Recombinant Protein SEY1 homolog (TP04_0477)

What is the SEY1 protein and what cellular functions does it perform?

SEY1 is a dynamin-like GTPase that mediates homotypic fusion of the endoplasmic reticulum (ER) membranes. It functions analogously to atlastins (ATL) in metazoans, though organisms typically have either SEY1 or ATL homologs, not both . The protein plays a critical role in maintaining the tubular ER network structure by facilitating the fusion of ER tubules. SEY1 consists of a dynamin-like GTPase domain with characteristic signature motifs, a helical bundle domain (significantly longer than that in ATL), two closely spaced transmembrane segments, and a C-terminal tail . In yeast, SEY1p interacts physically with tubule-shaping proteins Rtn1p and Yop1p, which are homologues of reticulons and DP1, respectively .

What experimental approaches are most effective for studying SEY1-mediated membrane fusion in vitro?

To study SEY1-mediated membrane fusion in vitro, researchers should consider a reconstituted liposome fusion assay. This approach involves:

• Protein purification: Express and purify the SEY1 protein (or the desired homolog) with intact transmembrane domains or a version with transmembrane domains replaced by a linker for studies of the cytosolic domain alone .

• Proteoliposome preparation: Incorporate the purified protein into liposomes with a lipid composition mimicking the ER membrane.

• Fusion assay setup: Two populations of proteoliposomes can be prepared—one containing fluorescent lipid dyes (e.g., NBD-PE and Rh-PE) at self-quenching concentrations and another unlabeled. Upon fusion, fluorescence dequenching occurs as the lipids diffuse into the unlabeled membrane, providing a quantitative measure of fusion .

• Controls and variables: Include conditions with different nucleotides (GTP, GDP, non-hydrolyzable GTP analogs) to assess nucleotide dependency. Comparing wild-type protein with GTPase-deficient mutants provides insights into the role of GTP hydrolysis .

This method demonstrated that proteoliposomes containing purified Sey1p fused in a GTP-dependent manner in vitro, confirming its direct role in membrane fusion .

How can researchers effectively design experiments to distinguish between SEY1-dependent and SEY1-independent ER fusion mechanisms?

To differentiate between SEY1-dependent and SEY1-independent ER fusion mechanisms, a multi-faceted experimental approach is recommended:

• Genetic manipulation:

  • Create SEY1 knockout/knockdown cell lines

  • Develop conditional expression systems for SEY1 and putative alternative fusion factors

  • Generate double or triple knockouts with related proteins (e.g., in yeast, SEY1 with RTN1 or YOP1)

• In vivo fusion assays:

  • Implement a cell fusion assay: Express different fluorescent proteins in separate cell populations (e.g., cytosolic GFP and ER-targeted RFP), then track fluorophore equilibration after cell fusion

  • Compare fusion kinetics between wild-type and SEY1-deficient cells

• Biochemical interaction studies:

  • Investigate physical interactions between SEY1 and other ER proteins

  • Examine dependencies on SNAREs (e.g., Ufe1p in yeast)

• Microscopy techniques:

  • Use high-resolution imaging to track ER network dynamics

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to measure ER connectivity

This comprehensive approach revealed that in yeast, while SEY1p is the primary mediator of ER fusion, a residual fusion mechanism exists that requires the ER SNARE Ufe1p . The observation that deletion of SEY1 alone produces subtle phenotypes while double deletions with RTN1 or YOP1 cause severe ER morphology defects further illustrates the importance of testing multiple genetic backgrounds .

What are the key considerations when designing structural studies of SEY1 and its homologs?

