Interaction of Leishmania PTS2 receptor peroxin 7 with the glycosomal protein import machinery

Leishmania proteins containing a peroxisomal targeting signal sequence 2 (PTS2) are selectively trafficked to the glycosome by associating with the peroxin 7 receptor protein (PEX7). The L. major PEX7 (LmPEX7) encodes a approximately 41 kDa protein that exhibits limited sequence identity with PEX7 homologues from other eukaryotic organisms. Functional characterization of recombinant and native LmPEX7 revealed that this receptor bound the PTS2 protein fructose-1,6-bisphosphate aldolase. Moreover, LmPEX7 also formed a tight association with the Leishmania PEX5, the cytosolic PTS1 receptor, and PEX14, a glycosomal peripheral membrane protein required for protein import into the glycosome. Mapping studies revealed that the Leishmania PEX7 binds to a domain on LdPEX5 encompassing residues 111-148 and to a site on LdPEX14 spanning residues 120-148. Finally, subcellular localization studies revealed that Leishmania PEX7 has a dual distribution within the cytosolic compartment and glycosomal lumen.     

Identification of a new complementation group of the peroxisome biogenesis disorders and PEX14 as the mutated gene

Peroxisome biogenesis disorders (PBD) are lethal hereditary diseases caused by abnormalities in the biogenesis of peroxisomes. At present, 12 different complementation groups have been identified and to date, all genes responsible for each of these complementation groups have been identified. The peroxisomal membrane protein PEX14 is a key component of the peroxisomal import machinery and may be the initial docking site for the two import receptors PEX5 and PEX7. Although PEX14 mutants have been identified in yeasts and CHO-cells, human PEX14 deficiency has apparently not been documented. We now report the identification of a new complementation group of the peroxisome biogenesis disorders with PEX14 as the defective gene. Indeed, human PEX14 rescues the import of a PTS1-dependent as well as a PTS2-dependent protein into the peroxisomes in fibroblasts from a patient with Zellweger syndrome belonging to the new complementation group. This patient was homozygous for a nonsense mutation in a putative coiled-coil region of PEX14, c.553C>T (p.Q185X). Furthermore, we showed that the patient's fibroblasts lacked PEX14 as determined by immunocytochemical analysis. These findings indicate that there are 13 genotypes in PBD and that the role of PEX14 is also essential in humans.     

Mutation analysis of PEX7 in 60 probands with rhizomelic chondrodysplasia punctata and functional correlations of genotype with phenotype

PEX7 encodes the cytosolic receptor for the set of peroxisomal matrix enzymes targeted to the organelle by the peroxisome targeting signal 2 (PTS2). Mutations in PEX7 cause rhizomelic chondrodysplasia punctata (RCDP), a distinct peroxisome biogenesis disorder. In previous work we described three novel PEX7 mutant alleles, including one, L292X, with a high frequency due to a founder effect. We have now extended our analysis to 60 RCDP probands and identified a total of 24 PEX7 alleles, accounting for 95% of the mutant PEX7 genes in our sample. Of these, 50% are L292X, 13% are IVS9+1G>C, and the remainder are mostly private. IVS9+1G>C occurs on at least three different haplotypes and thus appears to result from recurrent mutation. The phenotypic spectrum of RCDP is broader than commonly recognized and includes minimally affected individuals at the mild end of the spectrum. To relate PEX7 genotype and phenotype, we evaluated the consequence of the disease mutation on PEX7 RNA by Northern analysis and RT/PCR. We evaluated the function of the encoded Pex7 protein (Pex7p) by expressing selected alleles in fibroblasts from RCDP patients and assaying their ability to restore import of a PTS2 marker protein. We find that residual activity of mutant Pex7p and reduced amounts of normal Pex7p are associated with milder and variant phenotypes.     

