Pex7p is a WD40-containing protein involved in peroxisomal import of proteins containing an N-terminal peroxisome-targeting signal (PTS2). The interaction of human recombinant Pex7p expressed in different hosts/systems with its PTS2 ligand and other peroxins was analysed using various experimental approaches. Specific binding of human Pex7p to PTS2 could be demonstrated only when Pex7p was formed in vitro by a coupled transcription/translation system or synthesized in vivo in Chinese hamster ovary K1 cells transfected with a construct coding for a Pex7p-green fluorescent protein (GFP) fusion protein. Apparently, no cofactors are required and only monomeric Pex7p binds to PTS2. The interaction is reduced upon cysteine alkylation and is impaired upon truncation of the N-terminus of Pex7p. Interaction of Pex7p with other peroxins could not be demonstrated in bacterial or yeast two-hybrid screens, or in pull-down binding assays. The GFP fusion proteins, tagged at either the N- or C-terminus, were able to restore PTS2 import in rhizomelic chondrodysplasia punctata fibroblasts, and Pex7p-GFP was located both in the lumen of peroxisomes and in the cytosol.

Rhizomelic chondrodysplasia punctata (RCDP) is a rare autosomal recessive phenotype that comprises complementation group 11 of the peroxisome biogenesis disorders (PBD). PEX7, a candidate gene for RCDP identified in yeast, encodes the receptor for peroxisomal matrix proteins with the type-2 peroxisome targeting signal (PTS2). By homology probing we identified human and murine PEX7 genes and found that expression of either corrects the PTS2-import defect characteristic of RCDP cells. In a collection of 36 RCDP probands, we found two inactivating PEX7 mutations: one, L292ter, was present in 26 of the probands, all with a severe phenotype; the second, A218V, was present in three probands, including two with a milder phenotype. A third mutation, G217R, whose functional significance is yet to be determined, was present in five probands, all compound heterozygotes with L292ter. We conclude that PEX7 is responsible for RCDP (PBD CG11) and suggest a founder effect may explain the high frequency of L292ter.

Pex7p is a WD40-containing protein involved in peroxisomal import of proteins containing an N-terminal peroxisome-targeting signal (PTS2). The interaction of human recombinant Pex7p expressed in different hosts/systems with its PTS2 ligand and other peroxins was analysed using various experimental approaches. Specific binding of human Pex7p to PTS2 could be demonstrated only when Pex7p was formed in vitro by a coupled transcription/translation system or synthesized in vivo in Chinese hamster ovary K1 cells transfected with a construct coding for a Pex7p-green fluorescent protein (GFP) fusion protein. Apparently, no cofactors are required and only monomeric Pex7p binds to PTS2. The interaction is reduced upon cysteine alkylation and is impaired upon truncation of the N-terminus of Pex7p. Interaction of Pex7p with other peroxins could not be demonstrated in bacterial or yeast two-hybrid screens, or in pull-down binding assays. The GFP fusion proteins, tagged at either the N- or C-terminus, were able to restore PTS2 import in rhizomelic chondrodysplasia punctata fibroblasts, and Pex7p-GFP was located both in the lumen of peroxisomes and in the cytosol.

Pex7p is a WD40-containing protein involved in peroxisomal import of proteins containing an N-terminal peroxisome-targeting signal (PTS2). The interaction of human recombinant Pex7p expressed in different hosts/systems with its PTS2 ligand and other peroxins was analysed using various experimental approaches. Specific binding of human Pex7p to PTS2 could be demonstrated only when Pex7p was formed in vitro by a coupled transcription/translation system or synthesized in vivo in Chinese hamster ovary K1 cells transfected with a construct coding for a Pex7p-green fluorescent protein (GFP) fusion protein. Apparently, no cofactors are required and only monomeric Pex7p binds to PTS2. The interaction is reduced upon cysteine alkylation and is impaired upon truncation of the N-terminus of Pex7p. Interaction of Pex7p with other peroxins could not be demonstrated in bacterial or yeast two-hybrid screens, or in pull-down binding assays. The GFP fusion proteins, tagged at either the N- or C-terminus, were able to restore PTS2 import in rhizomelic chondrodysplasia punctata fibroblasts, and Pex7p-GFP was located both in the lumen of peroxisomes and in the cytosol.

