SPBC30D10.09c Antibody

What is SPBC30D10.09c and what cellular functions does it perform?

SPBC30D10.09c encodes Yep1 (also known as Hva22 or Rop1), which is the ortholog of human REEP1-4 proteins in fission yeast. Yep1 plays essential roles in specific types of selective autophagy, particularly ER-phagy (degradation of endoplasmic reticulum) and nucleophagy (degradation of nuclear components), while being dispensable for bulk autophagy . This protein was identified through an imaging-based mutant screen designed to discover genes important for ER-phagy in S. pombe . When cells are treated with DTT or subjected to starvation, Yep1 has been observed forming puncta at approximately 30% of the sites where both Atg8 and the ER-phagy receptor Epr1 form puncta, suggesting its participation in autophagosome assembly at these specific degradation sites .

How should Yep1 antibodies be validated for specificity in immunological applications?

For proper validation of SPBC30D10.09c antibodies, researchers should:

• Perform Western blot analysis comparing wild-type and yep1Δ deletion strains

• Include additional controls with:

  • Epitope-tagged Yep1 strains (e.g., Yep1-GFP) to confirm antibody recognition

  • Point mutation strains (such as the T17M mutant identified in screens)

  • Quantitative comparison of signal intensity between samples

The antibody should show specific binding to a protein of the expected molecular weight in wild-type samples while showing no signal in yep1Δ strains. Cross-reactivity with other S. pombe proteins should be minimal or absent. The same validation approach should be applied for immunofluorescence applications, with additional assessment of subcellular localization patterns matching those observed with tagged Yep1 constructs.

What is the subcellular localization pattern of Yep1 and how can it be visualized?

Based on fluorescence microscopy studies, Yep1 forms distinct puncta that colocalize with a subset of Atg8 and Epr1 puncta during ER-phagy and nucleophagy processes . These puncta represent sites of autophagosome assembly. To visualize this pattern:

• Fix cells with 4% paraformaldehyde for 15-20 minutes

• Permeabilize with 0.1% Triton X-100

• Block with 3% BSA for 30 minutes

• Incubate with primary SPBC30D10.09c antibody overnight at 4°C

• Wash 3× with PBS

• Incubate with fluorophore-conjugated secondary antibody

• Counterstain with DAPI to visualize nuclei

• Mount and image using confocal microscopy

For proper co-localization studies, combine with antibodies against Atg8 or use strains expressing fluorescently tagged Epr1. Treatment with 2mM DTT for 4 hours or nitrogen starvation will induce the formation of these autophagy-related structures.

How can SPBC30D10.09c antibodies be effectively used in immunoprecipitation experiments?

For successful immunoprecipitation of Yep1:

Lysis Buffer Composition:

• 50 mM HEPES-KOH, pH 7.5

• 150 mM NaCl

• 1 mM EDTA

• 1% Triton X-100

• 10% glycerol

• Protease inhibitor cocktail

• Phosphatase inhibitors (if studying phosphorylation)

Procedure:

• Harvest 50-100 mL of yeast culture (OD600 ~0.8-1.0)

• Lyse cells using glass beads in lysis buffer (8 cycles of 30 sec vortexing, 30 sec on ice)

• Clear lysate by centrifugation (13,000 × g, 15 min, 4°C)

• Pre-clear with Protein A/G beads for 1 hour

• Incubate cleared lysate with SPBC30D10.09c antibody (2-5 μg) overnight at 4°C

• Add Protein A/G beads and incubate for 2 hours

• Wash 5× with lysis buffer containing reduced detergent (0.1% Triton X-100)

• Elute bound proteins by boiling in SDS sample buffer

This method can be optimized for studying Yep1 interactions with autophagy machinery components or for identifying post-translational modifications.

What are the key considerations when using SPBC30D10.09c antibodies for autophagy studies?

