The peroxin pex3p initiates membrane assembly in peroxisome biogenesis

Rat cDNA encoding a 372-amino-acid peroxin was isolated, primarily by functional complementation screening, using a peroxisome-deficient Chinese hamster ovary cell mutant, ZPG208, of complementation group 17. The deduced primary sequence showed approximately 25% amino acid identity with the yeast Pex3p, thereby we termed this cDNA rat PEX3 (RnPEX3). Human and Chinese hamster Pex3p showed 96 and 94% identity to rat Pex3p and had 373 amino acids. Pex3p was characterized as an integral membrane protein of peroxisomes, exposing its N- and C-terminal parts to the cytosol. A homozygous, inactivating missense mutation, G to A at position413, in a codon (GGA) for Gly(138) and resulting in a codon (GAA) for Glu was the genetic cause of peroxisome deficiency of complementation group 17 ZPG208. The peroxisome-restoring activity apparently required the full length of Pex3p, whereas its N-terminal part from residues 1 to 40 was sufficient to target a fusion protein to peroxisomes. We also demonstrated that Pex3p binds the farnesylated peroxisomal membrane protein Pex19p. Moreover, upon expression of PEX3 in ZPG208, peroxisomal membrane vesicles were assembled before the import of soluble proteins such as PTS2-tagged green fluorescent protein. Thus, Pex3p assembles membrane vesicles before the matrix proteins are translocated.     

PEX12, the Pathogenic Gene of Group III Zellweger Syndrome: cDNA Cloning by Functional Complementation on a CHO Cell Mutant, Patient Analysis, and Characterization of Pex12p

Rat PEX12 cDNA was isolated by functional complementation of peroxisome deficiency of a mutant CHO cell line, ZP109 (K. Okumoto, A. Bogaki, K. Tateishi, T. Tsukamoto, T. Osumi, N. Shimozawa, Y. Suzuki, T. Orii, and Y. Fujiki, Exp. Cell Res. 233:11–20, 1997), using a transient transfection assay and an ectopic, readily visible marker, green fluorescent protein. This cDNA encodes a 359-amino-acid membrane protein of peroxisomes with two transmembrane segments and a cysteine-rich zinc finger, the RING motif. A stable transformant of ZP109 with the PEX12 was morphologically and biochemically restored for peroxisome biogenesis. Pex12p was shown by expression of bona fide as well as epitope-tagged Pex12p to expose both N- and C-terminal regions to the cytosol. Fibroblasts derived from patients with the peroxisome deficiency Zellweger syndrome of complementation group III (CG-III) were also complemented for peroxisome biogenesis with PEX12. Two unrelated patients of this group manifesting peroxisome deficiency disorders possessed homozygous, inactivating PEX12 mutations: in one, Arg180Thr by one point mutation, and in the other, deletion of two nucleotides in codons for ²⁹¹Asn and ²⁹²Ser, creating an apparently unchanged codon for Asn and a codon 292 for termination. These results indicate that the gene encoding peroxisome assembly factor Pex12p is a pathogenic gene of CG-III peroxisome deficiency. Moreover, truncation and site mutation studies, including patient PEX12 analysis, demonstrated that the cytoplasmically oriented N- and C-terminal parts of Pex12p are essential for biological function.     

