihood of successful editing, and verify edits through sequencing.

What are the key considerations when designing deletion mutants of GEP7 in C. lusitaniae?

When designing deletion mutants of GEP7 in C. lusitaniae, researchers should consider the following key factors:

• Deletion strategy design:

  • Complete ORF deletion vs. disruption (full deletion is preferable to avoid truncated proteins)

  • Marker selection (nourseothricin, hygromycin B, or other appropriate selectable markers)

  • Retention or removal of the selection marker after confirmation (recyclable marker systems)

• Genetic background selection:

  • Use well-characterized laboratory strains with known genotypes

  • Consider the mating type if sexual reproduction studies are planned

  • Baseline phenotypic characterization, especially mitochondrial function

• Confirmation methods:

  • PCR verification of deletion with primers outside the homology regions

  • Southern blotting to confirm single integration at the correct locus

  • RNA-seq or RT-qPCR to confirm absence of transcript

  • Whole genome sequencing to identify potential off-target effects

• Control strain generation:

  • Create complemented strains by reintroducing GEP7 at its native locus or at a neutral site

  • Include wild-type controls processed through the same transformation procedure

• Phenotypic characterization:

  • Respiratory growth on non-fermentable carbon sources

  • Mitochondrial morphology and function assessments

  • Synthetic genetic interaction testing with prohibitin (phb1) and other related genes

Based on experiences with similar genes in C. lusitaniae, deletion strategies that employ the CRISPR-Cas9 system with homology-directed repair have shown higher efficiency compared to traditional homologous recombination approaches .

How can gene complementation studies be designed to verify GEP7 function in C. lusitaniae?

Effective gene complementation studies for GEP7 in C. lusitaniae should follow this methodological framework:

• Vector construction:

  • Design vectors containing the native GEP7 gene with its endogenous promoter and terminator regions (approximately 1kb upstream and 500bp downstream)

  • Include an alternative selection marker different from that used for the deletion

  • Create versions with epitope tags (C-terminal tags preferred if N-terminus contains targeting signals)

• Integration strategies:

  • Reintegration at the native locus using CRISPR-Cas9 to ensure physiological expression levels

  • Integration at neutral genomic loci (e.g., RPS1 or similar safe harbor sites) for controlled expression

  • Use of autonomously replicating plasmids for temporary complementation

• Heterologous complementation:

  • Express GEP7 from related species (e.g., S. cerevisiae GEP7) in C. lusitaniae GEP7 deletion strain

  • Express C. lusitaniae GEP7 in S. cerevisiae gep7Δ to assess functional conservation

• Expression validation:

  • RT-qPCR to confirm transcription levels

  • Western blotting with epitope-tagged versions to confirm protein expression

  • Subcellular localization studies using fluorescent protein fusions

• Phenotypic rescue assessment:

  • Growth assays on fermentable and non-fermentable carbon sources

  • Mitochondrial function tests (oxygen consumption, membrane potential)

  • Genetic interaction tests with prohibitin and other interactors

• Mutational analysis:

  • Generate point mutations in conserved residues to identify critical functional domains

  • Create chimeric proteins with domains from other species to map functional regions

This comprehensive approach not only verifies the specific function of GEP7 but also provides insights into its evolutionary conservation and functional domains.

What methodologies are most effective for determining the role of GEP7 in mitochondrial function in C. lusitaniae?

The most effective methodologies for determining GEP7's role in mitochondrial function in C. lusitaniae include:

• Respiratory growth assessment:

  • Growth curve analysis on non-fermentable carbon sources (glycerol, ethanol, lactate)

  • Oxygen consumption measurements using respirometry

  • Measurement of mitochondrial membrane potential using fluorescent dyes (e.g., JC-1, TMRM)

• Mitochondrial morphology analysis:

  • Live-cell imaging using mitochondria-targeted fluorescent proteins

  • Transmission electron microscopy for ultrastructural analysis

  • Super-resolution microscopy for detailed morphological assessment

• Biochemical function assessment:

  • Measurement of activities of electron transport chain complexes

  • Assessment of reactive oxygen species (ROS) production

  • Analysis of mitochondrial protein import efficiency

• Genetic interaction mapping:

  • Synthetic genetic array analysis with known mitochondrial genes

  • Focused analysis of interactions with prohibitins and other GEP family proteins

  • Double knockout studies with genes implicated in similar pathways

• Omics approaches:

  • Proteomics analysis of purified mitochondria from wild-type and gep7Δ strains

  • Metabolomics analysis focusing on TCA cycle intermediates and related metabolites

  • Transcriptomics to identify compensatory responses to GEP7 deletion

• Protein interaction studies:

  • Co-immunoprecipitation with tagged GEP7 followed by mass spectrometry

  • Proximity labeling approaches (BioID or APEX) for in vivo interaction mapping

  • Yeast two-hybrid screening against mitochondrial protein libraries

Given the reported respiratory growth defects in S. cerevisiae gep7Δ mutants , tracking mitochondrial function through oxygen consumption rates and membrane potential would likely provide immediate insights into the role of GEP7 in C. lusitaniae.

How can researchers investigate the potential interaction between GEP7 and prohibitin in C. lusitaniae?

To investigate the potential interaction between GEP7 and prohibitin in C. lusitaniae, researchers should employ a multi-faceted approach:

• Genetic interaction analysis:

  • Generate single and double deletion mutants (gep7Δ, phb1Δ, gep7Δ/phb1Δ)

  • Compare growth phenotypes under various stress conditions

  • Quantitative fitness analysis under respiratory and fermentative conditions

• Direct protein interaction studies:

  • Co-immunoprecipitation using epitope-tagged GEP7 and PHB1

  • Bimolecular fluorescence complementation (BiFC) to visualize interactions in vivo

  • Förster resonance energy transfer (FRET) with fluorescent protein-tagged versions

• Mitochondrial complex analysis:

  • Blue native PAGE to identify prohibitin complexes with and without GEP7

  • Sucrose gradient fractionation to determine co-migration in complexes

  • Crosslinking mass spectrometry to map interaction interfaces

• Functional rescue experiments:

  • Overexpression of GEP7 in phb1Δ strains and vice versa

  • Domain mapping to identify interaction regions

  • Point mutation analysis to identify critical residues for interaction

• Localization studies:

  • Co-localization analysis using differently colored fluorescent tags

  • Super-resolution microscopy to determine spatial relationships within mitochondria

  • Immunogold electron microscopy for ultrastructural localization

• Transcriptional response analysis:

  • RNA-seq comparison of wild-type, gep7Δ, phb1Δ, and double mutants

  • ChIP-seq to identify any transcriptional regulatory relationships

  • qPCR validation of key differentially expressed genes

Based on studies in S. cerevisiae showing synthetic interactions between GEP7 and prohibitin (phb1) , similar interactions are likely to exist in C. lusitaniae and can be investigated using these methodologies.

What techniques are recommended for studying the potential role of GEP7 in drug resistance pathways in C. lusitaniae?

To investigate GEP7's potential role in drug resistance pathways in C. lusitaniae, the following techniques are recommended:

• Drug susceptibility testing:

  • Standardized broth microdilution assays for antifungal susceptibility testing

  • Time-kill kinetic assays to assess rate of fungicidal activity

  • Biofilm formation and drug penetration assays

• Resistance development monitoring:

  • Serial passage experiments in sub-inhibitory drug concentrations

  • Whole genome sequencing before and after resistance development

  • Transcriptomic analysis during resistance acquisition

• Gene expression correlation studies:

  • qRT-PCR analysis of GEP7 expression in drug-resistant versus susceptible isolates

  • Correlation analysis between GEP7 and known resistance genes (MRR1, MFS7, FKS1)

  • RNA-seq analysis of global transcriptional changes in gep7Δ mutants exposed to antifungals

• Genetic manipulation approaches:

  • Overexpression of GEP7 and assessment of resulting drug susceptibility profiles

  • Deletion of GEP7 in resistant strains to determine if resistance is reversed

  • Double mutant analysis with known resistance genes

• Mitochondrial function in resistance:

  • Assessment of mitochondrial membrane potential in resistant strains

  • Measurement of ROS production in response to antifungal exposure

  • Analysis of metabolic adaptations using metabolomics

• Protein interaction studies:

  • Pull-down experiments to identify interactions between GEP7 and resistance-associated proteins

  • Phosphoproteomic analysis to identify signaling pathways involving GEP7

  • Localization studies during drug exposure

Since C. lusitaniae can develop resistance to multiple antifungals , investigating whether GEP7's mitochondrial function contributes to drug resistance mechanisms, particularly through interactions with energy metabolism or stress response pathways, could reveal novel resistance mechanisms.

