Recombinant Schizosaccharomyces pombe Peroxisomal membrane protein pex13 (pex13)

What is Pex13 in Schizosaccharomyces pombe and what is its significance?

Pex13 is a key component of the peroxisomal docking complex in yeasts and mammals, playing an essential role in peroxisomal biogenesis and matrix protein import. In S. pombe, Pex13 functions as a membrane-bound receptor that facilitates the import of proteins containing peroxisomal targeting signals (PTS) into the peroxisomal matrix. This protein is critical for normal peroxisomal function, which affects various cellular processes including fatty acid metabolism and response to oxidative stress .

The significance of Pex13 extends beyond basic cell biology, as homologues in pathogenic fungi like Magnaporthe oryzae have been shown to be indispensable for pathogenicity, sexual reproduction, and resistance to reactive oxygen species . Understanding S. pombe Pex13 provides insights into conserved mechanisms of peroxisomal import across eukaryotes while taking advantage of the genetic tractability of fission yeast.

How does Pex13 function in the peroxisomal protein import machinery?

Pex13 functions as part of the peroxisomal docking complex along with Pex14 to facilitate the import of matrix proteins. The process works through several key steps:

• Recognition: Pex13 contains an SH3 domain that recognizes and binds to WxxxF/Y peptide motifs in the N-terminal domain of Pex5, the cytosolic receptor for PTS1-containing proteins .

• Docking: This interaction enables the docking of cargo-loaded Pex5 at the peroxisomal membrane.

• Translocation: Following docking, matrix proteins are translocated across the peroxisomal membrane into the organelle lumen.

• Receptor recycling: After cargo release, Pex5 is recycled back to the cytosol.

Notably, the SH3 domain of Pex13 demonstrates autoinhibitory properties through interaction with a proximal FxxxF motif, which can modulate its binding to Pex5 and fine-tune the import process . This regulatory mechanism appears to be important for optimal peroxisomal function.

What structural domains characterize S. pombe Pex13?

S. pombe Pex13 contains several key structural domains that define its function:

• SH3 domain: Located in the C-terminal region, this domain is critical for recognizing and binding to WxxxF/Y motifs in Pex5 . Unlike the SH3 domains in some other organisms, the S. pombe Pex13 SH3 domain shows binding preferences that differ from its orthologs.

• Transmembrane domains: These anchor Pex13 in the peroxisomal membrane with the SH3 domain facing the cytosol.

• FxxxF motif: Present in the C-terminal region, this motif mediates autoinhibitory interactions with the SH3 domain and can also interact with Pex14 .

The functional importance of these domains is evidenced by the fact that mutations affecting the SH3 domain can disrupt peroxisomal import without affecting the physical Pex13-Pex14 interaction, suggesting distinct functional roles for different domains of the protein .

How is Pex13 localized within S. pombe cells?

Pex13 is primarily localized to the peroxisomal membrane in S. pombe cells. Visualization studies using GFP-tagged versions of Pex proteins have shown that Pex13 displays a punctate pattern that overlaps with peroxisomal matrix markers (such as those tagged with PTS1 signals) . In enlarged microscopy images, Pex13 can be observed surrounding the peroxisomal matrix, confirming its membrane localization .

This membrane distribution is essential for Pex13's function as part of the docking complex that facilitates the import of matrix proteins. The proper localization of Pex13 to peroxisomes is dependent on the peroxisomal membrane protein import machinery, which is distinct from the matrix protein import pathway in which Pex13 itself functions.

How evolutionarily conserved is Pex13 across species?

Pex13 shows significant conservation across fungal species and between fungi and mammals, with both structural and functional similarities:

What are the optimal methods for expressing and purifying recombinant S. pombe Pex13?

The expression and purification of functional S. pombe Pex13 requires careful consideration of its membrane protein nature and domain organization. Based on successful approaches with other Pex proteins, the following protocol is recommended:

Expression Strategy:

• Expression system selection: E. coli BL21(DE3) cells transformed with a pET-based vector containing the Pex13 sequence optimized for bacterial expression work well for the SH3 domain. For full-length Pex13, a eukaryotic expression system like Pichia pastoris may be more suitable due to proper membrane insertion.