Novel PEX1 coding mutations and 5' UTR regulatory polymorphisms

Zellweger syndrome and its milder variants--neonatal adrenoleukodystrophy and infantile Refsum disease--comprise a clinical continuum of diseases referred to as the Zellweger spectrum. Mutations in the PEX1 gene, which consists of 24 exons and encodes a AAA ATPase protein required for peroxisomal protein import, account for approximately two-thirds of the known Zellweger spectrum patient mutations. In this paper, we report on four novel PEX1 mutations and two polymorphisms in an Australasian cohort. Two of the mutations--c.1108_1109insA and c.2391_2392delTC--that lead to the introduction of a premature termination codon in exons 5 and 14, respectively, are associated with the severe Zellweger phenotype. One patient with a milder disease phenotype was a compound heterozygote for two missense mutations (I989T and R998Q), both affecting amino acids in the second, C-terminal AAA domain of the protein. PTS1 protein import levels in cultured skin fibroblasts from this patient were almost 20% of normal control levels. We have also characterized two co-segregating polymorphisms in the 5' UTR of the PEX1 gene. Based on reporter assays, the c.-137T>C polymorphism leads to reduced PEX1 expression, whereas the c.-53C>G polymorphism leads to increased expression. When present together, these regulatory polymorphisms lead to near-normal PEX1 expression. Altered PEX1 expression due to the presence of either the c.-137T>C or the c.-53C>G variant could impact on residual PEX1 function if another co-allelic mutation was present which did not completely abolish PEX1 function. It also follows that the presence of polymorphisms in the PEX1 promoter region could have implications for patients with mutations in other PEX proteins known to interact with PEX1, such as PEX6. Thus, although not deleterious in control individuals, these polymorphisms could contribute to phenotypic heterogeneity among Zellweger spectrum patients.     

Peroxisomal targeting protein 14 (PEX14) from Leishmania donovani. Molecular,biochemical, and immunocytochemical characterization

Pathogens of the Leishmania and Trypanosoma genera compartmentalize glycolytic and other nutritional pathways in glycosomes, unique subcellular organelles related to the peroxisomes of mammals and yeasts. Most glycosomal proteins are targeted to the glycosomes by a COOH-terminal tripeptide signal similar to the peroxisomal targeting signal-1 (PTS-1). It has been proposed that PTS-1 forms a complex with the PEX5 receptor protein which then docks to the glycosomal membrane through interactions with the membrane associated PEX14 protein. To analyze the role of PEX14 in glycosomal protein import, the gene encoding the L. donovani PEX14 (LdPEX14) was isolated and shown to encode a 464 amino acid protein that exhibited very limited sequence homology with peroxisomal PEX14 proteins. In vitro binding experiments with purified recombinant LdPEX14 and LdPEX5 confirmed that LdPEX14-LdPEX5 interacted with a K(d) of 2.75 microM. When LdPEX5 was preloaded with a PTS-1 peptide, the affinity of the LdPEX14-LdPEX5 interaction affinity increased. Furthermore, binding experiments with truncated forms of LdPEX5 and LdPEX14 showed that the interaction domains localized to the amino terminal region of both proteins. Finally, confocal microscopy, subcellular fractionation, and differential extraction experiments indicated that LdPEX14 is a soluble protein that associates tightly with the glycosomal membrane and further support the role of LdPEX14 in forming a docking complex involved in glycosome biogenesis.     

Phenotype-genotype relationships in PEX10-deficient peroxisome biogenesis disorder patients

The peroxisome biogenesis disorders (PBD) are characterized by neural, hepatic, and renal deficiencies, severe mental retardation, and are often lethal. These disorders are genetically and phenotypically heterogeneous and are caused by defective peroxisomal protein import and decreased peroxisomal metabolic function. Mutations in PEX10 have been identified in patients from complementation group 7 (CG7) of the PBDs and we report here an analysis of the genotypes and phenotypes of PEX10-deficient patients. All four PEX10-deficient Zellweger Syndrome (ZS) patients were found to have nonsense, frameshift, or splice site mutations that remove large portions of the PEX10 coding region. In contrast, a more mildly affected PEX10-deficient neonatal adrenoleukodystrophy patient expressed a PEX10 allele with a missense mutation, H290Q, affecting the C-terminal zinc-binding domain of the PEX10 product. These results support the hypothesis that severe, loss-of-function mutations in PEX genes cause more severe clinical phenotypes, whereas mildly affected PBD patients have PEX gene mutations that retain residual function. To quantitate the effects of the PEX10 mutations identified here and elsewhere we employed a functional complementation assay. Surprisingly, we observed that nonsense and frameshift mutations predicted to delete the C-terminal 2/3 (R125X) or 1/3 (c.704insA) of the protein displayed nearly normal PEX10 activity. Even more surprising, we found that the unexpectedly high PEX10 activity displayed by these cDNAs could be eliminated by removing or mutating segments of the PEX10 cDNA downstream of the mutations. Although these results demonstrate serious flaws in the PEX10 functional complementation assay, they do suggest that the C-terminal zinc-binding domain is critical for PEX10 function.     

