Recombinant Naumovozyma castellii Protein transport protein SEC24-2 (SEC242), partial

What is SEC24-2 in Naumovozyma castellii and what is its role in protein transport?

SEC24-2 in Naumovozyma castellii is a protein transport protein that functions as a component of the coat protein complex II (COPII). It plays a critical role in mediating the recruitment of transmembrane cargos or cargo adaptors into newly forming COPII vesicles on the endoplasmic reticulum (ER) membrane. The protein is part of the fundamental process of cellular protein trafficking, where it helps concentrate and package newly synthesized proteins into vesicles at specific ER exit sites. SEC24-2 forms a complex with SEC23 in the cytosol, and this heterodimer is recruited to ER exit sites upon activation of the GTPase SAR1, where it interacts with ER exit signals on the cytoplasmic tail of protein cargoes to facilitate vesicle formation .

How does Naumovozyma castellii SEC24-2 compare structurally and functionally to SEC24 paralogs in other yeast species?

Naumovozyma castellii (formerly known as Saccharomyces castellii or Naumovia castellii) SEC24-2 shares structural and functional similarities with SEC24 paralogs in other yeasts, particularly Saccharomyces cerevisiae . Based on sequence identity patterns observed in SEC24 proteins, yeast SEC24 paralogs typically contain several highly conserved C-terminal domains and a hypervariable N-terminal segment comprising approximately one-third of the protein sequence . The functional role remains consistent across species, as Sec24 serves as the principal subunit of the COPII coat responsible for incorporating cargo proteins into vesicles through direct or indirect interactions . The evolutionary relationship between yeast SEC24 paralogs suggests ancient and more recent gene duplications that have led to functional diversification while maintaining core transport functions .

What are the optimal storage and reconstitution conditions for recombinant Naumovozyma castellii SEC24-2?

For optimal storage of recombinant Naumovozyma castellii SEC24-2 (product code CSB-MP803178NAY), the protein's shelf life is dependent on several factors including storage state, buffer ingredients, storage temperature, and the protein's inherent stability. The recommended storage conditions are:

• Liquid form: 6 months shelf life at -20°C/-80°C

• Lyophilized form: 12 months shelf life at -20°C/-80°C

For reconstitution, the following protocol is recommended:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add 5-50% glycerol (final concentration) and aliquot for long-term storage at -20°C/-80°C (50% glycerol is the default recommendation)

• For working aliquots, store at 4°C for up to one week

Importantly, repeated freezing and thawing is not recommended as it may compromise protein integrity .

How can Naumovozyma castellii SEC24-2 be used to study vesicular trafficking pathways in yeast models?

To study vesicular trafficking pathways using Naumovozyma castellii SEC24-2:

• Cargo-specific transport assays: Design experiments to track the movement of specific cargo proteins known to interact with SEC24-2. This can be achieved by:

  • Creating fluorescently-tagged cargo constructs

  • Performing pulse-chase experiments with SEC24-2 and cargo proteins

  • Using live-cell imaging to visualize transport dynamics

• Comparative paralog studies: Since SEC24 proteins show varying degrees of cargo specificity ranging from exclusive paralog dependence to partial redundancy , researchers can:

  • Create SEC24-2 mutants with altered cargo-binding sites

  • Perform complementation studies with SEC24 paralogs from other species

  • Measure differential transport efficiencies of various cargoes

• Reconstitution of COPII vesicle formation in vitro: Using the purified recombinant protein (>85% purity by SDS-PAGE) , researchers can:

  • Combine SEC24-2 with other COPII components

  • Add artificial membranes and cargo proteins

  • Measure vesicle budding efficiency through electron microscopy or biochemical assays

These approaches provide insights into the specific role of SEC24-2 in the broader context of the cellular secretory pathway.

What experimental controls should be included when studying SEC24-2 function in protein transport?

When studying SEC24-2 function in protein transport, several key controls should be included:

• Paralog controls: Include experiments with other SEC24 paralogs to determine specificity versus redundancy in cargo selection. SEC24 paralogs within the same subgroup (e.g., SEC24C/D) often share 60% identity and may have overlapping functions .

