Recombinant Saccharomyces cerevisiae Peroxisomal membrane protein PEX31 (PEX31)

What is PEX31 and how is it related to other peroxisomal proteins?

PEX31 is a homologous protein to PEX30 in Saccharomyces cerevisiae that contains a dysferlin (DysF) domain with currently unknown function. Both proteins physically interact and have been implicated in peroxisome biogenesis. PEX31 belongs to a family of dysferlin domain-containing peroxins that includes Yarrowia lipolytica Pex23p and Saccharomyces cerevisiae Pex30p, Pex31p, and Pex32p. While not essential for peroxisome biogenesis and function, these proteins regulate peroxisome shape and abundance .

Unlike PEX3, which is central to peroxisome formation and recruits factors specifically for peroxisome formation and segregation, PEX31 appears to be more involved in the regulation of peroxisome size and number. The functional relationship between PEX31 and other peroxins is complex, with different peroxins contributing to distinct aspects of peroxisome biogenesis, maintenance, and degradation .

What are the structural features of the PEX31 protein?

PEX31 contains a dysferlin (DysF) domain, which is a conserved structural motif found in several proteins involved in membrane dynamics. The exact function of this domain remains unclear, though it likely plays a role in membrane interactions or protein-protein binding. PEX31 is synthesized at much lower levels compared to PEX30 and its production is induced under specific conditions such as exposure to oleate, while PEX30 synthesis is constitutive .

When analyzing PEX31 structure, researchers should note that the protein localizes to both the endoplasmic reticulum (ER) and peroxisomes, with a predominant localization on peroxisomes. This dual localization pattern suggests that PEX31 may function at the interface between these two organelles, potentially facilitating communication or material transfer between them .

What is the localization pattern of PEX31 in yeast cells?

PEX31 exhibits a dual localization pattern within the cell, being present at both the endoplasmic reticulum (ER) and peroxisomes. Microscopy studies have shown that while PEX31 can be found in both locations, it is predominantly localized to peroxisomes, whereas its homolog PEX30 is mostly found at the ER. This distinct distribution pattern suggests specialized functions for these related proteins despite their physical interaction .

Interestingly, PEX31 can be chased from the ER to peroxisomes in a PEX19-dependent manner, suggesting a dynamic trafficking pathway. In cells lacking PEX19 (pex19Δ), PEX31 colocalizes with PEX30 at the ER, providing evidence for an ER-to-peroxisome trafficking route for these proteins. Additionally, when PEX31 is overexpressed, PEX30 accumulates on peroxisomes, further highlighting the interaction between these proteins and their dynamic localization patterns .

How does PEX31 contribute to peroxisome biogenesis?

PEX31, together with PEX30, plays a crucial role in shaping and generating specialized regions of the endoplasmic reticulum (ER) where preperoxisomal vesicle (PPV) biogenesis occurs. These ER subdomains serve as the sites where most PPVs are generated, highlighting the importance of PEX31 in the early stages of peroxisome formation. Research indicates that PEX31 has membrane-shaping abilities, with experimental evidence showing that overexpression or deletion of PEX31 alters the size, shape, and number of PPVs .

To study PEX31's role in peroxisome biogenesis, researchers typically employ genetic approaches, including gene deletion and overexpression studies. Microscopy techniques, particularly fluorescence microscopy with tagged proteins, allow visualization of PPV formation and monitoring of changes in peroxisome morphology and abundance in response to manipulation of PEX31 levels. Biochemical assays can also be used to measure peroxisome function and integrity in cells with altered PEX31 expression .

What effect does PEX31 deletion have on peroxisome morphology and function?

The deletion of PEX31 in Pichia pastoris does not affect the function or division of methanol-induced peroxisomes but results in fewer and enlarged oleate-induced peroxisomes. This observation suggests that PEX31 plays a specific role in regulating peroxisome size and number under certain metabolic conditions rather than being essential for peroxisome function in general. The functional peroxisomes in PEX31-deletion mutants indicate that basic peroxisomal metabolic activities remain intact despite alterations in organelle morphology .

When investigating the effects of PEX31 deletion, researchers should consider using different induction conditions (such as methanol versus oleate) to fully characterize the phenotype. Quantitative analysis of peroxisome size and number using fluorescence microscopy and image analysis software provides valuable data. Additionally, functional assays measuring the activity of peroxisomal enzymes help determine whether the morphological changes affect metabolic capabilities .

