Recombinant Schizosaccharomyces pombe Cytochrome oxidase assembly protein shy1 (shy1)

What is the structural composition of Shy1 protein in S. pombe?

Shy1 in Schizosaccharomyces pombe is a mitochondrial inner membrane protein comprising 290 amino acids with a predicted molecular weight of 33 kDa and an isoelectric point (pI) of 10.67. Bioinformatics analysis has revealed that it contains a conserved SURF1 domain essential for the biogenesis of cytochrome c oxidase (complex IV). The protein features two transmembrane domains that anchor it to the inner mitochondrial membrane (IMM) .

When compared with homologs in other species, S. pombe Shy1 shares 24% identity and 36% similarity with Saccharomyces cerevisiae SHY1 and 27% identity and 37% similarity with human SURF1. Despite these moderate sequence similarities, tertiary structure predictions using AlphaFold demonstrate significant structural conservation, suggesting functional conservation despite sequence divergence .

How does Shy1 contribute to cytochrome c oxidase assembly in S. pombe?

Shy1 plays a pivotal role in the assembly of cytochrome c oxidase (complex IV) in S. pombe by:

• Facilitating the expression of mitochondrial DNA (mtDNA)-encoded genes essential for complex IV assembly .

• Physically interacting with structural subunits and assembly factors of complex IV, forming stable intermediate complexes that are crucial for the stepwise assembly process .

• Potentially participating in the formation of supercomplexes involving both complex III and complex IV, as evidenced by co-immunoprecipitation with Rip1, a subunit of ubiquinone-cytochrome c oxidoreductase (complex III) .

Unlike its homologs in other species, deletion of shy1 in S. pombe does not completely disrupt respiratory chain assembly, suggesting the existence of compensatory mechanisms specific to this organism .

What experimental approaches can verify Shy1's subcellular localization?

To verify Shy1's subcellular localization in S. pombe, researchers have successfully employed multiple complementary approaches:

• Fluorescent microscopy: By tagging Shy1 with fluorescent proteins (e.g., GFP), researchers can visualize its mitochondrial localization in living cells .

• Subcellular fractionation and Western blotting: This approach involves:

  • Isolating mitochondria through differential centrifugation

  • Separating mitochondrial compartments (outer membrane, intermembrane space, inner membrane, matrix)

  • Detecting Shy1 using specific antibodies in Western blot analysis

• Protease protection assays: Treatment with proteinase K in the presence or absence of detergents (e.g., Triton X-100) helps determine the membrane topology of Shy1 .

Using these methods, researchers have confirmed that Shy1 is an integral inner mitochondrial membrane protein with its functional domains oriented toward the intermembrane space.

What are the optimal methods for generating recombinant Shy1 in S. pombe?

For generating recombinant Shy1 in S. pombe, several methodological approaches have proven effective:

In-frame Integration Using Overlap Extension PCR:

• Design primers to amplify the shy1 gene with appropriate restriction sites

• Use overlap extension PCR to add the desired tag (e.g., FLAG, HA) to the C-terminus

• Clone the construct into an appropriate S. pombe expression vector

• Transform the construct into wild-type S. pombe strain (e.g., yHL6381)

Plasmid-based Expression Systems:
Several plasmid systems have been successfully used with S. pombe:

• Episomal vectors based on ars1 sequence

• Integrative vectors targeting specific genomic loci

Growth Conditions for Optimal Expression:

• For respiratory function studies: YES rich medium with 3% glycerol and 0.1% glucose

• For fermentative growth: YES rich medium with 3% glucose

The choice of method depends on research objectives, with genomic integration providing more physiological expression levels, while plasmid-based systems may offer higher expression levels suitable for protein purification.

What protein tagging strategies are most effective for studying Shy1 interactions?

