Recombinant Schizosaccharomyces pombe Microsomal signal peptidase subunit 1 (new19)

What is Schizosaccharomyces pombe Microsomal signal peptidase subunit 1 (new19)?

Microsomal signal peptidase subunit 1, encoded by the new19 gene (ORF name SPBC887.22), is a component of the signal peptidase complex in S. pombe. This small protein consists of 78 amino acids with the sequence "MNYLEGTIDFAGQLRCQKYMNYGLCTSAVISYIYGYLVQDSYCVIKLFLILASLVALVCLPAWSMYNKNPLKFQKKKE" . It functions in the cleavage of signal peptides from secretory and membrane proteins as they are translocated into the endoplasmic reticulum. New19 was identified through genome reanalysis efforts that detected previously unannotated genes in the S. pombe genome, particularly those encoding smaller proteins . It has the UniProt accession number G2TRR4 and is also known as Spc1 (Signal peptidase complex subunit 1) .

How was the new19 gene discovered?

The new19 gene was discovered through a systematic reappraisal of the S. pombe genome that specifically looked beyond the arbitrary 100-amino acid cutoff threshold initially used for genome annotation . The discovery methodology involved:

• Translating the entire S. pombe genome in all six reading frames

• Partitioning at stop codons to create a database of candidate sequences (783,342 candidates)

• Comparing these sequences to proteomic data from vegetatively growing and sexually differentiating cells

• Using comparative genomics approaches to identify conserved sequences across fungal genomes

• Confirming transcription through RNA-Seq data

This approach identified 39 novel loci, including new19, that had been overlooked in the initial genome annotation due to size constraints . New19 was specifically identified through comparative genomics methods and was confirmed to be transcribed through RNA-Seq evidence, though PCR evidence was not reported .

What is the functional role of signal peptidase complexes in fission yeast?

Signal peptidase complexes play an essential role in the secretory pathway of eukaryotic cells, including fission yeast. Their primary functions include:

• Cleaving N-terminal signal peptides from nascent proteins during their co-translational translocation into the endoplasmic reticulum

• Contributing to proper protein folding and trafficking by removing signal sequences that would otherwise interfere with protein maturation

• Ensuring correct localization of proteins to their target compartments (secretory pathway, cell membrane, or extracellular space)

• Maintaining ER homeostasis by preventing accumulation of unprocessed proteins

In S. pombe, the signal peptidase complex is composed of multiple subunits, with new19 serving as one component. The complex recognizes specific features in signal peptides, including a positively charged N-terminal region, a hydrophobic core, and a C-terminal region containing the cleavage site . Dysfunction in this system can lead to protein mislocalization, ER stress, and potential growth defects.

What experimental evidence confirms new19 as a genuine gene?

Multiple lines of evidence support the annotation of new19 as a functional gene rather than a pseudogene or false positive prediction:

• Transcriptional evidence: RNA-Seq data confirmed that the new19 locus is actively transcribed in S. pombe cells

• Comparative genomics: The new19 sequence shows conservation across fungal species, suggesting functional importance maintained through evolutionary pressure

• Structural features: The gene contains appropriate start and stop codons and follows canonical gene structure patterns

• Protein domain analysis: The sequence contains features consistent with signal peptidase complex components

• Systematic validation: new19 was part of a rigorously validated set of novel gene predictions that underwent multiple confirmation steps

This multi-faceted validation approach provides strong confidence that new19 encodes a functional protein despite its small size, which had previously caused it to be overlooked in genome annotation.

What is known about the expression pattern of new19?

While the specific expression pattern of new19 is not detailed in the provided materials, the study mentioned that expression levels of 14 of the 39 newly identified transcripts fluctuated during meiosis . This suggests that new19 might exhibit regulated expression during sexual differentiation, though whether it is among these 14 genes is not explicitly stated. Further expression studies would be needed to characterize its regulation under various conditions, including vegetative growth, meiosis, and stress responses.