Peroxisome biogenesis and molecular defects in peroxisome assembly disorders

Peroxisome assembly in mammals requires more than 14 genes. So far, we have isolated seven complementation groups (CGs) of peroxisome biogenesis-defective Chinese hamster ovary (CHO) cell mutants, Z65, Z24/ZP107, ZP92, ZP105/ZP139, ZP109, ZP110, ZP114. Two peroxin cDNAs, PEX2 and PEX6, were first cloned by genetic phenotype-complementation assay using Z65 and ZP92, respectively, and were shown to be responsible for peroxisome biogenesis disorders (PBD) such as Zellweger syndrome, of CG-F (the same as CG-X in U.S.A.) and CG-C (the same as CG-IV), respectively. Pex2p is a RING zinc finger membrane protein of peroxisomes and Pex6p is a member of the AAA ATPase family. We likewise isolated PEX12 encoding a peroxisomal integral membrane protein in the RING family, by functional complementation of ZP109, demonstrating PEX12 to be responsible for CG-III PBD. We also cloned PEX1 by screening of human liver cDNA library, using ZP107. PEX1 mutation was delineated to be the genetic cause of PBD in the most highest incidence group, CG-E (the same a CG-I). Moreover, we recently found that Pex5p, using PEX5-defective ZP105 and ZP139. Thus, CHO cell mutants defective in peroxisome biogenesis are indeed shown to be very useful for the studies of peroxisome assembly and delineating pathogenic genes in PBD. Furthermore, we have isolated novel CGs of CHO mutants, ZP119 and ZP126.     

Disruption of the interaction of the longer isoform of Pex5p, Pex5pL, with Pex7p abolishes peroxisome targeting signal type 2 protein import in mammals. Study with a novel Pex5-impaired Chinese hamster ovary cell mutant

We isolated peroxisome biogenesis-defective Chinese hamster ovary cell mutants from TKaG2 cells, wild-type CHO-K1 cells transformed with two cDNAs encoding rat Pex2p and peroxisome targeting signal (PTS) type 2-tagged green fluorescent protein, by the 9-(1'-pyrene)nonanol/UV selection method. Ten mutant clones showed cytosolic PTS2-green fluorescent protein, indicative of a defect in PTS2 import, and were classified in five complementation groups, i.e. pex1, pex2, pex5, pex14, and group A. One PEX5-deficient mutant, ZPG231, showed a novel phenotype: PTS2 proteins in the cytosol, but PTS1 proteins and catalase in peroxisomes. In ZPG231, two isoforms of the PTS1 receptor Pex5p, a shorter Pex5pS and a longer Pex5pL, were expressed as in wild-type cells, but possessed the missense point mutation S214F in both Pex5p isoforms, termed Pex5pS-S214F and Pex5pL-S214F, respectively. The S214F mutation was located only one amino acid upstream of the Pex5pL-specific 37-amino acid insertion site. Pex5pS-S214F and Pex5pL-S214F interacted with peroxisomal proteins, including PTS1 protein, catalase, and Pex14p, as efficiently as normal Pex5p. In contrast, the S214F mutation severely affected the binding of Pex5pL to the PTS2 receptor Pex7p. Expression of Pex5pL-S214F in pex5 cell mutants defective in PTS1 and PTS2 transport restored peroxisomal import of PTS1, but not PTS2. Together, the results indicate that ZPG231 is the first cell mutant providing evidence that disruption of the Pex5pL-Pex7p interaction completely abolishes PTS2 import in mammals.     

Isolation and characterization of novel peroxisome biogenesis-defective Chinese hamster ovary cell mutants using green fluorescent protein

We developed an improved method for isolation of peroxisome biogenesis-defective somatic animal cell mutants, using a combination of green fluorescent protein (GFP) expression and the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/UV) selection method. We used TKaG1 and TKaG2 cells, the wild-type Chinese hamster ovary (CHO) cells, CHO-K1, that had been stably transfected with cDNAs each encoding rat Pex2p as well as GFP tagged at the C-terminus with peroxisome targeting signal type 1 (PTS1) or N-terminally PTS2-tagged GFP. P9OH/UV-resistant cell colonies were examined for intracellular location of GFP on unfixed cells, by fluorescence microscopy. Seven each of the mutant cell clones isolated from TKaG1 and TKaG2 showed cytosolic GFP-PTS1 and PTS2-GFP, respectively, indicating the defect in peroxisome assembly. By transfection of PEX2, PEX5, PEX6, and PEX12 cDNAs and cell fusion analysis between the CHO cell mutants, five different complementation groups (CGs) were identified. Two mutant clones, ZPG207 and ZPG208, belonged to novel CGs. Further CG analysis using fibroblasts from patients with peroxisome biogenesis disorders, including rhizomelic chondrodysplasia punctata (RCDP), revealed that ZPG208 belonged to none of human CGs. ZPG207 was classified into the same CG as RCDP. Taken together, ZPG208 is in a newly identified, the 12th, CG in peroxisome-deficient CHO mutants reported to date and represents a novel mammalian CG.     