Effective protein tagging strategies for studying Shy1 interactions in S. pombe include:

C-terminal Tagging:

• Most widely used approach as it typically preserves protein function

• Tags: FLAG, HA, GFP, TAP (Tandem Affinity Purification)

• Method: Integrate tagging cassettes by homologous recombination

• Verification: Western blotting to confirm expression and expected size

Tag Selection Based on Experimental Purpose:

• For co-immunoprecipitation: FLAG or HA tags

• For localization studies: GFP or other fluorescent protein tags

• For purification: His6 tag or TAP tag

• For studying protein dynamics: Conditional degron tags

Dual Tagging System for Interaction Studies:
When studying interactions between Shy1 and other proteins, complementary tags can be used:

• Example: FLAG-tagged Shy1 and HA-tagged potential interactors

• This approach allows for sequential or reciprocal co-immunoprecipitation experiments to validate interactions

Important considerations include confirming that the tag does not interfere with Shy1 function by assessing respiratory growth and complex IV activity in the tagged strain compared to wild-type.

How can protein-protein interactions involving Shy1 be effectively analyzed?

Multiple complementary techniques can be employed to analyze Shy1 protein-protein interactions in S. pombe:

Co-immunoprecipitation (Co-IP):

• Prepare mitochondrial lysates using mild detergents (e.g., digitonin, 1%)

• Immunoprecipitate tagged Shy1 using appropriate antibodies

• Analyze co-precipitated proteins by Western blotting or mass spectrometry

This approach has successfully identified interactions between Shy1 and both structural subunits and assembly factors of complex IV, as well as with Rip1, a component of complex III .

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE):

• Preserves native protein complexes

• Enables identification of Shy1-containing assembly intermediates

• Can be combined with second-dimension SDS-PAGE for detailed subunit analysis

Yeast Two-Hybrid Screening:

• Allows detection of direct protein-protein interactions

• Has been successfully used to identify interactions in S. pombe

• Example shown in search result for identifying histone H4-like TAF in S. pombe

Mass Spectrometry-Based Approaches:

• Quantitative proteomics can identify interaction partners

• SILAC (Stable Isotope Labeling with Amino acids in Cell culture) approaches enable comparison between wild-type and mutant conditions

• Cross-linking mass spectrometry (XL-MS) can map interaction interfaces

Each method has strengths and limitations, so combining multiple approaches provides the most reliable characterization of Shy1's interaction network.

How does Shy1 contribute to mitochondrial respiratory chain supercomplex assembly?

Recent studies have revealed that Shy1 likely plays a critical role in the assembly of mitochondrial respiratory chain supercomplexes in S. pombe through several mechanisms:

Physical Interaction with Complex III Components:

• Co-immunoprecipitation experiments have demonstrated that Shy1 can physically interact with Rip1, a subunit of complex III (ubiquinone-cytochrome c oxidoreductase or cytochrome bc1 complex) .

• This interaction suggests Shy1 may function as a bridging factor between assembly intermediates of complex IV and complex III.

BN-PAGE Analysis Evidence:

• Blue Native PAGE analysis of mitochondrial complexes has corroborated the involvement of Shy1 in supercomplex formation

• In these analyses, Shy1 was detected in high-molecular-weight complexes corresponding to the size of supercomplexes .

Comparison with S. cerevisiae Homolog:
Similar findings have been reported for the S. cerevisiae homolog, where:

• "Shy1 associates with the subcomplexes of complex IV that are potential assembly intermediates"

• "Partially assembled forms of complex IV bound to Shy1 and Cox14 can associate with the bc1 complex"

These findings expand our understanding of Shy1 beyond its established role in complex IV assembly, suggesting it also coordinates the formation of higher-order respiratory chain structures that optimize electron transport efficiency in mitochondria.

What are the phenotypic consequences of shy1 deletion in S. pombe?

Deletion of shy1 in S. pombe produces several distinct phenotypic consequences that differ somewhat from its homologs in other species:

Impact on Respiratory Chain Assembly:
Unlike its homologs in S. cerevisiae and humans, deletion of shy1 in S. pombe does not critically disrupt respiratory chain assembly, suggesting the presence of compensatory mechanisms specific to S. pombe .

Effects on Mitochondrial Function:

• Shy1 is indispensable for optimal mitochondrial respiration

• Deletion affects the maintenance of stable levels of core subunits of the electron transport chain

• Impaired expression of mtDNA-encoded genes

Growth Phenotypes:
When grown under respiratory conditions (glycerol medium), shy1Δ strains show:

• Reduced growth rates compared to wild-type

• Viability remains largely intact, unlike the severe growth defects observed in S. cerevisiae shy1Δ strains

Molecular Consequences:

• Reduction in complex IV activity

• Altered assembly of respiratory chain complexes

• Changes in mitochondrial morphology

• Potential activation of retrograde signaling pathways

These findings highlight species-specific differences in the reliance on Shy1 for respiratory function and suggest that S. pombe may possess alternative mechanisms to ensure mitochondrial functionality even in the absence of Shy1.