When designing structural studies of SEY1 homologs, researchers should consider:

• Protein construct design:

  • Create multiple truncation constructs to identify stable, crystallizable domains

  • For full structural understanding, separately examine the cytosolic domain and the transmembrane regions

  • Consider replacing transmembrane domains with a linker for cytosolic domain studies

• Nucleotide states:

  • Examine different nucleotide-bound states: apo (nucleotide-free), GDP-bound, GTP/GTP analog-bound, and transition state mimics (GDP+AlF₄⁻)

  • These different states reveal conformational changes during the GTPase cycle

• Oligomerization analysis:

  • Include sedimentation velocity analysis to study oligomeric states

  • Compare oligomerization in different nucleotide states (SEY1p forms monomers with GDP but dimers with GDP/AlF₄⁻)

• Comparative approach:

  • Study homologs from multiple species to identify conserved structural features

  • Compare with structurally related but functionally distinct proteins (e.g., ATLs)

This approach revealed that SEY1p forms side-by-side dimers in complex with GMP-PNP or GDP/AlF₄⁻ but remains monomeric with GDP, providing key insights into its fusion mechanism .

How does the GTP hydrolysis cycle of SEY1 correlate with its membrane fusion activity?

The relationship between GTP hydrolysis and membrane fusion by SEY1 reveals nuanced mechanics:

Research has shown that SEY1p can mediate fusion without GTP hydrolysis, although fusion is much more efficient with GTP . This suggests that GTP binding induces a conformational change sufficient for minimal fusion activity, but GTP hydrolysis significantly enhances the process. The formation of side-by-side dimers in complex with GMP-PNP (a non-hydrolyzable GTP analog) or GDP/AlF₄⁻ indicates that dimerization is a key step in the fusion mechanism .

The transition from GTP-bound to post-hydrolysis states likely drives a power stroke that pulls membranes together, similar to the mechanism proposed for ATLs but with structural distinctions specific to SEY1 .

What functional redundancies exist between SEY1 and other membrane fusion machinery in different organisms?

The functional redundancies between SEY1 and other membrane fusion machinery vary across organisms and reveal evolutionary adaptations:

• Cross-kingdom complementation:

  • SEY1p from yeast can partially replace ATL1 function in mammalian cells, demonstrating functional conservation despite structural differences

  • This suggests a fundamental conservation of the fusion mechanism across evolutionary distance

• Multiple fusion pathways:

  • In S. cerevisiae, residual ER fusion occurs in cells lacking SEY1p, which requires the ER SNARE Ufe1p

  • This indicates parallel fusion pathways with partial redundancy

• Organism-specific patterns:

  • No organism appears to have both ATL and SEY1p homologues

  • Yeast and plants utilize SEY1/RHD3 (a plant homolog), while metazoans utilize ATLs

• Interaction with ER-shaping proteins:

  • Deletion of SEY1 alone produces subtle phenotypes

  • Double deletion of SEY1 and either RTN1 or YOP1 causes severe ER morphology defects

  • This suggests functional cooperation between fusion and shaping machinery

This complex pattern of redundancies suggests that membrane fusion systems evolved multiple solutions to similar biological problems, with organisms adopting either SEY1-like or ATL-like mechanisms, often with backup pathways involving SNAREs.

How do post-translational modifications regulate SEY1 function in different cellular contexts?

While the search results do not provide direct information about post-translational modifications (PTMs) of SEY1, this represents an important area for advanced research. Based on knowledge of related GTPases, researchers should consider:

• Potential regulatory PTMs:

  • Phosphorylation sites that might regulate GTPase activity

  • Ubiquitination events potentially controlling protein turnover

  • Glycosylation that might influence protein-protein interactions

• Cellular context variations:

  • Changes in PTM patterns during cell cycle progression

  • Stress-induced modifications affecting ER remodeling

  • Tissue-specific modification patterns in multicellular organisms

• Experimental approaches:

  • Mass spectrometry to map modification sites

  • Phospho-mimetic and phospho-dead mutants to test functional consequences

  • Cell-type specific proteomic profiling

• Comparative analysis:

  • Examination of conservation of potential modification sites across species

  • Comparison with known regulatory mechanisms for related GTPases

This represents a critical frontier in understanding the contextual regulation of SEY1 function, particularly given the protein's essential role in ER network maintenance under different physiological conditions.

What are the optimal conditions for expressing and purifying recombinant SEY1 homolog proteins for structural and functional studies?

