Recombinant Candida glabrata Mitochondrial import inner membrane translocase subunit TIM22 (TIM22)

What is the basic structure and function of TIM22 in Candida glabrata?

TIM22 in C. glabrata functions as the core channel-forming component of the mitochondrial inner membrane translocase complex. Similar to other fungal species, it likely contains four transmembrane domains that form a hydrophobic lateral cave exposed to the lipid bilayer . The TIM22 complex mediates the insertion of multipass transmembrane proteins, particularly members of the SLC25A family of metabolite carrier proteins, into the mitochondrial inner membrane .

The TIM22 complex in most fungal species has an apparent molecular weight of approximately 300-440 kDa when assessed by blue native PAGE . Structurally, TIM22 is expected to have four hydrophobic transmembrane segments with conserved cysteine residues that form a disulfide bond critical for protein stability and function .

How does the TIM22 complex in C. glabrata compare to those in other yeast species?

While specific data on C. glabrata TIM22 is limited in the provided search results, we can infer similarities based on conservation patterns. In Saccharomyces cerevisiae, the TIM22 complex consists of four membrane-integrated subunits (Tim22, Tim54, Tim18, and Sdh3) and a peripheral chaperone complex of small TIM proteins (Tim9-Tim10-Tim12) .

It's worth noting that metazoan TIM22 complexes contain additional subunits not found in yeast, such as Tim29 , highlighting evolutionary divergence in this machinery.

What are the recommended methods for recombinant expression and purification of C. glabrata TIM22?

Based on successful approaches with TIM22 from other organisms, the following methodology is recommended:

• Expression system selection:

  • For structural studies, a HEK293F cell expression system has proven effective for human TIM22 complex expression

  • For biochemical studies, yeast or E. coli expression systems may be suitable

• Purification strategy:

  • Affinity purification using a Flag-tag followed by gel filtration has successfully yielded well-behaved TIM22 complex

  • Blue native PAGE can be used to assess complex integrity, with an expected molecular weight of approximately 440 kDa

• Quality control:

  • Mass spectrometry confirmation of protein identity

  • Circular dichroism or thermal shift assays to verify proper folding

  • Size exclusion chromatography to assess oligomeric state

What techniques are most effective for structural characterization of recombinant C. glabrata TIM22?

Based on recent advances in structural biology of membrane proteins:

• Cryo-electron microscopy (cryo-EM):

  • This has proven highly effective for human TIM22 complex structure determination, yielding a 3.7 Å resolution map

  • Sample preparation involves grid preparation with purified complex

• Crosslinking mass spectrometry:

  • Useful for mapping protein-protein interactions within the complex

  • Can identify intersubunit contacts that stabilize the complex

• Functional reconstitution:

  • Reconstitution of purified TIM22 into liposomes to assess channel activity

  • Electrophysiological measurements can verify channel formation and characterize conductance properties

How can the channel activity of recombinant C. glabrata TIM22 be assessed in vitro?

Reconstitution experiments have demonstrated that TIM22 forms a hydrophilic, high-conductance channel with distinct opening states and pore diameters . To assess channel activity:

• Liposome reconstitution:

  • Purified TIM22 protein can be incorporated into liposomes of defined lipid composition

  • The protein-to-lipid ratio should be optimized to ensure proper insertion

• Electrophysiological measurements:

  • Planar lipid bilayer experiments can measure conductance properties

  • TIM22 channels exhibit voltage-dependent gating and specific responses to substrate proteins with internal targeting signals, but not to presequences

• Substrate protein interaction assays:

  • Fluorescently labeled substrate proteins can be used to measure binding affinity

  • Changes in channel conductance upon substrate addition can indicate functional interactions

What is the significance of the conserved cysteine residues in C. glabrata TIM22?

Based on studies in other yeast species, the conserved cysteine residues in TIM22 form an intramolecular disulfide bond that is critical for protein stability and function . Specifically:

• Structural stability:

  • The disulfide bond likely stabilizes the conformation of the transmembrane segments (particularly TM1 and TM2)

  • This stabilization is essential for maintaining the quaternary structure of the TIM22 complex

• Functional implications:

  • Mutations of these cysteine residues (Cys→Ser) result in destabilization of the TIM22 complex

  • The destabilized complex shows reduced efficiency in assembly of polytopic inner membrane proteins, especially when handling excess substrate proteins

  • Growth defects become apparent in yeast cells with Cys→Ser mutations when carrier proteins are overexpressed

A disulfide bond between Cys69 and Cys141 has been identified in structural studies, which appears to stabilize transmembrane helices . These cysteines are likely conserved in C. glabrata TIM22 as well.

How might TIM22 function relate to C. glabrata pathogenicity and drug resistance?

Candida glabrata has emerged as a significant pathogen with innate resistance to azole antifungal agents . While direct evidence linking TIM22 to pathogenicity is not established in the provided search results, several potential connections can be hypothesized:

• Metabolic adaptation:

  • The TIM22 complex is responsible for inserting metabolite carriers into the mitochondrial inner membrane

  • These carriers are essential for mitochondrial metabolism, which may influence C. glabrata's ability to adapt to diverse host environments

• Stress response:

  • Mitochondrial function is implicated in cellular stress responses

  • Proper protein import via TIM22 may be critical for responding to host immune defenses or antifungal drugs

• Drug resistance mechanisms:

  • Altered mitochondrial function has been linked to antifungal resistance in some fungi

  • TIM22-dependent protein import might influence the expression or localization of drug efflux pumps or other resistance factors

Could C. glabrata TIM22 serve as a potential antifungal target?

