Recombinant Zygosaccharomyces rouxii Protein SEY1 (SEY1)

What is the structural composition of Zygosaccharomyces rouxii Sey1p?

Sey1p from Z. rouxii, like its homologs in other yeasts, is a dynamin-like GTPase that mediates homotypic fusion of the endoplasmic reticulum. Crystal structure analyses of the cytosolic domain of Sey1p (derived from Candida albicans) reveal a distinctive structure consisting of a GTPase domain followed by a stalk-like, helical bundle domain. This helical bundle represents a previously unidentified configuration within the dynamin superfamily . The stalk-like domain is significantly longer than that found in metazoan atlastins (ATLs) and is critical for the fusion process. The protein also contains transmembrane domains that anchor it to the ER membrane .

How does the GTPase activity of Sey1p contribute to its function?

Interestingly, while most dynamin-like proteins strictly require GTP hydrolysis for their function, Sey1p has been shown to mediate membrane fusion even without GTP hydrolysis, though the efficiency is significantly enhanced with GTP. This suggests a unique mechanism of action compared to other dynamin superfamily members. The punctate localization of Sey1p in cells depends on its GTPase activity, indicating that this activity is essential for proper targeting within the ER network .

What expression systems are recommended for producing recombinant Z. rouxii Sey1p?

For recombinant expression of Z. rouxii Sey1p, several systems can be considered:

When designing expression constructs, consider including affinity tags (such as His-tag or GST) for purification purposes, and ensure appropriate codon optimization depending on the host system used.

How does the osmotolerant nature of Z. rouxii influence the functional properties of its Sey1p compared to homologs from other yeasts?

Z. rouxii is known for its remarkable osmotolerance, allowing it to thrive in environments with high salt or sugar concentrations . This adaptation is likely reflected in the properties of its cellular proteins, including Sey1p.

When investigating the functional properties of Z. rouxii Sey1p compared to homologs from non-osmotolerant yeasts, researchers should consider several aspects:

• Membrane composition effects: Z. rouxii adapts to osmotic stress partly through modifications in membrane composition, including changes in unsaturated fatty acid content. The interaction between Sey1p and these specialized membranes may differ from interactions in other yeasts .

• Experimental approaches: To investigate these differences, researchers can:

  • Express recombinant Z. rouxii Sey1p and its homologs from non-osmotolerant yeasts in a common host

  • Compare their GTPase activities and membrane fusion efficiencies under varying osmotic conditions

  • Assess structural stability using circular dichroism or thermal shift assays across different salt/sugar concentrations

• Potential adaptations: Given Z. rouxii's ability to maintain proper cell function under extreme conditions, its Sey1p might exhibit enhanced stability or modified activity parameters compared to homologs from osmosensitive yeasts. These could include altered GTP binding affinity, modified dimerization properties, or enhanced interaction with specific lipid compositions characteristic of stress-adapted membranes.

What are the recommended approaches for studying the in vitro membrane fusion activity of recombinant Z. rouxii Sey1p?

For studying the membrane fusion activity of recombinant Z. rouxii Sey1p in vitro, researchers can employ several complementary approaches:

• Liposome fusion assays: Reconstitute purified recombinant Sey1p into liposomes of defined composition. Monitor fusion using:

  • Fluorescence-based assays (lipid mixing: using NBD-rhodamine FRET pairs)

  • Content mixing assays (using self-quenching fluorescent dyes)

  • Light scattering to monitor changes in liposome size

• GTPase activity assays: Measure GTP hydrolysis rates using:

  • Colorimetric phosphate release assays

  • HPLC-based nucleotide separation

  • Coupled enzyme assays

• Microscopy-based approaches:

  • Observe fusion events using giant unilamellar vesicles (GUVs) and fluorescence microscopy

  • Apply super-resolution techniques to track individual fusion events

• Control conditions to consider:

  • Compare GTP versus non-hydrolyzable GTP analogs (GMP-PNP)

  • Test GDP and GDP/AlF4- effects (mimicking transition state)

  • Vary lipid compositions to mimic different stress conditions

  • Compare activities to Sey1p homologs from non-osmotolerant yeasts

Given the finding that Sey1p can mediate fusion without GTP hydrolysis (though less efficiently) , carefully designed controls are essential to distinguish between hydrolysis-dependent and independent activities.

How can researchers effectively study the interaction between Z. rouxii Sey1p and membrane lipids under varying osmotic conditions?