Acyl-CoA:dihydroxyacetonephosphate acyltransferase: cloning of the human cDNA and resolution of the molecular basis in rhizomelic chondrodysplasia punctata type 2

Rhizomelic chondrodysplasia punctata (RCDP) is a genetic disorder which is clinically characterized by rhizomelic shortening of the upper extremities, typical dysmorphic facial appearance, congenital contractures and severe growth and mental retardation. Patients with RCDP can be subdivided into three subgroups based on biochemical analyses and complementation studies. The largest subgroup contains patients with mutations in the PEX7 gene encoding the PTS2 receptor. This results in multiple peroxisomal abnormalities which includes a deficiency of acyl-CoA:dihydroxyacetonephosphate acyltransferase (DHAPAT), alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase), peroxisomal 3-ketoacyl-CoA thiolase and phytanoyl-CoA hydroxylase, although there are differences in the extent of the deficiencies observed. Patients in the two other subgroups have been reported to be either deficient in the activity of DHAPAT (RCDP type 2) or alkyl-DHAP synthase (RCDP type 3) while no other abnormalities could be observed. To examine whether the gene encoding DHAPAT is mutated in patients with RCDP type 2, we determined the N-terminal amino acid sequence of the enzyme isolated from human placenta. Using this sequence as a query, we identified a 2040 bp open reading frame (ORF) in the human database of expressed sequence tags. Expression of this ORF in the yeast Saccharomyces cerevisiae showed that we have identified the DHAPAT cDNA. The deduced amino acid sequence revealed no PTS2 consensus sequence. In contrast DHAPAT appears to contain a putative PTS1 at the extreme C-terminus. All RCDP type 2 patients analyzed were found to contain mutations in their DHAPAT cDNA. This demonstrates that RCDP type 2 is the result of mutations in DHAPAT.      

Novel PEX1 mutations and genotype-phenotype correlations in Australasian peroxisome biogenesis disorder patients

The peroxisome biogenesis disorders (PBDs) are a group of neuronal migration/neurodegenerative disorders that arise from defects in PEX genes. A major subgroup of the PBDs includes Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD). These three disorders represent a clinical continuum with Zellweger syndrome the most severe. Mutations in the PEX1 gene, which encodes a protein of the AAA ATPase family involved in peroxisome matrix protein import, account for the genetic defect in more than half of the patients in this PBD subgroup. We report here on the results of PEX1 mutation detection in an Australasian cohort of PEX1-deficient PBD patients. This screen has identified five novel mutations, including nonsense mutations in exons 14 and 19 and single nucleotide deletions in exons 5 and 18. Significantly, the allele carrying the exon 18 frameshift mutation is present at moderately high frequency (approx. 10%) in this patient cohort. The fifth mutation is a missense mutation (R798G) that attenuates, but does not abolish PEX1 function. We have evaluated the cellular impact of these novel mutations, along with that of the two most common PEX1 mutations (c.2097-2098insT and G843D), in PBD patients by determining the levels of PEX1 mRNA, PEX1 protein, and peroxisome protein import. The findings are consistent with a close correlation between cellular phenotype, disease severity, and PEX1 genotype.     

Nonsense and temperature-sensitive mutations in PEX13 are the cause of complementation group H of peroxisome biogenesis disorders.

Peroxisome biogenesis disorders, including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease, are lethal hereditary diseases caused by abnormalities in peroxisomal assembly. To date, 12 genotypes have been identified. We now have evidence that the complete human cDNA encoding Pex13p, an SH3 protein of a docking factor for the peroxisome targeting signal 1 receptor (Pex5p), rescues peroxisomal matrix protein import and its assembly in fibroblasts from PBD patients of complementation group H. In addition, we detected mutations on the human PEX13 cDNA in two patients of group H. A severe phenotype of a ZS patient (H-02) was homozygous for a nonsense mutation, W234ter, which results in the loss of not only the SH3 domain but also the putative transmembrane domain of Pex13p. A more mildly affected NALD patient (H-01), whose fibroblasts showed the temperature-sensitive (TS) phenotype, was homozygous for a missense mutation in the SH3 domain of Pex13p, I326T. This mutant PEX13 cDNA expression in a PEX13-defective CHO mutant showed I326T to be a TS mutation and thus suggested that Pex13p with the I326T mutation in the SH3 domain is stable at 30 degrees C but is somewhat unstable at 37 degrees C.     