Newly identified Chinese hamster ovary cell mutants defective in peroxisome assembly represent complementation group A of human peroxisome biogenesis disorders and one novel group in mammals

We isolated peroxisome biogenesis-defective mutants from rat PEX2-transformed Chinese hamster ovary (CHO) cells, using the 9-(1'-pyrene)nonanol/ultraviolet method. A total of 18 mutant cell clones showing cytosolic localization of catalase were isolated. By complementation group (CG) analysis by means of PEX cDNA transfection and cell fusion, cell mutants, ZP124 and ZP126, were found to belong to two novel CGs of CHO mutants. Mutants, ZP135 and ZP167, were also classified to the same CG as ZP124. Further cell fusion analysis using 12 CGs fibroblasts from patients with peroxisome deficiency disorders such as Zellweger syndrome revealed that ZP124 belonged to human CG-A, the same group as CG-VIII in the United States. ZP126 could not be classified to any of human and CHO CGs. These mutants also showed typical peroxisome assembly-defective phenotypes such as severe loss of catalase latency and impaired biogenesis of peroxisomal enzymes. Collectively, ZP124 represents CG-A, and ZP126 is in a newly identified CG distinct from the 14 mammalian CGs previously characterized.     

Molecular anatomy of the peroxin Pex12p: ring finger domain is essential for Pex12p function and interacts with the peroxisome-targeting signal type 1-receptor Pex5p and a ring peroxin, Pex10p

The three peroxin genes, PEX12, PEX2, and PEX10, encode peroxisomal integral membrane proteins with RING finger at the C-terminal part and are responsible for human peroxisome biogenesis disorders. Mutation analysis in PEX12 of Chinese hamster ovary cell mutants revealed a homozygous nonsense mutation at residue Trp263Ter in ZP104 cells and a pair of heterozygous nonsense mutations, Trp170Ter and Trp114Ter, in ZP109. This result and domain mapping of Pex12p showed that RING finger is essential for peroxisome-restoring activity of Pex12p but not necessary for targeting to peroxisomes. The N-terminal region of Pex12p, including amino acid residues at positions 17-76, was required for localization to peroxisomes, while the sequence 17-76 was not sufficient for peroxisomal targeting. Peroxins interacting with RING finger of Pex2p, Pex10p, and Pex12p were investigated by yeast two-hybrid as well as in vitro binding assays. The RING finger of Pex12p bound to Pex10p and the PTS1-receptor Pex5p. Pex10p also interacted with Pex2p and Pex5p in vitro. Moreover, Pex12p was co-immunoprecipitated with Pex10p from CHO-K1 cells, where Pex5p was not associated with the Pex12p-Pex10p complex. This observation suggested that Pex5p does not bind to, or only transiently interacts with, Pex10p and Pex12p when Pex10p and Pex12p are in the oligomeric complex in peroxisome membranes. Hence, the RING finger peroxins are most likely to be involved in Pex5p-mediated matrix protein import into peroxisomes.     

