Recombinant Debaryomyces hansenii Genetic interactor of prohibitins 3, mitochondrial (GEP3), partial

What is Debaryomyces hansenii and why is it relevant to mitochondrial research?

Debaryomyces hansenii is a non-conventional oleaginous budding yeast found in natural salty environments and on salted foods such as cheeses and cured meats. It is considered a highly valuable organism for both fundamental and biotechnological research due to its unique characteristics:

• It is an osmotolerant, stress-tolerant, and oleaginous microbe with considerable biotechnological potential

• D. hansenii is metabolically versatile, non-pathogenic, and represents an attractive target for applied biotechnological research

• Most D. hansenii strains are haploid but can temporarily become diploid through autogamy

Its relevance to mitochondrial research stems from its unusual stress tolerance mechanisms, which involve specific mitochondrial adaptations. These adaptations make it an excellent model organism for studying the role of mitochondrial membrane proteins like prohibitins under various stress conditions.

What are prohibitins and how do they function in mitochondria?

Prohibitins are evolutionarily conserved membrane proteins essential for cell proliferation and development in higher eukaryotes:

• They form large, multimeric ring complexes in the inner membrane of mitochondria composed of PHB1 and PHB2 subunits

• Prohibitins function as protein and lipid scaffolds that ensure the integrity and functionality of the mitochondrial inner membrane

• They regulate the processing of the dynamin-like GTPase OPA1, which controls mitochondrial fusion and cristae morphogenesis

• In their absence, cells exhibit increased reactive oxygen species generation, disorganized mitochondrial nucleoids, abnormal cristae morphology, and increased sensitivity to apoptotic stimuli

Prohibitins have a fundamental functional link with membrane phospholipids, particularly cardiolipin and phosphatidylethanolamine, highlighting their role in maintaining mitochondrial membrane integrity .

How does GEP3 interact with the prohibitin complex in yeast mitochondria?

GEP3 (Genetic interactor of prohibitins 3) functions as a critical mitochondrial protein that genetically and functionally interacts with prohibitin complexes. Based on available research:

• GEP3 was identified through genetic screens as a synthetic lethal interactor with prohibitin deletions in yeast

• It likely participates in the assembly or stability of the prohibitin scaffold complex in the inner mitochondrial membrane

• The protein appears to play a role in maintaining mitochondrial morphology and function, particularly when prohibitin function is compromised

• GEP3 may be involved in the regulation of OPA1 processing, which is a key process regulated by prohibitins

What techniques are most effective for targeting the GEP3 gene in D. hansenii?

Recent advances have made gene targeting in D. hansenii significantly more efficient:

• PCR-based gene targeting using 50 bp homology flanks has been shown to achieve integration through homologous recombination at frequencies exceeding 75%

• The method employs a simple PCR-based amplification that extends a completely heterologous selectable marker with 50 bp flanks identical to the target site in the genome

• Two effective selectable marker cassettes have been developed for D. hansenii:

  • A hygromycin B resistance cassette containing the CTG codon-adapted Klebsiella pneumoniae hygromycin B phosphotransferase (hph) ORF placed between S. stipitis TEF1 promoter and terminator

  • A G418/Geneticin resistance cassette containing the CTG codon-adapted bacterial kanamycin resistance (kanr) ORF from E. coli transposon Tn903, placed under the control of S. stipitis ACT1 promoter and terminator

How can transformation efficiency be optimized when working with D. hansenii?

Optimizing transformation efficiency for D. hansenii requires attention to several key parameters:

The PCR product should be purified to remove any residual template DNA and primers that might interfere with the transformation process .

What are the recommended protocols for verifying successful GEP3 targeting?

Verification of successful GEP3 targeting in D. hansenii should follow these steps:

• Colony PCR screening:

  • Design primers that span the junction between the integrated cassette and genomic DNA

  • One primer should bind within the marker cassette and the other to genomic DNA outside the homology region

  • This confirms proper integration at the intended locus

• Southern blot analysis:

  • To confirm single integration event and rule out random integrations

  • Use probes specific to the marker cassette and to the targeted locus

• Whole-genome sequencing:

  • For comprehensive verification of integration and detection of potential off-target effects

  • Particularly valuable for strains destined for detailed functional studies

• RT-PCR or RNA-seq:

  • To confirm the absence of GEP3 transcript in knockout strains

  • Or to verify expression levels in gene replacement variants

• Western blot:

  • For protein-level verification in tagged versions of GEP3

  • Requires development of specific antibodies or use of epitope tags

How does the deletion of GEP3 affect mitochondrial function in relation to prohibitins?

