Recombinant Human Peroxisomal membrane protein 11C (PEX11G)

What is PEX11G and how does it relate to other PEX11 family members?

PEX11G (PEX11γ) belongs to the PEX11Z subfamily of peroxins involved in peroxisome proliferation. Based on phylogenetic reconstructions, the PEX11 family can be divided into two distinct subfamilies: PEX11Y (which includes fungal PEX11 and mammalian PEX11α/β) and PEX11Z (which includes metazoan PEX11γ, fungal PEX11C, and the GIM5A/B proteins in Trypanosoma brucei) . While most studies have focused on members of the PEX11Y subfamily, much less is known about the PEX11Z subfamily proteins including PEX11G .

Unlike its paralogs PEX11α and PEX11β, which are well-characterized for their direct involvement in peroxisome proliferation, PEX11G appears to play a more specialized role in coordinating peroxisomal growth and division through heterodimerization with other mammalian PEX11 paralogs and interactions with proteins like Mff and Fis1 . This functional specialization makes PEX11G a particularly interesting target for researchers studying the molecular mechanisms of peroxisome dynamics.

What are the key structural features of PEX11G that researchers should be aware of?

When working with recombinant PEX11G, researchers should consider several key structural features that affect its function:

• PEX19 binding sites: Like other PEX11 proteins, PEX11G contains conserved PEX19 binding sites that are essential for proper targeting to peroxisomes. These binding sites form part of the membrane protein targeting signal (mPTS) .

• N-terminal region: PEX11 proteins contain several conserved helices, particularly in the N-terminal region . The N-terminal region often contains an amphiphilic helix that can interact with membranes and may be involved in membrane remodeling .

• Transmembrane domains: PEX11G, as a membrane protein, contains transmembrane domains that anchor it to the peroxisomal membrane. When designing expression constructs, these domains must be preserved to maintain proper localization and function .

• Potential cryptic targeting signals: Evidence from related PEX11 proteins suggests that they may contain secondary targeting signals. For example, in Trypanosoma brucei PEX11, the N-terminal region contains cryptic signals that can direct the protein to mitochondria if its normal targeting to glycosomes (specialized peroxisomes) is blocked .

What expression systems are optimal for producing functional recombinant PEX11G?

When expressing recombinant PEX11G for research purposes, several expression systems have been successfully used for various PEX11 family proteins:

To verify correct expression, researchers typically utilize epitope tagging strategies, such as fusion with GFP or smaller tags like FLAG or HA. When designing such constructs, care must be taken to ensure that the tag does not interfere with targeting signals, particularly the PEX19 binding sites .

What techniques are most effective for studying PEX11G localization and dynamics?

To study the localization and dynamics of PEX11G in cellular contexts, researchers have employed several powerful techniques:

• Fluorescence microscopy with GFP-tagged PEX11G: This approach allows for visualization of PEX11G localization in living cells. Studies with PEX11-GFP fusion proteins have revealed distinct localization patterns under various conditions .

• Immunofluorescence microscopy: Using antibodies against PEX11G or epitope tags, this technique can visualize the protein's distribution relative to other cellular markers.

• Cell fractionation and Western blotting: These biochemical approaches can quantitatively assess the distribution of PEX11G between different cellular compartments.

• Live-cell imaging: For studying dynamic processes such as peroxisome fission, researchers use time-lapse microscopy of fluorescently tagged PEX11G to track its behavior during peroxisome proliferation events.

• Automated image analysis: Tools like CellProfiler have been used to quantify morphological features and localization patterns of PEX11-GFP, allowing for statistical comparison between different experimental conditions .

How do PEX19 binding sites in PEX11G affect its localization and function?

PEX19 binding sites are critical determinants of PEX11G localization and function. Studies of related PEX11 proteins from both Trypanosoma brucei and Saccharomyces cerevisiae have demonstrated that:

• PEX11 proteins typically contain one or more PEX19 binding sites. In T. brucei PEX11, two distinct binding sites have been identified: one near the N-terminus (BS1) and another near the first transmembrane domain (BS2) .

• The N-terminal PEX19 binding site (BS1) is highly conserved across different organisms and is required for maintaining appropriate protein levels and efficient targeting to peroxisomes .

• Deletion or mutation of PEX19 binding sites in PEX11 proteins results in mislocalization to mitochondria rather than peroxisomes . This suggests that PEX11G localization depends on a hierarchy of targeting signals, with the PEX19-dependent pathway being dominant over potential mitochondrial targeting signals.

For researchers studying human PEX11G, preserving the integrity of PEX19 binding sites is essential for ensuring proper localization. When designing truncated versions or point mutations of PEX11G, researchers should carefully consider how these modifications might affect interaction with PEX19 and subsequent targeting to peroxisomes.

What is the relationship between PEX11G and the ERMES complex?