What methods are recommended for studying new19 function in vivo?

To effectively investigate new19 function in S. pombe, researchers should consider these methodological approaches:

• Gene deletion and complementation:

  • Generate knockout strains using homologous recombination with selectable markers

  • Create conditional mutants if deletion is lethal (e.g., using thiamine-repressible promoters)

  • Perform cross-species complementation with homologs to assess functional conservation

• Protein localization and dynamics:

  • Tag new19 with fluorescent proteins or epitope tags for microscopy and biochemical studies

  • Use photo-convertible tags to study protein trafficking and turnover

  • Implement super-resolution microscopy to precisely localize within ER subdomains

• Interaction studies:

  • Perform co-immunoprecipitation with other signal peptidase components

  • Use proximity labeling approaches (BioID, TurboID) to identify the interactome

  • Implement crosslinking mass spectrometry to map interaction interfaces

• Functional assays:

  • Develop reporter systems to monitor signal peptide cleavage efficiency

  • Analyze the secretome in wild-type versus mutant strains

  • Measure ER stress markers to assess consequences of new19 dysfunction

• High-throughput genetic approaches:

  • Conduct synthetic genetic interaction screens to identify functional relationships

  • Perform multicopy suppressor screens to identify related pathway components

  • Implement CRISPR interference/activation to modulate new19 expression levels

These approaches should be integrated to build a comprehensive understanding of new19's role in the signal peptidase complex and its importance for S. pombe cellular function.

How can researchers generate recombinant new19 protein for in vitro studies?

Producing recombinant new19 protein presents several challenges due to its small size (78 amino acids) and likely membrane association. Based on information from commercial recombinant protein production and general methodological considerations, the following protocol is recommended:

• Expression system selection:

  • E. coli: BL21(DE3) or Rosetta strains for cytoplasmic expression

  • Yeast: Pichia pastoris for proper folding of eukaryotic proteins

  • Cell-free systems: Consider for membrane proteins to avoid toxicity issues

• Vector design:

  • Include a fusion tag to improve solubility and purification (His6, GST, MBP, or SUMO)

  • Add a cleavable linker between the tag and new19

  • Consider a C-terminal tag to avoid interfering with N-terminal processing

• Expression optimization:

  • Test multiple induction conditions (temperature, inducer concentration, time)

  • For membrane proteins, use lower temperatures (16-20°C) to improve folding

  • Include membrane-mimetic environments (detergents, lipids) during extraction

• Purification strategy:

  • Employ affinity chromatography based on the chosen tag

  • Follow with size exclusion chromatography to remove aggregates

  • For proper folding, consider on-column refolding if the protein forms inclusion bodies

• Storage considerations:

  • Based on commercial preparations, store in Tris-based buffer with 50% glycerol

  • Aliquot to avoid freeze-thaw cycles

  • Consider storage at -80°C for long-term stability

• Validation methods:

  • Confirm identity by mass spectrometry

  • Verify structural integrity using circular dichroism

  • Assess functional activity in reconstituted membrane systems

This methodological framework can be adapted based on specific research goals and available resources.

What approaches are effective for studying interactions between new19 and other signal peptidase complex components?

Investigating the interactions between new19 and other signal peptidase complex components requires specialized techniques suitable for membrane protein complexes:

• In vivo interaction mapping:

  • Split fluorescent protein complementation to visualize interactions in living cells

  • Förster resonance energy transfer (FRET) to detect proximity between tagged components

  • Immunoprecipitation using stringently validated antibodies or epitope tags

  • Chemical crosslinking followed by mass spectrometry (XL-MS) to identify interaction interfaces

• Reconstitution approaches:

  • Co-expression of multiple subunits in heterologous systems

  • Step-wise reconstitution to determine assembly hierarchy

  • Nanodiscs or liposome reconstitution to provide native-like membrane environment

