Recombinant Vanderwaltozyma polyspora Protein SEY1 (SEY1)

What is SEY1 protein and what is its evolutionary relationship to other dynamin-like GTPases?

SEY1 from Vanderwaltozyma polyspora is a dynamin-like GTPase that functions analogously to the atlastin family of proteins found in mammalian systems. These proteins form a distinct evolutionary group that is conserved across most eukaryotic organisms, with each organism typically possessing either atlastin or SEY1 homologues, but not both . The SEY1 protein consists of characteristic GTPase domains with signature motifs, a helical bundle domain (notably longer than that in atlastins), two closely spaced transmembrane segments, and a C-terminal tail . This structural arrangement is optimized for its membrane fusion capabilities, making it a crucial component of cellular architecture.

What is the primary function of SEY1 protein in cellular contexts?

SEY1 protein primarily functions as a mediator of homotypic endoplasmic reticulum (ER) fusion. Research in Saccharomyces cerevisiae has demonstrated that Sey1p (the S. cerevisiae homolog) plays a critical role in maintaining proper ER morphology, particularly the tubular ER network . When SEY1 is deleted in combination with other ER-shaping proteins such as Yop1p or Rtn1p, cells exhibit significant reductions in ER tubules and an increased presence of ER sheets . The protein's GTPase activity is essential for this function, as mutations in the GTP-binding domain render the protein inactive in restoring normal ER morphology . Functionally, SEY1 can be considered the yeast ortholog of mammalian atlastins, which perform similar roles in higher eukaryotes.

How does SEY1 protein interact with other ER-shaping proteins?

SEY1 protein physically interacts with tubule-shaping proteins like Rtn1p and Yop1p, which are homologues of mammalian reticulons and DP1, respectively . These interactions are crucial for maintaining proper ER morphology. Research has shown that while deletion of SEY1 alone does not produce obvious ER morphology defects, the combined deletion of SEY1 with either YOP1 or RTN1 results in the conversion of tubular cortical ER into sheets . This indicates that SEY1 collaborates with these ER-shaping proteins in a partially redundant manner to maintain the tubular ER network. The physical interaction between SEY1 and these proteins likely facilitates coordinated regulation of ER membrane curvature and fusion events necessary for maintaining the dynamic ER network.

What mechanism does SEY1 use to mediate ER membrane fusion?

SEY1-mediated ER membrane fusion follows a GTP-dependent mechanism similar to that proposed for atlastins. The process begins with GTP-dependent dimerization of GTPase domains positioned in opposing membranes (trans-interaction) . This dimerization brings the membranes into close proximity. Following GTP hydrolysis, a significant conformational change occurs in the SEY1 protein complex, which actively pulls the apposing membranes together and forces them to fuse .

Research using a combination of in vivo and in vitro approaches has provided substantial evidence for this mechanism:

• Sedimentation velocity analysis of SEY1-ΔTM (with transmembrane domains replaced by a linker) reveals that the protein runs predominantly as a monomer in the absence of nucleotide or in the presence of GDP alone, but dimerizes in the presence of GDP and AlFx (a transition state mimic) .

• In liposome fusion assays, SEY1 can mediate GTP-dependent fusion of proteoliposomes, requiring the presence of SEY1 in both membranes for efficient fusion .

• An in vivo assay tracking the equilibration of ER-localized fluorescent proteins during yeast mating showed that homotypic ER fusion requires SEY1 expression in both mating partners .

The coiled-coil region of SEY1 is significantly longer than that of atlastins, suggesting potential differences in the exact conformational changes that drive membrane fusion . This structural difference may reflect adaptations to specific cellular environments or regulatory mechanisms.

How do regulatory proteins like Lunapark affect SEY1-mediated fusion?

Lunapark proteins (Lnp1p in yeast) function as critical negative regulators of SEY1-mediated ER fusion. Research demonstrates that:

• Sey1p-mediated ER microsome fusion was markedly increased by the deletion of the LNP1 gene and decreased by the overexpression of Lnp1p .

