Recombinant Neurospora crassa Vacuolar protein sorting-associated protein 27 (vps-27), partial

What is Vacuolar protein sorting-associated protein 27 (vps-27) in Neurospora crassa?

Vacuolar protein sorting-associated protein 27 (vps-27) in Neurospora crassa is a key component of the endosomal sorting complex required for transport (ESCRT) pathway, specifically functioning within the ESCRT-0 complex. The protein plays a critical role in the recognition and sorting of ubiquitinated cargo proteins destined for the vacuole or for degradation. As in other fungi, N. crassa VPS27 likely contains functional domains including a VHS (Vps27/Hrs/STAM) domain for binding to ubiquitinated proteins and a FYVE domain that binds to phosphatidylinositol 3-phosphate (PI3P) on endosomal membranes . The gene encoding VPS27 in N. crassa is identified as NCU04015, with alternative designations including 49D12.130 . Understanding VPS27's structure and function in N. crassa contributes to broader knowledge of eukaryotic intracellular trafficking mechanisms.

How does VPS27 function in the ESCRT pathway?

VPS27 functions as the initial component in the sequential assembly of the ESCRT machinery, which mediates protein sorting and multivesicular body (MVB) formation. Methodologically, the function can be elucidated through the following sequential events:

• VPS27 is recruited to endosomal membranes through its FYVE domain, which specifically binds to PI3P generated by the class III PI3-kinase VPS34

• Once localized to endosomes, VPS27 recognizes and binds ubiquitinated cargo proteins via its ubiquitin-binding domains

• VPS27 then recruits subsequent ESCRT complexes (ESCRT-I, II, and III) to initiate the formation of intraluminal vesicles within MVBs

• These processes facilitate either the degradation of cargo proteins in the vacuole or their transport to other cellular destinations

Studies in Cryptococcus neoformans demonstrated that deletion of VPS27 results in the accumulation of multivesicular bodies with vacuolar fragmentation and mistargeting of vacuolar proteins such as carboxypeptidase Y (CPY/Prc1) . In N. crassa, VPS27 likely performs similar functions, as the protein is highly conserved across fungal species. Research approaches examining phenotypic changes in vps27 deletion mutants provide valuable insights into its specific roles in protein trafficking and vacuolar function.

What experimental phenotypes are associated with VPS27 deletion?

VPS27 deletion produces multiple observable phenotypes that can be assessed through specific experimental approaches:

• Vacuolar morphology: Fluorescence microscopy using vacuole-specific dyes (e.g., FM4-64, MDY-64) reveals that vps27Δ mutants typically display fragmented, irregular vacuoles compared to the large, round vacuoles seen in wild-type cells

• Protein trafficking: The mislocalization of vacuolar proteins can be monitored using:

  • CPY/Prc1 secretion assays, which demonstrate extracellular localization of normally vacuolar proteins

  • Fluorescently-tagged protein trafficking analysis showing accumulation in aberrant intracellular compartments

• Endocytosis kinetics: Time-course experiments with FM4-64 staining show delayed endocytic trafficking in vps27Δ mutants, with statistical analysis revealing significantly fewer cytoplasmic puncta (1.0 ± 1.0) compared to wild-type cells (5 ± 2 vesicles)

• Growth characteristics: While basic growth may not be affected at standard conditions, vps27Δ strains often show compromised growth under specialized conditions, such as alkaline pH (pH 8)

These phenotypes are consistent across multiple fungal species, suggesting functional conservation of VPS27 among fungi including N. crassa.

How does the structure of N. crassa VPS27 contribute to its function?

N. crassa VPS27 contains several conserved structural domains that directly contribute to its functional capabilities:

The functional importance of these domains can be investigated through complementation studies using domain deletion or point mutation constructs. For example, mutations in the FYVE domain would be expected to prevent endosomal localization, while UIM motif alterations would disrupt cargo recognition. Visualization of these effects can be achieved using fluorescently tagged VPS27 variants and co-localization studies with endosomal markers. Comparative analysis of N. crassa VPS27 with homologs from other fungi reveals high conservation of these domains, suggesting evolutionary preservation of the protein's function in the ESCRT pathway .

What is the relationship between VPS27 and VPS34 in the ESCRT pathway?