• Cargo specificity controls:

  • Positive controls: Include cargoes known to depend on SEC24-2

  • Negative controls: Use cargoes known to utilize other SEC24 paralogs exclusively

  • General secretory pathway markers: Include cargoes that use multiple pathways

• Functional domain controls:

  • Wild-type protein

  • Domain-deletion mutants

  • Point mutations in cargo-binding sites

  • Chimeric constructs with domains from other SEC24 paralogs

• Environmental condition controls:

  • Standard growth conditions

  • ER stress conditions (e.g., tunicamycin treatment)

  • Temperature sensitivity tests

• Localization controls:

  • ER markers

  • COPII coat markers

  • Golgi apparatus markers

These controls help validate experimental findings and distinguish between SEC24-2-specific effects and general secretory pathway phenomena.

How can researchers investigate the cargo specificity of Naumovozyma castellii SEC24-2 compared to other SEC24 paralogs?

Investigating cargo specificity of Naumovozyma castellii SEC24-2 requires a multi-faceted experimental approach:

• In vitro binding assays:

  • Immobilize purified SEC24-2 on beads or biosensor chips

  • Expose to potential cargo proteins with various ER export motifs

  • Measure binding affinities using surface plasmon resonance or pull-down assays

  • Compare with other SEC24 paralogs tested under identical conditions

• Structural analysis of cargo-binding sites:

  • Perform homology modeling based on known SEC24 structures

  • Identify putative cargo-binding pockets

  • Design mutagenesis experiments targeting these regions

  • Validate through crystallography of SEC24-2/cargo complexes

• Interactome mapping:

  • Perform crosslinking mass spectrometry to identify interacting partners

  • Use proximity labeling approaches (BioID or APEX) in vivo

  • Compare the SEC24-2 interactome with that of other paralogs

  • Identify unique and shared cargo proteins

• Cargo packaging assays:

  • Reconstitute COPII vesicle formation with SEC24-2 in vitro

  • Analyze vesicle content using proteomics

  • Compare cargo profiles with vesicles formed using other SEC24 paralogs

This approach provides a comprehensive understanding of the unique cargo specificity profile of Naumovozyma castellii SEC24-2 within the context of evolutionary divergence of SEC24 paralogs .

What experimental approaches can be used to study the functional redundancy between SEC24-2 and other paralogs in Naumovozyma castellii?

To study functional redundancy between SEC24-2 and other paralogs in Naumovozyma castellii:

• Gene deletion and complementation studies:

  • Create SEC24-2 knockout strains

  • Rescue phenotypes by expressing different SEC24 paralogs

  • Quantify the efficiency of complementation

  • This approach mimics studies in mice where SEC24D can substitute for SEC24C during embryonic development

• Domain swapping experiments:

  • Create chimeric proteins with domains from different paralogs

  • Test functionality in vivo and in vitro

  • Identify which domains contribute to specific versus shared functions

• Cargo trafficking analysis in paralog mutants:

  • Track specific cargo proteins in single and double paralog mutants

  • Quantify trafficking defects using microscopy and biochemical assays

  • Create a matrix of cargo-paralog dependencies

• Evolutionary analysis:

  • Compare with Saccharomyces cerevisiae, where SEC24 paralogs show different levels of functional redundancy

  • Assess conservation of cargo-binding sites across paralogs

  • Determine if N. castellii SEC24 paralogs fall into the same subgroups as mammalian SEC24A/B and SEC24C/D

• Double and triple paralog knockouts:

  • Create combinatorial paralog deletions

  • Assess synthetic phenotypes

  • Map genetic interactions between paralogs

These approaches can reveal whether N. castellii SEC24 paralogs evolved through neofunctionalization (acquiring new functions) or subfunctionalization (splitting ancestral functions) .

How does phosphorylation or other post-translational modifications affect SEC24-2 function in vesicular transport?