• When co-reconstituted into liposomes with Sey1p, Lnp1p directly inhibits Sey1p-mediated liposome fusion, suggesting a direct interaction that prevents Sey1p function .

• The mechanistic basis for this inhibition involves interference with the formation of trans-Sey1p complexes, which are prerequisites for fusion between two ER tubules .

• Specifically, experimental evidence shows that levels of trans-Sey1p complexes were markedly increased when LNP1 was deleted and decreased when Lnp1p was overexpressed .

• The N-terminal region of Lnp1p appears to be responsible for preventing the formation of trans-Sey1p complexes .

This regulatory mechanism provides cells with a sophisticated control system for ER network formation and maintenance, allowing for dynamic responses to cellular needs by modulating the rate of ER tubule fusion events.

What are the experimental approaches to study SEY1-mediated membrane fusion in vitro?

Several robust experimental approaches have been developed to study SEY1-mediated membrane fusion:

Liposome Fusion Assays

Liposome fusion assays involve reconstituting purified SEY1 protein into proteoliposomes and monitoring their fusion. This typically employs fluorescent lipid mixing assays where donor proteoliposomes contain fluorescently labeled lipids that undergo fluorescence dequenching upon fusion with unlabeled acceptor proteoliposomes . Key methodological considerations include:

• Protein:lipid ratios must be carefully optimized to achieve physiologically relevant densities.

• The GTP concentration and buffer conditions significantly impact fusion efficiency.

• Control experiments using GTPase-defective mutants (e.g., SEY1-K50A) are essential to confirm the GTP-dependence of fusion .

ER Microsome Fusion Assays

This approach involves isolating ER microsomes from yeast cells with different genotypes (e.g., wild-type, LNP1 deletion, or Lnp1p overexpression) and comparing their fusion efficiency in vitro . This system allows for assessment of how different proteins impact SEY1-mediated fusion in a more native membrane environment.

Trans-SEY1 Complex Formation Assay

A specialized assay has been developed to monitor the formation of trans-Sey1p complexes, which are prerequisites for fusion . This involves:

• Using conditions that permit complex formation but arrest the fusion process (e.g., GMP-PNP or GDP/AlF4⁻).

• Quantifying the levels of trans-complexes formed between SEY1 proteins on opposing membranes.

• Analyzing how regulatory proteins or mutations affect complex formation.

This approach has been particularly valuable in dissecting the inhibitory mechanism of Lnp1p on SEY1-mediated fusion .

How can researchers design effective experiments to resolve contradictions in SEY1 research data?

When addressing contradictions in SEY1 research data, researchers should implement a comprehensive experimental design strategy:

Identify Specific Contradictions

For example, research has shown that trans-Sey1p complexes can become post-fusion cis-Sey1p complexes, yet other data indicates distinct regulatory mechanisms for these states . Such apparent contradictions require careful experimental design to resolve.

Multi-technique Validation

Implement complementary approaches to validate findings:

Temporal Resolution Studies

Implement time-course experiments to distinguish between sequential events in the fusion process. This is particularly important when resolving contradictions between snapshot observations that may represent different stages of a dynamic process .

Quantitative Analysis

Apply rigorous quantitative methods to measure reaction kinetics, binding affinities, and fusion efficiencies. Statistical validation should include biological replicates (n≥3) with appropriate statistical tests to determine significance .

How can recombinant Vanderwaltozyma polyspora SEY1 be effectively used in research applications?

Recombinant Vanderwaltozyma polyspora SEY1 protein provides a valuable tool for numerous research applications:

Reconstitution Studies

The His-tagged recombinant protein can be purified to >90% purity and reconstituted into proteoliposomes for fusion assays . For optimal reconstitution:

• Use freshly purified protein stored appropriately to maintain activity.

• Reconstitute into liposomes with lipid compositions mimicking the ER membrane.

• Validate protein orientation in the membrane using protease protection assays.