The relationship between VPS27 and VPS34 represents a critical functional interaction in the ESCRT pathway that can be experimentally demonstrated through several approaches:

• VPS34 is a class III PI 3-kinase responsible for generating PI3P on endosomal membranes, which serves as a binding site for the FYVE domain of VPS27

• Genetic studies reveal that VPS27 is epistatic to VPS34, as demonstrated by the following findings:

  • VPS34 deletion results in more severe phenotypes than VPS27 deletion due to VPS34's involvement in multiple cellular processes beyond the ESCRT pathway

  • Overexpression of VPS27 in vps34Δ mutants partially rescues certain phenotypes, including CPY/Prc1 sorting and laccase activity

• Microscopy analysis shows that fluorescently labeled VPS27-YFP forms distinct puncta in wild-type cells but displays a diffuse pattern in vps34Δ strains, confirming the dependence of VPS27 localization on VPS34 activity

This hierarchical relationship indicates that VPS34 functions upstream of VPS27 in the endosomal sorting pathway. In N. crassa, similar interactions likely occur, with VPS34-generated PI3P serving as the initial recruitment signal for VPS27 to endosomal membranes.

How can researchers utilize recombinant VPS27 to study protein-protein interactions?

Recombinant VPS27 serves as a valuable tool for investigating protein-protein interactions within the ESCRT pathway through multiple methodological approaches:

• In vitro binding assays: Purified recombinant VPS27 (>90% purity) can be used in pull-down experiments to identify direct binding partners . These assays typically involve:

  • Immobilizing recombinant VPS27 on an affinity matrix

  • Incubating with cellular lysates or purified candidate interacting proteins

  • Washing to remove non-specific binding

  • Eluting and analyzing bound proteins by mass spectrometry or immunoblotting

• Surface Plasmon Resonance (SPR): This technique measures binding kinetics and affinity constants between VPS27 and potential interaction partners, providing quantitative data on:

  • Association rates (kon)

  • Dissociation rates (koff)

  • Equilibrium dissociation constants (KD)

• Structural studies: High-purity recombinant VPS27 enables:

  • X-ray crystallography of VPS27 alone or in complex with binding partners

  • Cryo-electron microscopy to visualize VPS27 within larger protein complexes

  • Nuclear magnetic resonance (NMR) spectroscopy for analyzing dynamic interactions

• Reconstitution experiments: Using recombinant VPS27 and other ESCRT components to:

  • Reconstitute ESCRT-mediated vesicle formation on artificial membranes

  • Measure the effects of VPS27 mutations on complex assembly and function

  • Test competitive inhibitors of specific protein-protein interactions

When designing such experiments, researchers should consider using the E. coli-expressed recombinant N. crassa VPS27 with appropriate tags for purification and detection, while ensuring that the storage conditions (glycerol-containing buffer at -20°C or -80°C) maintain protein stability and activity .

What are the optimal conditions for expressing and purifying recombinant N. crassa VPS27?

Successful expression and purification of recombinant N. crassa VPS27 requires careful optimization of multiple parameters:

Expression Systems:

• E. coli: Most commonly used for basic structural studies, offering high yield but potential issues with folding of eukaryotic proteins

• Yeast: Provides post-translational modifications, improving functionality for interaction studies

• Baculovirus: Optimal for large-scale production of functionally active protein

• Mammalian cell lines: Best for maintaining native conformation and modifications

Purification Protocol:

• Cell lysis: Sonication or mechanical disruption in buffer containing protease inhibitors

• Initial capture: Affinity chromatography using histidine, GST, or other fusion tags

• Intermediate purification: Ion exchange chromatography to separate based on charge properties

• Polishing: Size exclusion chromatography to achieve >90% purity

• Quality control: SDS-PAGE and Western blotting to confirm identity and purity

Critical Parameters for Optimization:

• Induction conditions: Temperature (typically 16-25°C for complex proteins), inducer concentration, and duration

• Buffer composition: pH optimization, salt concentration, reducing agents to maintain stability

• Tag selection: N-terminal vs. C-terminal tags based on structural predictions

• Protease inhibitor cocktails: To prevent degradation during purification

Storage Conditions:

• Store in glycerol-containing buffer (typically 10-20%)

• Maintain at -20°C for short-term or -80°C for long-term storage

• Avoid repeated freeze-thaw cycles which can cause protein denaturation

For functional studies, researchers should validate the activity of purified VPS27 through binding assays with known interactors or phosphoinositide binding tests before proceeding to experimental applications.

How can CRISPR-Cas9 be used to generate VPS27 deletion or modification in N. crassa?