How do PEX30 and PEX31 cooperate to regulate ER subdomain formation?

PEX30 and PEX31 physically interact and cooperatively shape and generate regions of the ER where preperoxisomal vesicle (PPV) biogenesis occurs. Research indicates that PEX30 can tubulate membranes both in cells and when reconstituted into proteoliposomes, highlighting its role as an ER-shaping protein. PEX31, which interacts with PEX30, appears to modulate this activity, with the two proteins working together to maintain the proper structure of ER subdomains involved in peroxisome formation .

The cooperation between PEX30 and PEX31 is evident from the observation that PEX30 is required for PEX31 stability, suggesting a regulatory relationship. When studying this interaction, co-immunoprecipitation experiments can identify binding regions, while microscopy techniques can visualize the colocalization of these proteins at ER subdomains. Functional studies using mutants lacking one or both proteins help elucidate the specific contributions of each protein to ER subdomain formation and PPV biogenesis .

What are the optimal methods for expressing and purifying recombinant PEX31?

For the expression and purification of recombinant PEX31, a systematic approach is essential due to the protein's membrane localization and relatively low expression levels. The recommended expression system is Escherichia coli, using vectors with strong inducible promoters such as T7 or tac. Fusion tags, particularly His6 or GST, significantly facilitate purification and can be added to either the N- or C-terminus, though care should be taken to ensure the tag doesn't interfere with protein function or localization.

The following table outlines a standardized protocol for PEX31 expression and purification:

Protein quality should be assessed by SDS-PAGE, Western blotting, and functional assays to verify the recombinant protein's integrity and activity. For membrane proteins like PEX31, maintaining proper folding can be challenging, and the addition of stabilizing agents such as glycerol (10%) to purification buffers is recommended.

What techniques are most effective for studying PEX31 localization and trafficking?

For studying PEX31 localization and trafficking, fluorescence microscopy techniques are most effective, particularly when combined with genetic approaches. Based on research findings, PEX31 exhibits dual localization to both the ER and peroxisomes, with a predominant localization to peroxisomes. To accurately visualize this distribution pattern, researchers should employ high-resolution confocal or super-resolution microscopy techniques .

For fluorescent tagging, GFP fusion constructs have proven effective, though care must be taken to ensure that the tag doesn't disrupt protein function or localization. Colocalization studies using markers for the ER (such as Sec63-RFP) and peroxisomes (such as PEX3-RFP) help confirm the dual localization of PEX31. Live-cell imaging enables the investigation of dynamic trafficking events, such as the PEX19-dependent movement of PEX31 from the ER to peroxisomes .

Genetic approaches, including the use of mutants lacking specific trafficking components (such as pex19Δ), provide valuable insights into the mechanisms of PEX31 movement between organelles. Additionally, pulse-chase experiments, where PEX31 synthesis is induced and its movement tracked over time, can reveal the kinetics of protein trafficking from the ER to peroxisomes.

How can researchers quantify PEX31's effects on peroxisome morphology?

Quantifying PEX31's effects on peroxisome morphology requires a combination of imaging techniques and analytical methods. Given that PEX31 has been shown to influence peroxisome size and number, particularly in oleate-induced conditions, a systematic approach to measuring these parameters is essential .

Fluorescence microscopy using peroxisome-targeted fluorescent proteins (such as GFP-SKL) provides the foundation for such analysis. Z-stack confocal imaging followed by 3D reconstruction offers the most accurate representation of peroxisome morphology. For quantification, automated image analysis software like ImageJ with appropriate plugins can measure peroxisome number, size, and shape parameters.

The following data table illustrates typical measurements for comparing wild-type and pex31Δ cells:

When conducting these analyses, it's important to examine a sufficient number of cells (typically >100) across multiple independent experiments to ensure statistical robustness. Different growth conditions, particularly methanol versus oleate induction, should be compared to fully characterize PEX31's effects on peroxisome morphology.

How does PEX31 interact with PEX30 and what is the functional significance?

PEX31 physically interacts with PEX30, and this interaction has significant functional implications for peroxisome biogenesis and regulation. Research has shown that PEX30 is required for PEX31 stability, suggesting a regulatory relationship. The interaction likely occurs through specific domains of both proteins, though the exact binding regions have not been fully characterized. At the functional level, the PEX30-PEX31 complex appears to regulate the formation of ER subdomains where preperoxisomal vesicles are generated .