For optimal expression and purification of recombinant SEY1 homolog proteins:

• Expression systems:

  • For full-length protein: Insect cell expression systems (Sf9, Hi5) typically yield better results for membrane proteins

  • For cytosolic domains: E. coli expression with appropriate solubility tags (MBP, SUMO, GST)

• Purification approach:

  • Two-step affinity purification followed by size exclusion chromatography

  • For the transmembrane-containing versions, detergent selection is critical (mild non-ionic detergents like DDM or LMNG)

  • Consider fluorescence-based thermostability assays to optimize buffer conditions

• Storage considerations:

  • Store in Tris-based buffer with 50% glycerol at -20°C for standard storage

  • For extended storage, conserve at -80°C

  • Avoid repeated freeze-thaw cycles

  • Prepare working aliquots for storage at 4°C for up to one week

• Quality control:

  • Verify protein activity via GTPase assays

  • Assess oligomeric state by analytical ultracentrifugation

  • Check protein folding by circular dichroism spectroscopy

These conditions have been successfully used for structural studies of the cytosolic domain of Sey1p from Candida albicans and functional studies of Sey1p from Saccharomyces cerevisiae .

How can researchers control for potential confounding variables when studying SEY1-mediated membrane dynamics?

When studying SEY1-mediated membrane dynamics, controlling for confounding variables is essential:

• Experimental design considerations:

  • Use both between-subjects and within-subjects designs as appropriate

  • Implement proper control groups (positive, negative, and vehicle controls)

  • Blind experimenters to treatment conditions when possible

• Specific confounders in SEY1 studies:

  • Expression levels: Use inducible expression systems to maintain consistent protein levels

  • Membrane composition: Standardize lipid compositions in reconstitution experiments

  • Interacting proteins: Account for variations in levels of binding partners (Rtn1p, Yop1p)

  • GTP/GDP ratios: Maintain consistent nucleotide concentrations and regeneration systems

• Controls for in vivo studies:

  • When using fluorescent protein fusion constructs, verify that tagging doesn't disrupt function

  • Include GTPase-deficient mutants as controls

  • Monitor ER stress markers to ensure phenotypes aren't secondary to stress responses

• Statistical approach:

  • Pre-determine sample sizes based on power analysis

  • Use appropriate statistical tests based on data distribution

  • Consider hierarchical or nested experimental designs for complex cellular studies

By systematically addressing these variables, researchers can increase the reliability and reproducibility of their findings on SEY1-mediated membrane dynamics.

What are the most common technical challenges when investigating SEY1 homolog function and how can they be overcome?

Researchers studying SEY1 homologs frequently encounter these technical challenges, with corresponding solutions:

• Protein solubility and stability issues:

  • Challenge: Full-length SEY1 contains transmembrane domains that complicate expression and purification

  • Solutions:

    • Express the cytosolic domain for initial characterization

    • Use optimized detergents for membrane protein extraction

    • Consider nanodiscs or amphipols for maintaining native-like membrane environment

• Reconstituting physiologically relevant membrane fusion:

  • Challenge: In vitro systems may not recapitulate the complexity of cellular environments

  • Solutions:

    • Incorporate physiologically relevant lipid compositions

    • Include regulatory binding partners in reconstitution experiments

    • Validate findings with complementary in vivo approaches

• Redundant fusion mechanisms:

  • Challenge: Alternative fusion pathways may mask SEY1-specific effects

  • Solutions:

    • Use multiple genetic backgrounds (e.g., SNARE mutants)

    • Combine genetic approaches with acute protein inactivation

    • Design kinetic experiments that can distinguish primary and secondary effects

• Quantifying dynamic membrane events:

  • Challenge: Membrane fusion events are rapid and sometimes difficult to capture

  • Solutions:

    • Implement high-speed confocal or TIRF microscopy

    • Develop robust quantification algorithms for fusion events

    • Use complementary assays (e.g., content mixing and lipid mixing) to validate observations

By anticipating these challenges and implementing the suggested solutions, researchers can substantially improve their investigations of SEY1 homolog function and contribute more robust findings to the field.