How can researchers analyze Shy1's role in the expression of mtDNA-encoded genes?

To analyze Shy1's role in the expression of mtDNA-encoded genes in S. pombe, researchers can employ several sophisticated techniques:

RNA Analysis Techniques:

• RT-qPCR: For quantitative analysis of mtDNA-encoded transcript levels

  • Total RNA isolation from wild-type and shy1Δ strains using specialized kits (e.g., High Pure RNA Isolation kit)

  • cDNA synthesis followed by qPCR with mitochondrial gene-specific primers

  • Normalization to nuclear-encoded reference genes (e.g., act1)

• Northern Blotting: For visualization of specific mtDNA transcripts

  • RNA extraction followed by gel electrophoresis

  • Transfer to membrane and hybridization with gene-specific probes

  • Allows detection of processing defects and precursor accumulation

Protein-Level Analysis:

• Western Blotting: To assess steady-state levels of mtDNA-encoded proteins

  • Mitochondrial isolation from wild-type and shy1Δ strains

  • SDS-PAGE separation followed by immunoblotting with specific antibodies

  • Quantification of relative protein levels

• Pulse-Chase Labeling: To study synthesis and turnover rates

  • In vivo labeling of newly synthesized mitochondrial proteins with [35S]methionine

  • Chase with unlabeled methionine

  • Analysis by SDS-PAGE and autoradiography

Translational Activity Assessment:

• Analysis of mitochondrial polysomes

• Study of mitochondrial ribosome association with mRNAs

• Investigation of translational activators and their interaction with Shy1

These approaches have revealed that Shy1 influences mitochondrial gene expression at multiple levels, potentially coordinating the synthesis of mtDNA-encoded subunits with the assembly process of respiratory complexes.

How does S. pombe Shy1 structurally compare to its homologs in other species?

S. pombe Shy1 shares structural features with its homologs while displaying species-specific characteristics:

Sequence Comparison:

• 24% identical and 36% similar to S. cerevisiae SHY1

• 27% identical and 37% similar to human SURF1

• Contains the conserved SURF1 domain present in all homologs

Structural Analysis:
Studies using AlphaFold to predict tertiary structures reveal:

Domain Organization:

• Two transmembrane domains anchoring the protein to the inner mitochondrial membrane

• Conserved SURF1 domain linking to complex IV biogenesis

• Species-specific variations in N- and C-terminal regions

This structural conservation despite sequence divergence underscores the evolutionary importance of Shy1/SURF1 proteins in mitochondrial function across eukaryotic species.

What are the functional differences between S. pombe Shy1 and its homologs?

Despite structural similarities, S. pombe Shy1 exhibits notable functional differences from its homologs:

Impact of Gene Deletion:

• S. pombe: Deletion of shy1 does not critically disrupt respiratory chain assembly, suggesting compensatory mechanisms

• S. cerevisiae: SHY1 deletion causes severe respiratory defects

• Humans: SURF1 mutations lead to Leigh Syndrome, a severe neurometabolic disorder associated with complex IV deficiency

Complex IV Assembly Role:

• S. pombe Shy1: Participates in complex IV assembly but appears less critical for complex completion

• S. cerevisiae SHY1: Essential for complex IV assembly, couples Cox1 translational regulation to assembly

• Human SURF1: Critical for complex IV assembly, mutations cause >90% reduction in complex IV activity

Interaction with Other Complexes:

• S. pombe Shy1: Strong evidence for interaction with complex III components (Rip1)

• S. cerevisiae SHY1: "Partially assembled forms of complex IV bound to Shy1 and Cox14 can associate with the bc1 complex"

• Human SURF1: Primarily characterized in the context of complex IV assembly

These functional differences highlight the evolutionary adaptation of Shy1/SURF1 proteins to species-specific requirements for mitochondrial respiration and energy metabolism.

How can researchers leverage knowledge from other species to advance S. pombe Shy1 research?