How does GEP7 in C. lusitaniae compare structurally and functionally to homologs in other Candida species?

Structural and functional comparison of GEP7 across Candida species reveals important evolutionary insights:

Structural comparison:

*Estimated values based on homology with S. cerevisiae GEP7

Functional comparison across species:

• Conservation of mitochondrial localization:

  • GEP7 homologs across fungal species localize to mitochondria, suggesting conserved function

  • Targeting sequences and transmembrane domains show higher variability than functional domains

• Genetic interaction conservation:

  • Interaction with prohibitin (PHB1) is conserved in S. cerevisiae and likely in Candida species

  • Synthetic interactions with mitochondrial morphology genes (e.g., GEM1) observed in multiple species

• Phenotypic impacts of deletion:

  • Respiratory growth defects are common across species when GEP7 is deleted

  • Severity of phenotypes varies, suggesting species-specific adaptations

• Divergence in regulatory mechanisms:

  • Promoter regions show significant divergence, indicating species-specific regulation

  • Stress response elements in promoters differ between pathogenic and non-pathogenic species

The GEP7 family appears to be well-conserved across fungal species, maintaining its association with mitochondrial function through interactions with prohibitins . Further comparative genomic analyses would help elucidate species-specific adaptations of this gene family in relation to pathogenicity and drug resistance.

What experimental approaches can determine if GEP7 has a role in the distinctive drug resistance mechanisms of C. lusitaniae?

To determine if GEP7 contributes to C. lusitaniae's distinctive drug resistance mechanisms, researchers should implement the following experimental approaches:

• Gene expression analysis in resistant isolates:

  • Compare GEP7 expression levels in clinical isolates with different resistance profiles

  • Analyze correlation between GEP7 expression and known resistance genes (MRR1, MFS7, FKS1)

  • Perform time-course expression analysis during resistance development

• Genetic modification studies:

  • Generate GEP7 deletion in both susceptible and resistant backgrounds

  • Create overexpression strains to assess if elevated GEP7 levels confer resistance

  • Introduce specific mutations in GEP7 to identify potential functional residues

• Drug susceptibility profiling:

  • Perform comprehensive antifungal susceptibility testing (AFST) for multiple drug classes:

    • Azoles (fluconazole, voriconazole)

    • Echinocandins (caspofungin, micafungin)

    • Polyenes (amphotericin B)

    • 5-flucytosine

• Mitochondrial function assessment:

  • Compare mitochondrial membrane potential in wild-type vs. gep7Δ strains during drug exposure

  • Measure ROS production and oxidative stress responses

  • Assess energy metabolism adaptations during drug exposure

• Transcriptional network analysis:

  • Perform RNA-seq comparing wild-type and gep7Δ strains with and without drug exposure

  • Analyze differential expression of known resistance genes like MRR1 and MFS7

  • Identify potential regulatory relationships between GEP7 and resistance pathways

• Protein interaction studies:

  • Investigate interactions between GEP7 and known resistance factors

  • Perform co-immunoprecipitation followed by mass spectrometry in drug-treated cells

  • Use proximity labeling to identify drug-induced changes in GEP7 interactome

• Comparative analysis with resistant clinical isolates:

  • Sequence GEP7 in clinical isolates showing different resistance profiles

  • Compare transcriptional profiles of GEP7 pathway genes between resistant isolates

C. lusitaniae is known to rapidly develop multidrug resistance through various mechanisms, including FKS1 mutations for echinocandin resistance and MFS7 overexpression for azole and 5-flucytosine resistance . Investigating whether GEP7's mitochondrial function intersects with these established pathways could reveal novel resistance mechanisms.

How can researchers effectively use phylogenetic analysis to understand the evolution of GEP7 in pathogenic yeasts?