When investigating Yep1's role in selective autophagy using specific antibodies:

• Treatment conditions: Compare DTT-induced ER stress (2 mM DTT, 4 hours) and nitrogen starvation (EMM-N medium, 4 hours), as these elicit different autophagy responses

• Monitoring autophagy flux:

  • Track the processing of fluorescent protein-tagged reporters (e.g., Pus1-mECitrine for nucleophagy)

  • Use western blotting to monitor the generation of free fluorescent protein fragments

  • Include wild-type, atg5Δ, and yep1Δ controls to distinguish autophagy-dependent phenotypes

• Autophagosome visualization:

  • Compare Yep1 antibody staining with markers for autophagosomes (Atg8) and specific cargo receptors (Epr1)

  • Use TEM to visualize double-ring structures characteristic of ER/nuclear-derived autophagosomes

• Degron protection assays: Consider implementing auxin-inducible degron (AID) tag systems to distinguish between cytosolic exposure and membrane-protected localization of cargo proteins

How can transmission electron microscopy complement immunofluorescence when studying Yep1?

TEM provides valuable ultrastructural information that complements antibody-based fluorescence studies:

• Sample preparation protocol:

  • Fix S. pombe cells with 2% glutaraldehyde in 0.1M phosphate buffer

  • Post-fix with 1% osmium tetroxide

  • Dehydrate through an ethanol series (50-100%)

  • Embed in epoxy resin

  • Section at 70-90 nm thickness

  • Stain with uranyl acetate and lead citrate

• Structures to identify:

  • In yep1Δ cells, look for accumulation of ring-shaped membrane structures in the cytoplasm

  • In fsc1Δ cells (defective in autophagosome-vacuole fusion), identify double-ring structures representing ER/nuclear-derived autophagosomes

  • Compare the frequency of these structures between DTT treatment and starvation conditions

• Quantitative analysis:

  • Count the number of double-ring structures per cell section

  • Measure the size distribution of these structures

  • Compare wild-type, yep1Δ, and various autophagy mutants (e.g., atg5Δ, fsc1Δ)

• Immunogold labeling option:

  • For more precise localization, adapt the protocol for post-embedding immunogold labeling using SPBC30D10.09c antibodies

  • Use 10-15 nm gold particles conjugated to secondary antibodies

How can SPBC30D10.09c antibodies be utilized in ChIP-seq studies?

While Yep1 is primarily studied for its role in autophagy, investigating potential chromatin-associated functions requires specialized ChIP-seq approaches:

Protocol modifications for optimal results:

• Crosslinking optimization:

  • Test both formaldehyde (1%, 10 min) and dual crosslinking (1.5 mM EGS followed by 1% formaldehyde)

  • For membrane-associated proteins like Yep1, additional optimization may be required

• Sonication parameters:

  • Use Bioruptor or similar device: 30 sec ON/30 sec OFF, 20-25 cycles

  • Verify fragment size distribution (200-500 bp) by agarose gel electrophoresis

• Chromatin preparation:

  • Include detergents suitable for membrane protein extraction (0.1% SDS, 1% Triton X-100)

  • Consider treatment with micrococcal nuclease to improve chromatin accessibility

• Immunoprecipitation:

  • Use 2-5 μg of SPBC30D10.09c antibody per IP

  • Include IgG control and input samples

  • Perform additional technical replicates compared to standard ChIP-seq

• Data analysis considerations:

  • Apply stringent peak calling parameters

  • Look for enrichment at nuclear envelope proximity

  • Compare with ChIP-seq profiles of known nuclear envelope proteins

This approach can be particularly relevant when studying potential roles of Yep1 in nucleophagy mechanisms that might involve chromatin interactions .

What approaches can resolve contradictory data when analyzing Yep1 function in different autophagy contexts?

