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Modification of proteins with small ubiquitin-related modifier (SUMO; SUMOylation) is involved in the regulation of various biological processes. Recent studies have demonstrated that noncovalent associations between SUMOylated proteins and co-operative proteins containing SUMO-interacting motifs (SIMs) are important for the spatiotemporal organization of many protein complexes. In this study, we demonstrate that interactions between lamin A, a major component of the nuclear lamina, and SUMO isoforms are dependent on one of the four SIMs (SIM3) resided in lamin A polypeptide in vitro. Live cell imaging and immunofluorescence staining showed that SIM3 is required for accumulation of lamin A on the chromosomes during telophase, and subsequent evaluation of a panel of deletion mutants determined that a 156-amino acid region spanning the carboxyl-terminal Ig-fold domain of lamin A is sufficient for this accumulation. Notably, mutation of SIM3 abrogated the dephosphorylation of mitosis-specific phosphorylation at Ser-22 of lamin A, which normally occurs during telophase, and the subsequent nuclear lamina reorganization. Furthermore, expression of a conjugation-defective SUMO2 mutant, which was previously shown to inhibit endogenous SUMOylation in a dominant-negative manner, also impaired the accumulation of wild type lamin A on telophase chromosomes. These findings suggest that interactions between SIM3 of lamin A and a putative SUMO2-modified protein plays an important role in the reorganization of the nuclear lamina at the end of mitosis.

The microenvironment of cells, which includes basement proteins, shear stress, and extracellular stimuli, should be taken into consideration when examining physiological cell behavior. Although microfluidic devices allow cellular responses to be analyzed with ease at the single-cell level, few have been designed to recover cells. We herein demonstrated that a newly developed microfluidic device helped to improve culture conditions and establish a clonality-validated human pluripotent stem cell line after tracing its growth at the single-cell level. The device will be a helpful tool for capturing various cell types in the human body that have not yet been established in vitro.

Chanarin–Dorfman syndrome (CDS) is a rare autosomal recessive disease of lipid metabolism; it is associated with congenital ichthyosis typed as non-bullous congenital ichthyosiform erythroderma (NCIE). CDS is characterized by the presence of an abnormally large number of cytosolic lipid droplets containing triacylglycerol (TG) in various tissues such as the skin, liver, and leukocytes. Mutations in the CGI-58 (also called ABHD5) gene encoding a 39-kDa protein of the α/β hydrolase domain subfamily have been shown to be responsible for this disorder. In adipocytes, CGI-58 is involved in TG degradation on lipid droplets; in doing so, it coordinates with several lipolytic factors including perilipin, a member of the PAT protein family, and ATGL, a putative rate-limiting lipase in adipocytes. In quiescent adipocytes, CGI-58 interacts with perilipin on the surfaces of lipid droplets. Upon hormonal stimulation, CGI-58 facilitates massive lipolysis by activating ATGL. Some CGI-58 mutations found in CDS patients cancel the ability to interact with perilipin or activate ATGL, indicating that the loss of these interactions is physiologically important. However, based on the tissue distributions of these lipolytic factors, there are likely multiple molecular targets of CGI-58 actions. This in turn gives rise to the multiple phenotypes of CDS, such as ichthyosis, liver steatosis, or neurosensory diseases.

