Recombinant Mouse Peroxisomal membrane protein 11C (Pex11g)

What is Pex11g and how does it differ from other Pex11 family proteins?

Pex11g (also known as Pex11γ) is one of the three mammalian paralogs of the Pex11 family, alongside Pex11α and Pex11β. The Pex11 proteins are integral peroxisomal membrane proteins containing at least two alpha-helical transmembrane domains with both terminal regions facing the cytosol . While Pex11β is widely expressed in mammalian tissues with a well-established function in the initial phase of peroxisomal fission (membrane elongation and remodeling), the specific functions of Pex11α and Pex11γ are less clearly defined .

Methodologically, researchers distinguish between these paralogs through:

• Gene expression analysis across different tissues

• Knockout studies comparing phenotypic effects

• Protein localization studies using paralog-specific antibodies or epitope tags

• Complementation assays to determine functional redundancy

What targeting mechanisms ensure proper localization of Pex11g to peroxisomal membranes?

Pex11g, like other peroxisomal membrane proteins, contains specific targeting signals called mPTS (membrane Peroxisomal Targeting Signal). These signals include PEX19 binding sites that are essential for proper targeting to peroxisomes . Based on studies of Pex11 in various organisms, proper targeting relies on:

• Recognition of the mPTS by the cytosolic chaperone PEX19

• PEX19-dependent delivery to the peroxisomal membrane

• Interaction with the peroxisomal membrane receptor PEX3

Research has shown that deletion or mutation of PEX19 binding sites in Pex11 results in mislocalization to mitochondria . Specifically, Pex11 contains an N-terminal PEX19 binding site (BS1) that is highly conserved across different organisms and is required for maintaining proper steady-state concentration and efficient targeting to peroxisomes .

What experimental techniques are recommended for studying Pex11g expression levels in different mouse tissues?

For the most comprehensive analysis, researchers should employ a combination of these techniques. When using GFP-tagging strategies for subcellular localization studies, it is recommended to use high-content microscopy approaches that allow for quantitative analysis of localization patterns, as demonstrated in yeast Pex11 studies .

How does Pex11g function as a GTPase Activating Protein (GAP) in peroxisomal fission, and what experimental approaches can verify this activity?

Pex11 proteins play a dual role in peroxisomal fission: initial membrane remodeling and activation of dynamin-like proteins (DLPs) that mediate the final membrane scission step . Research has demonstrated that:

• Pex11p functions as a GTPase Activating Protein (GAP) for Dynamin-related 1 (Dnm1p) in yeast

• This GAP activity is conserved from yeast to mammals, with mammalian Pex11β activating the corresponding DLP Drp1

• Pex11p physically interacts with Dnm1p, and inhibiting this interaction compromises peroxisomal fission

To experimentally verify Pex11g GAP activity, researchers should:

• Perform in vitro GTPase assays with purified recombinant Pex11g and Drp1

• Conduct co-immunoprecipitation experiments to confirm physical interaction

• Generate point mutations in predicted GAP domains to identify essential residues

• Employ live-cell imaging to visualize the recruitment of Drp1 to peroxisomal constriction sites

• Develop peroxisome fission assays in cells with wild-type versus mutant Pex11g

What is the relationship between Pex11g and the ERMES complex in maintaining peroxisome-mitochondria contact sites?

Studies in yeast have revealed intriguing connections between Pex11 localization and the Endoplasmic Reticulum-Mitochondria Encounter Structure (ERMES) complex . Genome-wide localization studies showed that:

• Deletion of mitochondrial and cytosolic ERMES components (Mdm10, Mdm12, and Mdm34) significantly altered Pex11-GFP localization patterns

• Pex11-GFP localization in mdm10Δ and mdm12Δ strains showed numerous additional but weaker puncta compared to wild-type cells

• The mdm34Δ mutant showed fewer focal highly intense Pex11-GFP signal puncta

• Interestingly, absence of the ER component of the ERMES complex (Mmm1) did not affect Pex11-GFP localization

These findings suggest complex interorganellar communication mechanisms involving Pex11. For mouse Pex11g research, investigators should:

• Examine localization patterns in cells with disrupted mitochondria-peroxisome contact sites

• Identify potential mammalian counterparts of the ERMES complex that might interact with Pex11g

• Use proximity labeling techniques (BioID, APEX) to map the Pex11g interactome at contact sites

• Employ super-resolution microscopy to visualize Pex11g distribution at organelle contact sites

How can researchers distinguish between properly targeted and mistargeted Pex11g in experimental systems?