• Domain-based approach: Express the soluble SH3 domain (C-terminal region) separately from the transmembrane regions. For structural studies, the construct boundaries for the SH3 domain should be carefully optimized based on secondary structure predictions.

• Fusion tags: N-terminal 6xHis-GST fusion with a TEV protease cleavage site facilitates both solubility and purification.

Purification Protocol:

• Cell lysis in buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, and protease inhibitors

• Affinity chromatography using Ni-NTA resin

• On-column tag cleavage with TEV protease

• Size exclusion chromatography using a Superdex 75 column

• Concentration using centrifugal filters (10 kDa cutoff)

For membrane-associated regions of Pex13, detergent screening is crucial, with typical starting points being mild detergents like DDM (n-dodecyl-β-D-maltoside) or LDAO (lauryldimethylamine oxide).

The quality of purified protein should be assessed by SDS-PAGE, Western blotting, and dynamic light scattering to ensure monodispersity before functional assays.

How can researchers effectively study Pex13-Pex14 interactions in S. pombe?

Multiple complementary approaches can be employed to study Pex13-Pex14 interactions in S. pombe:

In vitro binding assays:

• Pull-down assays: Using recombinant GST-tagged Pex13 SH3 domain and His-tagged Pex14 N-terminal domain (NTD) to quantify direct binding interactions.

• Surface Plasmon Resonance (SPR): For determining binding kinetics and affinity constants. Immobilize one protein (typically the smaller domain) on a sensor chip and flow the partner protein across it at varying concentrations.

• Isothermal Titration Calorimetry (ITC): To measure the thermodynamic parameters of the interaction, particularly useful for studying how the FxxxF motif of Pex13 interacts with the Pex14 NTD .

In vivo approaches:

• Co-immunoprecipitation: Using epitope-tagged versions of Pex13 and Pex14 expressed in S. pombe.

• Fluorescence microscopy: Employing split-GFP or FRET-based approaches to visualize interactions in living cells.

• Yeast two-hybrid assays: With appropriate modifications for membrane proteins (such as using soluble domains only).

• Bimolecular Fluorescence Complementation (BiFC): To visualize the subcellular localization of protein interactions.

When designing experiments to study these interactions, researchers should be aware that:

• The interaction interface may differ significantly from that observed in S. cerevisiae, as human PEX13 SH3 domain does not recognize PxxP motifs in PEX14 unlike in S. cerevisiae .

• The autoinhibitory FxxxF motif in Pex13 competes with WxxxF/Y motifs for the same binding surface on the SH3 domain .

• Controls for membrane protein interactions should include both positive interactions and negative controls testing for non-specific membrane protein associations.

What phenotypes result from Pex13 mutations or deletion in S. pombe?

Mutations or deletion of Pex13 in S. pombe and related fungi result in multiple distinct phenotypes that reflect the importance of peroxisomal function:

Metabolic phenotypes:

• Fatty acid utilization defects: Inability to grow on media with fatty acids as the sole carbon source, due to impaired β-oxidation .

• Altered lipid metabolism: Changes in lipid composition and distribution.

Cellular stress responses:

• Increased sensitivity to oxidative stress: Higher susceptibility to hydrogen peroxide and other reactive oxygen species .

• Cell wall integrity issues: Sensitivity to cell wall-disrupting compounds .

Developmental phenotypes:

• Impaired sexual reproduction: Defects in mating and sporulation processes .

• Growth defects: Slower growth rates under specific conditions.

In pathogenic fungi related to S. pombe, additional phenotypes include:

• Loss of pathogenicity: Inability to infect host organisms .

• Appressorial defects: Reduction in turgor pressure necessary for host penetration .

A systematic phenotypic analysis approach should include:

• Growth assays on different carbon sources

• Microscopy to assess peroxisome number, size, and distribution

• PTS1 and PTS2 reporter localization assays

• Biochemical assays for peroxisomal enzyme activities

• Lipidomic analysis to assess changes in lipid metabolism

These phenotypes collectively demonstrate that Pex13 functions are essential for peroxisomal metabolism, stress responses, and developmental processes in S. pombe.