Recent research has uncovered intriguing connections between peroxisomal proteins and the Endoplasmic Reticulum-Mitochondria Encounter Structure (ERMES) complex, which facilitates contact between the ER and mitochondria. Studies in yeast have shown that:

• Deletion of mitochondrial and cytosolic ERMES complex components (Mdm10, Mdm12, and Mdm34) significantly alters the localization pattern of Pex11-GFP compared to wild-type cells .

• In Mdm10Δ and Mdm12Δ strains, Pex11-GFP shows both intense puncta (as seen in wild-type cells) and numerous additional weaker puncta .

• The Mdm34Δ mutant displays fewer focal highly intense Pex11-GFP signal puncta compared to other ERMES component deletions .

• Interestingly, deletion of the ER component of the ERMES complex (Mmm1) does not affect Pex11-GFP localization .

These findings suggest that PEX11G may participate in organelle contact sites, potentially mediating interactions between peroxisomes and other cellular compartments. Researchers investigating human PEX11G should consider examining its potential role in similar inter-organelle contact sites, which may represent an important but understudied aspect of its cellular function.

How can researchers distinguish between the functions of different PEX11 paralogs?

Distinguishing the specific functions of PEX11G from other PEX11 family members presents several challenges:

• Functional redundancy: Many PEX11 proteins share similar functions in peroxisome proliferation, making it difficult to isolate the specific contribution of PEX11G.

• Species-specific nomenclature confusion: The naming of PEX11 proteins does not always reflect evolutionary relationships. For example, plant PEX11A is not equivalent to human PEX11α, and fungal PEX11C belongs to the same subfamily as human PEX11γ, but PEX11C from Arabidopsis thaliana does not .

To address these challenges, researchers can employ several strategies:

• Paralog-specific knockdown/knockout: Using RNA interference or CRISPR-Cas9 to specifically target PEX11G while leaving other paralogs intact.

• Complementation studies: Expressing human PEX11G in model systems lacking their native PEX11 homologs to determine which functions can be rescued.

• Protein-protein interaction mapping: Identifying interaction partners unique to PEX11G versus other PEX11 paralogs can provide insights into specific functions.

• Domain swap experiments: Creating chimeric proteins with domains from different PEX11 paralogs to determine which regions confer specific functions.

What methods are effective for studying the role of PEX11G in peroxisome membrane dynamics?

PEX11G, like other PEX11 family members, is believed to play a role in peroxisome membrane remodeling and fission. To study these dynamic processes:

• Membrane curvature assays: In vitro assays using purified recombinant PEX11G and artificial membrane systems can assess its ability to induce membrane curvature.

• Peroxisome morphology analysis: Quantitative analysis of peroxisome number, size, and shape in cells with modified PEX11G expression can reveal its role in controlling peroxisome morphology.

• Protein interaction studies: Techniques such as proximity labeling (BioID or APEX) can identify proteins that interact with PEX11G during different stages of peroxisome division.

• Super-resolution microscopy: Advanced imaging techniques can visualize PEX11G distribution on the peroxisomal membrane during different stages of peroxisome proliferation.

• Live-cell imaging of membrane dynamics: Using fluorescent lipid probes in conjunction with tagged PEX11G can reveal how this protein affects membrane fluidity and organization during peroxisome division.

How does PEX11G coordinate with the mitochondrial fission machinery?

The peroxisomal fission process shares several components with the mitochondrial fission machinery, including the dynamin-related protein Drp1/DLP1, Fis1, and Mff . Research has shown that:

• Human PEX11β (a paralog of PEX11G) recruits DRP1 to the peroxisomal membrane and functions as a GTPase activating protein (GAP) for Drp1 .

• PEX11γ (PEX11G) has been suggested to coordinate peroxisomal growth and division through heterodimerization with other mammalian PEX11 paralogs and interaction with components of the fission machinery (Mff and Fis1) .

For researchers investigating PEX11G's role in this process, several approaches can be valuable:

• Co-immunoprecipitation assays to detect direct interactions between PEX11G and components of the fission machinery.

• In vitro GTPase assays to determine whether PEX11G, like PEX11β, has GAP activity toward Drp1.

• Structured illumination microscopy to visualize the spatial relationship between PEX11G and fission machinery components during peroxisome division events.

What are the potential implications of PEX11G research for human disease?

• Screening for PEX11G mutations in patients with peroxisomal disorders of unknown genetic origin.

• Investigating whether PEX11G expression or function is altered in conditions associated with peroxisome dysfunction, such as metabolic disorders or neurodegenerative diseases.

• Exploring whether PEX11G could be a potential therapeutic target for diseases characterized by peroxisome dysfunction.

• Studying the effects of environmental factors or drugs on PEX11G expression and function, which could provide insights into toxicological mechanisms affecting peroxisome homeostasis.