Distinguishing between properly targeted and mistargeted Pex11g is critical for studying peroxisomal biogenesis and quality control mechanisms. Research has shown that quality control systems exist to prevent accumulation of mistargeted peroxisomal proteins:

• The AAA ATPase Msp1 in yeast (and its mammalian homolog ATAD1) prevents accumulation of mistargeted tail-anchored proteins, including peroxisomal membrane proteins

• Peroxisomal proteins can be protected from Msp1-dependent degradation through interactions with resident peroxisomal proteins (e.g., Pex15 interacts with Pex3)

For experimental distinction between properly targeted and mistargeted Pex11g, researchers should:

What are the critical experimental controls when investigating Pex11g-mediated peroxisome proliferation?

When studying Pex11g-mediated peroxisome proliferation, several critical controls must be included:

• Expression level controls: Since overexpression can cause artifactual effects, researchers should:

  • Use endogenous tagging approaches when possible

  • Include titration experiments with inducible expression systems

  • Compare effects with physiological versus non-physiological expression levels

• Functional redundancy controls:

  • Single knockout/knockdown of Pex11g

  • Double/triple knockouts of Pex11 paralogs (Pex11α/β/γ)

  • Complementation with wild-type versus mutant constructs

• Specificity controls:

  • Mutation of key functional domains (PEX19 binding sites, transmembrane domains)

  • Analysis of membrane curvature effects versus proliferation-specific effects

  • Validation with multiple peroxisomal markers to confirm genuine proliferation

• Metabolic status controls:

  • Growth in different carbon sources that influence peroxisome abundance

  • Analysis under conditions of induced peroxisome proliferation (e.g., fibrate treatment)

  • Comparison between different metabolic states that affect peroxisomal functions

How do mutations in PEX19 binding sites of Pex11g affect its function and what methods can detect these effects?

The PEX19 binding sites in Pex11 proteins are critical for their proper localization and function. Research has shown that:

• Pex11 contains a highly conserved N-terminal PEX19 binding site (BS1) required for maintaining steady-state concentration and efficient targeting to peroxisomes

• Deletion or mutations of PEX19 binding sites result in mislocalization of Pex11 to mitochondria

To investigate the effects of mutations in PEX19 binding sites of mouse Pex11g, researchers should employ:

• Site-directed mutagenesis approaches:

  • Alanine scanning of predicted PEX19 binding motifs

  • Complete deletion of binding sites

  • Chimeric constructs swapping binding sites between paralogs

• Binding assays:

  • In vitro binding assays with recombinant PEX19 and wild-type versus mutant Pex11g

  • Surface plasmon resonance to determine binding kinetics

  • Isothermal titration calorimetry for thermodynamic analysis

• Cellular localization studies:

  • Live-cell imaging of fluorescently-tagged wild-type versus mutant Pex11g

  • Co-localization analysis with peroxisomal versus mitochondrial markers

  • Quantitative image analysis to measure mislocalization rates

• Functional assays:

  • Peroxisome proliferation assays in Pex11g knockout cells complemented with wild-type versus mutant constructs

  • Analysis of peroxisome morphology and distribution

  • Assessment of peroxisomal metabolic functions

What are the optimal conditions for expressing and purifying recombinant mouse Pex11g for in vitro studies?

For successful expression and purification of recombinant mouse Pex11g, researchers should consider the following optimized protocol:

Recommended purification workflow:

• Express with N-terminal His-SUMO or His-MBP tag to enhance solubility

• Solubilize using mild detergents (DDM, LMNG, or amphipols)

• Purify using Ni-NTA affinity chromatography

• Remove fusion tag with SUMO or TEV protease

• Perform secondary purification via size exclusion chromatography

• Verify protein quality by SDS-PAGE, Western blot, and circular dichroism

For functional studies, consider reconstitution into liposomes or nanodiscs to maintain the native membrane environment of Pex11g.

What genomic engineering approaches are most effective for studying Pex11g function in mouse models?

For the most informative studies of Pex11g, researchers should consider:

• Generating conditional knockout models to bypass potential embryonic lethality

• Creating knock-in reporter lines with minimal tags (e.g., HA, FLAG) at the endogenous locus

• Developing allelic series with mutations in key functional domains (PEX19 binding sites, transmembrane regions)

• Implementing tissue-specific deletion models focusing on metabolically active tissues (liver, brain, muscle)

How can researchers quantitatively assess Pex11g-mediated peroxisome fission in live cells?