Promoters of Drosophila melanogaster genes related to DNA replication, such as those for the 180-kDa catalytic subunit of DNA polymerase α and proliferating cell nuclear antigen (PCNA), contain a common 8-bp palindromic sequence (5′-TATCGATA-3′), named the DNA replication-related element (DRE) (12). These DREs are required for promoter activities both in cultured cells and in flies in vivo (41). We have purified the DRE-binding factor (DREF) from cultured Drosophila cells, consisting of an 86-kDa polypeptide homodimer specifically binding to DRE, and isolated a cDNA (12, 13). The importance of Drosophila DREF in development has been demonstrated from studies using transgenic flies (11, 14, 44). For example, ectopic expression of Drosophila DREF in eye imaginal disc cells behind the morphogenetic furrow, which are normally postmitotic, induced ectopic DNA synthesis and apoptosis and abolished photoreceptor specifications (11). More recently, we and Hyun et al. have succeeded in knocking down Drosophila DREF expression in various tissues (16, 45). Decreased levels of DREF in developing wing and eye imaginal discs were associated with reduction in wing size with smaller cells and drastically aberrant small and rough eyes, respectively. These lines of evidence indicate that the Drosophila DRE/DREF system performs important roles in regulation of cell growth as well as cell proliferation during development. How many and what kind of genes other than those described above are under control of the Drosophila DRE/DREF system? Immunostaining of polytene chromosomes of salivary glands revealed that Drosophila DREF binds to hundreds of loci (8, 10), and recent computational analysis of core promoters in the Drosophila genome showed DRE to be the second most frequent motif apparent in core promoter sequences from −60 to +40, with a frequency higher than those for the TATA box and initiator sequences (4, 24). Already, we and others have demonstrated that the Drosophila DRE/DREF system regulates a number of Drosophila genes involved in DNA replication as well as those involved in cell cycle progression through S (dE2F1) (33) and G2/M (D-raf, D-myb, and cyclin A) phases (25, 29, 34). Despite much progress in understanding the Drosophila DRE/DREF system, little is known about the corresponding mammalian DRE/DREF system. We have identified a human homologue of DREF (hDREF) and a binding consensus sequence [hDRE; 5′-TGTCG(C/T)GA(C/T)A-3′] (26). Our previous study showed that the expression level of hDREF is elevated during the G1-to-S transition and reaches a maximum at S phase in normal human fibroblasts. Moreover, RNA interference experiments targeting hDREF pointed to an important role in cell cycle progression. We also demonstrated that the histone H1 gene carrying an hDRE is regulated by hDREF (26). However, other functions and target genes of hDREF remain to be clarified. The ribosome is a vital organelle which is responsible for protein synthesis in all living organisms. Production of mature ribosomes consisting of rRNAs and ribosomal proteins (RPs) requires a highly coordinated multistep process, and many reports have shown that the initiation of rRNA transcription is tightly linked to cell cycle progression, with synthesis of rRNA increasing during G1 phase, becoming maximal in S and G2 phases, and being repressed during mitosis (7, 20, 22). Similarly, coordinated synthesis of all RP genes during the cell cycle, leading to the precise and equimolar production of the 79 RPs necessary for ribosome biogenesis and translation control, is required to support adequate protein synthesis (37). Despite the obvious importance of RP gene expression for cell proliferation, only a limited number of experimental studies of mammalian RP gene promoter structures and transcriptional regulation have been performed so far. Recent determination of a complete map and nucleotide sequences now allows searches for regulatory elements common to a number of human RP genes, and Perry has identified “box A” [5′-TCTCGCGA(G/T)-3′] as one of the most conserved sequences in RP gene promoters (28). In this report, we document evidence that hDREF is a transcription factor essential for cell proliferation which binds to box A and positively regulates a set of human RP genes. We also show that mammalian RP gene expression is induced during late G1 and S phases and that fluctuation of hDREF expression during the cell cycle is in line with cell cycle-dependent expression of RP genes.

The human transcription factor DNA replication-related element-binding factor (hDREF) is essential for the transcription of a number of housekeeping genes. The mechanisms underlying constitutively active transcription by hDREF were unclear. Here, we provide evidence that hDREF possesses small ubiquitin-like modifier (SUMO) ligase activity and can specifically SUMOylate Mi2α, an ATP-dependent DNA helicase in the nucleosome remodeling and deacetylation complex. Moreover, immunofluorescent staining and biochemical analyses showed that coexpression of hDREF and SUMO-1 resulted in dissociation of Mi2α from chromatin, whereas a SUMOylation-defective Mi2α mutant remained tightly bound to chromatin. Chromatin immunoprecipitation and quantitative RT-PCR analysis demonstrated that Mi2α expression diminished transcription of the ribosomal protein genes, which are positively regulated by hDREF. In contrast, coexpression of hDREF and SUMO-1 suppressed the transcriptional repression by Mi2α. These data indicate that hDREF might incite transcriptional activation by SUMOylating Mi2α, resulting in the dissociation of Mi2α from the gene loci. We propose a novel mechanism for maintaining constitutively active states of a number of hDREF target genes through SUMOylation.

Peroxisome proliferator-activated receptors (PPARs) constitute a subfamily of nuclear receptor superfamily. A wide variety of compounds including hypolipidemic agents, antidiabetic drugs, and long-chain fatty acids are the potential ligands of PPARs. To approach the regulatory mechanisms of PPARs, we studied on two subjects in this work. First, we identified a functional PPAR-binding site in the spacer region between the PEX11alpha and perilipin genes, which are arranged in tandem on the mouse genome. By gene reporter assays and in vivo as well as in vitro binding assays, we show that these genes are regulated tissue-selectively through this common binding site: The PEX11alpha gene is activated by PPARalpha in the liver, whereas the perilipin gene by PPARgamma in the adipose tissue. As the second subject, we found that PPARgamma2 is conjugated with small ubiquitin-related modifier (SUMO) at a specific lysine residue in the amino-terminal region. By site-directed mutagenesis combined with gene reporter assays and sumoylation analyses, we show that sumoylation represses the ligand-independent transactivating function carried by this region, and hence negatively regulates the whole transactivating competence of PPARgamma2. In addition, phosphorylation at a specific site in the amino-terminal region represses the transactivation by PPARgamma2 possibly through enhancing sumoylation.