Resolving contradictions in experimental data requires systematic troubleshooting:

• Comparative analysis of bulk vs. selective autophagy:

  • Use parallel assays for bulk autophagy (CFP-Atg8 and Tdh1-FP processing) and selective autophagy (Pus1-mECitrine for nucleophagy, Ost4-GFP for ER-phagy)

  • The observed discrepancy where yep1Δ cells show normal bulk autophagy but defective selective autophagy requires careful interpretation

• Resolution of conflicting microscopy and biochemical data:

  • When fluorescent protein cleavage assays show different results from TEM quantification (as noted with DTT treatment) , consider:

    • DTT may inhibit vacuolar proteolysis, affecting fluorescent protein processing

    • Perform time-course experiments to distinguish between formation and clearance defects

    • Use multiple independent cargo markers to verify pathway-specific effects

• Methodological approach to resolve discrepancies:

• Interpretation framework:

  • When autophagosome formation appears normal but cargo processing is impaired, focus on cargo recognition and sequestration mechanisms

  • When morphological assays and biochemical assays disagree, consider the kinetics and sensitivity of each method

  • Develop mathematical models incorporating temporal dynamics of each process being measured

How can phospho-specific antibodies against Yep1 advance our understanding of its regulation?

Developing and utilizing phospho-specific antibodies against key Yep1 residues can provide valuable insights:

• Identification of regulatory phosphorylation sites:

  • Perform phosphoproteomic analysis of Yep1 under various conditions (normal, starvation, DTT)

  • Focus on serine/threonine residues conserved between yeast and human orthologs

  • Generate phospho-specific antibodies against the identified sites

• Validation of phospho-antibodies:

  • Test against wild-type and phospho-mutant (S→A or S→E) Yep1 variants

  • Confirm phosphorylation dynamics using phosphatase treatment controls

  • Verify signal specificity in yep1Δ strains

• Applications to study regulatory mechanisms:

  • Monitor phosphorylation status during autophagy induction

  • Identify kinases responsible through kinase inhibitor screens

  • Correlate phosphorylation with Yep1 puncta formation and function

  • Map phosphorylation status to structural domains and protein interaction interfaces

• Advanced experimental design:

  • Combine with proximity labeling approaches (BioID or APEX) to capture transient interactions

  • Develop FRET-based biosensors to monitor Yep1 conformational changes driven by phosphorylation

  • Create computational models predicting how phosphorylation affects Yep1's role in autophagosome formation

These approaches can elucidate the molecular switches controlling Yep1 activity during different cellular stresses and explain the selectivity of its function in specific autophagy pathways.

What technical innovations could improve detection of low-abundance Yep1 interactions?

For capturing transient or low-abundance interactions of Yep1:

• Proximity-dependent labeling techniques:

  • Express Yep1-BioID2 or Yep1-TurboID fusion proteins

  • Induce labeling with biotin during autophagy stimulation

  • Purify biotinylated proteins and identify by mass spectrometry

  • This approach can capture weak or transient interactions missed by conventional IP

• Advanced imaging approaches:

  • Implement super-resolution microscopy (SIM, STED, or PALM/STORM)

  • Use with optimized SPBC30D10.09c antibodies for single-molecule localization

  • Apply FRET or FLIM to detect protein-protein interactions in situ

  • Consider correlative light and electron microscopy (CLEM) to precisely locate Yep1 at ultrastructural level

• Quantitative crosslinking mass spectrometry (QCLMS):

  • Apply protein crosslinkers of different lengths

  • Enrich for Yep1-containing complexes

  • Identify crosslinked peptides by mass spectrometry

  • Map interaction interfaces at amino acid resolution

• Split reporter systems:

  • Develop split GFP, split luciferase, or split dihydrofolate reductase fusions

  • Test candidate interactors systematically

  • Monitor interaction dynamics during autophagy induction

  • Validate with biochemical approaches

These technical innovations can provide new insights into how Yep1 functions in the selective recognition and sequestration of ER and nuclear components during specific autophagy processes.

How can researchers address poor signal-to-noise ratio when using SPBC30D10.09c antibodies?