Effective phylogenetic analysis of GEP7 in pathogenic yeasts requires a comprehensive methodological approach:

• Sequence acquisition and alignment:

  • Collect GEP7 homolog sequences from diverse fungal species (pathogenic and non-pathogenic)

  • Include sequence data from clinical isolates when available

  • Perform multiple sequence alignment using MAFFT or T-Coffee with iterative refinement

  • Manually curate alignments to correct for insertion/deletion errors

• Phylogenetic tree construction:

  • Employ multiple tree-building methods:

    • Maximum Likelihood (RAxML or IQ-TREE)

    • Bayesian Inference (MrBayes)

    • Distance-based methods (Neighbor-Joining)

  • Use appropriate evolutionary models selected by ModelTest or similar tools

  • Implement bootstrap analysis (>1000 replicates) to assess node support

• Selective pressure analysis:

  • Calculate dN/dS ratios across the gene to identify regions under selection

  • Perform branch-site tests to detect episodic selection on specific lineages

  • Use PAML or HyPhy for selection analysis across branches and sites

• Domain architecture analysis:

  • Map conserved domains onto the phylogenetic tree

  • Identify lineage-specific domain gain/loss events

  • Correlate domain architecture changes with species pathogenicity

• Synteny and gene neighborhood analysis:

  • Compare genomic context of GEP7 across species

  • Identify conservation or changes in neighboring genes

  • Correlate synteny breaks with major evolutionary transitions

• Integration with phenotypic data:

  • Map drug resistance profiles onto the phylogenetic tree

  • Correlate GEP7 sequence variations with mitochondrial phenotypes

  • Identify convergent evolution patterns in pathogenic lineages

• Ancestral sequence reconstruction:

  • Infer ancestral GEP7 sequences at key nodes

  • Experimentally test ancestral proteins through heterologous expression

  • Identify critical mutations that coincide with functional shifts

This integrated phylogenetic approach would reveal whether GEP7 has undergone adaptive evolution in pathogenic yeasts like C. lusitaniae compared to non-pathogenic relatives, potentially correlating with the emergence of drug resistance mechanisms or host adaptation strategies.

What are the most promising approaches for developing GEP7-targeted antifungal strategies against drug-resistant C. lusitaniae?

Development of GEP7-targeted antifungal strategies against drug-resistant C. lusitaniae should focus on these promising approaches:

• Structure-based drug design:

  • Solve the crystal structure of GEP7 and identify druggable pockets

  • Perform in silico screening of compound libraries against identified pockets

  • Design small molecule inhibitors that specifically target GEP7-prohibitin interactions

• Synthetic lethality exploitation:

  • Identify genes that show synthetic lethality with GEP7

  • Target these genes in strains with compromised GEP7 function

  • Screen for compounds that selectively target cells with altered GEP7 activity

• Mitochondrial function targeting:

  • Develop compounds that disrupt mitochondrial function in a GEP7-dependent manner

  • Target the electron transport chain in cells with altered GEP7 function

  • Exploit metabolic vulnerabilities revealed by GEP7 deletion studies

• Combination therapy strategies:

  • Test GEP7-targeting compounds in combination with existing antifungals

  • Focus on combinations with echinocandins and azoles where resistance mechanisms are well-characterized

  • Identify synergistic combinations that prevent resistance development

• Immunomodulatory approaches:

  • Develop strategies to enhance host recognition of C. lusitaniae with altered GEP7

  • Target surface changes that occur in response to GEP7 disruption

  • Enhance neutrophil and macrophage responses to fungal cells with compromised GEP7

• Peptide-based approaches:

  • Design peptide inhibitors that mimic GEP7 interaction domains

  • Develop cell-penetrating peptides that disrupt GEP7-prohibitin interactions

  • Screen for naturally occurring antimicrobial peptides with enhanced activity against gep7Δ strains

Since C. lusitaniae can rapidly develop resistance to multiple antifungal classes , targeting a mitochondrial protein like GEP7 could provide an alternative mechanism of action less susceptible to existing resistance mechanisms. The relationship between mitochondrial function and drug resistance in Candida species makes this approach particularly promising.

How can researchers design experiments to elucidate the relationship between GEP7 and multidrug resistance transport systems like MFS7?

To elucidate the relationship between GEP7 and multidrug resistance transport systems like MFS7 in C. lusitaniae, researchers should design experiments following this strategic framework:

• Genetic interaction studies:

  • Generate single and double deletion mutants (gep7Δ, mfs7Δ, gep7Δ/mfs7Δ)

  • Create strains with overexpression of one gene and deletion of the other

  • Perform epistasis analysis to determine genetic pathway relationships

  • Compare drug susceptibility profiles across these genetic backgrounds

• Transcriptional regulation analysis:

  • Examine MFS7 expression levels in gep7Δ mutants with and without drug exposure

  • Investigate GEP7 expression in strains with MRR1 gain-of-function mutations known to upregulate MFS7

  • Perform ChIP-seq to identify potential transcription factors linking these pathways