CRISPR-Cas9 technology offers precise genetic manipulation of VPS27 in N. crassa through the following methodological steps:

• sgRNA design:

  • Target sequences within the VPS27 gene (NCU04015) with minimal off-target effects

  • Design primers to incorporate the sgRNA sequence into a suitable expression vector

  • Optimize the sgRNA sequence using N. crassa codon usage patterns

• Repair template construction:

  • For gene deletion: Design homology arms (500-1000 bp) flanking the VPS27 coding sequence

  • For point mutations: Include the desired mutation within a repair template containing homology regions

  • For tagging: Incorporate fluorescent protein or epitope tag sequences in-frame with VPS27

• Transformation protocol:

  • Prepare N. crassa protoplasts using standard enzymatic digestion methods

  • Co-transform Cas9 expression construct, sgRNA, and repair template

  • Select transformants using appropriate markers

  • Screen using PCR, sequencing, or phenotypic analysis

• Validation approaches:

  • Genomic PCR to confirm integration

  • RT-PCR and Western blotting to verify absence or modification of VPS27

  • Phenotypic characterization using vacuolar staining (FM4-64) and protein trafficking assays

  • Complementation with wild-type VPS27 to confirm phenotype specificity

This approach offers significant advantages over traditional homologous recombination methods, including higher efficiency and precision. Researchers investigating VPS27 should consider creating conditional mutants (rather than complete deletions) if VPS27 function is essential, using inducible promoters to control expression levels.

What imaging techniques are most effective for studying VPS27 localization and dynamics?

Advanced imaging techniques provide crucial insights into VPS27 localization, dynamics, and function:

• Confocal Microscopy with Fluorescent Protein Fusions:

  • Construction of VPS27-GFP/YFP/mCherry fusions for live-cell imaging

  • Time-lapse imaging to track VPS27 recruitment to endosomal membranes

  • Co-localization studies with markers for early endosomes, late endosomes, and MVBs

  • Quantification of puncta formation and dynamics

• Super-Resolution Microscopy Approaches:

  • Stimulated Emission Depletion (STED) microscopy to resolve VPS27-positive structures below the diffraction limit

  • Single-Molecule Localization Microscopy (PALM/STORM) for precise localization and tracking of individual VPS27 molecules

  • Structured Illumination Microscopy (SIM) for improved resolution of VPS27-containing complexes

• Correlative Light and Electron Microscopy (CLEM):

  • Combining fluorescence microscopy of VPS27-tagged proteins with electron microscopy

  • Providing ultrastructural context to fluorescence observations

  • Visualizing MVB formation in relation to VPS27 localization

• Fluorescence Recovery After Photobleaching (FRAP):

  • Measuring the dynamics of VPS27 association with endosomal membranes

  • Determining the mobile and immobile fractions of VPS27

  • Comparing wild-type dynamics with mutant variants

The choice of imaging technique should be guided by the specific research question. For example, in studies examining VPS27 behavior in vps34Δ backgrounds, researchers observed the transition from distinct puncta to a diffuse pattern, requiring high-resolution imaging to accurately characterize this phenotype . When designing these experiments, proper controls including known endosomal markers and careful consideration of tag effects on protein function are essential.

How can contradictory results in VPS27 functional studies be reconciled?

When confronted with contradictory results in VPS27 studies, researchers should implement a systematic approach to reconcile discrepancies:

• Strain and genetic background analysis:

  • Compare the exact genetic backgrounds used in different studies

  • Sequence the VPS27 locus and surrounding regions to identify potential second-site mutations

  • Cross contradictory strains to determine if phenotypes segregate with the VPS27 locus

• Experimental condition variations:

  • Standardize growth conditions (media composition, pH, temperature)

  • Control for cell density and growth phase effects

  • Document exact buffer compositions and incubation times

• Complementation studies:

  • Express wild-type VPS27 in mutant backgrounds to confirm phenotype rescue

  • Use cross-species complementation to assess functional conservation

  • Create domain-specific mutations to isolate conflicting functions

• Methodological validation:

  • Apply multiple independent techniques to measure the same parameter

  • For trafficking studies, compare direct fluorescence, immunolocalization, and biochemical fractionation

  • Validate antibody specificity using knockout controls

A structured approach to resolving contradictions can be seen in studies of Cryptococcus neoformans, where VPS27 deletion produced complex phenotypes affecting multiple cellular processes. Researchers reconciled these by carefully examining the epistatic relationship between VPS27 and VPS34, demonstrating that VPS27 overexpression could partially rescue vps34Δ phenotypes . Similar approaches applied to N. crassa VPS27 studies can help resolve contradictory findings.