  • Single-molecule techniques to analyze complex dynamics

• Structural studies:

  • Cryo-electron microscopy of the intact complex

  • NMR studies of individual components and their interactions

  • X-ray crystallography of subcomplexes or the entire assembly

  • Hydrogen-deuterium exchange mass spectrometry to map binding interfaces

• Genetic approaches:

  • Suppressor screens to identify residues that can compensate for mutations

  • Synthetic genetic interaction mapping to identify functional relationships

  • Alanine-scanning mutagenesis to identify critical interaction residues

• Computational methods:

  • Molecular dynamics simulations of component interactions

  • Protein-protein docking to predict interaction interfaces

  • Coevolution analysis to identify co-varying residues between subunits

These complementary approaches can provide a comprehensive picture of how new19 integrates into the signal peptidase complex and contributes to its function.

How can researchers assess the substrate specificity of new19-containing signal peptidase complexes?

To characterize the substrate specificity of signal peptidase complexes containing new19, researchers should implement a multi-faceted experimental strategy:

• Comparative proteomics approaches:

  • Quantitative secretome analysis comparing wild-type and new19 mutant strains

  • N-terminal proteomics to directly identify cleaved signal peptides

  • SILAC labeling to quantify differences in processing efficiency

  • Pulse-chase experiments to track kinetics of signal peptide removal

• In vitro enzymatic assays:

  • Reconstitute signal peptidase complexes with and without new19

  • Use synthetic fluorogenic peptide substrates with varying signal sequences

  • Determine kinetic parameters (Km, kcat) for different substrates

  • Implement high-throughput screening of peptide libraries to identify sequence preferences

• Reporter systems:

  • Design fusion proteins with varied signal peptides linked to easily detected reporters

  • Monitor processing efficiency in vivo in different genetic backgrounds

  • Develop split reporter systems activated upon successful signal peptide cleavage

  • Use flow cytometry for quantitative single-cell analysis of processing efficiency

• Structural biology approaches:

  • Obtain structures of the complex bound to substrate peptides

  • Map the substrate binding pocket and identify new19's contribution

  • Use molecular dynamics simulations to understand substrate recognition

  • Implement crosslinking to capture transient enzyme-substrate interactions

• Bioinformatic analysis:

  • Compare processing of different classes of signal peptides

  • Identify sequence motifs that correlate with new19-dependent processing

  • Develop predictive models for substrate preference

These methodologies will help determine whether new19 plays a direct role in catalysis, contributes to substrate recognition, or serves a structural/regulatory function within the complex.

What phenotypes might be expected in new19 mutant strains?

While the search results do not specify phenotypes associated with new19 deletion or mutation , several predictions can be made based on its function as a signal peptidase component:

• Growth and viability phenotypes:

  • Potential lethality if new19 is essential for signal peptidase function

  • Growth retardation if partial function is maintained

  • Temperature sensitivity if complex stability is compromised

  • Synthetic lethality with mutations in other secretory pathway components

• Cellular stress responses:

  • Activation of the unfolded protein response (UPR)

  • Induction of ER-associated degradation (ERAD) pathways

  • Upregulation of chaperones and protein quality control machinery

  • Possible autophagy induction to clear protein aggregates

• Protein trafficking defects:

  • Accumulation of unprocessed precursors in the ER

  • Mislocalization of secretory and membrane proteins

  • Altered glycosylation patterns due to delayed ER exit

  • Changes in cell wall composition and integrity

• Stress sensitivity phenotypes:

  • Increased sensitivity to ER stressors (tunicamycin, DTT)

  • Hypersensitivity to cell wall-targeting compounds

  • Defects in response to nutrient limitation

  • Potential mating and sporulation deficiencies

• Morphological changes:

  • Altered cell shape or size due to cell wall/membrane defects

  • ER morphology changes (expansion, fragmentation)

  • Vacuolar abnormalities as secondary consequences of secretory defects

  • Possible cell separation defects if septum formation is compromised

Systematic phenotypic characterization under various conditions would be necessary to fully understand the consequences of new19 dysfunction.

