Recombinant Arabidopsis thaliana Peroxisomal membrane protein 11C (PEX11C)

What is Arabidopsis thaliana PEX11C and what is its primary function?

Arabidopsis thaliana PEX11C (At1g01820) is a member of the phylogenetically distinct PEX11 protein family that functions as an integral membrane protein in peroxisomes . Its primary role involves promoting peroxisome proliferation, contributing to the regulation of peroxisome abundance in plant cells. PEX11C belongs to one of three phylogenetically distinct subfamilies of Arabidopsis PEX11 proteins, which collectively have a single evolutionary origin but evolved independently after the separation of plant, animal, and fungal kingdoms .

The function of PEX11C is directly tied to peroxisome dynamics, as demonstrated in overexpression and gene silencing experiments. When overexpressed, PEX11C induces peroxisome proliferation, while reduction in its expression decreases peroxisome abundance . Simultaneous silencing of PEX11c, PEX11d, and PEX11e results in approximately 40% reduction in peroxisome number, highlighting their collective importance in maintaining proper peroxisome populations .

How does PEX11C compare functionally to other members of the Arabidopsis PEX11 family?

While all five Arabidopsis PEX11 proteins target to peroxisomes and promote peroxisome proliferation, they display distinct functional characteristics. Most notably, PEX11c and PEX11e, but not PEX11a, PEX11b, or PEX11d, can complement to significant degrees the growth phenotype of the Saccharomyces cerevisiae pex11 null mutant on oleic acid . This functional complementation suggests that PEX11C possesses conserved functional elements that are recognized across species boundaries.

The five Arabidopsis PEX11 proteins likely play specific roles in distinct peroxisomal subtypes, environmental conditions, and possibly in different steps of peroxisome proliferation . Expression profile analysis has revealed that, unlike PEX11a, transcripts of PEX11b through PEX11e are detected in roots, leaves, and suspension cultured cells, suggesting tissue-specific functions for different family members .

What is known about the subcellular localization of PEX11C?

PEX11C is exclusively localized to peroxisomes, as demonstrated through multiple complementary approaches. Fluorescence microscopy of CFP-PEX11C fusion proteins in transgenic seedlings has shown clear colocalization with the peroxisomal marker YFP-PTS1 (yellow fluorescent protein fused with the peroxisomal targeting signal type 1) . This peroxisomal localization has been further confirmed through immunobiochemical analysis using highly purified leaf peroxisomes .

Importantly, PEX11C behaves as an integral protein of the peroxisome membrane, rather than as a peripheral or matrix protein . This membrane integration is consistent with its role in peroxisome proliferation, as membrane remodeling is a critical aspect of organelle division.

What is the evolutionary history of PEX11C within the broader PEX11 protein family?

The PEX11 protein family has a complex evolutionary history characterized by independent paralogizations in different eukaryotic lineages. Phylogenetic analysis demonstrates that all PEX11 sequences from various species form a monophyletic group, which can be separated into plant, animal, and fungal subclades . This suggests that PEX11 genes had a single origin and later evolved independently after the separation of these kingdoms .

Within this broader evolutionary context, two main groups can be tentatively distinguished within the Pex11 protein family: one containing fungal PEX11 and vertebrate PEX11α/β, and another containing fungal PEX11C and vertebrate PEX11γ . Interestingly, plants apparently do not have orthologs from the group with fungal PEX11C , indicating potential functional divergence in plant peroxisome biology.

What molecular mechanisms underlie PEX11C function in peroxisome proliferation?

The precise molecular mechanisms by which PEX11C mediates peroxisome proliferation are still being elucidated, but current evidence suggests it acts within a coordinated network of proteins involved in peroxisome division. PEX11C, along with other PEX11 family members, participates in a process where peroxisomes sequentially enlarge, elongate, and then divide . This process correlates with expression patterns of PEX11, FIS1 (FISSION1), and DRP3A (DYNAMIN-RELATED PROTEIN3A) genes, particularly during the G2 phase of the cell cycle .

At the molecular level, PEX11C likely contributes to membrane remodeling required for peroxisome division. Its integration into the peroxisome membrane positions it ideally to influence membrane curvature and/or recruit other division factors. The observation that simultaneous silencing of PEX11c-e results in reduced peroxisome numbers suggests functional redundancy among these family members , potentially providing robustness to the peroxisome division machinery.

How does PEX11C interact with other proteins involved in peroxisome dynamics?

Protein-protein interaction studies have revealed that PEX11C engages in both homooligomerization and heterooligomerization. Bimolecular fluorescence complementation assays in Arabidopsis thaliana suspension cells demonstrated that PEX11C can form homooligomers with itself and heterooligomers with FIS1b (FISSION1b), but not with FIS1a or DRP3A . This selective interaction pattern suggests functional specialization in the recruitment of division machinery components.

The interaction with FIS1b is particularly significant, as FIS1 proteins are known adaptor proteins that help recruit dynamin-related proteins to organelle division sites in various organisms. The specific interaction with FIS1b, but not FIS1a, indicates that PEX11C may participate in a defined subset of peroxisome division events, potentially those associated with specific developmental stages or environmental conditions .

What role does PEX11C play in cell cycle-associated peroxisome replication?