Post-translational modifications (PTMs) of SEC24-2 can significantly alter its function in vesicular transport:

• Identification of modification sites:

  • Perform mass spectrometry analysis of purified SEC24-2

  • Map PTM sites to functional domains

  • Compare with known modification sites in SEC24 from other species

  • Based on known patterns in related proteins, look for potential phosphorylation, ubiquitination, and SUMOylation sites (similar to the SUMOylation observed in S. cerevisiae Rad52)

• Functional analysis of modifications:

  • Create phosphomimetic and phospho-dead mutations

  • Test cargo binding and vesicle formation efficiency

  • Analyze changes in SEC24-2 localization and dynamics

• Regulation of modifications:

  • Identify kinases and phosphatases that act on SEC24-2

  • Determine conditions that trigger modifications (cell cycle, stress)

  • Analyze the temporal dynamics of modifications during vesicle formation

• Crosstalk between different modifications:

  • Investigate how one modification affects others

  • Determine the hierarchical relationship between modifications

  • Create modification-specific antibodies to track modification status

• Structural consequences of modifications:

  • Perform structural modeling to predict how modifications alter protein conformation

  • Use limited proteolysis to detect conformational changes

  • Analyze changes in protein-protein interactions

This multi-layered approach would provide insights into how the cell regulates SEC24-2 function through dynamic post-translational modifications.

What are the best approaches for studying SEC24-2 interactions with cargo proteins in Naumovozyma castellii?

For studying SEC24-2 interactions with cargo proteins in Naumovozyma castellii, several complementary approaches are recommended:

• In vivo crosslinking and co-immunoprecipitation:

  • Express tagged versions of SEC24-2 in N. castellii

  • Apply membrane-permeable crosslinkers to stabilize transient interactions

  • Purify SEC24-2 complexes and identify associated cargo proteins by mass spectrometry

  • Validate with reciprocal co-immunoprecipitation using tagged cargo proteins

• Proximity-based labeling:

  • Fuse SEC24-2 to enzymes like BioID or APEX2

  • Allow in vivo biotinylation of proteins in close proximity

  • Purify biotinylated proteins and identify by mass spectrometry

  • Compare with results from other SEC24 paralogs

• Fluorescence-based interaction assays:

  • Implement split-GFP or FRET-based assays for specific cargo-SEC24-2 pairs

  • Visualize interactions at ER exit sites in real-time

  • Quantify interaction dynamics during vesicle formation

• Reconstituted in vitro systems:

  • Purify recombinant SEC24-2 (>85% purity by SDS-PAGE)

  • Add synthetic liposomes and purified cargo proteins

  • Measure cargo incorporation into COPII-coated vesicles

  • Use microscopy and biochemical assays to quantify interactions

• Genetic screens for interaction partners:

  • Create SEC24-2 mutant libraries

  • Screen for defects in transport of specific cargoes

  • Map mutations to cargo-binding interfaces

  • Validate through direct binding assays

These methodologies provide complementary data that together create a comprehensive picture of SEC24-2's cargo selection mechanisms.

What are the recommended protocols for expressing and purifying functional recombinant Naumovozyma castellii SEC24-2 for in vitro studies?

For expressing and purifying functional recombinant Naumovozyma castellii SEC24-2:

• Expression system selection:

  • Mammalian cell expression systems are recommended based on commercial production of this protein

  • HEK293 or CHO cells typically provide proper folding and post-translational modifications

  • Alternative: Insect cell expression (Sf9 or High Five cells) using baculovirus

  • Consider codon optimization for the expression host

• Expression construct design:

  • Include appropriate purification tags (His, GST, or MBP)

  • Consider including a cleavable tag for tag removal after purification

  • Ensure proper signal peptide if secretion is desired

  • The tag type will be determined during the manufacturing process

• Purification protocol:

  • Affinity chromatography based on the chosen tag

  • Ion exchange chromatography as a secondary purification step

  • Size exclusion chromatography for final polishing

  • Aim for >85% purity as assessed by SDS-PAGE

• Quality control:

  • SDS-PAGE and Western blotting to confirm identity

  • Mass spectrometry to verify protein integrity

  • Dynamic light scattering to assess aggregation state

  • Circular dichroism to verify proper folding

• Storage and stability:

  • Store purified protein in buffer containing 5-50% glycerol

  • Aliquot and store at -20°C/-80°C for long-term storage

  • Avoid repeated freeze-thaw cycles

  • Working aliquots can be stored at 4°C for up to one week

• Functional verification:

  • Cargo binding assays

  • SEC23 interaction assays

  • Liposome binding tests

  • COPII vesicle reconstitution assays

This protocol ensures the production of high-quality, functional SEC24-2 suitable for detailed in vitro studies.