When experiencing high background or weak specific signals:

• Optimization strategies for Western blotting:

  • Test blocking agents: 5% non-fat milk vs. 3-5% BSA vs. commercial blockers

  • Optimize antibody concentration: Perform titration from 1:250 to 1:5000

  • Increase washing stringency: Add 0.1-0.3% Tween-20 to wash buffers

  • Consider membrane type: PVDF may provide better signal-to-noise than nitrocellulose for some antibodies

  • Enhance detection sensitivity with signal enhancers compatible with your detection system

• Immunofluorescence optimization:

  • Test fixation methods: 4% PFA vs. methanol vs. combined approaches

  • Optimize permeabilization: Test 0.1-0.5% Triton X-100 or 0.1-0.2% Saponin

  • Implement antigen retrieval if needed: Citrate buffer (pH 6.0) at 95°C for 10-20 minutes

  • Use image acquisition settings that maximize dynamic range

  • Apply careful background subtraction during image analysis

• Systematic testing of conditions:

What strategies help distinguish between direct and indirect effects when studying Yep1 knockout phenotypes?

To establish causality and distinguish direct from indirect effects:

• Complement with structure-function analysis:

  • Generate point mutations in specific domains of Yep1

  • Create chimeric proteins swapping domains with human REEP orthologs

  • Identify separation-of-function mutants that affect only specific aspects of Yep1 activity

• Implement acute depletion systems:

  • Construct auxin-inducible degron (AID) tagged Yep1

  • Use temperature-sensitive alleles if available

  • Compare fast-responding phenotypes (likely direct) vs. delayed effects (potentially indirect)

• Establish temporal sequences:

  • Perform time-course experiments after autophagy induction

  • Monitor the order of recruitment of Yep1 and other factors

  • Use live-cell imaging with tagged proteins to establish causality

• Use epistasis analysis:

  • Determine the genetic relationship between Yep1 and other autophagy factors

  • Create double mutants and analyze phenotypes

  • Map Yep1 in the hierarchy of autophagy pathways

These approaches can help establish the direct mechanistic contributions of Yep1 to ER-phagy and nucleophagy processes.

How can comparative analysis between yeast Yep1 and human REEP1-4 inform therapeutic development?

Investigating the functional conservation between yeast Yep1 and human REEP proteins:

• Evolutionary conservation analysis:

  • Perform detailed sequence alignment of functional domains

  • Identify conserved residues critical for autophagy functions

  • Map disease-associated mutations in human REEP genes onto the yeast ortholog

• Cross-species complementation:

  • Express human REEP1-4 in yep1Δ S. pombe

  • Assess rescue of ER-phagy and nucleophagy defects

  • Determine which human isoforms best complement yeast functions

• Disease relevance:

  • Human REEP1 mutations cause hereditary spastic paraplegia (HSP)

  • Investigate whether these mutations affect autophagy functions

  • Develop yeast models expressing human disease variants

• Application to antibody development:

  • Identify epitopes conserved between species for broad-spectrum antibodies

  • Develop isoform-specific antibodies targeting divergent regions

  • Create conformation-specific antibodies recognizing active vs. inactive states

This comparative approach can accelerate understanding of human disease mechanisms and identify potential therapeutic targets in REEP-related disorders.

What novel technological approaches could enhance spatial resolution in Yep1 localization studies?

Advancing beyond conventional microscopy for precise localization:

• Expansion microscopy:

  • Physically expand fixed samples using swellable polymers

  • Achieve ~70 nm resolution with standard confocal microscopy

  • Particularly useful for resolving Yep1 puncta relative to autophagosome formation sites

• Lattice light-sheet microscopy:

  • Enable high-speed 3D imaging with minimal phototoxicity

  • Capture dynamic processes of autophagosome formation

  • Track Yep1 recruitment in living cells with unprecedented temporal resolution

• Cryo-electron tomography:

  • Visualize Yep1 in its native cellular environment at molecular resolution

  • Use immunogold labeling with SPBC30D10.09c antibodies

  • Generate 3D reconstructions of autophagosome formation sites

• DNA-PAINT super-resolution:

  • Achieve 5-10 nm localization precision

  • Use antibody fragments conjugated to DNA oligonucleotides

  • Perform multiplexed imaging of multiple autophagy components simultaneously

These approaches can provide new insights into the precise spatial organization of Yep1 during autophagosome formation and cargo sequestration.