Pex12p of Saccharomyces cerevisiae is a component of a multi-protein complex essential for peroxisomal matrix protein import

We have isolated the Saccharomyces cerevisiae pex12-1 mutant from a screen to identify mutants defective in peroxisome biogenesis. The pex12delta deletion strain fails to import peroxisomal matrix proteins through both the PTS1 and PTS2 pathway. The PEX12 gene was cloned by functional complementation of the pex12-1 mutant strain and encodes a polypeptide of 399 amino acids. ScPex12p is orthologous to Pex12 proteins from other species and like its orthologues, S. cerevisiae Pex12p contains a degenerate RING finger domain of the C3HC4 type in its essential carboxy-terminus. Localization studies demonstrate that Pex12p is an integral peroxisomal membrane protein, with its NH2-terminus facing the peroxisomal lumen and with its COOH-terminus facing the cytosol. Pex12p-deficient cells retain particular structures that contain peroxisomal membrane proteins consistent with the existence of peroxisomal membrane remnants ("ghosts") in pex12A null mutant cells. This finding indicates that pex12delta cells are not impaired in peroxisomal membrane biogenesis. In immunoisolation experiments Pex12p was co-purified with the RING finger protein Pex10p, the PTS1 receptor Pex5p and the docking proteins for the PTS1 and the PTS2 receptor at the peroxisomal membrane, Pex13p and Pex14p. Furthermore, two-hybrid experiments suggest that the two RING finger domains are sufficient for the Pex10p-Pex12p interaction. Our results suggest that Pex12p is a component of the peroxisomal translocation machinery for matrix proteins.     

A novel pex2 mutant: catalase-deficient but temperature-sensitive PTS1 and PTS2 import

We searched for Chinese hamster ovary (CHO) cell mutants defective in peroxisome biogenesis by using peroxisome targeting sequence (PTS) of Pex3p (amino acid residues 1-40)-fused enhanced green fluorescent protein (EGFP). From mutagenized wild-type CHO-K1 cells stably expressing rat Pex2p and Pex3p(1-40)-EGFP, cell colonies resistant to the 9-(1(')-pyrene)nonanol/ultraviolet treatment were examined for intracellular location of peroxisomal proteins, including EGFP chimera, catalase, and matrix proteins with PTS types 1 and 2. One clone, ZPEG309, showed a distinct phenotype: import defect of catalase, but normal transport of PTS1 and PTS2 proteins at 37 degrees C. PTS1 and PTS2 import was abrogated when ZPEG309 was cultured at 39 degrees C. Genetic defect of ZPEG309 was a nonsense point mutation in a codon for Arg50 in CHO PEX2 and a mutation resulting in a C-terminal truncation of the introduced rat Pex2p. Therefore, ZPEG309 is a novel pex2, catalase-deficient mutant with temperature-sensitive PTS1 and PTS2 import.     

Functional heterogeneity of C-terminal peroxisome targeting signal 1 in PEX5-defective patients.

To investigate mechanisms related to functions of the peroxisome targeting signal (PTS) 1 receptor, Pex5p, we analyzed peroxisome matrix protein import in fibroblasts from three patients with peroxisome biogenesis disorders, all with different mutations in the PEX5 gene. The patients 2-01 (Zellweger syndrome) and 2-05 (neonatal adrenoleukodystrophy) have the reported mutations, R390X and N489K, and patient 2-03 (infantile Refsum disease) has a newly identified mutation, S563W. Fibroblasts from 2-03 (S563W) were detected in both PTS1 and PTS2 imports despite the PEX5 defect, findings in contrast with fibroblasts from 2-05 (N489K) severely defective in PTS1 import and those from 2-01 (R390X) severely defective in both PTS1 and PTS2. The PTS1 receptor in 2-03 is functional for only the C-terminal -SKL sequence (acyl-CoA oxidase) and had little or no function for C-terminal -AKL (D-bifunctional protein and sterol carrier protein 2) and -KANL (catalase) sequences, respectively. After transfection of these mutated PEX5 cDNA into the PEX5-defective CHO mutant, transformants of ZP102 revealed that each mutation was responsible for each dysfunction of the PTS1 import. It seems apparent that -AKL and -KANL are poorer variants of PTS1 and are likely to be more susceptible to effects of mutation of its receptor, Pex5p.     

The ubiquitin-conjugating enzyme Pex4p of Hansenula polymorpha is required for efficient functioning of the PTS1 import machinery.

