PEX19 Human

What is human PEX19 and what is its primary function?

Human PEX19 encodes a hydrophilic protein (Pex19p) comprising 299 amino acids with a molecular weight of approximately 32,806 Da. The protein contains a prenylation motif known as a CAAX box at its C-terminus. Pex19p is primarily involved in the early stages of peroxisome biogenesis, specifically in peroxisomal membrane assembly before the import of matrix proteins occurs .

As demonstrated through functional complementation studies, PEX19 is essential for the initial stage of peroxisome membrane formation. When expressed in peroxisome-deficient cells (such as the Chinese hamster ovary cell line ZP119), human PEX19 restores both morphological and biochemical aspects of peroxisome biogenesis . This indicates its fundamental role in establishing peroxisomal membrane structures that later support matrix protein import.

How is PEX19 protein structured and where is it localized in cells?

PEX19 encodes a predominantly hydrophilic protein with no apparent membrane-spanning regions. A key structural feature is the prenylation motif (CAAX box) at the C-terminus, which undergoes farnesylation. This post-translational modification is crucial for the protein's biological function .

Regarding subcellular localization, farnesylated Pex19p is partly anchored in the peroxisomal membrane, with its N-terminal portion exposed to the cytosol . This dual localization pattern allows Pex19p to function effectively as an import receptor for peroxisomal membrane proteins - interacting with newly synthesized proteins in the cytosol and facilitating their targeting to the peroxisomal membrane.

What genetic disorders are associated with PEX19 mutations?

PEX19 mutations are associated with peroxisome biogenesis disorders (PBDs) of complementation group J (CG-J), including Zellweger syndrome, which represents the most severe form of these disorders .

A specific example comes from patient PBDJ-01 with Zellweger syndrome, who possessed a homozygous inactivating mutation in PEX19. The mutation involved a 1-base insertion (A764) in the codon for Met255, resulting in a frameshift that produced a 24-amino acid sequence entirely different from normal Pex19p . This alteration in the C-terminal region, which includes the critical CAAX box, rendered the protein non-functional and prevented normal peroxisome biogenesis, demonstrating that the C-terminal part of PEX19 is essential for its biological activity .

What model systems are commonly used to study PEX19 function?

Several model systems have proven effective for studying PEX19 function:

• Chinese hamster ovary (CHO) cell mutants: Cell lines such as ZP119 and ZP165, which are defective in peroxisome biogenesis, have been extensively used for functional complementation studies with human PEX19 cDNA .

• Patient fibroblasts: Skin fibroblasts from patients with Zellweger syndrome of complementation group J (such as PBDJ-01) provide valuable models for studying the effects of PEX19 mutations in human cells .

• Reporter systems: ZP119EG1 cells (stably expressing "enhanced" green fluorescent protein tagged with peroxisomal targeting signal 1) allow for visualization of peroxisome formation and protein import through fluorescence microscopy .

• Expression systems: Plasmid vectors such as pUcD2Hyg have been used to express wild-type and mutant forms of PEX19 in both CHO cell mutants and patient fibroblasts .

The following table shows complementation results when transfecting various peroxisome-deficient cell lines with human PEX19:

What methodologies are most effective for characterizing PEX19 mutations?

Multiple complementary approaches can be employed to characterize PEX19 mutations:

• Reverse Transcription-PCR (RT-PCR): Using PEX19-specific primers covering the full-length open reading frame, followed by cloning and sequencing of the PCR products. This method successfully identified the frameshift mutation in patient PBDJ-01 .

• Genomic DNA analysis: PCR amplification of specific regions within the PEX19 gene using genomic DNA prepared from cultured fibroblasts, followed by direct sequencing. This approach can determine the zygosity of specific alleles, as demonstrated for the PEX19A764ins allele in patient PBDJ-01 .

• Functional complementation assays: Expressing wild-type PEX19 cDNA in patient fibroblasts or model cell lines to assess rescue of peroxisome biogenesis. This approach confirms the pathogenicity of identified mutations and demonstrates that the defect is indeed due to PEX19 dysfunction .

• Morphological analysis: Visualizing peroxisomes using indirect immunofluorescence microscopy with antibodies against peroxisomal markers (catalase, PTS1, and PMP70). This technique allows assessment of whether mutant forms of PEX19 can restore peroxisome assembly .

• Multiple sequence alignment: Comparing the mutated sequence with wild-type PEX19 sequences from different species to evaluate conservation of the affected region and predict functional significance .

What is the significance of the CAAX box in PEX19 function?