What are common pitfalls when working with recombinant VPS27 in experimental settings?

Researchers should be aware of several common pitfalls when working with recombinant VPS27:

• Protein stability and activity issues:

  • Recombinant VPS27 may lose activity during purification or storage

  • Solution: Include functionality tests (e.g., PI3P binding assays) after purification

  • Monitor batch-to-batch variation using activity-based quality control

• Tag interference with protein function:

  • N-terminal or C-terminal tags may disrupt critical domains or interactions

  • Solution: Compare multiple tag positions and types (His, GST, MBP)

  • Include tag-removal options via protease cleavage sites

• Solubility challenges:

  • Membrane-associated proteins like VPS27 often have solubility issues

  • Solution: Optimize buffer conditions with different detergents or solubilizing agents

  • Consider expressing only soluble domains for specific applications

• Non-specific binding in interaction studies:

  • VPS27 may exhibit non-physiological interactions in vitro

  • Solution: Include stringent controls and validation in cellular contexts

  • Use competition assays with known binding partners

• Storage-related degradation:

  • Improper storage can lead to protein degradation and loss of activity

  • Solution: Store in glycerol-containing buffers at -20°C or -80°C

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots

These challenges can be mitigated through careful experimental design and appropriate controls. For example, when studying VPS27-PI3P interactions, researchers should confirm that recombinant VPS27 retains its FYVE domain structure and binding capacity, potentially through comparative analysis with well-characterized FYVE domains from other proteins.

How can researchers accurately quantify VPS27-dependent phenotypes?

Accurate quantification of VPS27-dependent phenotypes requires rigorous methodological approaches:

• Vacuolar morphology analysis:

  • Automated image analysis of vacuole size, number, and shape

  • Establish clear morphological categories and blind scoring by multiple observers

  • Report statistical measures including variance and confidence intervals

  • Example metric: In vps27Δ studies, vacuolar fragmentation can be quantified by counting the number of vacuolar structures per cell (typically increased from 1-2 to >5)

• Protein trafficking quantification:

  • For secreted proteins: ELISA or activity assays of media samples

  • For intracellular trafficking: Quantitative co-localization analysis with compartment markers

  • Time-course studies to measure kinetics rather than endpoints

  • Example approach: Quantifying CPY/Prc1 activity in culture supernatants provides a measure of mistargeting in vps27Δ mutants

• Endocytosis and membrane trafficking:

  • Pulse-chase experiments with fluorescent markers like FM4-64

  • Quantify fluorescence intensity in defined cellular regions over time

  • Example data: Statistical analysis of FM4-64 staining showing vps27Δ strains contained 1.0 ± 1.0 cytoplasmic puncta compared to 5 ± 2 vesicles in wild-type cells (P < 0.05)

• Growth and viability measurements:

  • High-resolution growth curves using automated plate readers

  • Colony forming unit (CFU) assays under different stress conditions

  • Cell viability staining to distinguish metabolically active cells

• Molecular interaction quantification:

  • Förster Resonance Energy Transfer (FRET) to measure protein-protein interactions in vivo

  • Bioluminescence Resonance Energy Transfer (BRET) for sensitive detection of interactions

  • Quantitative mass spectrometry with SILAC labeling for interaction proteomics

How conserved is VPS27 function across fungal species?

VPS27 exhibits remarkable functional conservation across diverse fungal species, with important implications for evolutionary biology and comparative cell biology:

Functional conservation is demonstrated through complementation studies, where VPS27 from one species can rescue defects in another. For example, C. neoformans VPS27 can restore proper CPY/Prc1 localization in S. cerevisiae vps27Δ strains . This cross-species functionality indicates conservation of:

• Core structural elements required for endosomal binding

• Interaction interfaces with other ESCRT components

• Cargo recognition mechanisms for ubiquitinated proteins

Despite this conservation, species-specific adaptations exist. For instance, in pathogenic fungi like C. neoformans, VPS27 has acquired additional functions related to virulence, including roles in laccase trafficking and melanin production . These specialized functions likely represent evolutionary adaptations to specific ecological niches.

The conservation of VPS27 function across evolutionary distance suggests its fundamental importance in eukaryotic cellular processes and provides valuable insights for researchers studying protein trafficking in N. crassa or other fungal models.

What unique features distinguish N. crassa VPS27 from its homologs in other fungi?