Patients affected with Refsum disease (RD) have elevated levels of phytanic acid due to a deficiency of the peroxisomal enzyme phytanoyl-CoA hydroxylase (PhyH). In most patients with RD, disease-causing mutations in the PHYH gene have been identified, but, in a subset, no mutations could be found, indicating that the condition is genetically heterogeneous. Linkage analysis of a few patients diagnosed with RD, but without mutations in PHYH, suggested a second locus on chromosome 6q22-24. This region includes the PEX7 gene, which codes for the peroxin 7 receptor protein required for peroxisomal import of proteins containing a peroxisomal targeting signal type 2. Mutations in PEX7 normally cause rhizomelic chondrodysplasia punctata type 1, a severe peroxisomal disorder. Biochemical analyses of the patients with RD revealed defects not only in phytanic acid alpha-oxidation but also in plasmalogen synthesis and peroxisomal thiolase. Furthermore, we identified mutations in the PEX7 gene. Our data show that mutations in the PEX7 gene may result in a broad clinical spectrum ranging from severe rhizomelic chondrodysplasia punctata to relatively mild RD and that clinical diagnosis of conditions involving retinitis pigmentosa, ataxia, and polyneuropathy may require a full screen of peroxisomal functions.

The rhizomelic form of chondrodysplasia punctata (RCDP) is an autosomal recessive disease of peroxisome biogenesis characterized by deficiencies in several peroxisomal proteins, including the peroxisomal enzymes of plasmalogen biosynthesis and peroxisomal 3-ketoacyl thiolase. In cultured fibroblasts from patients with this disorder, both the peroxisomal targeting and proteolytic removal of the amino-terminal type 2 peroxisomal targeting sequence (PTS2) of thiolase are defective, whereas the biogenesis of proteins targeted by carboxyterminal type 1 peroxisomal targeting sequences (PTS1) is unimpaired. We have previously isolated a Saccharomyces cerevisiae peroxisomal biogenesis mutant, pex7 (formerly peb1/pas7), which demonstrates a striking similarity to the cellular phenotype of RCDP fibroblasts in that PTS1 targeting is functional, but the peroxisomal packaging of PTS2 targeted thiolase is lacking. Complementation of this mutant has led to the identification of the protein ScPex7p, a PTS2 receptor. In this paper we report cloning of the human orthologue of ScPEX7, and demonstrate that this is the defective gene in RCDP. We show that expression of human PEX7 in RCDP cells rescues PTS2 targeting and restores some activity of dihydroxyacetone phosphate acyltransferase (DHAP-AT), a peroxisomal enzyme of plasmalogen biosynthesis, and we identify the mutations responsible for loss of function of PEX7 in a compound heterozygote RCDP patient. These results imply that several peroxisomal proteins are targeted by PTS2 signals and that the various biochemical and clinical defects in RCDP result from a defect in the receptor for this class of PTS.

Rhizomelic chondrodysplasia punctata (RCDP) is a rare autosomal recessive phenotype that comprises complementation group 11 of the peroxisome biogenesis disorders (PBD). PEX7, a candidate gene for RCDP identified in yeast, encodes the receptor for peroxisomal matrix proteins with the type-2 peroxisome targeting signal (PTS2). By homology probing we identified human and murine PEX7 genes and found that expression of either corrects the PTS2-import defect characteristic of RCDP cells. In a collection of 36 RCDP probands, we found two inactivating PEX7 mutations: one, L292ter, was present in 26 of the probands, all with a severe phenotype; the second, A218V, was present in three probands, including two with a milder phenotype. A third mutation, G217R, whose functional significance is yet to be determined, was present in five probands, all compound heterozygotes with L292ter. We conclude that PEX7 is responsible for RCDP (PBD CG11) and suggest a founder effect may explain the high frequency of L292ter.

Patients affected with Refsum disease (RD) have elevated levels of phytanic acid due to a deficiency of the peroxisomal enzyme phytanoyl-CoA hydroxylase (PhyH). In most patients with RD, disease-causing mutations in the PHYH gene have been identified, but, in a subset, no mutations could be found, indicating that the condition is genetically heterogeneous. Linkage analysis of a few patients diagnosed with RD, but without mutations in PHYH, suggested a second locus on chromosome 6q22-24. This region includes the PEX7 gene, which codes for the peroxin 7 receptor protein required for peroxisomal import of proteins containing a peroxisomal targeting signal type 2. Mutations in PEX7 normally cause rhizomelic chondrodysplasia punctata type 1, a severe peroxisomal disorder. Biochemical analyses of the patients with RD revealed defects not only in phytanic acid alpha-oxidation but also in plasmalogen synthesis and peroxisomal thiolase. Furthermore, we identified mutations in the PEX7 gene. Our data show that mutations in the PEX7 gene may result in a broad clinical spectrum ranging from severe rhizomelic chondrodysplasia punctata to relatively mild RD and that clinical diagnosis of conditions involving retinitis pigmentosa, ataxia, and polyneuropathy may require a full screen of peroxisomal functions.