How does Naumovozyma castellii SEC24-2 differ from SEC24 proteins in other yeast and mammalian systems?

Naumovozyma castellii SEC24-2 exhibits both conserved and divergent features compared to SEC24 proteins in other systems:

• Evolutionary context:

  • Naumovozyma castellii was previously known as Saccharomyces castellii or Naumovia castellii

  • It belongs to the Saccharomycetaceae family of budding yeasts

  • Based on patterns observed in related proteins, it likely shows moderately high sequence similarity with S. cerevisiae homologs

• Structural comparison:

  • Mammalian genomes encode four SEC24 paralogs that form two subgroups: SEC24A/B and SEC24C/D

  • Yeast typically have fewer SEC24 paralogs than mammals (three in S. cerevisiae)

  • All SEC24 proteins share conserved C-terminal domains with a hypervariable N-terminal segment

  • Based on the pattern in related proteins, N. castellii SEC24-2 likely has higher sequence conservation in functional domains

• Functional differences:

  • Cargo specificity varies between paralogs and across species

  • SEC24 paralog specificity ranges from exclusive dependence to partial redundancy

  • N. castellii SEC24-2 likely has a unique cargo recognition profile that reflects its evolutionary history

• Expression and regulation:

  • Different organisms regulate SEC24 expression through varied mechanisms

  • Species-specific stress responses may affect SEC24 function differently

  • Post-translational modifications likely differ between species

• Interaction with pathogenic factors:

  • The SEC24 secretion system can be hijacked by viral and bacterial pathogens

  • Species-specific differences in SEC24 may influence pathogen interactions

This comparative analysis helps place N. castellii SEC24-2 in an evolutionary context and provides insights into the functional diversification of SEC24 proteins across species.

What can the study of Naumovozyma castellii SEC24-2 tell us about the evolution of the COPII trafficking system across yeast species?

Studying Naumovozyma castellii SEC24-2 provides valuable insights into COPII trafficking system evolution:

• Paralog diversification:

  • SEC24 gene duplications led to functional specialization

  • Comparing N. castellii SEC24-2 with paralogs from other yeasts helps determine whether paralogs evolved through neofunctionalization (new functions) or subfunctionalization (splitting ancestral functions)

  • The degree of redundancy between paralogs indicates evolutionary pressure on vesicular transport

• Cargo adaptation:

  • Different yeast species transport varied cargoes

  • SEC24 cargo-binding sites evolve to accommodate species-specific secretory proteins

  • Comparing binding specificities across species reveals adaptive evolution

• Structural conservation:

  • Core functional domains remain highly conserved across species

  • Variable regions evolve more rapidly to accommodate species-specific needs

  • N. castellii SEC24-2 likely shows a pattern similar to other proteins in this species, where certain domains show high conservation (like the 95.9% similarity observed in the N-terminal domain of Rad52)

• Regulatory evolution:

  • Comparison of expression patterns and post-translational modifications

  • Evolution of interaction networks with other COPII components

  • Species-specific regulatory mechanisms for vesicular trafficking

• Response to cellular stresses:

  • Different yeasts inhabit varied ecological niches

  • SEC24 function during stress likely adapted to species-specific challenges

  • Comparative studies reveal how trafficking systems adapted to different environments

These evolutionary insights contribute to our understanding of how fundamental cellular processes diversify while maintaining core functions across species.

What are the common technical challenges when working with recombinant Naumovozyma castellii SEC24-2 and how can they be addressed?