While VPS27 functions are broadly conserved, N. crassa VPS27 exhibits several distinctive features that may reflect adaptation to its specific ecological niche and cellular biology:

• Sequence divergence in non-catalytic regions:

  • While core functional domains (VHS, FYVE) show high conservation, linker regions and regulatory sequences display greater divergence

  • These variations may fine-tune VPS27 activity to N. crassa-specific trafficking requirements

  • Comparative sequence analysis revealing N. crassa-specific motifs can guide functional studies

• Expression patterns and regulation:

  • N. crassa exhibits unique developmental stages including conidiation and protoperithecia formation

  • VPS27 expression may be differentially regulated during these stages compared to other fungi

  • Transcriptomic data analysis can reveal condition-specific expression patterns unique to N. crassa

• Protein interaction networks:

  • While core ESCRT interactions are conserved, N. crassa VPS27 may interact with species-specific partners

  • Interactome studies using techniques like BioID or proximity labeling can identify N. crassa-specific interactors

  • These unique interactions may connect VPS27 to N. crassa-specific cellular processes

• Subcellular distribution patterns:

  • The distribution of VPS27-positive endosomal structures may differ in the highly polarized hyphal cells of N. crassa

  • This may relate to the specialized trafficking requirements of filamentous growth

  • Advanced imaging of VPS27 localization during hyphal extension can reveal these patterns

Understanding these distinctive features provides valuable insights into both the core functions of VPS27 and its adaptation to specific cellular contexts. Researchers studying N. crassa VPS27 should consider these unique aspects when designing experiments and interpreting results in comparison to data from other fungal systems.

What emerging technologies will advance our understanding of VPS27 function?

Several cutting-edge technologies are poised to revolutionize our understanding of VPS27 function:

• Cryo-electron tomography:

  • Enables visualization of VPS27-containing protein complexes in their native cellular environment

  • Reveals the 3D architecture of ESCRT assemblies at endosomal membranes

  • Provides insights into how VPS27 initiates membrane deformation

• Proximity labeling proteomics:

  • BioID or APEX2 fusions with VPS27 to identify transient or weak interactors

  • TurboID for rapid labeling to capture dynamic interaction networks

  • Quantitative spatial proteomics to map VPS27's molecular neighborhood

• Optogenetic control of VPS27 function:

  • Light-inducible dimerization to trigger VPS27 recruitment to specific membranes

  • Photocleavable protein domains to acutely inactivate VPS27 function

  • Allows precise temporal control for studying VPS27's role in dynamic processes

• Single-molecule tracking in living cells:

  • Monitors individual VPS27 molecules in real-time

  • Reveals dynamic behaviors including residence times at endosomes

  • Provides insights into how VPS27 cooperatively assembles ESCRT machinery

• AlphaFold2 and integrative structural biology:

  • AI-predicted structures of full-length VPS27 and its complexes

  • Integration with experimental data from X-ray crystallography, NMR, and cryo-EM

  • Creates comprehensive structural models of VPS27 in different functional states

These technologies will enable researchers to address fundamental questions about VPS27 dynamics, assembly mechanisms, and regulatory control that remain challenging with conventional approaches. For N. crassa specifically, these methods can elucidate how VPS27 functions within the unique cellular architecture of filamentous fungi.

What are the implications of VPS27 research for understanding human diseases?

Research on fungal VPS27 has significant translational implications for understanding human diseases related to protein trafficking:

• Neurodegenerative disorders:

  • The human VPS27 homolog (HRS/HGS) is implicated in protein aggregation diseases

  • Insights from fungal models inform mechanisms of protein clearance defects in conditions like Alzheimer's and Parkinson's diseases

  • Comparative studies between fungal VPS27 and human HRS can identify conserved functional principles

• Cancer biology:

  • Dysregulation of the ESCRT pathway affects receptor tyrosine kinase signaling and tumor suppressor trafficking

  • Understanding fundamental VPS27 mechanisms in simple eukaryotes provides insights into dysregulated trafficking in cancer cells

  • Fungal models allow high-throughput screening for compounds affecting ESCRT function

• Infectious disease mechanisms:

  • VPS27's role in pathogenic fungi like C. neoformans informs host-pathogen interactions

  • Many human pathogens exploit or inhibit the ESCRT machinery during infection

  • Comparative analysis between non-pathogenic N. crassa and pathogenic fungi reveals virulence-associated adaptations

• Lysosomal storage disorders:

  • VPS27's function in protein sorting affects lysosomal delivery

  • Mutations in human ESCRT components cause lysosomal dysfunction diseases

  • Fungal models provide platforms for testing therapeutic approaches targeting ESCRT function