We have cloned the Hansenula polymorpha PEX4 gene by functional complementation of a peroxisome-deficient mutant. The PEX4 translation product, Pex4p, is a member of the ubiquitin-conjugating enzyme family. In H.polymorpha, Pex4p is a constitutive, low abundance protein. Both the original mutant and the pex4 deletion strain (Deltapex4) showed a specific defect in import of peroxisomal matrix proteins containing a C-terminal targeting signal (PTS1) and of malate synthase, whose targeting signal is not yet known. Import of the PTS2 protein amine oxidase and the insertion of the peroxisomal membrane proteins Pex3p and Pex14p was not disturbed in Deltapex4 cells. The PTS1 protein import defect in Deltapex4 cells could be suppressed by overproduction of the PTS1 receptor, Pex5p, in a dose-response related manner. In such cells, Pex5p is localized in the cytosol and in peroxisomes. The peroxisome-bound Pex5p specifically accumulated at the inner surface of the peroxisomal membrane and thus differed from Pex5p in wild-type peroxisomes, which is localized throughout the matrix. We hypothesize that in H. polymorpha Pex4p plays an essential role for normal functioning of Pex5p, possibly in mediating recycling of Pex5p from the peroxisome to the cytosol.     

Dynamic and functional assembly of the AAA peroxins, Pex1p and Pex6p, and their membrane receptor Pex26p

Two AAA peroxins, Pex1p and Pex6p, are encoded by PEX1 and PEX6, the causal genes for peroxisome biogenesis disorders of complementation group 1 (CG1) and CG4, respectively. PEX26 responsible for peroxisome biogenesis disorders of CG8 encodes Pex26p, the recruiter of Pex1p.Pex6p complexes to peroxisomes. We herein assigned the binding regions between human Pex1p and Pex6p and elucidated pivotal roles of the AAA cassettes, called D1 and D2 domains, in Pex1p-Pex6p interaction and peroxisome biogenesis. ATP binding in both AAA cassettes but not ATP hydrolysis in D2 of both Pex1p and Pex6p was prerequisite for Pex1p-Pex6p interaction and their peroxisomal localization. The AAA cassettes, D1 and D2, were essential for peroxisome-restoring activity of Pex1p and Pex6p. In HEK293 cells, endogenous Pex1p was partly localized likely as a homo-oligomer in the cytoplasm, while Pex6p and Pex26p were predominantly localized on peroxisomes. Interaction of Pex1p with Pex6p conferred a conformational change and dissociation of the Pex1p oligomer. These results suggested that Pex1p possesses two distinct oligomeric forms, a homo-oligomer in the cytosol and a hetero-oligomer on peroxisome membranes, possibly playing distinct functions in peroxisome biogenesis.     

Multiple PEX genes are required for proper subcellular distribution and stability of Pex5p, the PTS1 receptor: evidence that PTS1 protein import is mediated by a cycling receptor

PEX5 encodes the type-1 peroxisomal targeting signal (PTS1) receptor, one of at least 15 peroxins required for peroxisome biogenesis. Pex5p has a bimodal distribution within the cell, mostly cytosolic with a small amount bound to peroxisomes. This distribution indicates that Pex5p may function as a cycling receptor, a mode of action likely to require interaction with additional peroxins. Loss of peroxins required for protein translocation into the peroxisome (PEX2 or PEX12) resulted in accumulation of Pex5p at docking sites on the peroxisome surface. Pex5p also accumulated on peroxisomes in normal cells under conditions which inhibit protein translocation into peroxisomes (low temperature or ATP depletion), returned to the cytoplasm when translocation was restored, and reaccumulated on peroxisomes when translocation was again inhibited. Translocation inhibiting conditions did not result in Pex5p redistribution in cells that lack detectable peroxisomes. Thus, it appears that Pex5p can cycle repeatedly between the cytoplasm and peroxisome. Altered activity of the peroxin defective in CG7 cells leads to accumulation of Pex5p within the peroxisome, indicating that Pex5p may actually enter the peroxisome lumen at one point in its cycle. In addition, we found that the PTS1 receptor was extremely unstable in the peroxin-deficient CG1, CG4, and CG8 cells. Altered distribution or stability of the PTS1 receptor in all cells with a defect in PTS1 protein import implies that the genes mutated in these cell lines encode proteins with a direct role in peroxisomal protein import.     