  • Use reporter gene assays to assess MFS7 promoter activity in different GEP7 backgrounds

• Protein localization and trafficking studies:

  • Track MFS7 localization in wild-type versus gep7Δ strains using fluorescent protein fusions

  • Examine membrane domain organization and potential co-localization

  • Investigate protein stability and turnover rates of MFS7 in different GEP7 backgrounds

  • Analyze post-translational modifications of both proteins during drug exposure

• Mitochondria-plasma membrane communication:

  • Investigate mitochondrial membrane potential effects on MFS7 activity

  • Examine lipid raft composition and plasma membrane fluidity in gep7Δ strains

  • Analyze calcium signaling between mitochondria and plasma membrane in relation to drug efflux

  • Measure ATP production and its impact on ABC and MFS transporter function

• Metabolic flux analysis:

  • Compare metabolic profiles between wild-type, gep7Δ, and mfs7Δ strains

  • Investigate how metabolic changes in gep7Δ affect drug detoxification pathways

  • Measure intracellular drug accumulation in different genetic backgrounds

  • Analyze redox status and its impact on drug efflux capacity

• Systems biology approach:

  • Perform integrative multi-omics analysis (transcriptomics, proteomics, metabolomics)

  • Construct network models connecting mitochondrial function to drug resistance

  • Use mathematical modeling to predict combined effects of GEP7 and MFS7 alterations

  • Validate model predictions through targeted experimental interventions

Research has shown that MFS7 upregulation contributes to fluconazole and 5-flucytosine resistance in C. lusitaniae . Since GEP7 is involved in mitochondrial function, investigating how mitochondrial status affects drug efflux transporter activity could reveal novel connections between energy metabolism and drug resistance mechanisms.

What methodological considerations are essential when using CRISPR-Cas9 to create precise point mutations in GEP7 for structure-function studies?

Creating precise point mutations in C. lusitaniae GEP7 using CRISPR-Cas9 requires specific methodological considerations for successful structure-function studies:

• sgRNA design for maximum specificity:

  • Select target sites that minimize off-target effects using predictive algorithms

  • Choose sgRNAs with the cut site within 10-20 bp of the desired mutation site

  • Design multiple sgRNAs per target to increase success rates

  • Validate sgRNA efficiency using in vitro Cas9 cleavage assays before cell transformation

• Repair template optimization:

  • Design ssODN (single-stranded oligodeoxynucleotide) repair templates for point mutations

  • Include 30-60 nucleotides of homology on each side of the cut site

  • Introduce silent mutations in the PAM site or sgRNA binding region to prevent re-cutting

  • Incorporate additional silent mutations as markers for screening

• CRISPR delivery system selection:

  • Use transient expression systems optimized for C. lusitaniae

  • Consider RNP (ribonucleoprotein) complex delivery for higher efficiency and lower off-target effects

  • Optimize transformation protocols specifically for C. lusitaniae (electroporation parameters, cell wall weakening)

  • Balance expression levels to minimize toxicity while maintaining editing efficiency

• Mutation design strategy:

  • Target conserved residues identified through multiple sequence alignments

  • Focus on residues within predicted functional domains or protein interaction interfaces

  • Create alanine scanning libraries across regions of interest

  • Design phosphomimetic mutations for potential regulatory sites (S/T to D/E)

• Screening and validation approach:

  • Implement high-throughput screening methods appropriate for C. lusitaniae

  • Use restriction enzyme digestion screening when mutations create or abolish sites

  • Design PCR strategies that can distinguish between wild-type and mutant sequences

  • Perform whole genome sequencing to verify on-target editing and detect potential off-target effects

• Phenotypic characterization plan:

  • Develop assays specific to GEP7 function (mitochondrial activity, prohibitin interaction)

  • Include growth assays under respiratory and fermentative conditions

  • Test drug susceptibility profiles using standardized methods

  • Examine synthetic genetic interactions with known GEP7 interactors

• Control design:

  • Generate revertant strains to confirm phenotype-genotype relationships

  • Create synonymous mutations at the same sites as controls

  • Include wild-type strains processed through the same transformation protocol

Since C. lusitaniae has been historically recalcitrant to genetic manipulation, the optimization of a transient CRISPR-Cas9 system specifically for this species represents a significant methodological advancement . Application of this technology for precise point mutations in GEP7 will enable detailed structure-function analysis of this mitochondrial protein and its potential roles in drug resistance.