Recombinant Saccharomyces cerevisiae Sensitive to high expression protein 9, mitochondrial (SHE9)

What is SHE9 and where is it localized in yeast cells?

SHE9 (Sensitive to High Expression protein 9) is a protein localized to the inner mitochondrial membrane of Saccharomyces cerevisiae. It plays an essential role in maintaining normal mitochondrial morphology. While the protein has been characterized structurally as spanning amino acids 31-456, its precise molecular function remains under investigation . Research has established that SHE9 is critical for proper mitochondrial functioning and homeostasis, particularly under respiratory growth conditions, suggesting its involvement in energy metabolism pathways within the mitochondria .

What phenotypes are observed in SHE9 deletion mutants?

SHE9 deletion mutants (she9Δ) display several distinctive phenotypes that provide insights into the protein's function:

• Viability despite abnormal mitochondrial morphology

• Formation of large ring-like mitochondrial structures

• Extremely elongated mitochondria that can extend through half of the cell

• Decreased mitochondrial fission activity while maintaining fusion capabilities

• Significantly reduced growth during diauxic shift, especially when cultivated on glycerol media (YPG) compared to glucose media (YPD)

• Exacerbated growth defects at elevated temperatures (37°C), suggesting temperature sensitivity

These phenotypes collectively indicate that SHE9 plays important roles in mitochondrial dynamics, particularly in maintaining proper balance between fusion and fission processes.

How does SHE9 expression level affect mitochondrial structure?

SHE9 expression levels dramatically influence mitochondrial structure in opposing ways:

When SHE9 is deleted (she9Δ):

• Mitochondria form large ring-like structures

• Mitochondrial fission appears compromised while fusion remains functional

• Extremely elongated mitochondria extend through half the cell

Conversely, when SHE9 is overexpressed:

• Mitochondria form membranous partitions/septa that separate the inner compartment into distinct chambers

• Some mitochondria become largely devoid of cristae

• There is a measurable decrease in phospholipids cardiolipin (CL) and phosphatidylethanolamine (PE)

This bidirectional effect suggests SHE9 acts as a key regulator of mitochondrial structure, potentially through its influence on membrane organization and phospholipid composition.

What is the role of SHE9 during diauxic shift in yeast?

SHE9 appears particularly critical during diauxic shift - the metabolic transition from fermentative to respiratory metabolism. Research demonstrates:

• she9Δ cells display significantly decreased growth on glycerol media (YPG), which forces cells to rely on oxidative phosphorylation rather than glycolysis

• The growth defect becomes more pronounced at elevated temperatures (37°C)

• During diauxic shift, when mitochondria primarily employ oxidative phosphorylation to generate ATP, SHE9 appears essential for this metabolic adaptation

These findings suggest SHE9 plays a specialized role in optimizing mitochondrial function specifically during respiratory growth conditions, potentially through its effects on mitochondrial structure and membrane composition that support efficient oxidative phosphorylation.

How might SHE9 interact with phospholipid metabolism pathways?

Several lines of evidence suggest SHE9 plays a regulatory role in mitochondrial phospholipid metabolism:

• Overexpression of SHE9 leads to decreased levels of phospholipids cardiolipin (CL) and phosphatidylethanolamine (PE), suggesting negative regulation of their synthesis or stability

• Bioinformatics analyses have identified potential functional associations between SHE9 and proteins involved in phospholipid metabolism, particularly the Mdm31/Mdm32/Por1 complex

• In non-fermentable carbon sources, SHE9 may function as an inhibitor of the Ups-1 independent CL accumulation pathway

• Epistasis studies show that she9Δ mutants are epistatic to many genes encoding mitochondrial structure and dynamics, except for deletions of genes involved in lipid metabolism (FMP30, GEM1, MDM10, MDM12, MDM31, MDM34, or MMM1), where the she9Δ phenotype was largely suppressed

A proposed model suggests that under non-fermentable carbon sources, SHE9 participates as an inhibitor of phospholipid metabolism pathways that are particularly important during respiratory growth, potentially explaining the growth defects observed in she9Δ mutants during diauxic shift.

What molecular mechanisms have been proposed for SHE9 function?

While the exact molecular function of SHE9 remains to be fully elucidated, several mechanisms have been proposed:

• Phospholipid metabolism regulation: SHE9 may inhibit the Ups-1 independent cardiolipin (CL) accumulation pathway. According to a proposed model, under non-fermentable carbon sources, SHE9 inhibits this pathway, and its deletion might lead to increased phosphatidylethanolamine (PE), which in turn inhibits CL production.

• Mitochondrial fission regulation: she9Δ cells show decreased mitochondrial fission while maintaining fusion capability, suggesting SHE9 might promote or regulate fission processes.

• Interaction with membrane organization complexes: Bioinformatics analyses suggest interactions between SHE9 and proteins involved in membrane organization, such as Mdm31/Mdm32 (MIM proteins) and Por1 (MOM protein).

• Nucleic acid transport: Reports indicate she9Δ reduces nucleic acid uptake by mitochondria, suggesting SHE9 might facilitate this transport process, potentially through connections to outer membrane complexes .

These proposed mechanisms provide a foundation for future research to establish SHE9's precise molecular function.

What genetic interactions have been identified for SHE9?

Several important genetic interactions have been identified for SHE9:

• Epistatic relationships: she9Δ mutants are epistatic to many genes encoding mitochondrial structure and dynamics

• Exception to epistasis: When combined with deletions of genes involved in lipid metabolism (FMP30, GEM1, MDM10, MDM12, MDM31, MDM34, or MMM1), the she9Δ phenotype is largely suppressed

• Potential functional associations: Bioinformatics analyses using GeneMANIA have identified both genetic (functional) and physical interactions of SHE9

• Notable interactions: Particular attention has been drawn to interactions with MIM proteins Mdm31/Mdm32 and MOM protein Por1, which play crucial roles in phospholipid metabolism

These genetic interactions provide valuable clues about the biological pathways in which SHE9 participates and highlight the importance of phospholipid metabolism in understanding SHE9 function.