Human mesenchymal stem cells (hMSCs) are capable of differentiating into several cell types including adipocytes, osteoblasts, and chondrocytes, under appropriate culture conditions. We found that SP600125, an inhibitor of Jun N-terminal kinase (JNK), promoted adipogenesis whereas it repressed osteogenesis from hMSCs. SP600125 increased the expression of adipogenic transcription factors, CCAAT/enhancer-binding proteins alpha and beta as well as peroxisome proliferator-activated receptor gamma2, which suggested that the chemical acted on the early steps of transcriptional regulatory cascade in adipogenesis. A gene reporter assay showed that SP600125 and a dominant negative JNK promoted a transcriptional activity dependent on the cAMP-response element (CRE). Thus, JNK represses adipogenesis from hMSCs probably by, at least in part, inhibiting the transactivating function of CRE-binding protein. Another action of JNK, phosphorylation at Ser(307) of insulin receptor substrate-1, was also predicted to contribute to the repression of adipogenesis.

SKIP has been described as a transcriptional coregulator as well as a spliceosome component, but the relationship between these functions is not clear. We found that SKIP activated reporter gene expression from the basal promoters of viral origin. SKIP exhibited more prominent effect on the promoters with stronger activities, in an experiment employing a series of reporter constructs carrying different numbers of GC boxes. We also found that SKIP suppressed aberrant splicing at a cryptic splice donor site in the luciferase reporter gene. In addition, SKIP suppressed splicing of an extra intron created by a beta-thalassemia mutation in the human beta-globin gene. In the transfection experiment, an intronless reporter exhibited a higher level of expression, but was less significantly activated by SKIP, than the intron-containing reporter. These results indicate that SKIP affects gene expression by both transcriptional activation and regulation of pre-mRNA splicing.

Most cis-acting regulatory elements have generally been assumed to activate a single nearby gene. However, many genes are clustered together, raising the possibility that they are regulated through a common element. We show here that a single peroxisome proliferator response element (PPRE), located between the mouse PEX11 alpha and perilipin genes, confers on both genes activation by peroxisome proliferator-activated receptor alpha (PPAR alpha) and PPAR gamma. A functional PPRE 8.4 kb downstream of the promoter of PEX11 alpha, a PPAR alpha target gene, was identified by a gene transfection study. This PPRE was positioned 1.9 kb upstream of the perilipin gene and also functioned with the perilipin promoter. In addition, this PPRE, when combined with the natural promoters of the PEX11 alpha and perilipin genes, conferred subtype-selective activation by PPAR alpha and PPAR gamma 2. The PPRE sequence specifically bound to the heterodimer of RXR alpha and PPAR alpha or PPAR gamma 2, as assessed by electrophoretic gel mobility shift assays. Furthermore, tissue-selective binding of PPAR alpha and PPAR gamma to the PPRE was demonstrated in hepatocytes and adipocytes, respectively, by chromatin immunoprecipitation assay. Hence, the expression of these genes is induced through the same PPRE in the liver and adipose tissue, where the two PPAR subtypes are specifically expressed.

Pex6p belongs to the AAA family of ATPases. Its CHO mutant, ZP92, lacks normal peroxisomes but contains peroxisomal membrane remnants, so called peroxisomal ghosts, which are detected with anti–70-kDa peroxisomal membrane protein (PMP70) antibody. No peroxisomal matrix proteins were detected inside the ghosts, but exogenously expressed green fluorescent protein (GFP) fused to peroxisome targeting signal-1 (PTS-1) accumulated in the areas adjacent to the ghosts. Electron microscopic examination revealed that PMP70-positive ghosts in ZP92 were complex membrane structures, rather than peroxisomes with reduced matrix protein import ability. In a typical case, a set of one central spherical body and two layers of double-membraned loops were observed, with endoplasmic reticulum present alongside the outer loop. In the early stage of complementation by PEX6 cDNA, catalase and acyl-CoA oxidase accumulated in the lumen of the double-membraned loops. Biochemical analysis revealed that almost all the peroxisomal ghosts were converted into peroxisomes upon complementation. Our results indicate that 1) Peroxisomal ghosts are complex membrane structures; and 2) The complex membrane structures become import competent and are converted into peroxisomes upon complementation with PEX6.