Common technical challenges and solutions when working with recombinant N. castellii SEC24-2:

• Protein stability issues:

  • Challenge: Protein degradation during storage or experiments

  • Solution: Add 5-50% glycerol to storage buffer; store at -20°C/-80°C; avoid repeated freeze-thaw cycles; add protease inhibitors during handling

• Functionality assessment:

  • Challenge: Determining if recombinant protein maintains native activity

  • Solution: Develop functional assays such as cargo binding tests, liposome tubulation assays, or reconstituted vesicle formation systems

• Aggregation problems:

  • Challenge: Protein forms aggregates during storage or experiments

  • Solution: Optimize buffer conditions (pH, salt concentration); centrifuge briefly before use; consider adding stabilizing agents like trehalose

• Co-factor requirements:

  • Challenge: Protein may require co-factors for proper function

  • Solution: Include GTP for SAR1 activation; ensure presence of SEC23; test different lipid compositions for membrane-binding assays

• Protein concentration optimization:

  • Challenge: Determining optimal protein concentration for experiments

  • Solution: Perform concentration titration experiments; follow recommendation to reconstitute to 0.1-1.0 mg/mL

• Expression and purification yield:

  • Challenge: Low yield of functional protein

  • Solution: Optimize expression conditions; consider different tags; use mammalian or insect cell expression systems

• Cargo interaction detection:

  • Challenge: Detecting transient or weak interactions

  • Solution: Use crosslinking approaches; optimize buffer conditions; employ multiple complementary detection methods

• Reconstitution efficiency:

  • Challenge: Poor reconstitution of lyophilized protein

  • Solution: Follow recommended protocol: briefly centrifuge vial, use deionized sterile water, add glycerol to final concentration of 5-50%

These solutions can significantly improve experimental outcomes when working with recombinant N. castellii SEC24-2.

How can researchers validate that recombinant Naumovozyma castellii SEC24-2 maintains its native conformation and activity after purification?

To validate that recombinant N. castellii SEC24-2 maintains native conformation and activity:

• Structural validation:

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • Thermal shift assays to measure protein stability

  • Size exclusion chromatography to confirm monomeric/oligomeric state

  • Limited proteolysis to verify proper folding

• Functional validation:

  • SEC23 binding assay: SEC24-2 should form a stable complex with SEC23

  • Membrane binding: Verify interaction with lipid membranes using liposome flotation assays

  • GTPase activation: Test ability to stimulate SAR1 GTPase activity

  • Cargo binding: Verify interaction with known cargo peptides

• Comparative approaches:

  • Compare activity with commercially available standard (e.g., CSB-MP803178NAY)

  • Benchmark against SEC24 from well-characterized species like S. cerevisiae

  • Use western blotting with conformation-specific antibodies if available

• In vitro reconstitution assays:

  • Reconstitute COPII vesicle formation with purified components

  • Measure budding efficiency from synthetic liposomes

  • Verify cargo selection and incorporation into vesicles

  • Assess vesicle morphology by electron microscopy

• Activity after storage:

  • Test functional parameters after storage at different conditions

  • Compare fresh vs. stored protein activity

  • Develop stability-indicating assays for routine quality control

These validation approaches ensure that experimental results accurately reflect the native properties of N. castellii SEC24-2 rather than artifacts of the recombinant production process.

What are the emerging research areas involving Naumovozyma castellii SEC24-2 and other COPII components?

Emerging research areas involving N. castellii SEC24-2 and COPII components include:

• Systems biology of vesicular trafficking:

  • Integrating proteomics, genomics, and computational modeling

  • Mapping the complete interactome of SEC24-2

  • Understanding how trafficking networks respond to environmental changes

• Evolutionary genomics of COPII systems:

  • Comparative analysis across yeast species to trace functional divergence

  • Investigating how gene duplication and specialization shaped modern trafficking systems

  • Similar to studies on RAD52 in N. castellii that revealed both conserved and divergent features

• Structural biology applications:

  • Cryo-electron microscopy of entire COPII coat assemblies

  • Structure-guided design of mutations to alter cargo specificity

  • Molecular dynamics simulations of SEC24-cargo interactions

• Synthetic biology approaches:

  • Engineering SEC24 variants with novel cargo specificities

  • Creating minimal COPII systems for biotechnology applications

  • Developing SEC24-based biosensors for cargo trafficking

• Pathogen-host interactions:

  • Investigating how viral and bacterial pathogens exploit SEC24

  • Developing interventions to block pathogen hijacking of the secretory pathway

  • Understanding species-specific differences in susceptibility to pathogen interference

• Stress response regulation:

  • Exploring how SEC24 function adapts during cellular stress

  • Understanding the role of post-translational modifications in stress adaptation

  • Investigating the unfolded protein response connection to COPII function

These research directions represent frontier areas where N. castellii SEC24-2 studies could make significant contributions to our understanding of fundamental cellular processes.