Researchers can strategically leverage knowledge from other species to advance S. pombe Shy1 research through several approaches:

Comparative Genomic Strategies:

• Identify conserved interaction partners from S. cerevisiae and human studies

• Target these for investigation in S. pombe through co-immunoprecipitation or yeast two-hybrid

• Compare protein complexes across species using BN-PAGE and mass spectrometry

Functional Complementation Studies:

• Express human SURF1 or S. cerevisiae SHY1 in S. pombe shy1Δ strains

• Assess rescue of phenotypes (growth, complex IV activity)

• Identify domains responsible for functional conservation through chimeric proteins

Translational Insights from Human Disease Models:

• Introduce Leigh Syndrome-associated SURF1 mutations into corresponding residues in S. pombe Shy1

• Evaluate functional consequences to establish S. pombe as a model for human disease

• Screen for suppressor mutations that restore function, potentially identifying therapeutic targets

Evolutionary Analysis Approaches:

• Perform comprehensive phylogenetic analysis of Shy1/SURF1 proteins

• Identify species-specific adaptations versus conserved elements

• Use this information to guide mutagenesis studies targeting functionally important regions

By integrating knowledge from diverse species, researchers can develop more targeted hypotheses about S. pombe Shy1 function and potentially uncover novel aspects of mitochondrial biology with relevance to human health and disease.

What experimental design principles should be followed when studying Shy1 function?

When designing experiments to study Shy1 function in S. pombe, researchers should adhere to several key principles:

Control Selection and Validation:

• Include appropriate positive controls (wild-type strains) and negative controls (shy1Δ strains)

• For tagged proteins, verify that the tag does not interfere with function

• Validate antibody specificity using shy1Δ strains4

Growth Condition Considerations:

• Test phenotypes under both fermentative (glucose) and respiratory (glycerol) conditions

• Consider carbon source effects on mitochondrial function

• Include time-course analyses to capture dynamic processes

Genetic Background Management:

• Use isogenic strains to minimize confounding genetic variables

• Document all genetic modifications comprehensively

• Consider potential synthetic interactions with other mutations

Quantitative Assessment:

• Employ multiple independent biological replicates (minimum n=3)

• Use appropriate statistical analyses for data interpretation

• Quantify results using image analysis software for consistent measurement4

Complementary Methodologies:

• Combine genetic approaches with biochemical and cell biological techniques

• Use both in vivo and in vitro systems when appropriate

• Validate key findings using orthogonal methods4

Following these principles ensures robust, reproducible results when investigating Shy1 function in S. pombe.

What are common technical challenges in Shy1 research and how can they be addressed?

Research on Shy1 in S. pombe presents several technical challenges that can be addressed with specific strategies:

Challenge 1: Mitochondrial Protein Extraction

• Problem: Maintaining native protein complexes during extraction

• Solution: Use gentle detergents (digitonin 1-2%), maintain cold temperatures throughout, add protease inhibitors, and optimize buffer conditions (pH, salt concentration)

Challenge 2: Variability in Growth Phenotypes

• Problem: Inconsistent growth phenotypes between experiments

• Solution: Standardize inoculum density, growth phase of starter cultures, media preparation, and incubation conditions; use quantitative growth measurements (e.g., plate readers) rather than qualitative assessments

Challenge 3: Low Abundance of Shy1 Protein

• Problem: Difficulty detecting low-abundance Shy1

• Solution: Optimize protein extraction, use sensitive detection methods (chemiluminescence, fluorescence), consider enrichment steps (mitochondrial isolation), and potentially enhance expression using stronger promoters for specific experiments

Challenge 4: Distinguishing Direct from Indirect Effects

• Problem: Determining whether phenotypes are directly due to Shy1 loss

• Solution: Use acute depletion systems (e.g., auxin-inducible degron tags), conduct time-course analyses, complement with wild-type Shy1, and identify separation-of-function mutations

Challenge 5: Analysis of Transient Protein Interactions

• Problem: Capturing dynamic/transient interactions

• Solution: Use in vivo crosslinking approaches, employ proximity labeling techniques (BioID, TurboID), and analyze under different physiological conditions that may stabilize specific interactions

These strategies can significantly improve the reliability and reproducibility of Shy1 research in S. pombe.