Mutation of the lamin A gene (LMNA) causes a diverse range of diseases referred to as laminopathies. Because most laminopathies have a dominant inheritance pattern and progress gradually, cultured cells stably expressing mutant lamin A at the same level as endogenous wild-type cells are required for chronological analysis. In this study, we showed that an expression system involving a lentiviral vector that carries the human metallothionein gene basal promoter ensures stable and basal-level expression of proteins and is thus suitable for investigating the properties of lamin A mutants. The small ubiquitin-related modifier (SUMO) modification (SUMOylation)-defective E203G mutant that is associated with familial dilated cardiomyopathy exhibited abnormal subnuclear distribution and inhibited normal localization of WT lamin A in a dominant-negative manner. Low-level and long-term expression of the E203G mutant resulted in multinucleated giant cells, aberrant lipid droplet accumulation in the cytoplasm and premature senescence. Expression of another SUMOylation-defective mutant (K201R) did not induce any phenotypes observed in cells expressing E203G. These results indicate that the E203G mutant may inhibit the normal functions of wild-type lamin A in a dominant-negative manner, but a defect in SUMOylation itself may not be involved in disease pathogenesis.

Peroxisome biogenesis disorders (PBDs) contain various clinical phenotypes; Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD), decreasing in the clinical severity in this order. We found that all IRD cell lines and some NALD lines belonging to several different complementation groups are temperature-sensitive in peroxisome assembly; that is, they lacked catalase-positive peroxisomes at 37 degrees C, but do gain the peroxisomes at 30 degrees C. We identified heterozygous mutations E55K/R119Stop in the PEX2 gene of an IRD patient of complementation group F. The E55K mutation was the direct cause of the temperature-sensitivity because similar phenotypes could be transferred to PEX2-defective CHO cells by transfecting the mutant gene. Thus, temperature-sensitive peroxisome assembly is representative of milder forms of PBDs.

We have found that peroxisome assembly is temperature-sensitive (ts) in mild forms of peroxisome biogenesis disorders (PBDs), that is all infantile Refsum disease (IRD) patients and a few neonatal adrenoleukodystrophy patients of several complementation groups. The number of peroxisomes increased daily in incubation at 30 degrees C in the ts cells. Oxidation of very long-chain fatty acids, processing of acyl-CoA oxidase and dihydroxyacetonephosphate acyltransferase activity also improved after 8 days incubation at 30 degrees C in the IRD fibroblasts. These biochemical functions of the peroxisome did not change at 30 degrees C in Zellweger fibroblasts. Number of peroxisomes gradually decreased after 4 days when the temperature shifted from 30 to 37 degrees C in the ts cells. These results indicate that the biochemical functions of peroxisome are also restored by incubation at 30 degrees C in the mild and ts phenotype of PBDs, and the results will aid to predict the severity and the prognosis of affected children.

Nuclear matrix is a complex intranuclear network supposed to be involved in the various nuclear functions. In order to identify the nuclear matrix proteins, we isolated a cDNA clone from a human placenta cDNA library. This clone was partially represented a known cDNA clone HA1237. HA1237 encoded a 631-amino-acid peptide, which we designated NXP-1. NXP-1 was related to yeast Rad21/Scc1/Mcd1, Xenopus XRAD21, and mouse PW29, and identical with HR21spA isolated from a human testis cDNA library. We developed a polyclonal antibody to the purified NXP-1 bacterially expressed as a fusion protein with GST. Western blot analysis with anti-NXP-1 polyclonal antibody showed nuclear matrix localization of NXP-1 in HeLa cells. Indirect immunofluorescence staining also showed nuclear and nuclear matrix localization of the NXP-1. Results of in vitro binding assays employing nuclear matrix preparations indicated that the N-terminal region (16–128 amino acid) of NXP-1 has an important role in nuclear matrix distribution.

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.

We thank T. Hashimoto and N. Usuda for anti-human and anti-rat catalase antibodies, respectively, and we thank H. W. Moser, A. B. Moser, R. J. A. Wanders, G. T. N. Besley, B. C. Paton, and A. Poulos for patients' fibroblasts. This work was supported in part by Grants-in-Aid for Scientific Research and Exploratory Research, from the Ministry of Education, Science, Sports and Culture of Japan, and by grants from the Sumitomo Foundation and Uehara Memorial Foundation.