How might CRISPR/Cas9 gene editing be used to study SEC24-2 function in Naumovozyma castellii?

CRISPR/Cas9 gene editing offers powerful approaches for studying SEC24-2 function in N. castellii:

• Knockout and knockdown studies:

  • Generate complete SEC24-2 deletion mutants

  • Create conditional knockdowns using inducible promoters

  • Similar to targeted gene replacement transformations used for RAD52 in N. castellii

  • Analyze phenotypes related to growth, morphology, and stress sensitivity

• Domain modification studies:

  • Introduce precise mutations in cargo-binding domains

  • Create truncation variants to assess domain functionality

  • Generate chimeric proteins with domains from other paralogs

  • Implement domain swapping between species to test evolutionary conservation

• Endogenous tagging:

  • Add fluorescent protein tags for live-cell imaging

  • Introduce epitope tags for immunoprecipitation studies

  • Create split-protein complementation systems for interaction studies

  • Add degron tags for rapid protein depletion

• Regulatory element engineering:

  • Modify promoters to alter expression levels

  • Create reporter constructs to monitor expression dynamics

  • Implement CRISPR interference/activation to modulate transcription

• High-throughput screening:

  • Generate libraries of SEC24-2 variants

  • Screen for altered cargo specificity or trafficking efficiency

  • Identify suppressors of SEC24-2 mutant phenotypes

  • Create synthetic genetic interaction maps

• Base editing applications:

  • Introduce precise amino acid changes without double-strand breaks

  • Modify potential phosphorylation or ubiquitination sites

  • Create series of point mutations for structure-function analysis

These CRISPR-based approaches would significantly advance our understanding of SEC24-2 function in N. castellii and provide insights applicable to vesicular trafficking across species.

What are the implications of SEC24-2 research for understanding human diseases related to protein trafficking defects?

Research on N. castellii SEC24-2 has important implications for understanding human diseases:

• Neurodegenerative disorders:

  • Many neurodegenerative diseases involve defects in protein trafficking

  • SEC24 paralogs in mammals are critical for proper neuronal function

  • Insights from yeast models can inform therapeutic approaches for protein misfolding diseases

• Developmental disorders:

  • SEC24 mutations in mammals cause developmental defects

  • Studies in mice show that SEC24D can substitute for SEC24C during embryonic development, demonstrating functional redundancy that might be therapeutically exploitable

  • Yeast models help elucidate the basic mechanisms disrupted in human developmental disorders

• Metabolic diseases:

  • SEC24A-deficient mice show reduced plasma cholesterol due to impaired PCSK9 secretion

  • Understanding cargo selection mechanisms can inform approaches to modulating secretion of disease-relevant proteins

  • Yeast studies reveal evolutionarily conserved trafficking mechanisms relevant to metabolic regulation

• Infectious diseases:

  • Viral and bacterial pathogens hijack the SEC24 secretion system

  • Insights from yeast models can help develop strategies to block pathogen exploitation of host trafficking

  • Comparative studies across species reveal conserved mechanisms of pathogen-host interaction

• Cancer biology:

  • Altered secretory pathway function contributes to cancer progression

  • SEC24-dependent trafficking affects tumor microenvironment formation

  • Basic mechanisms of cargo selection identified in yeast inform cancer biology

• Therapeutic development:

  • Fundamental understanding of SEC24 function enables targeted drug design

  • Small molecules targeting specific SEC24-cargo interactions could modulate protein secretion

  • Gene therapy approaches might exploit paralog redundancy for disease correction