Given the essential nature of TIM22 in mitochondrial biogenesis, it represents a potential target for novel antifungal development:

• Target validation considerations:

  • TIM22 is the only essential membrane-integrated subunit of the insertion complex in yeast

  • It combines three essential functions: signal recognition, channel formation, and energy transduction

• Selectivity potential:

  • Structural or functional differences between fungal and human TIM22 complexes could be exploited

  • The human TIM22 complex contains additional subunits like Tim29 not present in fungi , suggesting divergent mechanisms

• Experimental approaches:

  • High-throughput screening of compound libraries against recombinant C. glabrata TIM22

  • Structure-based drug design targeting unique features of the fungal protein

  • In vitro reconstitution assays to identify compounds that disrupt channel activity

How does C. glabrata TIM22 differ from human TIM22, and what are the implications for research?

Based on the available information about yeast and human TIM22 complexes:

• Compositional differences:

  • The human TIM22 complex includes metazoan-specific subunits like Tim29, which are absent in fungi

  • Tim29 creates a link between the TIM22 and TOM complexes in humans, facilitating cooperation between these complexes

• Structural variations:

  • Human TIM22 complex has an apparent molecular weight of approximately 440 kDa , compared to the 300 kDa complex in yeast

  • Recent cryo-EM structures of human TIM22 complex revealed only one Tim22 subunit per complex, forming a lateral hydrophobic cave rather than a closed pore channel

• Functional implications:

  • These differences suggest divergent mechanisms of substrate recognition and membrane insertion

  • They may offer opportunities for selective targeting of fungal TIM22 for therapeutic purposes

What experimental controls are essential when comparing TIM22 function across different yeast species?

When conducting comparative studies between C. glabrata TIM22 and its homologs in other yeast species:

• Expression level normalization:

  • Ensure comparable expression levels of TIM22 across different species

  • Western blotting with antibodies against conserved epitopes or tagged constructs

• Substrate selection:

  • Use conserved substrate proteins that are imported via TIM22 in multiple species

  • The ADP/ATP carrier (AAC) and phosphate carrier (PiC) are well-characterized substrates

• Growth condition standardization:

  • Culture all yeast strains under identical conditions

  • Test multiple conditions, including stress conditions that might reveal functional differences

• Genetic complementation assays:

  • Test whether C. glabrata TIM22 can complement S. cerevisiae TIM22 deletion

  • Analyze growth rates and mitochondrial protein import efficiency in complemented strains

How does the disulfide bond in TIM22 influence conformational dynamics during protein import?

The disulfide bond between conserved cysteine residues in TIM22 plays a crucial role in protein stability and function . Advanced research questions include:

• Conformational changes during import:

  • The disulfide bond may restrict conformational flexibility of Tim22

  • Molecular dynamics simulations could reveal how this constraint affects channel gating

• Redox regulation:

  • The redox state of the mitochondrial intermembrane space might regulate TIM22 activity

  • Investigation of whether transient reduction/oxidation of the disulfide bond occurs during the import cycle

• Experimental approaches:

  • Site-directed spin labeling and electron paramagnetic resonance spectroscopy to track conformational changes

  • Single-molecule FRET to monitor distance changes between domains during substrate binding

What is the role of TIM22 in regulating mitochondrial one-carbon metabolism in C. glabrata?

Recent studies have implicated the TIM22 complex in regulating mitochondrial one-carbon metabolism , which could have significant implications for C. glabrata physiology:

• Metabolic dependencies:

  • Investigation of how TIM22 function affects one-carbon metabolic enzymes

  • The TIM22 complex may influence levels of key proteins like MTHFD2 and SFXN family transporters

• Potential research directions:

  • Metabolomic profiling of wild-type versus TIM22-depleted C. glabrata

  • Analysis of how alterations in one-carbon metabolism affect virulence and drug resistance

  • Comparison with metabolic adaptations in other pathogenic Candida species

• Experimental approaches:

  • CRISPR-based manipulation of TIM22 expression levels

  • Proteomics analysis of mitochondrial carrier protein abundance

  • 13C-labeled substrate tracing to monitor metabolic flux

What are the common challenges in expressing and purifying functional recombinant C. glabrata TIM22?

Membrane protein expression and purification present numerous challenges:

• Expression systems:

  • Toxicity when overexpressed in heterologous systems

  • Improper folding or aggregation

  • Solution: Test multiple expression systems (E. coli, yeast, insect cells) and optimize induction conditions

• Solubilization and stability:

  • Finding appropriate detergents for extraction from membranes

  • Maintaining stability during purification

  • Solution: Screen detergent panels and consider addition of lipids or stabilizing agents

• Quality assessment:

  • Verifying proper folding and oligomeric state

  • Solution: Combine size exclusion chromatography, BN-PAGE, and thermal shift assays

• Functional verification:

  • Confirming that purified protein forms channels

  • Solution: Liposome reconstitution and electrophysiology measurements

How can researchers overcome difficulties in studying protein-protein interactions within the C. glabrata TIM22 complex?

Studying interactions within membrane protein complexes presents unique challenges:

• Co-immunoprecipitation strategies:

  • Use epitope tags at positions that don't disrupt complex formation

  • Carefully optimize detergent conditions to maintain interactions

  • Cross-validation with multiple tag positions and reciprocal pull-downs

• Crosslinking approaches:

  • Chemical crosslinking combined with mass spectrometry can map interaction interfaces

  • In vivo photo-crosslinking with genetically encoded crosslinkers provides spatial precision

• Genetic interaction mapping:

  • Synthetic genetic array analysis to identify functional relationships

  • Suppressor screening to identify compensatory mutations

• Split reporter assays:

  • Bimolecular fluorescence complementation to visualize interactions in living cells

  • Split luciferase assays for quantitative interaction assessment