HumanPEX1(HsPEX1) is the causative gene for peroxisome-deficiency disorders such as Zellweger syndrome of complementation group I, encoding the peroxin, Pex1p, a member of AAA family. Pex1p tagged with an epitope flag was expressed in wild-type Chinese hamster ovary (CHO) cell, CHO-K1. Pex1p was localized in the cytoplasm, as assessed by immunofluorescent microscopy. Cell-lysate ofHsPEX1-transfected CHO-K1 was incubated within vitrosynthesized35S-labelled Pex6p, an AAA family peroxin. Immunoprecipitation of Pex1p using anti-Pex1p antibody resulted in concomitant recovery of35S-Pex6p. Conversely,35S-Pex1p was obtained in immunoprecipitate from CHO-K1 expressing human Pex6p, using anti-Pex6p antibody. These results strongly suggest that Pex1p and Pex6p interact with each other.

Ten complementation groups of generalized peroxisome biogenesis disorders (PBD), (excluding rhizomelic chondrodysplasia punctata) have been identified using complementation analysis. Four of the genes involved have been identified using two different methods of (1) genetic functional complementation of peroxisome deficient CHO cell mutants and (2) homology searches for human dbEST, based on yeast genes involved in peroxisome biogenesis (PEX genes). We report here the first identification of a new complementation group which is genetically different from peroxisome deficient CHO mutants. There were no complementations by the human PEX 13 gene. The nature of the related gene is being investigated.

We thank K. Hori for technical assistance, M. Ohara for helpful comments, and K. Kamijo for human PMP70 cDNA and an anti-ALDP antibody. This study was supported in part by a grant-in-aid for scientific research (08670870) from the Ministry of Education, Science, Sports, and Culture of Japan, by a research grant from the National Center of Neurology and Psychiatry of the Ministry of Health and Welfare of Japan, and by a research grant from Ono Medical Research Foundation.

Polymethylene-interrupted (PMI)-polyunsaturated fatty acids (PUFA) are fatty acids present largely in gymnosperm. Sciadonic acid (SciA, 20:3 Δ-5,11,14) and juniperonic acid (JA, 20:4 Δ-5,11,14,17) are typical C20 PMI-PUFA with an isolated double bond at Δ5. Previously, we found that SciA and JA are converted to linoleic acid (LNA) and α-linolenic acid (ΑLA), respectively. The conversion process includes chain-shortening step by peroxisomal β-oxidation for elimination a double bond at Δ5, and subsequent chain-elongation step in microsomes. In this study, we examined the substrate specificity of this metabolism in rodent and human cells. Supplementation of SciA, eicosadienoic acid (EDA, 20:2 Δ-11,14) or JA to CHO-K1 cells (wild type) induced an accumulation of LNA, LNA or ALA, respectively, in cellular lipids. These changes were not observed in the peroxisomes-deficient CHO cells, indicating involvement of peroxisomes in the metabolism. Two types of human cells (MKN74 and HepG2) also converted the C20 PMI-PUFA and EDA to the respective essential fatty acids. In contrast, no chain-shortened metabolite of pinolenic acid (18:3 Δ-5,9,12) was detected in any cell lines tested. From these results, C20 PMI-PUFA and EDA, but not C18 PMI-PUFA, are suggested as being effectively converted to essential fatty acids by the fatty acid remodeling system in rodent and human cells.

Using a probe obtained by PCR amplification, a full-length cDNA encoding squalene synthase was isolated from a mouse liver cDNA library. Its nucleotide sequence had an open reading frame for a 416 amino acid polypeptide (calculated molecular mass, 48 kDa). In vitro transcription of the cDNAfollowed by in vitro translation produced a protein of the expected size. The deduced amino acid sequence was 93%, 88% and 46% identical to those of the rat, human and budding yeast squalene synthases, respectively. Blotting analyses showed that the mRNA is 1.6 kb in size and that less than two copies of the gene are present in the mouse genome. To establish the enzyme activity, the entire coding region was subcloned into an expression plasmid so that it was in frame with the N-terminal region of β-galactosidase. Escherichia coli , which was transformed with the recombinant plasmid, expressed high activity of converting farnesyl diphosphate into squalene.

The role of the histidine residue at position −17 of the amino-terminal signal peptide of rat peroxisomal 3-ketoacyl-CoA thiolase was studied in vivo , employing site-directed mutagenesis. Among the nine amino acids tested, only glutamine could partially substitute for the histidine. Mutants carrying basic amino acids, arginine and lysine, and hydrophobic residues, leucine and valine, in place of histidine were all translocated to mitochondria, but not to peroxisomes. These results indicate that the signal peptide of the thiolase is recognized by a mechanism totally different from that for the SKL motif, a known peroxisomal targeting signal. Relationship of the thiolase signal peptide to those of mitochondrial proteins is discussed.