Deletion of GEP3 in yeast models has revealed significant impacts on mitochondrial function, particularly when prohibitin function is also compromised:

• Loss of prohibitins leads to destabilization of the mitochondrial genome and respiratory deficiencies in aged neurons

• In the absence of prohibitins, mitochondria show abnormal cristae morphology and perinuclear clustering

• GEP3 deletion, particularly in combination with prohibitin deficiency, likely exacerbates these phenotypes

• The processing of OPA1, which regulates mitochondrial fusion and cristae morphogenesis, is significantly affected when either prohibitins or their interactors are compromised

These observations suggest that GEP3 works in concert with prohibitins to maintain proper mitochondrial ultrastructure and function.

What are the functional implications of GEP3-prohibitin interactions under osmotic stress?

D. hansenii is known for its exceptional osmotolerance, making it an ideal model to study mitochondrial adaptations under salt stress conditions:

• Prohibitin complexes ensure the integrity of the mitochondrial inner membrane , which is particularly important under stress conditions

• GEP3 likely contributes to this regulatory network, helping maintain mitochondrial function during osmotic stress

• The yeast's ability to produce and assimilate a wide variety of polyols is part of its osmoadaptation strategy

• Transport systems, including polyol/H+ symporters, play critical roles in D. hansenii's osmoadaptation

Understanding how GEP3-prohibitin interactions respond to osmotic stress could reveal novel aspects of stress tolerance mechanisms in this extremophilic yeast.

How do GEP3-prohibitin interactions differ between D. hansenii and other yeast models?

Comparative analysis reveals important differences in GEP3-prohibitin interactions across yeast species:

These differences highlight the evolutionary adaptations of mitochondrial regulatory networks across yeast species with different ecological niches and metabolic capabilities.

What approaches can be used to study prohibitin complex assembly in D. hansenii?

Several methodologies are applicable for studying prohibitin complex assembly:

• Blue Native PAGE:

  • Allows visualization of intact prohibitin complexes

  • Can detect changes in complex size and composition when GEP3 is modified

• Fluorescence microscopy with tagged proteins:

  • GFP tagging of prohibitins and GEP3 enables visualization of their localization

  • Five distinct D. hansenii polyol/H+ symporters have been successfully tagged with GFP and characterized

  • Similar approaches could be applied to GEP3 and prohibitins

• Co-immunoprecipitation followed by mass spectrometry:

  • Identifies proteins interacting with prohibitins or GEP3

  • Can reveal novel components of these complexes

• Cryo-electron microscopy:

  • Provides structural insights into prohibitin complex organization

  • Can detect structural changes induced by GEP3 deletion

How can mitochondrial function be assessed in GEP3-modified D. hansenii strains?

Assessment of mitochondrial function in GEP3-modified strains should include:

• Oxygen consumption measurements:

  • Respirometry to assess oxidative phosphorylation capacity

  • Compare basal, maximal, and reserve respiratory capacity

• Membrane potential assays:

  • Using fluorescent dyes like JC-1 or TMRM

  • Quantify changes in mitochondrial membrane potential

• ROS production:

  • Measure reactive oxygen species using specific probes

  • Assess impact of GEP3 modification on oxidative stress

• mtDNA stability:

  • PCR-based assays to detect mtDNA deletions or copy number changes

  • Loss of prohibitins leads to destabilization of the mitochondrial genome

• Mitochondrial morphology:

  • Electron microscopy to examine cristae structure

  • Confocal microscopy with mitochondrial markers to assess network morphology

  • Loss of prohibitins affects mitochondrial ultrastructure and induces perinuclear clustering

What are promising approaches for studying GEP3-prohibitin interaction under industrial conditions?

Future research should focus on understanding how GEP3-prohibitin interactions contribute to D. hansenii's industrial applications:

• Study GEP3 function under conditions relevant to food fermentation (cheese ripening, meat curing)

• Investigate how GEP3-prohibitin interactions contribute to D. hansenii's resistance to perchlorate in bioreactors

• Develop optimized strains with enhanced stress tolerance through targeted modification of GEP3 and prohibitins

• Explore the connection between polyol metabolism (a key feature of D. hansenii ) and mitochondrial function mediated by GEP3-prohibitin interactions

How can CRISPR-Cas9 technology be adapted for targeting GEP3 in D. hansenii?

While the search results don't specifically mention CRISPR-Cas9 use in D. hansenii, this technology could potentially be adapted:

• Codon optimization:

  • Adapt Cas9 and guide RNA expression for D. hansenii's genetic code

  • Consider the CTG codon usage difference, as was done for other heterologous genes

• Delivery methods:

  • Develop efficient transformation protocols specifically for CRISPR components

  • Consider using ribonucleoprotein (RNP) complexes to avoid integration

• Guide RNA design:

  • Optimize for D. hansenii's genome composition

  • Select target sites with minimal off-target potential

• Homology-directed repair templates:

  • Leverage the high efficiency of homologous recombination in D. hansenii

  • Use the established 50 bp homology flanks that have proven effective