The CAAX box at the C-terminus of PEX19 is a prenylation motif that is critical for protein function. Research has demonstrated that farnesylated Pex19p is partly anchored in the peroxisomal membrane through this post-translational modification .

The functional importance of the CAAX box is highlighted by the case of patient PBDJ-01 with Zellweger syndrome. This patient possessed a frameshift mutation that altered the C-terminal 24 amino acids of the protein, affecting the CAAX box region . The resulting protein was non-functional, suggesting that the C-terminal part, including the CAAX homology box, is required for the biological activity of Pex19p .

Experimental approaches to verify farnesylation and study its functional significance include:

• Protein mobility shift assays to detect differences between farnesylated and non-farnesylated forms

• Site-directed mutagenesis of the cysteine residue within the CAAX box to prevent farnesylation

• Treatment with farnesyltransferase inhibitors to assess effects on PEX19 localization and function

• Subcellular fractionation to compare membrane association of wild-type versus CAAX box mutant proteins

How can comparative genomics enhance our understanding of PEX19 evolution and function?

Comparative genomics approaches provide valuable insights into PEX19 evolution and function:

• Ortholog detection methods: Two systematic approaches have proven effective for identifying PEX19 orthologs across species - reciprocal searches of single protein sequences and reciprocal searches based on protein profiles (Hidden Markov models) . These methods help identify conserved features that may indicate functional importance.

• Multiple sequence alignment: Manual filtering and alignment using tools like Mafft enable detailed comparison of PEX19 sequences across species, highlighting conserved residues or motifs that are likely functionally important .

• Phylogenetic analysis: Construction of phylogenetic trees using methods such as IQ-TREE with ultrafast bootstrap support helps clarify evolutionary relationships between PEX19 proteins from different organisms .

• Structural and functional domain annotation: Computational tools for annotating functional domains (Pfam), predicting protein disorder (IUPRED), and identifying transmembrane helices (TMHMM) reveal conserved features that may be critical for PEX19 function .

Human Pex19p shows 20% amino acid identity with Saccharomyces cerevisiae Pex19p (which is 51 amino acids longer) and 92% identity with Chinese hamster Pex19p of the same length . These evolutionary relationships provide insights into functionally conserved regions that have been maintained throughout eukaryotic evolution.

How does PEX19 coordinate with other PEX proteins in the peroxisome biogenesis pathway?

PEX19 functions within a complex network of peroxisome biogenesis proteins. Comparative genomic studies have identified 37 known PEX proteins across eukaryotes . PEX19 appears to function at the initial stage of peroxisome membrane assembly, before the import of matrix proteins occurs .

The complementation studies summarized in Table 2 show that PEX19 specifically complements the defect in complementation group J (CG-J) cells but not cells from other complementation groups with defects in different PEX genes (PEX1, PEX5, PEX12, PEX6, etc.) . This indicates PEX19 has a specific, non-redundant role in the peroxisome biogenesis pathway.

Based on its function in peroxisomal membrane protein import, PEX19 likely interacts with:

• Newly synthesized peroxisomal membrane proteins in the cytosol (acting as a chaperone)

• Membrane-bound docking factors that facilitate the integration of these proteins into the peroxisomal membrane

• Other components of the peroxisomal import machinery

Understanding these interactions requires comparative analysis with other peroxins and examining the sequence conservation patterns across species to identify interaction interfaces and functional domains.

What experimental approaches are most effective for studying PEX19 interactions with peroxisomal membrane proteins?

Several experimental approaches are particularly effective for investigating PEX19 interactions:

• Functional complementation assays: As demonstrated in the isolation of human PEX19 cDNA through complementation of peroxisome deficiency in ZP119 cells . This approach can reveal whether specific domains affect PEX19's ability to facilitate membrane protein import.

• cDNA library screening: The screening of human liver cDNA library divided into small pools, followed by transfection into peroxisome-deficient cells, has proven successful in identifying functional PEX19 .

• Mutation analysis and domain mapping: Creating mutant forms of PEX19 with deletions or point mutations in specific domains can help identify regions critical for interaction with binding partners. The importance of the C-terminal region including the CAAX box was demonstrated through patient mutation analysis .

• Immunofluorescence microscopy: Indirect immunofluorescence using antibodies against peroxisomal markers (catalase, PTS1, PMP70) allows visualization of peroxisome formation and the localization of both PEX19 and potential interaction partners .

• Structural protein analysis: Computational prediction of functional domains, protein disorder, and transmembrane regions can guide experimental studies by highlighting potential interaction interfaces .