How might studying SEY1 homologs contribute to understanding diseases associated with ER dysfunction?

Studying SEY1 homologs offers valuable insights into diseases involving ER dysfunction:

• Neurodegenerative disorders:

  • Mutations in the human functional equivalent of SEY1, the atlastin proteins (particularly ATL1), cause hereditary spastic paraplegia (HSP)

  • Understanding the fundamental mechanisms of SEY1-mediated ER fusion could illuminate pathogenic processes in these disorders

  • The ability of SEY1p to partially replace ATL1 in mammalian cells suggests therapeutic potential in compensating for defective atlastin function

• ER stress-related conditions:

  • Disruptions in ER network structure contribute to ER stress, which is implicated in diabetes, obesity, and certain inflammatory disorders

  • SEY1's role in maintaining proper ER morphology suggests it might influence cellular stress responses

• Infectious disease mechanisms:

  • As TP04_0477 is from Theileria parva, a parasite causing East Coast fever in cattle, understanding this protein could reveal how parasites modify host cell processes

  • The conservation of these GTPases across species suggests they represent essential cellular machinery that could be targeted therapeutically

• Experimental approaches:

  • Develop disease models incorporating SEY1/atlastin mutations

  • Screen for small molecule modulators of SEY1/atlastin activity

  • Use comparative studies between SEY1 and atlastins to identify critical functional domains

These investigations could ultimately lead to novel therapeutic strategies targeting ER fusion machinery in various disease contexts.

What computational approaches can enhance our understanding of SEY1 structure-function relationships?

Advanced computational methods offer powerful tools for exploring SEY1 structure-function relationships:

• Molecular dynamics simulations:

  • Simulate GTP hydrolysis-induced conformational changes

  • Model membrane interactions and deformations during fusion

  • Predict effects of mutations on protein stability and function

• Structure prediction and refinement:

  • Use AlphaFold2 or RoseTTAFold to predict structures of understudied SEY1 homologs

  • Refine models with experimental constraints from cross-linking or SAXS data

  • Model complete protein including transmembrane domains in lipid environments

• Network analysis:

  • Identify co-evolving residues across SEY1 homologs to predict functional interactions

  • Map evolutionary conservation onto structural models to identify critical regions

  • Analyze interaction networks to predict functional partners

• Virtual screening and drug design:

  • Identify potential binding pockets for small molecule modulators

  • Screen virtual libraries for compounds that could enhance or inhibit function

  • Design peptide mimetics that could regulate SEY1 activity

These computational approaches complement experimental work and can guide hypothesis generation, especially in areas difficult to address experimentally, such as transient conformational states during membrane fusion.

What are the implications of SEY1 research for synthetic biology applications in membrane remodeling?

SEY1 research holds significant potential for synthetic biology applications:

• Engineered organelle morphology:

  • Design synthetic SEY1 variants with altered fusion properties to create customized ER networks

  • Develop inducible systems to dynamically control organelle morphology in response to cellular needs

  • Create chimeric fusion proteins combining domains from different organisms for novel functions

• Membrane vesicle technologies:

  • Engineer SEY1-based fusion systems for targeted delivery of therapeutic cargo

  • Develop controlled release mechanisms based on GTPase activity triggers

  • Create synthetic vesicles with programmable fusion specificity

• Cell-free biotechnology:

  • Reconstitute minimal membrane systems with defined fusion machinery

  • Develop microfluidic platforms incorporating SEY1-mediated fusion for on-demand reactions

  • Create artificial cell-like systems with dynamic membrane compartments

• Cross-disciplinary applications:

  • Biomaterial development with self-healing properties inspired by membrane fusion mechanisms

  • Biosensors utilizing conformational changes in SEY1 to detect nucleotides or interaction partners

  • Biocomputing elements using membrane fusion events as computational operations

These applications could transform numerous fields, from drug delivery to biomanufacturing, by harnessing the fundamental membrane remodeling capabilities of SEY1-like proteins.