How do the binding mechanisms of S. pombe Pex13 differ from its orthologs in other species?

S. pombe Pex13 exhibits several key differences in binding mechanisms compared to its orthologs in other species:

WxxxF/Y peptide binding:
While both yeast and human Pex13 SH3 domains bind to WxxxF/Y motifs in Pex5, the binding surfaces and affinities may differ. In humans, this interaction occurs through a non-canonical surface on the SH3 domain , and evidence suggests this is evolutionarily conserved from yeast to humans, including S. pombe.

Autoinhibitory mechanisms:
The human PEX13 SH3 domain exhibits autoinhibition through interaction with an FxxxF motif in its C-terminal region . This autoinhibitory mechanism competes with WxxxF/Y binding and appears to be a regulatory feature that may be conserved in S. pombe but potentially with species-specific characteristics.

The table below summarizes key binding differences:

These differences highlight the evolutionary divergence in the peroxisomal import machinery and suggest that S. pombe may serve as a better model for human peroxisomal disorders than S. cerevisiae in some aspects.

What approaches can be used to visualize Pex13 localization and dynamics in S. pombe cells?

Several advanced imaging approaches can be employed to visualize Pex13 localization and dynamics in S. pombe:

Fluorescent protein tagging strategies:

• N- or C-terminal GFP fusion: GFP-tagged versions of Pex13 can be expressed to visualize its distribution . Care must be taken to ensure the tag doesn't interfere with targeting or function.

• Internal tagging: For proteins where terminal tags disrupt function, internal tagging at flexible loop regions based on structural predictions can be employed.

• Split fluorescent protein complementation: To visualize Pex13 interactions with other peroxisomal proteins in vivo.

Microscopy techniques:

• Confocal microscopy: For high-resolution imaging of fixed cells to determine precise localization patterns.

• Super-resolution microscopy: Techniques such as STORM or PALM can provide nanoscale resolution of Pex13 distribution on the peroxisomal membrane.

• Live cell imaging: To track dynamics of peroxisomes and associated Pex13.

• FRAP (Fluorescence Recovery After Photobleaching): To measure the mobility and turnover rate of Pex13 on the peroxisomal membrane.

Co-localization studies:

• Dual-color imaging: Using mCherry-PTS1 as a peroxisomal matrix marker alongside GFP-Pex13 to confirm peroxisomal membrane localization .

• Triple-color imaging: To simultaneously visualize Pex13, Pex14, and matrix proteins.

Experimental considerations:

• Expression levels should be kept close to endogenous to avoid artifacts from overexpression.

• Both N- and C-terminal tags should be tested, as membrane topology may affect tag accessibility.

• Controls should include known peroxisomal markers and cytosolic proteins.

• Time-lapse imaging can reveal dynamic processes such as peroxisome biogenesis, division, or degradation.

These approaches have revealed that Pex13 displays a punctate distribution pattern that surrounds the peroxisomal matrix, confirming its membrane localization .

What experimental approaches can be used to study the autoinhibitory properties of S. pombe Pex13?

Based on findings from human PEX13, S. pombe Pex13 likely possesses autoinhibitory properties mediated by an FxxxF motif . Several experimental approaches can be employed to study this mechanism:

Biochemical and biophysical approaches:

• Isothermal Titration Calorimetry (ITC): To compare binding affinities between:

  • Pex13 SH3 domain and Pex5 WxxxF/Y peptides

  • Pex13 SH3 domain and its own FxxxF peptide

  • Pex13 SH3-CTR (containing the FxxxF motif) and Pex5 WxxxF/Y peptides

• NMR spectroscopy: To map the binding interfaces and identify chemical shift perturbations upon peptide binding. This approach successfully identified the autoinhibitory mechanism in human PEX13 .

• X-ray crystallography: To determine high-resolution structures of:

  • Pex13 SH3 domain alone

  • Pex13 SH3 domain bound to FxxxF peptide

  • Pex13 SH3 domain bound to WxxxF/Y peptides

• Site-directed mutagenesis: Introducing mutations in the FxxxF motif to disrupt autoinhibition and testing the effects on binding to Pex5 and Pex14.