Quantitative assessment of peroxisome fission dynamics requires sophisticated imaging and analysis techniques:

• Live-cell imaging setup:

  • Spinning disk or lattice light-sheet microscopy for rapid 3D acquisition

  • Fluorescent labeling: peroxisomal matrix marker (e.g., GFP-SKL) and tagged Pex11g

  • Temperature and CO2 control for physiological conditions

  • Acquisition rate: 1-5 frames/minute for at least 30 minutes

• Analysis pipeline:

  • Automated peroxisome segmentation and tracking

  • Quantification of elongation events (aspect ratio changes)

  • Measurement of constriction and fission events

  • Correlation of Pex11g enrichment with fission sites

• Key parameters to measure:

  • Peroxisome number per cell over time

  • Rate of fission events (events/minute/cell)

  • Duration of pre-fission elongation phase

  • Correlation between Pex11g levels and fission rates

  • Co-localization dynamics of Pex11g with Drp1 recruitment

• Software recommendations:

  • CellProfiler for automated image analysis

  • Imaris or TrackMate (ImageJ) for 3D tracking

  • Custom MATLAB or Python scripts for event detection

This quantitative approach allows researchers to detect subtle phenotypes and kinetic differences between wild-type and mutant Pex11g that might be missed by endpoint assays.

What is the evidence linking Pex11g dysfunction to human peroxisomal disorders?

While mutations in PEX11β have been directly linked to human neurological disorders , the specific role of PEX11γ (Pex11g) in human disease is less well characterized. Current evidence suggests:

• The PEX11 family as a whole is essential for proper peroxisome proliferation and metabolism

• Dysfunction in peroxisomal fission machinery contributes to a spectrum of peroxisomal disorders

• The conserved role of Pex11 proteins as GTPase activating proteins for dynamin-like proteins suggests potential involvement in diseases with defective organelle dynamics

Researchers investigating the role of Pex11g in human disease should:

• Screen patient cohorts with peroxisomal disorders of unknown genetic origin for PEX11γ mutations

• Analyze PEX11γ expression levels in tissues from patients with peroxisomal disorders

• Develop cellular and animal models with Pex11g mutations that mimic potential human variants

• Investigate potential functional redundancy between Pex11 paralogs in disease contexts

How can mouse models of Pex11g dysfunction inform therapeutic strategies for peroxisomal disorders?

Mouse models of Pex11g dysfunction can provide valuable insights for therapeutic development:

• Phenotypic characterization:

  • Metabolic profiling (very long-chain fatty acids, branched-chain fatty acids, plasmalogens)

  • Behavioral testing for neurological abnormalities

  • Histopathological analysis of affected tissues

  • Lifespan and developmental progression

• Intervention testing:

  • Dietary modifications (e.g., Lorenzo's oil-type interventions)

  • Pharmacological induction of peroxisome proliferation

  • Gene therapy approaches for complementation

  • Small molecule screens for compounds that bypass Pex11g requirements

• Mechanistic investigations:

  • Identification of compensatory pathways activated in Pex11g deficiency

  • Analysis of cross-talk between peroxisomes and other organelles

  • Determination of tissue-specific requirements for Pex11g function

These approaches can identify potential therapeutic targets and intervention strategies that might be applicable to human peroxisomal disorders.

What emerging technologies could advance our understanding of Pex11g structure and function?

Combining these technologies will provide multiscale understanding of Pex11g function from molecular mechanisms to physiological significance.

How might synthetic biology approaches be used to engineer Pex11g for enhanced peroxisome proliferation?

Synthetic biology offers innovative approaches to engineering Pex11g for research and potential therapeutic applications:

• Engineered variants:

  • Constitutively active Pex11g through targeted mutations

  • Chemically-inducible dimerization systems to control Pex11g activity

  • Domain swapping between paralogs to create hybrids with enhanced activity

  • Fusion with lipid-binding domains to enhance membrane association

• Regulatory systems:

  • Optogenetic control of Pex11g activity/localization

  • Metabolite-responsive expression systems

  • Cell type-specific expression cassettes

  • Tunable degradation systems for precise control of protein levels

• Applications:

  • Enhanced peroxisomal metabolism for detoxification in environmental applications

  • Improved fatty acid oxidation for metabolic disease models

  • Engineered organelle contacts for studying interorganelle communication

  • Synthetic organelle biogenesis systems for biotechnology

These approaches could provide both research tools and potential therapeutic strategies for peroxisomal disorders.