Genetic heterogeneity in peroxisome-deficient disorders, including Zellweger's cerebrohepatorenal syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease, was investigated. Fibroblasts from 17 patients were fused using polyethylene glycol, cultivated on cover slips, and the formation of peroxisomes in the fused cells was visualized by immunofluorescence staining, using anti-human catalase IgG. Two distinct staining patterns were observed: (1) peroxisomes appeared in the majority of multinucleated cells, and (2) practically no peroxisomes were identified. Single step 12-(1'-pyrene) dodecanoic acid/ultraviolet (P12/UV)-selection confirmed that the former groups were resistant to this selection, most of the surviving cells contained abundant peroxisomes, and the latter cells died. In the complementary matching, [1-14C]lignoceric acid oxidation and the biosynthesis of peroxisomal proteins were also normalized. Five complementation groups were identified. Group A: Zellweger syndrome and infantile Refsum disease; Groups B, C and D: Zellweger syndrome; Group E: Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease. We compared these groupings with those of Roscher and identified eight complementation groups. There was no obvious relation between complementation groups and clinical phenotypes. These results indicate that the transport, intracellular processing and function of peroxisomal proteins were normalized in the complementary matching and that at least eight different genes are involved in the formation of normal peroxisomes and in the transport of peroxisomal enzymes.

3-Ketothiolase deficiency (3KTD) stems from a deficiency of mitochondrial acetoacetyl-coenzyme A thiolase (T2). We analyzed the molecular basis of 3KTD in two generations of a family. A boy (patient 2, GK04), his father (patient 1, GK05), his mother, and his brother were studied; three mutant alleles of T2 gene were identified. Patient 1 is a compound heterozygote: one allele has a point mutation of G to A at position 547 on his T2 cDNA, causing Gly150 to Arg substitution of the mature T2 subunit, and the other allele has GT to TT transition at the 5' splice site of intron 8, causing exon 8's skipping of the T2 cDNA. Patient 2 is also a compound heterozygote: one allele inherited from his mother has AG to CG transition at the 3' splice site of intron 10, causing exon 11's skipping of the T2 cDNA, and the other allele derived from patient 1 has the G to A mutation (Gly to Arg). The brother of patient 2 is an obligatory carrier with the mutant allele causing the exon 8 skipping. This report seems to be the first complete molecular definition of 3KTD at the gene level.

To examine the function of the amino-terminal presequence of rat peroxisomal 3-ketoacyl-CoA thiolase precursor, fusion proteins of various amino-terminal regions of the precursor with non-peroxisomal enzymes were expressed in cultured mammalian cells. On immunofluorescence microscopy, all constructs carrying the presequence part exhibited punctate patterns of distribution, identical with that of catalase, a peroxisomal marker. Proteins lacking all or a part of the prepiece were found in the cytosol. These results indicate that the presequence of the thiolase has sufficient information for peroxisomal targeting.

To examine 3-ketothiolase deficiency at the gene level, we analyzed the structure of the human mitochondrial acetoacetyl-CoA thiolase (MAT; EC 2.3.1.9)-encoding gene ( MAT ). From the genomic library of a normal subject in λEMBL3, we isolated seven overlapping clones covering the entire length of MAT and the structural organization was determined. The gene spans approx. 27 kb and contains twelve exons interrupted by eleven introns. The 5′-flanking region of the gene lacks a conventional TATA box, but is G+C-rich and contains two CAAT boxes. Included are a putative binding site for the transcription factor, Sp1, and sequences resembling the binding sites of several other transcription factors, all features characteristics of housekeeping genes. A CAT assay revealed that a 101-bp DNA fragment immediately upstream from the cap site has promoter activity, and suggested that a DNA fragment from bp −8 to −102 probably contains a negative regulatory element(s).

The molecular basis of 3-ketothiolase deficiency (3KTD) was examined in a 3KTD family. Immunochemical analyses showed that mitochondrial acetoacetyl-CoA thiolase (T2) biosynthesized in the patient's fibroblasts (GK06) was unstable and that the parents and brother were obligatory carriers of 3KTD. When sequencing the PCR-amplified patient's T2cDNA, we noted a G to A replacement which caused 347 Ala to Thr substitution of the mature T2 subunit. Transfection analysis revealed that this substitution resulted in an instability of the T2 protein. Analyses of the T2 cDNA and gene of the family indicated that the patient was a compound heterozygote; the allele that derived from the mother had a point mutation ( 347 Ala to Thr) and the other allele from the father has a mutation which would abolish the T2 gene expression. This report is apparently the first definition of a mutant allele for 3KTD, at the gene level.