How can researchers resolve contradictory data in Shy1 functional studies?

When faced with contradictory data in Shy1 functional studies, researchers should employ a systematic approach to resolution:

Methodological Reconciliation:

• Compare experimental conditions in detail (media, temperature, growth phase)

• Standardize protocols across laboratories

• Determine if differences in strain backgrounds explain contradictions

• Consider whether tagged versus untagged proteins behave differently4

Hypothesis Refinement:

• Develop testable hypotheses that could explain apparent contradictions

• Design critical experiments specifically aimed at resolving contradictions

• Consider whether Shy1 may have context-dependent functions4

Technical Validation:

• Verify reagent quality and specificity (especially antibodies)

• Confirm genetic modifications by sequencing

• Assess protein expression levels across experimental conditions

• Use multiple independent methods to measure the same parameter

Statistical Reassessment:

• Increase sample size to improve statistical power

• Apply appropriate statistical tests

• Consider whether outliers are biologically meaningful or technical artifacts

• Use meta-analysis approaches for integrating multiple datasets

Collaborative Resolution:

• Engage with other researchers through material exchange

• Perform side-by-side experiments in different laboratories

• Consider joint publications that directly address and resolve contradictions

This systematic approach helps distinguish genuine biological complexities from technical issues and advances understanding of Shy1 function.

What are the most promising future directions in S. pombe Shy1 research?

Based on current knowledge and technological advances, several promising research directions for S. pombe Shy1 investigation emerge:

Compensatory Mechanism Identification:
Unlike its homologs in other species, shy1 deletion in S. pombe does not critically disrupt respiratory chain assembly, suggesting unique compensatory mechanisms. Identifying these through genetic screens or comparative proteomics could reveal novel assembly factors and pathways with potential therapeutic relevance for human mitochondrial diseases .

Supercomplex Assembly Regulation:
Further characterizing Shy1's role in supercomplex formation between complexes III and IV could provide insights into the coordination of respiratory chain assembly and the structural determinants of efficient electron transport .

Post-translational Modification Analysis:
Comprehensive mapping of Shy1 post-translational modifications and their functional significance could reveal regulatory mechanisms controlling complex IV assembly in response to cellular metabolic demands or stress conditions.

CRISPRi Applications for Temporal Studies:
Leveraging recently developed CRISPRi libraries for S. pombe to achieve controlled shy1 depletion would enable temporal studies of complex IV assembly dynamics that cannot be achieved with conventional knockout approaches .

Therapeutic Relevance Exploration:
Establishing S. pombe as a model system for testing interventions that might bypass SURF1 deficiency in human Leigh Syndrome could accelerate therapeutic development through high-throughput screening approaches.

These directions hold significant promise for advancing both basic understanding of mitochondrial biology and potential applications in human mitochondrial disease research.

How can integrative approaches enhance our understanding of Shy1 function?

Integrative approaches combining multiple experimental methodologies and computational analyses can significantly enhance our understanding of Shy1 function in S. pombe:

Multi-omics Integration:

• Combine proteomics, transcriptomics, and metabolomics data from wild-type and shy1Δ strains

• Map changes across biological scales to understand system-wide effects

• Identify key nodes in regulatory networks affected by Shy1 deletion

Structural Biology with Functional Genomics:

• Integrate structural predictions from AlphaFold with mutagenesis studies

• Map functional domains through systematic alanine scanning

• Correlate structure-function relationships with evolutionary conservation

Computational Modeling with Experimental Validation:

• Develop mathematical models of complex IV assembly incorporating Shy1

• Generate testable predictions about assembly kinetics and dependencies

• Iteratively refine models based on experimental results

Cross-species Comparative Analyses:

• Perform systematic comparisons of Shy1 function across yeast species

• Identify conserved versus divergent aspects of function

• Leverage evolutionary insights to predict functional motifs

Temporal and Spatial Resolution Studies:

• Combine live-cell imaging with biochemical analyses

• Track Shy1 dynamics during mitochondrial biogenesis and stress

• Correlate subcellular localization with functional states

By integrating these diverse approaches, researchers can develop a more comprehensive understanding of Shy1 function that spans from molecular mechanisms to cellular physiology, potentially revealing unexpected connections and novel therapeutic targets.