To study interactions between Z. rouxii Sey1p and membrane lipids under different osmotic conditions, researchers should consider these methodological approaches:

• Lipid binding assays:

  • Liposome flotation assays using density gradients

  • Surface plasmon resonance (SPR) with immobilized lipid layers

  • Microscale thermophoresis to measure binding affinities under varying salt/sugar concentrations

• Membrane composition variations:

  • Systematically vary lipid compositions in reconstitution experiments

  • Include unsaturated fatty acids typical of Z. rouxii under stress conditions

  • Test different membrane curvatures and tensions

• Structural studies:

  • Hydrogen-deuterium exchange mass spectrometry to identify lipid-interacting regions

  • Cryo-EM analysis of Sey1p in nanodiscs with different lipid compositions

  • Molecular dynamics simulations to predict lipid-protein interactions

• Functional correlation:

  • Compare lipid binding profiles with fusion efficiency data

  • Assess how varying osmolyte concentrations affect these interactions

This systematic approach can reveal how Z. rouxii Sey1p may have adapted to function efficiently in the specialized membrane environment of this osmotolerant yeast.

What considerations are important when designing site-directed mutagenesis studies for Z. rouxii Sey1p?

When designing site-directed mutagenesis studies for Z. rouxii Sey1p, researchers should consider:

• Critical functional domains:

  • GTPase domain: Target conserved motifs (G1-G4) essential for nucleotide binding and hydrolysis

  • Stalk-like helical bundle domain: Focus on residues that may contribute to the unique properties of Sey1p compared to atlastins

  • Dimerization interface: Identify and mutate residues involved in the side-by-side dimer formation

  • Membrane-proximal regions: Examine how these interact with the lipid bilayer

• Structure-guided approaches:

  • Use the crystal structure information available for Sey1p to identify critical residues

  • Perform sequence alignments between Z. rouxii Sey1p and homologs from other species to identify conserved and divergent residues

  • Consider both conservative and non-conservative substitutions

• Functional validation assays:

  • GTPase activity measurements (wild-type vs. mutant)

  • Membrane fusion efficiency in reconstituted systems

  • In vivo complementation assays in sey1Δ yeast strains

  • Subcellular localization studies to assess ER targeting

• Specific mutations to consider:

By carefully selecting mutation targets and employing appropriate validation assays, researchers can gain valuable insights into the structure-function relationships of Z. rouxii Sey1p and potentially identify adaptations that contribute to the osmotolerance of this yeast species.

How does the expression of recombinant Z. rouxii Sey1p in S. cerevisiae affect ER morphology and stress responses?

Investigating the effects of Z. rouxii Sey1p expression in S. cerevisiae provides valuable insights into both protein function and potential adaptations for osmotolerance:

• Experimental design considerations:

  • Express Z. rouxii Sey1p in wild-type S. cerevisiae (with endogenous Sey1p) and in sey1Δ strains

  • Use fluorescent protein tags to visualize the ER (e.g., Sec63-GFP)

  • Include proper controls: empty vector, S. cerevisiae Sey1p expression

  • Test under normal and stress conditions (osmotic, temperature, etc.)

• Expected effects on ER morphology:

  • In sey1Δ strains, expression of Z. rouxii Sey1p should rescue the fragmented ER phenotype if functionally compatible

  • In wild-type strains, any dominant effects would suggest functional differences from the endogenous protein

  • Under stress conditions, Z. rouxii Sey1p might maintain better ER connectivity than S. cerevisiae Sey1p

• Stress response analysis:

  • Monitor growth curves under various stressors

  • Assess unfolded protein response (UPR) activation using reporters

  • Measure membrane fluidity and integrity using fluorescent probes

• Comprehensive phenotypic assessment:

Research has shown that introducing unsaturated fatty acid synthesis genes from Z. rouxii into S. cerevisiae enhances stress tolerance and improves membrane functionality . Similarly, expression of Z. rouxii Sey1p might confer enhanced ER maintenance capabilities under stress conditions.

What are the key considerations for future research on Z. rouxii Sey1p?

Future research on Z. rouxii Sey1p should focus on several key areas:

• Structural adaptations for osmotolerance:

  • Comparative structural studies between Sey1p proteins from osmotolerant and non-osmotolerant yeasts

  • Investigation of potential post-translational modifications specific to stress responses

  • Analysis of interaction partners under various stress conditions

• Integration with cellular stress response systems:

  • Connections between ER membrane dynamics and other cellular processes in Z. rouxii

  • Potential coordination with specialized transporters such as ZrTrk1, which contributes to potassium homeostasis and osmotolerance

  • Exploration of how high glucose conditions, which stimulate stress resistance pathways in Z. rouxii , affect Sey1p function

• Biotechnological applications:

  • Potential use of Z. rouxii Sey1p to enhance stress tolerance in industrial yeast strains

  • Development of cellular models to study ER stress-related diseases

  • Engineering synthetic membrane systems with enhanced stability for biotechnological applications