Identification of trypanosomatid PEX19: functional characterization reveals impact on cell growth and glycosome size and number

Glycosomes are peroxisome-like organelles present in trypanosomatid pathogens. These organelles compartmentalize glycolysis, among other reactions, and are essential in both bloodstream and procyclic form Trypanosoma brucei. Peroxins (PEXs) are proteins necessary for biogenesis of peroxisomes and glycosomes. In each assembled trypanosomatid genome, we identified a predicted protein with approximately 20% sequence identity to human PEX19, a protein required for insertion of peroxisomal membrane proteins (PMPs) into the membrane. Functional analysis demonstrated that these proteins are indeed PEX19 orthologues. Like other PEX19s, T. brucei and Leishmania major PEX19 GFP fusion proteins are predominantly cytosolic. We further showed that LmPEX19 interacts with the glycosomal membrane protein PEX2 in the yeast two-hybrid system. Partial knockdown of TbPEX19 slowed parasite growth, particularly when glucose was present. Immunofluorescence and electron microscopic studies revealed biogenesis defect as evidenced by a sharp reduction in the number of glycosomes. Surprisingly, a four-fold increase in the size of the remaining glycosomes was observed. We propose that this phenotype of fewer but larger glycosomes results from the reduction in import of glycosomal membrane proteins.     

Defective PEX gene products correlate with the protein import, biochemical abnormalities, and phenotypic heterogeneity in peroxisome biogenesis disorders.

Peroxisome biogenesis disorders (PBD) comprise three phenotypes including Zellweger syndrome (ZS) (the most severe), neonatal adrenoleucodystrophy, and infantile Refsum disease (IRD) (the most mild), and can be classified into at least 12 genetic complementation groups, which are not predictive of the phenotypes. Several pathogenic genes for PBD groups have been identified, but the relationship between the defective gene products and phenotypic heterogeneity has remained unclear. We identified a mutation in the PEX2 gene in an IRD patient with compound heterozygosity for a missense mutation and the known nonsense mutation detected in ZS patients. In transfection experiments using the peroxisome deficient CHO mutant, Z65 with a nonsense mutation in the PEX2 gene, we noted the E55K mutation had mosaic activities of peroxisomal protein import machinery and residual activities of peroxisomal functions, including dihydroxyacetone phosphate acyltransferase and beta oxidation of very long chain fatty acids. The nonsense mutation severely affects these peroxisomal functions as well as the protein import. These data suggest that allelic heterogeneity of the PEX gene affects the peroxisomal protein import and functions and regulates the clinical severity in PBD.     

Identification of two novel type 1 peroxisomal targeting signals in Arabidopsis thaliana

Peroxisomes lack their own genetic material and must therefore import proteins encoded by genes in the nucleus. Amino acids within these proteins serve as targeting signals: they direct the delivery of the proteins to the organelle. The majority of soluble proteins destined for the peroxisomal matrix utilize a type 1 peroxisomal targeting signal (PTS1): a C-terminal tripeptide that follows the pattern small/basic/hydrophobic. We have discovered two new C-terminal tripeptides that target proteins to peroxisomes in Arabidopsis thaliana. The tripeptides PSL and KRR do not fit the major PTS1 consensus but cause green fluorescent protein to accumulate in peroxisomes of stably transformed Arabidopsis. We have identified forty-one proteins in the Arabidopsis genome that also bear these tripeptides at their C-termini and may therefore be peroxisomal.     