Comparative Studies with Homologs

Recombinant V. polyspora SEY1 can be used in comparative studies with homologs from other species to investigate evolutionary conservation and specialization:

• Express wild-type SEY1 and human ATL1 in sey1Δ yop1Δ yeast cells to assess functional complementation .

• Compare GTPase activities and membrane fusion efficiencies between different SEY1 homologs.

• Create chimeric proteins to identify domain-specific functions.

Structure-Function Analysis

The recombinant protein enables systematic structure-function analysis through site-directed mutagenesis:

• Target conserved residues in the GTPase domain (e.g., K50A mutation in the S. cerevisiae homolog).

• Modify the length or composition of the helical bundle domain.

• Alter transmembrane segments to investigate membrane insertion requirements.

When handling recombinant SEY1 protein, researchers should follow manufacturer recommendations for reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL and add 5-50% glycerol for long-term storage at -20°C/-80°C .

What are critical factors to control when designing experiments with recombinant SEY1?

When designing experiments with recombinant SEY1 protein, researchers must control several critical factors to ensure reliable and reproducible results:

Protein Quality and Stability

The recombinant protein's stability significantly impacts experimental outcomes. Consider:

• Avoiding repeated freeze-thaw cycles that can denature the protein .

• Storing working aliquots at 4°C for up to one week to maintain activity .

• Reconstituting lyophilized protein in appropriate buffers (Tris/PBS-based buffer with 6% Trehalose, pH 8.0 is recommended) .

• Adding 5-50% glycerol as a cryoprotectant for long-term storage .

GTPase Activity Preservation

The GTPase activity of SEY1 is essential for its function and must be preserved:

• Include appropriate nucleotides (GTP, GDP) in experimental buffers.

• Use GTPase-deficient mutants (e.g., K50A in S. cerevisiae Sey1p) as negative controls .

• Consider adding transition state mimics (GDP/AlFx) to capture specific conformational states .

Membrane Environment

SEY1 is a membrane protein, and its activity depends on the lipid environment:

• Use lipid compositions that mimic the ER membrane.

• Control protein:lipid ratios during reconstitution.

• Verify proper membrane insertion and orientation of the protein.

How can SEY1 research contribute to understanding human diseases related to the secretory pathway?

SEY1 research provides valuable insights into fundamental mechanisms of ER membrane dynamics that have direct implications for human diseases:

• Hereditary Spastic Paraplegia (HSP): Mutations in human atlastin (ATL1), the functional equivalent of SEY1, cause HSP. Research showing that human ATL1 can functionally replace Sey1p in yeast provides a valuable model system for studying disease-causing mutations .

• Neurodegenerative Disorders: Disruptions in ER morphology and function are implicated in various neurodegenerative diseases. Understanding SEY1-mediated ER fusion can illuminate pathogenic mechanisms.

• Cell Stress Responses: SEY1-mediated ER fusion is likely involved in cellular responses to stress, which are relevant to numerous disease states.

Researchers can leverage SEY1 studies by:

• Creating yeast models expressing disease-associated mutations in human ATL1.

• Developing high-throughput screens for compounds that modulate SEY1/atlastin activity.

• Investigating interactions between SEY1/atlastin and other disease-associated proteins.

What are emerging directions in SEY1 research?

Several promising research directions are emerging in the field of SEY1 biology:

• Regulatory Networks: Further exploration of how proteins like Lnp1p regulate SEY1 activity and how these regulatory mechanisms respond to cellular signals .

• Structural Dynamics: Detailed structural analysis of the conformational changes that drive SEY1-mediated membrane fusion, particularly focusing on the differences between SEY1 and atlastins.

• Systems Integration: Understanding how SEY1-mediated ER fusion integrates with other cellular processes such as ER-phagy, stress responses, and lipid metabolism.

• Therapeutic Applications: Exploring the potential of SEY1/atlastin as therapeutic targets for diseases associated with ER dysfunction.

• Evolutionary Conservation: Comparative studies of SEY1 homologs across different species to understand the evolution of ER fusion mechanisms.