Cellular and genetic approaches:

• Yeast complementation assays: Testing whether Pex13 variants with mutations in the FxxxF motif can rescue peroxisomal import in Pex13-deficient cells.

• PTS1 import assays: Using fluorescent reporters to measure peroxisomal import efficiency with wildtype versus mutant Pex13.

• FRET-based biosensors: Designing sensors to detect conformational changes associated with autoinhibition in living cells.

How can functional assays be designed to evaluate Pex13 activity in S. pombe?

Designing functional assays to evaluate Pex13 activity requires a multi-faceted approach that addresses both biochemical function and cellular phenotypes:

Peroxisomal import assays:

• Fluorescent reporter import: Express GFP-PTS1 or GFP-PTS2 reporters in wildtype and Pex13 mutant cells to quantify import efficiency.

• Biochemical fractionation: Separate peroxisomal and cytosolic fractions and measure the distribution of known peroxisomal matrix enzymes using enzyme activity assays or Western blotting.

• Pulse-chase assays: Monitor the kinetics of newly synthesized peroxisomal protein import using inducible expression systems.

Growth and metabolic assays:

• Carbon source utilization: Compare growth on glucose versus fatty acid media (e.g., oleate) to assess peroxisomal β-oxidation function.

• Stress resistance: Measure survival rates under oxidative stress conditions (e.g., hydrogen peroxide exposure) or cell wall-disrupting compounds .

• Metabolomic profiling: Analyze changes in metabolites related to peroxisomal function, particularly long-chain fatty acids and their derivatives.

Molecular interaction assays:

• Co-immunoprecipitation: Quantify Pex13 interactions with Pex5, Pex14, and Pex14/17 under different conditions.

• BiFC (Bimolecular Fluorescence Complementation): Visualize protein interactions in vivo.

• Protein proximity labeling: Use BioID or APEX2 fused to Pex13 to identify proximal proteins in the cellular environment.

Structure-function analysis:

• Domain deletion/mutation: Create a series of Pex13 variants with modifications in key domains (SH3, FxxxF motif) to map functional regions.

• Chimeric proteins: Exchange domains between S. pombe and other species' Pex13 to identify species-specific functions.

The table below summarizes key readouts for Pex13 functional assays:

What are the critical factors in resolving contradictory data about Pex13 function in S. pombe?

Resolving contradictory data about S. pombe Pex13 function requires systematic investigation of several critical factors:

Strain background considerations:

• Genetic background effects: All commonly used S. pombe strains are derived from a single culture , but accumulated mutations can affect results. Always compare mutants to their direct parental strain.

• Auxotrophic markers: Different nutritional requirements can influence peroxisomal function. Document all auxotrophies in experimental strains.

• Chromosome structure: S. pombe has unique chromosomal features including large centromeres and rDNA repeats that can vary between strains and affect recombination rates.

Methodological variables:

• Expression levels: Overexpression versus endogenous levels of Pex13 can lead to drastically different results. Use genomic integration at the native locus where possible.

• Tag interference: Different tags (GFP, HA, TAP) may interfere with function. Validate tagged versions by complementation assays.

• Growth conditions: Peroxisome number and function vary dramatically with carbon source and growth phase. Standardize culture conditions precisely.

• Assay sensitivity: Different methods for detecting protein interactions or import have varying sensitivities. Use multiple independent approaches.

Data interpretation frameworks:

• Redundancy and compensation: Related proteins may compensate for Pex13 deficiency. Consider double mutant analysis.

• Partial functions: Distinguish between complete loss versus partial impairment of specific Pex13 functions.

• Direct versus indirect effects: Separate primary consequences of Pex13 dysfunction from secondary cellular adaptations.

• Species-specific differences: Be cautious when extrapolating from other organisms, as the PxxP binding differences between yeast species and humans illustrate .

When confronted with contradictory data, a systematic approach would include:

• Repeating key experiments with standardized conditions

• Testing multiple independently generated mutants

• Using complementary assays that measure the same property

• Developing quantitative rather than qualitative readouts

• Considering whether discrepancies reveal new biological insights about context-dependent Pex13 functions