PEX3 is the causal gene responsible for peroxisome membrane assembly-defective Zellweger syndrome of complementation group G

Peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy are autosomal recessive diseases caused by defects in peroxisome assembly, for which 13 genotypes have been identified. Expression of the human peroxin Pex3p cDNA encoding a 373-amino-acid peroxisomal membrane protein morphologically and biochemically restored peroxisome biogenesis, including peroxisomal membrane assembly, in fibroblasts from PBDG-02, a patient with complementation group G (CG-G) ZS. Patient PBDG-02 carried a homozygous, inactivating mutation-a 97-bp deletion of nucleotide residues at positions 942-1038-resulting in a 32-amino-acid truncation and in a frameshift inducing both a 3-amino-acid substitution and a termination codon. Genomic PCR analysis revealed mutation of T-->G at eight bases upstream of the splicing site at the boundary of intron 10 and exon 11 of PEX3 gene, giving rise to a deletion of all of exon 11. When assessed by expression in a pex3 mutant of Chinese hamster ovary cells and the patient's fibroblasts, PBDG-02-derived PEX3 cDNA was found to be defective in peroxisome-restoring activity. These results provide evidence that PEX3 is a novel, pathogenic gene responsible for CG-G PBDs.     

The mammalian peroxin Pex5pL, the longer isoform of the mobile peroxisome targeting signal (PTS) type 1 transporter, translocates the Pex7p.PTS2 protein complex into peroxisomes via its initial docking site, Pex14p

In mammals, two isoforms of the peroxisome targeting signal (PTS) type 1 receptor Pex5p, i.e. Pex5pS and Pex5pL with an internal 37-amino acid insertion, have previously been identified. Expression of either type of Pex5p complements the impaired PTS1 import in Chinese hamster ovary pex5 mutants, but only Pex5pL can rescue the PTS2 import defect noted in a subgroup of pex5 mutants such as ZP105. In this work, we found that Pex5pL directly interacts with the PTS2 receptor Pex7p, carrying its cargo PTS2 protein in the cytosol. Pex5pL, but not Pex5pS, mediated the binding of PTS2 protein to Pex14p by translocating Pex7p, demonstrating that Pex5pL plays a pivotal role in peroxisomal PTS2 import. Pex5p was localized mostly in the cytosol in wild-type CHO-K1 and Pex14p-deficient mutant cells, whereas it accumulated in the peroxisomal remnants in cell mutants defective in Pex13p or the RING family peroxins such as Pex2p and Pex12p. Furthermore, overexpression of Pex14p, but not Pex10p, Pex12p, or Pex13p, caused accumulation of Pex5p in peroxisomal membranes, with concomitant interference with PTS1 and PTS2 import. Therefore, Pex5p carrying the cargoes most likely docks with the initial site (Pex14p) in a putative import machinery, subsequently translocating to other components such as Pex13p, Pex2p, Pex10p, and Pex12p.     

Isolation and characterization of novel phenotype CHO cell mutants defective in peroxisome assembly, using ICR191 as a potent mutagenic agent