Identification of PEX3 as the gene mutated in a Zellweger syndrome patient lacking peroxisomal remnant structures

Peroxisome biogenesis disorders, of which 13 complementation groups have been identified, are subdivided with regard to two major dysfunctions: peroxisomal matrix protein import and peroxisomal membrane synthesis. Detectable remnant membrane structures are evident only in the former. Molecular defects have been defined in 10 PEX genes, including eight related to protein import and two to membrane synthesis. We now have evidence that the human complete cDNA encoding Pex3p, a peroxisomal membrane protein (PMP) factor for the proper localization of PMPs, rescues the import of both PMP and the matrix protein in fibroblasts from a Zellweger syndrome patient of complementation group G. This patient was homozygous for a 1 base insertion in the codon for V182, which resulted in a change of codon (182-183) and introduced a termination codon (184), which inactivated PMP and matrix protein import by Pex3p. A PEX3-defective CHO mutant clone, ZPG208, was of the same complementation group as group G.     

Cholesterol biosynthesis is not defective in peroxisome biogenesis defective fibroblasts

To evaluate the presumed peroxisomal involvement in cholesterol/isoprenoid biosynthesis, we determined the protein levels and activities of five different enzymes of the presqualene segment of the cholesterol/isoprenoid biosynthetic pathway in primary skin fibroblasts of selected patients with a peroxisomal biogenesis disorder (PBD). These five enzymes all have been reported to be partly or exclusively peroxisomal and include HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, and isopentenyl pyrophosphate isomerase. To exclude that genetic differences, resulting in different defects in peroxisomal biogenesis, have differential effects on the activity of the cholesterol biosynthetic enzymes and on de novo cholesterol biosynthesis, we chose fibroblasts of patients with defined defects in one of four different PEX genes leading to Zellweger syndrome (PEX1, PEX5, PEX16 or PEX19). We found that all enzymes measured are at least as active in the peroxisome-deficient cells cultured in cholesterol-depleted medium as in identically cultured control cells. This indicates that if these presumed peroxisomal proteins are mislocalized to the cytosol they do not loose their activity, nor get degraded unlike most other authentic peroxisomal proteins. We also measured de novo cholesterol synthesis from radio-labeled acetate in all cell lines and found similar or even elevated rates for the PBD cells when compared to controls. Our results imply that functional peroxisomes are not a prerequisite for the functioning of enzymes involved in cholesterol/isoprenoid biosynthesis and as such raise doubts about the true involvement of peroxisomes therein.     

Genetic classification and mutational spectrum of more than 600 patients with a Zellweger syndrome spectrum disorder

The autosomal recessive Zellweger syndrome spectrum (ZSS) disorders comprise a main subgroup of the peroxisome biogenesis disorders and can be caused by mutations in any of 12 different currently identified PEX genes resulting in severe multisystemic disorders. To get insight into the spectrum of PEX gene defects among ZSS disorders and to investigate if additional human PEX genes are required for functional peroxisome biogenesis, we assigned over 600 ZSS fibroblast cell lines to different genetic complementation groups. These fibroblast cell lines were subjected to a complementation assay involving fusion by means of polyethylene glycol or a PEX cDNA transfection assay specifically developed for this purpose. In a majority of the cell lines we subsequently determined the underlying mutations by sequence analysis of the implicated PEX genes. The PEX cDNA transfection assay allows for the rapid identification of PEX genes defective in ZSS patients. The assignment of over 600 fibroblast cell lines to different genetic complementation groups provides the most comprehensive and representative overview of the frequency distribution of the different PEX gene defects. We did not identify any novel genetic complementation group, suggesting that all PEX gene defects resulting in peroxisome deficiency are currently known.     

Novel mutations in the PEX12 gene of patients with a peroxisome biogenesis disorder

The peroxisome biogenesis disorders (PBDs) form a genetically and clinically heterogeneous group of disorders due to defects in at least 11 distinct genes. The prototype of this group of disorders is Zellweger syndrome (ZS), with neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD) as milder variants. Liver disease, variable neurodevelopmental delay, retinopathy and perceptive deafness are common to PBDs. PBD patients belonging to complementation group 3 (CG3) have mutations in the PEX12 gene, which codes for a protein (PEX12) that contains two transmembrane domains, and a zinc-binding domain considered to be important for its interaction with other proteins of the peroxisomal protein import machinery. We report on the identification of five PBD patients belonging to CG3. Sequence analysis of their PEX12 genes revealed five different mutations, four of which have not been reported before. Four of the patients have mutations that disrupt the translation frame and/or create an early termination codon in the PEX12 open reading frame predicted to result in truncated protein products, lacking at least the COOH-terminal zinc-binding domain. All these patients display the more severe phenotypes (ZS or NALD). The fifth patient expresses two PEX12 alleles capable of encoding a protein that does contain the zinc-binding domain and displayed a milder phenotype (IRD). The three biochemical markers measured in fibroblasts (DHAPAT activity, C26:0 beta-oxidation and pristanic acid beta-oxidation) also correlated with the genotypes. Thus, the genotypes of our CG3 patients show a good correlation with the biochemical and clinical phenotype of the patients.     