During the cell cycle, particularly in the G2 phase, peroxisomes undergo a coordinated sequence of morphological changes leading to their duplication. This process involves sequential enlargement, elongation, and division of peroxisomes, which correlates with peaks in the expression of PEX11, FIS1, and DRP3A genes . PEX11C, along with PEX11d and PEX11e, appears to be particularly important in this cell cycle-associated peroxisome replication.

The timing of peroxisome division relative to cell division ensures proper inheritance of these essential organelles by daughter cells. The fact that simultaneous silencing of PEX11c-e resulted in approximately 40% reduction in peroxisome number indicates that these proteins play crucial roles in maintaining appropriate peroxisome populations during cell division. This connection between peroxisome proliferation and the cell cycle represents an important area for further investigation to understand how organelle inheritance is coordinated with cell division.

How is PEX11C expression regulated in response to environmental conditions?

The regulation of PEX11C expression in response to environmental conditions provides insights into how peroxisome proliferation adapts to changing cellular needs. Analysis of web-based microarray databases and RT-PCR experiments has been used to study PEX11 gene expression in response to various stresses . Additionally, the Arabidopsis coresponse database has been searched for genes that are coexpressed with PEX11 family members, potentially identifying regulatory networks controlling PEX11C expression .

What expression systems are most effective for producing recombinant Arabidopsis thaliana PEX11C?

Several expression systems have been successfully employed for producing recombinant Arabidopsis thaliana PEX11C, each with distinct advantages depending on the research objectives:

• Plant-based expression systems: PEX11C has been expressed in Arabidopsis plants using the 35S constitutive promoter, allowing for in vivo functional studies . This approach is particularly valuable for localization studies and for investigating effects on peroxisome proliferation in the native cellular environment.

• Heterologous expression in yeast: The functional complementation of Saccharomyces cerevisiae pex11 null mutants with Arabidopsis PEX11C has demonstrated the feasibility of expressing this protein in yeast systems . This provides opportunities for more controlled biochemical characterization and functional analysis.

• Cell-free expression systems: While not explicitly mentioned in the search results, cell-free systems could potentially be useful for producing recombinant PEX11C, especially for initial biochemical characterization, though the membrane protein nature of PEX11C presents challenges for this approach.

The choice of expression system should consider PEX11C's nature as an integral membrane protein, which may necessitate specialized approaches for solubilization and purification.

What techniques are most suitable for studying PEX11C-mediated peroxisome proliferation?

Quantitative assessment of PEX11C-mediated peroxisome proliferation requires robust methodological approaches:

• Fluorescence microscopy with peroxisome markers: The use of fluorescent proteins targeted to peroxisomes (such as YFP-PTS1) provides a powerful tool for visualizing and quantifying peroxisomes in cells with normal, overexpressed, or silenced PEX11C . This approach allows for direct observation of changes in peroxisome number and morphology.

• Statistical analysis of peroxisome populations: Quantitative analysis of peroxisome numbers should include proper statistical methods, as demonstrated in studies with other PEX11 family members where significant differences were determined using Student's t-test (P values < 0.05) . The data can be presented in tables, as shown in Table I below:

*Note: This table shows data from studies in Hansenula polymorpha , provided as an example of quantitative peroxisome analysis methodology.

• Synchronized cell cultures: Synchronizing cell populations allows for tracking peroxisome changes throughout the cell cycle, particularly during G2 when peroxisomes sequentially enlarge, elongate, and then double in number .

What approaches are effective for studying PEX11C protein-protein interactions?

Understanding PEX11C's interactions with other proteins is crucial for elucidating its function in peroxisome proliferation. Several techniques have proven effective:

• Bimolecular fluorescence complementation (BiFC): This approach has successfully demonstrated homooligomerization of all five PEX11 isoforms and heterooligomerizations with FIS1b . BiFC provides the advantage of visualizing protein interactions in their native cellular context.

• Yeast two-hybrid assays: While not explicitly mentioned in the search results for PEX11C, this approach could be valuable for screening potential interaction partners and mapping interaction domains.

• Co-immunoprecipitation: This biochemical approach could complement the fluorescence-based methods to verify protein-protein interactions under different conditions or in response to various stimuli.

• Protein targeting experiments: Intracellular protein targeting experiments have demonstrated that FIS1b, but not FIS1a nor DRP3A, targeted to peroxisomes only when coexpressed with PEX11d or PEX11e . Similar approaches could be applied to investigate PEX11C-mediated recruitment of division factors.

What strategies are recommended for gene silencing studies of PEX11C?

Gene silencing approaches provide valuable insights into PEX11C function by revealing the consequences of its absence or reduced expression:

• Simultaneous silencing of related genes: Given the potential functional redundancy among PEX11 family members, simultaneous silencing of PEX11c, PEX11d, and PEX11e has proven effective in revealing their collective importance in peroxisome proliferation . This approach demonstrates approximately 40% reduction in peroxisome number, which might not be observed when silencing individual genes.

• Individual gene silencing: For understanding the specific contribution of PEX11C, individual silencing approaches can be employed, though the effects may be subtler due to compensation by other family members.

• CRISPR/Cas9 gene editing: While not explicitly mentioned in the search results, modern genome editing techniques could provide more precise tools for generating PEX11C knockouts or specific mutations to study structure-function relationships.

• Conditional silencing systems: Inducible silencing approaches would allow for temporal control over PEX11C expression, potentially revealing stage-specific requirements during development or in response to environmental changes.