To elucidate molecular and cellular mechanisms of peroxisome biogenesis, we have isolated Chinese hamster ovary (CHO) cell mutants defective in peroxisome biogenesis by making use of enhanced green fluorescent protein (EGFP) and a frameshift-inducing mutagen ICR191. CHO-TKa cells stably expressing Pex2p were transformed with a cDNA encoding EGFP fused with peroxisomal targeting signal type 2 (PTS2-EGFP), termed Tka/EG2. TKa/EG2 cells were mutagenized with ICR191 and cultured in the presence of P9OH (9-(1'-pyrene) nonanol) followed by an exposure to UV. P9OH/UV-resistant and morphologically peroxisome-deficient mutant cells were isolated by directly observing cytosolic localization of EGFP, without cell staining. By a combination of cell-fusion and PEX transfection, we determined complementation groups (CGs) of 16 cell mutants isolated here. The mutants were classified into five CGs, including pex2, pex3, pex5, pex6, and pex7 cell mutants. In contrast to typical pex6 mutants with the impaired import of both PTS1- and PTS2-proteins, two clones, ZPEG236 and ZPEG244, showed a distinct, novel phenotype where PTS1-protein import was normal despite the abrogated PTS2 import. Dysfunction of Pex3p in pex3 ZPEG 238 was due to one base (G) insertion in the codon for Asn7 resulting in a frameshift, thereby inducing a distinct 31 amino-acid sequence and a termination. pex2 ZPEG239 showed a mutation in codon GAG for Glu(201) to a nonsense mutation, TAG. Thus, the method developed here using ICR191 could be useful for isolation of further novel cell mutants impaired in peroxisome biogenesis.     

Mutations in the peroxin Pex26p responsible for peroxisome biogenesis disorders of complementation group 8 impair its stability, peroxisomal localization, and interaction with the Pex1p x Pex6p complex

Peroxisome biogenesis disorders (PBDs) are fatal autosomal recessive diseases and are caused by impaired peroxisome biogenesis. PBDs are genetically heterogeneous and classified into 13 complementation groups (CGs). CG8 is one of the most common groups and has three clinical phenotypes, including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy, and infantile Refsum disease (IRD). We recently isolated PEX26 as the pathogenic gene for PBD of CG8. Pex26p functions in recruiting to peroxisomes the complexes of the AAA ATPase peroxins, Pex1p and Pex6p. In the present work, we identified four distinct mutations in PEX26 from five patients of CG8 PBD including 2 with ZS and 3 with IRD, in addition to 7 mutant alleles in 8 patients in the first report describing the pathogenic PEX26 gene for CG8 PBD. Phenotype-genotype analyses revealed that temperature-sensitive (ts) peroxisome assembly gave rise to a milder IRD in contrast to the non-ts phenotype of the cells from ZS patients. Furthermore, we present several lines of evidence that show that the instability, insufficient binding to Pex1p x Pex6p complexes, or mislocalization of patient-derived Pex26p mutants is most likely responsible for the CG8 PBDs.     

The pathogenic peroxin Pex26p recruits the Pex1p-Pex6p AAA ATPase complexes to peroxisomes

Peroxisomes are ubiquitous organelles with a single membrane that contain over 50 different enzymes that catalyse various metabolic pathways, including beta-oxidation and lipid synthesis. Peroxisome biogenesis disorders (PBDs), such as Zellweger syndrome and neonatal adrenoleukodystrophy, are fatal genetic diseases that are autosomal recessive. Among the PBDs of the 12 complementation groups (CGs), 11 associated PEX genes have been isolated. Accordingly, only the PBD pathogenic gene for CG8 (also called CG-A) remains unidentified. Here we have isolated human PEX26 encoding a type II peroxisomal membrane protein of relative molecular mass 34,000 (M(r) 34K) by using ZP167 cells, a Chinese hamster ovary (CHO) mutant cell line. Expression of PEX26 restores peroxisomal protein import in the fibroblasts of an individual with PBD of CG8. This individual possesses a homozygous, inactivating pathogenic point mutation, Arg98Trp, in Pex26. Pex6 and Pex1 of the AAA ATPase family co-immunoprecipitate with Pex26. Epitope-tagged Pex6 and Pex1 are discernible as puncta in normal CHO-K1 cells, but not in PEX26-defective cells. PEX26 expression in ZP167 cells re-establishes colocalization of Pex6 and Pex1 with Pex26, in a Pex6-dependent manner. Thus, Pex26 recruits Pex6-Pex1 complexes to peroxisomes.