How evolutionarily conserved is new19 across fungal species?

Understanding the evolutionary conservation of new19 provides insights into its functional importance. Based on the identification of new19 through comparative genomics approaches , we can infer some degree of conservation, though specific details are not provided in the search results. To properly investigate this conservation:

• Sequence conservation analysis:

  • BLAST searches using S. pombe new19 sequence against fungal genome databases

  • Multiple sequence alignments to identify conserved residues and motifs

  • Calculation of sequence identity/similarity percentages across species

  • Conservation mapping onto predicted structural models

• Phylogenetic distribution:

  • Presence/absence patterns across major fungal lineages

  • Correlation with complexity of secretory systems

  • Identification of lineage-specific adaptations or losses

  • Comparison with distribution of other signal peptidase components

• Functional conservation assessment:

  • Cross-species complementation experiments

  • Comparison of biochemical properties of orthologs

  • Analysis of co-evolution with interacting partners

  • Identification of species-specific features that might reflect adaptive changes

• Structural conservation:

  • Prediction of secondary structure conservation despite sequence divergence

  • Identification of conserved motifs that might indicate functional sites

  • Analysis of selective pressure on different regions of the protein

This evolutionary perspective would help determine if new19 represents a core component of the signal peptidase complex or a fungal-specific adaptation, and could identify critical residues for function that have been maintained through evolutionary time.

What are the practical considerations for designing experiments with recombinant new19 protein?

When working with recombinant new19 protein, researchers should consider these practical aspects to ensure successful experiments:

• Protein stability and storage:

  • Store in Tris-based buffer with 50% glycerol as recommended for commercial preparations

  • Aliquot to minimize freeze-thaw cycles

  • Monitor stability using SDS-PAGE before critical experiments

  • Consider including protease inhibitors to prevent degradation

• Membrane protein handling:

  • Maintain in appropriate detergents or membrane mimetics

  • Avoid conditions that promote aggregation (high temperatures, low salt)

  • Use centrifugation steps to remove aggregates before experiments

  • Consider reconstitution into nanodiscs or liposomes for functional studies

• Concentration determination:

  • Due to small size (78 aa), standard Bradford or BCA assays may provide inaccurate results

  • Consider amino acid analysis or absorbance at 280nm with calculated extinction coefficient

  • Include multiple quantification methods for verification

  • Use internal standards when comparing different preparations

• Experimental controls:

  • Include heat-denatured protein as negative control

  • Use known signal peptidase substrates as positive controls

  • Compare activity with and without other complex components

  • Include background controls for non-specific binding in interaction studies

• Assay optimization:

  • Determine optimal pH, temperature, and ionic strength for activity

  • Identify cofactor requirements (if any)

  • Establish linear range for quantitative assays

  • Develop high-throughput assays for inhibitor screening or mutant analysis

Following these guidelines will help ensure reproducible results when working with this challenging membrane protein.

How does the identification of new19 impact our understanding of genome annotation?

The discovery of new19 through systematic genome reanalysis has significant implications for genome annotation practices:

• Size threshold reconsideration:

  • Challenges the arbitrary 100-amino acid cutoff commonly used in genome annotation

  • Demonstrates that small proteins can have important cellular functions

  • Suggests that thousands of functional small ORFs may be missed in current annotations

  • Encourages more sophisticated approaches to gene prediction beyond size thresholds

• Methodological advances:

  • Illustrates the value of integrating multiple evidence types (comparative genomics, transcriptomics, proteomics)

  • Highlights the importance of looking beyond computational prediction alone

  • Demonstrates the utility of targeted reanalysis of "complete" genomes

  • Provides a framework for discovering novel genes in other organisms

• Biological significance:

  • Expands our understanding of the minimal functional size for proteins

  • Suggests small proteins may be particularly important in certain cellular processes

  • Indicates that component parts of larger complexes (like signal peptidase) may be systematically under-annotated

  • Adds to growing evidence that genome complexity is underestimated

• Practical implications:

  • Necessitates reanalysis of existing genomes with improved methods

  • Suggests experimental validation should be prioritized for small ORF predictions

  • Indicates that transcriptome analysis should not discard small ORFs

  • Demonstrates that proteomics approaches need to be optimized for small protein detection

The identification of new19 and other novel small proteins in S. pombe serves as an important case study in how systematic reanalysis can improve genome annotation and discover functionally important genes previously overlooked due to arbitrary size constraints.

What techniques can be used to analyze the structural properties of new19?

Determining the structure of new19 presents challenges due to its small size (78 amino acids) and membrane association. A multi-technique approach is recommended:

• Computational structure prediction:

  • AlphaFold2 or RoseTTAFold for initial model generation

  • Molecular dynamics simulations to model membrane interactions

  • Ab initio modeling approaches optimized for small proteins

  • Threading against known signal peptidase components from other organisms

• Solution-state structural techniques:

  • NMR spectroscopy (suitable for small proteins)

  • Circular dichroism to determine secondary structure content

  • SAXS/SANS for low-resolution envelope determination

  • HDX-MS to probe solvent accessibility and dynamics

• Solid-state approaches for membrane context:

  • Solid-state NMR in lipid bilayers

  • EPR spectroscopy with site-directed spin labeling

  • X-ray crystallography of detergent-solubilized protein

  • Cryo-EM of the entire signal peptidase complex

• Hybrid structural approaches:

  • Integrate low-resolution data with computational models

  • Use crosslinking-MS to define distance constraints

  • Implement hydrogen-deuterium exchange to identify structured regions

  • Validate models through targeted mutagenesis of predicted structural elements

• Functional structural studies:

  • Alanine scanning to identify functionally important residues

  • Disulfide crosslinking to test structural predictions

  • Accessibility mapping using chemical modification

  • Protein engineering to test proposed structural models

These complementary approaches can overcome the challenges inherent in studying small membrane proteins and provide insights into how new19 contributes to signal peptidase complex structure and function.

How can researchers investigate if new19 has additional functions beyond signal peptidase activity?

To explore potential moonlighting functions of new19 beyond its role in the signal peptidase complex, researchers should implement these systematic approaches:

• Unbiased interaction screening:

  • Genome-wide yeast two-hybrid screening

  • BioID or TurboID proximity labeling in different cellular compartments

  • Co-immunoprecipitation followed by mass spectrometry under various conditions

  • Protein array screening to identify unexpected binding partners

• Phenotypic profiling:

  • Chemical-genetic profiling of new19 mutants with diverse compounds

  • Transcriptomic analysis under different stress conditions

  • Systematic growth assays in various media compositions

  • Microscopy-based phenotypic screens for subcellular abnormalities

• Localization studies:

  • Super-resolution microscopy to detect potential non-ER localization

  • Fractionation studies to identify presence in unexpected compartments

  • Time-lapse imaging to detect condition-dependent relocalization

  • Immunogold electron microscopy for precise ultrastructural localization

• Comparative genomics approaches:

  • Analysis of gene neighborhood conservation across species

  • Identification of co-evolved gene pairs suggesting functional relationships

  • Domain fusion events in other organisms that might suggest additional functions

  • Detection of lineage-specific adaptations in protein sequence

• Biochemical activity screening:

  • Testing for enzymatic activities beyond signal peptidase function

  • Nucleic acid binding assays

  • Lipid interaction studies

  • Metabolite binding assays

These approaches can reveal unexpected functions that might explain evolutionary conservation patterns or unexpected phenotypes observed in new19 mutants, potentially expanding our understanding of how small proteins contribute to cellular function beyond their primary annotated roles.