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 β 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. Keywords: Zellweger syndrome; infantile Refsum disease; PEX gene; mosaic

Perilipin is a protein localized on lipid droplet surfaces in adipocytes and steroidogenic cells, playing a central role in regulated lipolysis. Expression of the perilipin gene is markedly induced during adipogenesis. We found that transcription from the perilipin gene promoter is activated by an orphan nuclear receptor, estrogen receptor-related receptor (ERR)alpha. A response element to this receptor was identified in the promoter region by a gene reporter assay, the electrophoretic-gel mobility-shift assay and the chromatin immunoprecipitation assay. Peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha enhanced, whereas small heterodimer partner (SHP) repressed, the transactivating function of ERRalpha on the promoter. Thus, the perilipin gene expression is regulated by a transcriptional network controlling energy metabolism, substantiating the functional importance of perilipin in the maintenance of body energy balance.

The 70-kDa peroxisomal membrane protein (PMP70) is one of the major integral membrane proteins of rat liver peroxisomes. cDNA clones for PMP70 were isolated and sequenced. The predicted amino acid sequence (659 amino acid residues) revealed that the carboxyl-terminal region of PMP70 has strong sequence similarities to a group of ATP-binding proteins such as MalK and Mdr. These proteins form a superfamily and are involved in various biological processes including membrane transport. Limited protease treatment of peroxisomes showed that the ATP-binding domain of PMP70 is exposed to the cytosol. The hydropathy profile, in comparison with those of several other members of the ATP-binding protein superfamily, suggests that PMP70 is a transmembrane protein possibly forming a channel. Based on these results, we propose that PMP70 is involved in active transport across the peroxisomal membrane.

Estrogen receptor-related receptors (ERRs) are orphan members of the nuclear receptor superfamily. A single AGGTCA sequence element preceded by three conserved nucleotides has been identified as a specific recognition motif of ERRs. Here we performed systematic analyses of target sequences on all three ERR subtypes, alpha, beta and gamma. In electrophoretic gel-mobility shift assay and transcriptional reporter assays, they exhibited similar patterns of recognition specificities, showing extremely broad ranges of target sequences. We searched a mouse promoter database for a gene carrying possible ERR-binding sequences. The Rb-1 inducible coiled-coil 1 (Rb1cc1) gene was found to contain two putative ERR binding elements, named response element (RE)-1 and RE-2, in the promoter region. In gene reporter assays, RE-2, but not RE-1, functioned as an effective cis-regulatory element for transactivation by ERRalpha in the presence of a coactivator, peroxisome proliferator-activated receptor gamma coactivator-1alpha. Mutational analyses suggested that RE-2 is recognized by ERRalpha partly as a monovalent element, but also as a direct repeat motif separated by four spacer nucleotides. In vivo binding of ERRalpha to the Rb1cc1 promoter region was confirmed by the chromatin immunoprecipitation assay. Thus, Rb1cc1 is a target gene of ERRalpha, driven by a novel type of recognition sequence.

We previously demonstrated that hDREF, a human homologue of Drosophila DNA replication-related element binding factor (dDREF), is a DNA-binding protein predominantly distributed with granular structures in the nucleus. Here, glutathione S-transferase pulldown and chemical cross-linking assays showed that the carboxyl-terminal hATC domain of hDREF, highly conserved among hAT transposase family members, possesses self-association activity. Immunoprecipitation analyses demonstrated that hDREF self-associates in vivo, dependent on hATC domain. Moreover, analyses using a series of hDREF mutants carrying amino acid substitutions in the hATC domain revealed that conserved hydrophobic amino acids are essential for self-association. Immunofluorescence studies further showed that all hDREF mutants lacking self-association activity failed to accumulate in the nucleus. Self-association-defective hDREF mutants also lost association with endogenous importin beta1. Moreover, electrophoretic gel-mobility shift assays revealed that the mutations completely abolished the DNA binding activity of hDREF. These results suggest that self-association of hDREF via the hATC domain is necessary for its nuclear accumulation and DNA binding. We also found that ZBED4/KIAA0637, another member of the human hAT family, also self-associates, again dependent on the hATC domain, with deletion resulting in loss of efficient nuclear accumulation. Thus, hATC domains of human hAT family members appear to have conserved functions in self-association that are required for nuclear accumulation.