To study this interaction, co-immunoprecipitation experiments can detect the physical binding between PEX30 and PEX31, while yeast two-hybrid assays can help map the interaction domains. Mutational analysis, where specific regions of either protein are altered, can identify critical residues for the interaction. Additionally, microscopy techniques can visualize the colocalization of these proteins at ER subdomains and peroxisomes. The effect of disrupting this interaction on peroxisome morphology and abundance provides insights into its functional significance .

What role does the dysferlin domain play in PEX31 function?

The dysferlin (DysF) domain is a conserved structural motif present in PEX31 and related proteins, though its precise function remains enigmatic. Research on the PEX30 and PEX31 dysferlin domains suggests they may be involved in membrane interactions or protein-protein binding. Interestingly, studies on PEX31 in Pichia pastoris indicate that while the protein contains this domain, its exact role in peroxisome regulation is not fully understood .

To investigate the function of the dysferlin domain, researchers typically employ structure-function analyses using domain deletion or mutation approaches. By creating PEX31 variants lacking the dysferlin domain or containing specific mutations within it, the contribution of this domain to protein localization, stability, and function can be assessed. Complementation experiments, where mutant versions of PEX31 are expressed in pex31Δ cells, can determine whether the dysferlin domain is essential for the protein's role in regulating peroxisome morphology and number .

Additionally, structural studies using techniques such as X-ray crystallography or NMR spectroscopy could provide insights into the three-dimensional organization of the dysferlin domain and how it might contribute to PEX31 function. Comparative analyses with other dysferlin domain-containing proteins may also reveal conserved functional properties.

How is PEX31 expression regulated in response to metabolic conditions?

PEX31 expression is regulated in response to metabolic conditions, with studies in Pichia pastoris showing that PEX31 synthesis is oleate-induced, whereas PEX30 synthesis is constitutive. This differential regulation suggests that PEX31 expression is tailored to specific metabolic needs, particularly when fatty acid metabolism via peroxisomes becomes important. The induction of PEX31 under oleate conditions correlates with its role in regulating the size and number of oleate-induced peroxisomes .

To study PEX31 expression regulation, quantitative RT-PCR and Western blotting are essential techniques for measuring mRNA and protein levels, respectively, under different metabolic conditions. Promoter analysis using reporter gene assays can identify regulatory elements in the PEX31 promoter that respond to specific metabolic signals. Chromatin immunoprecipitation (ChIP) experiments can identify transcription factors that bind to the PEX31 promoter and regulate its expression in response to metabolic cues .

Understanding the regulation of PEX31 expression is crucial for interpreting its function in peroxisome biogenesis and maintenance under different physiological conditions. Researchers should consider testing multiple carbon sources and growth conditions to fully characterize the regulatory patterns governing PEX31 expression.

What approaches can be used to study the dynamics of PEX31 trafficking between the ER and peroxisomes?

To study the dynamics of PEX31 trafficking between the ER and peroxisomes, advanced live-cell imaging techniques combined with genetic and biochemical approaches provide comprehensive insights. Since PEX31 exhibits dual localization to the ER and peroxisomes and can traffic from the ER to peroxisomes in a PEX19-dependent manner, capturing this dynamic process requires sophisticated methodologies .

Fluorescence recovery after photobleaching (FRAP) and photoactivation experiments with fluorescently tagged PEX31 can measure the rate of protein movement between organelles. For these experiments, a photoactivatable GFP-PEX31 fusion can be activated specifically in one compartment (e.g., the ER) and its appearance in the other compartment (peroxisomes) monitored over time. Similarly, FRAP experiments can bleach fluorescence in one compartment and measure recovery rates, indicating protein trafficking.

Pulse-chase experiments using inducible expression systems allow researchers to follow newly synthesized PEX31 from its point of synthesis to its final destinations. By combining this with temperature-sensitive trafficking mutants or drug treatments that block specific trafficking steps, the pathway and mechanisms of PEX31 movement can be dissected. Additionally, correlative light and electron microscopy (CLEM) can provide ultrastructural details of PEX31 localization during trafficking between the ER and peroxisomes .

How can researchers resolve conflicting data regarding PEX31 function across different yeast species?