What methodological approaches can be used to study SHE9 function?

Multiple experimental approaches can be employed to investigate SHE9 function:

• Genetic manipulation:

  • Generation of she9Δ deletion strains

  • Overexpression systems

  • Creation of specific point mutations or domain deletions

  • Double deletion mutants for genetic interaction studies

• Phenotypic analysis:

  • Growth assays under different carbon sources (glucose vs. glycerol) and temperatures

  • Mitochondrial morphology visualization using fluorescent markers

  • Respirometry to measure oxidative phosphorylation capacity

• Biochemical approaches:

  • Mass spectrometry analysis of phospholipid composition

  • Protein-protein interaction studies

  • Subcellular fractionation to confirm localization

• Bioinformatics:

  • String analysis and other prediction tools to identify potential interactors

  • Structural modeling to predict functional domains

• Recombinant protein studies:

  • Expression and purification of recombinant SHE9 for in vitro studies

  • Structural analyses through techniques like crystallography

These methodologies, used in combination, can provide comprehensive insights into SHE9 function.

How can recombinant SHE9 protein be effectively produced and purified?

Based on available information about recombinant SHE9 production and practices for mitochondrial protein expression, the following approach is recommended:

• Expression system:

  • E. coli has been successfully used to express recombinant SHE9 protein (amino acids 31-456) with an N-terminal His-tag

  • For improved solubility, specialized E. coli strains like BL21(DE3) may be beneficial

• Construct design:

  • Using amino acids 31-456 appears effective, likely omitting the mitochondrial targeting sequence

  • N-terminal His-tag facilitates purification

  • Codon optimization for the expression host may improve yields

• Expression conditions:

  • Induction at lower temperatures (16-20°C) may improve protein folding

  • Extended expression times at lower temperatures

  • Use of specialized media such as Terrific Broth

• Purification approach:

  • Immobilized metal affinity chromatography (IMAC) using the His-tag

  • Gentle detergents to maintain native structure

  • Size exclusion chromatography as a secondary purification step

These conditions would need to be optimized for specific applications and downstream uses of the protein.

What techniques can validate SHE9's role in phospholipid metabolism?

To experimentally validate SHE9's potential role in phospholipid metabolism, researchers should consider:

• Lipidomic analysis:

  • Quantitative mass spectrometry of phospholipids (particularly CL and PE) in wild-type, she9Δ, and SHE9-overexpressing strains

  • Comparison of lipid profiles between cells grown in fermentable (glucose) versus non-fermentable (glycerol) carbon sources

  • Analysis of acyl chain composition and cardiolipin remodeling

• Metabolic labeling:

  • Pulse-chase experiments with isotope-labeled phospholipid precursors

  • Tracking incorporation rates in wild-type versus mutant strains

• Genetic interaction studies:

  • Construction of double mutants combining she9Δ with deletions of genes involved in phospholipid metabolism (e.g., CRD1, PSD1, UPS1, UPS2)

  • Testing whether manipulating phospholipid levels rescues she9Δ phenotypes

• Biochemical assays:

  • In vitro binding assays between recombinant SHE9 and phospholipids

  • Analysis of enzyme activities involved in CL and PE metabolism in she9Δ strains

These approaches would provide comprehensive evidence regarding SHE9's role in phospholipid metabolism, building on the observation that SHE9 overexpression decreases CL and PE levels.

How can microscopy techniques be optimized for visualizing SHE9 and mitochondrial morphology?

Several advanced microscopy approaches can be optimized for SHE9 visualization:

• Fluorescent protein tagging:

  • C-terminal fusion with GFP, YFP, or mCherry

  • Validation that the fusion protein remains functional through complementation testing

  • Co-localization with established mitochondrial markers

  • Live-cell imaging using confocal or super-resolution microscopy

• Immunofluorescence microscopy:

  • Generation of specific antibodies against SHE9

  • Use of epitope tags if antibodies are unavailable

  • Optimized fixation and permeabilization protocols for mitochondrial proteins

  • Co-staining with antibodies against known mitochondrial markers

• Electron microscopy approaches:

  • Immunogold labeling for precise sub-mitochondrial localization

  • Correlative light and electron microscopy (CLEM)

  • Particularly useful for analyzing the altered mitochondrial morphology in she9Δ or SHE9 overexpression conditions

These techniques would provide detailed insights into SHE9 localization and its effects on mitochondrial structure.

What are the most promising directions for future SHE9 research?

Based on current knowledge, several promising research directions emerge:

• Structural biology:

  • Determination of SHE9's three-dimensional structure

  • Identification of functional domains and critical residues

  • Structure-guided design of mutations to probe specific functions

• Mechanistic studies:

  • Detailed investigation of SHE9's role in phospholipid metabolism

  • Clarification of its function during diauxic shift and respiratory growth

  • Exploration of its role in mitochondrial fission/fusion balance

• Interaction network mapping:

  • Comprehensive identification of SHE9 binding partners

  • Validation of predicted interactions with Mdm31/Mdm32 and Por1

  • Investigation of conditional interactions that may occur specifically during respiratory growth

• Translational potential:

  • Exploration of homologs in higher eukaryotes

  • Investigation of whether SHE9 functions are conserved in mammalian cells

  • Potential relevance to mitochondrial diseases

These research directions would significantly advance our understanding of SHE9's role in mitochondrial function and potentially reveal new insights into fundamental aspects of mitochondrial biology.