Mouse PeP: a novel peroxisomal protein linked to myoblast differentiation and development.

The identification of several peroxisomal proteins in the past decade has deepened our understanding of the biology of peroxisomes and their involvement in human disorders. We report the cloning and expression pattern during the mouse development of a cDNA encoding a novel protein, named PeP, and show that its product is imported specifically to the peroxisome matrix in a variety of cell types. We also demonstrate that PeP is imported to the organelle through the PEX5 receptor pathway, which indicates that the C-terminal tripeptide SKI behaves as a type 1 peroxisomal targeting signal (PTS1). PeP expression is tightly regulated, as shown by Northern and in situ hybridization experiments. Thus during embryonic development in the mouse, PeP mRNA is detected almost exclusively in the skeletal muscle, whereas in adult mice, strong expression is also found in the heart and brain. In addition, PeP mRNA accumulation is induced after myoblast differentiation in vitro, when myotube formation is promoted. Sequence analysis reveals that PeP has no significant homology to any known protein, except for a short stretch of amino acids containing the fingerprint of the fibronectin type III superfamily, a domain present in proteins often related to molecular and cellular recognition and binding processes. Thus our data suggest a connection between the function of PeP and murine cell differentiation and development.     

Autosomal dominant Zellweger spectrum disorder caused by de novo variants in PEX14 gene

PurposeZellweger spectrum disorders (ZSDs) are known as autosomal recessive disorders caused by defective peroxisome biogenesis due to bi-allelic pathogenic variants in any of at least 13 different PEX genes. Here, we report 2 unrelated patients who present with an autosomal dominant ZSD.MethodsWe performed biochemical and genetic studies in blood and skin fibroblasts of the patients and demonstrated the pathogenicity of the identified PEX14 variants by functional cell studies.ResultsWe identified 2 different single heterozygous de novo variants in the PEX14 genes of 2 patients diagnosed with ZSD. Both variants cause messenger RNA mis-splicing, leading to stable expression of similar C-terminally truncated PEX14 proteins. Functional studies indicated that the truncated PEX14 proteins lost their function in peroxisomal matrix protein import and cause increased degradation of peroxisomes, ie, pexophagy, thus exerting a dominant-negative effect on peroxisome functioning. Inhibition of pexophagy by different autophagy inhibitors or genetic knockdown of the peroxisomal autophagy receptor NBR1 resulted in restoration of peroxisomal functions in the patients’ fibroblasts.ConclusionOur finding of an autosomal dominant ZSD expands the genetic repertoire of ZSDs. Our study underscores that single heterozygous variants should not be ignored as possible genetic cause of diseases with an established autosomal recessive mode of inheritance.     

Glaucoma-causing myocilin mutants require the Peroxisomal targeting signal-1 receptor (PTS1R) to elevate intraocular pressure

Glaucoma is a leading cause of worldwide irreversible visual impairment and blindness and is a clinically and genetically heterogenous group of optic neuropathies. Specific mutations in the myocilin (MYOC) gene cause primary open angle glaucoma (POAG) with varying age-of-onset and degree of severity. We show a mutation-dependent, gain-of-function association between human myocilin and the peroxisomal targeting signal type 1 receptor (PTS1R). There was correlation between the glaucoma phenotype and the specific MYOC mutations, with the more severe early-onset POAG mutations having a higher degree of association with PTS1R. Expression of human myocilin glaucomatous mutations in mouse eyes causes elevated intraocular pressure, which is a major phenotype of MYOC glaucoma. This is the first demonstration of a disease resulting from mutation-induced exposure of a cryptic signaling site that causes mislocalization of mutant protein to peroxisomes and the first disease-gene-based animal model of human POAG.