Resolving conflicting data regarding PEX31 function across different yeast species requires a systematic comparative approach. Studies have shown differences in PEX31 function between Saccharomyces cerevisiae and Pichia pastoris, particularly regarding its effects on peroxisome morphology and its regulation. In P. pastoris, deletion of PEX31 results in fewer and enlarged oleate-induced peroxisomes but does not affect methanol-induced peroxisomes, whereas studies in S. cerevisiae have suggested different phenotypes .

To address these discrepancies, researchers should conduct parallel experiments in multiple yeast species using standardized conditions and methodologies. This approach minimizes variables that could contribute to apparently conflicting results. Complementation studies, where the PEX31 gene from one species is expressed in a pex31Δ strain of another species, can determine functional conservation across species boundaries.

Molecular evolutionary analyses examining the sequence conservation and divergence of PEX31 across yeast species can identify regions that might explain functional differences. Additionally, creating chimeric proteins containing domains from different species' PEX31 homologs can pinpoint which protein regions are responsible for species-specific functions.

A comprehensive data table comparing key aspects of PEX31 across yeast species would look like this:

How might PEX31 function contribute to our understanding of human peroxisomal disorders?

While PEX31 itself doesn't have a direct human ortholog, studying its function in yeast contributes to our understanding of human peroxisomal disorders by elucidating fundamental mechanisms of peroxisome biogenesis and regulation. Many human peroxisomal disorders stem from defects in peroxisome formation, function, or maintenance, processes in which yeast PEX31 is involved. By understanding how PEX31 regulates peroxisome size, number, and formation from the ER in yeast, researchers can gain insights into similar processes in human cells .

The dysferlin domain present in PEX31 is found in some human proteins associated with membrane dynamics and repair. Studying the function of this domain in PEX31 might provide clues about the role of dysferlin domains in human health and disease. Additionally, the ER-to-peroxisome trafficking pathway, which involves PEX31 in yeast, has parallels in human cells, and disruptions in this pathway are associated with some peroxisomal biogenesis disorders.

Translational research approaches might include expressing human peroxisomal proteins in yeast pex31Δ mutants to test for functional complementation or screening for small molecules that affect PEX31 function as potential leads for therapeutic development. Additionally, CRISPR-based gene editing could be used to introduce mutations mimicking those found in human peroxisomal disorders into yeast PEX genes, creating model systems for studying disease mechanisms.

What are the most promising future research directions for PEX31 studies?

Several promising research directions for PEX31 studies could significantly advance our understanding of peroxisome biology. First, structural biology approaches, including cryo-electron microscopy and X-ray crystallography, could reveal the three-dimensional organization of PEX31, particularly its dysferlin domain, providing insights into its mechanism of action. Second, systems biology approaches combining proteomics, lipidomics, and transcriptomics could elucidate how PEX31 functions within the broader network of peroxisome biogenesis and regulation .

Investigating the potential role of PEX31 in responding to cellular stress represents another promising direction. Given that peroxisome abundance and function change in response to various stressors, determining whether PEX31 plays a role in stress-induced peroxisome remodeling could reveal new aspects of its function. Additionally, exploring the evolutionary conservation and divergence of PEX31 across fungal species might provide insights into the fundamental versus species-specific aspects of its function.

Finally, developing advanced imaging techniques to visualize PEX31 at the nanoscale level, particularly at the interface between the ER and forming peroxisomes, could reveal mechanistic details of how this protein contributes to peroxisome biogenesis. Technologies such as super-resolution microscopy and correlative light and electron microscopy are particularly promising for this purpose.

What methodological advances would most benefit PEX31 research?

Methodological advances that would significantly benefit PEX31 research include improved techniques for membrane protein purification and reconstitution, advanced imaging methods for visualizing protein dynamics, and new genetic tools for precise manipulation of protein expression and localization. For membrane protein studies, advances in native nanodiscs or styrene-maleic acid lipid particles (SMALPs) could allow purification of PEX31 in its native lipid environment, preserving its structural integrity and functional properties .

In imaging, the development of split fluorescent proteins or self-labeling tags compatible with super-resolution microscopy would enable more precise localization of PEX31 within cellular subdomains. Additionally, advances in correlative light and electron microscopy (CLEM) could provide ultrastructural context for fluorescence observations, particularly at the ER-peroxisome interface where PEX31 functions.