Perilipin is an adipocyte-specific protein associated with lipid droplets that is crucial for the regulation of storage and mobilization of lipids. We earlier reported that the mouse perilipin gene is regulated by peroxisome proliferator-activated receptor (PPAR) gamma through a peroxisome proliferator-response element (PPRE) positioned upstream of the perilipin promoter. Moreover, we showed that this PPRE also controls expression of the PEX11alpha gene, which is located further upstream. We show here that three elements, A, B, and C, in close proximity downstream of the PPRE, are essential for transactivation of the perilipin gene by PPARgamma. Electrophoretic gel-mobility shift assays demonstrated that nuclear factor (NF)-1 subtypes bind specifically to element B. Furthermore, chromatin immunoprecipitation using 3T3-L1 cells revealed that NF-1A and NF-1B bind to element B in a differentiation-dependent fashion, whereas binding is constitutive with NF-1C and NF-1X. Element C is likely to be a binding motif for nuclear receptors. With PPARalpha, elements A-C do not appear to be required for transactivation of the PEX11alpha gene, so that cooperation with other transcription factors may be differentially involved in selective transactivation of the PEX11alpha and perilipin genes by different PPAR subtypes.

Peroxisomes are ubiquitous organelles in eukaryotic cells and surrounded by a single membrane, and undergo considerable changes in size, shape and number. Peroxisomal disorders are classified into two categories: peroxisome biogenesis disorders (PBDs) and single-enzyme deficiencies (SEDs). Morphologically aberrant peroxisomes called 'peroxisomal ghosts' in PBDs are well known, however, a morphological approach to the study of peroxisomes in SEDs has been rarely reported. Here, we investigated the morphology of peroxisomes in cultured fibroblasts from patients lacking peroxisomal beta-oxidation enzymes, including acyl-CoA oxidase (AOX) or D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (D-BP). Morphological analysis by immunofluorescence examination using an antibody against catalase revealed a smaller number of large peroxisomes in fibroblasts from these patients. Moreover, immunoelectron microscopy using an antibody against the 70-kDa peroxisomal membrane protein (PMP70) showed large peroxisomes with various horseshoe-shaped membrane structures. These results give an important clue to elucidating the division of peroxisomes and how peroxisomes change in size, shape, number and position within cells, which are subjects for future study.

Peroxisome proliferator-activated receptor (PPAR)-gamma2, a member of the nuclear hormone receptor superfamily, plays a key role in adipocyte differentiation. Its amino-terminal region carries a ligand-independent gene-activating function, AF-1, and is composed of activation as well as repression domains. We have found PPARgamma2 and its isoform, PPARgamma1, to be modified by small ubiquitin-related modifier (SUMO)-1 in vivo, at a lysine residue in the repression domain. In reporter assays, a sumoylation-defective K107R mutant of PPARgamma2 exhibited much stronger transactivation than the wild-type, comparable with that of a mutant deleted for the repression domain. A close inverse correlation was observed between the levels of sumoylation and transactivation by PPARgamma2, in analyses employing PPARgamma2 forms with mutations in the sumoylation motif and a dominant-negative mutant of the SUMO conjugating enzyme, Ubc9. Studies with phosphorylation-defective mutants suggested that phosphorylation at S112 of PPARgamma2 promotes K107 sumoylation, and this latter exerts the more potent repressive effects. The K107R mutant PPARgamma2, when infected into NIH3T3 cells with a viral vector, promoted differentiation into adipocytes more efficiently than the wild-type. These observations provide evidence that sumoylation is involved in negative regulation of the transactivating function of PPARgamma2.

Estrogen receptor-related receptors (ERRs) constitute a subfamily of the nuclear hormone receptor superfamily. ERRs are closely related to estrogen receptors (ERs), but apparently lack ligand dependence. In this study, we cloned rat ERRgamma as an interacting partner of an orphan nuclear receptor, small heterodimer partner (SHP). ERRgamma exhibited significant binding affinities with a wide spectrum of sequences: inverted and direct repeat motifs composed of AGGTCA half-sites with various spacings, as well as a monovalent motif of the same sequence carrying extra T(C/G)A trinucleotides on the 5' side. On the other hand, inverted repeat spaced by three nucleotides was dominantly efficient for the binding of ERalpha. These results were mostly consistent with those of gene reporter assays. ERRgamma bound as a homodimer to all binding sequences tested, including a monovalent binding site, and ERRgamma did not heterodimerize with ERalpha. Taken together, ERRgamma recognizes a tremendously broad range of sequences as a homodimer. Finally, we found that SHP efficiently represses the transcriptional activity of ERRgamma, even at a far lower concentration than that of ERRgamma.

Lipid droplets (LDs) are a class of ubiquitous cellular organelles that are involved in lipid storage and metabolism. Although the mechanisms of the biogenesis of LDs are still unclear, a set of proteins called the PAT domain family have been characterized as factors associating with LDs. Perilipin, a member of this family, is expressed exclusively in the adipose tissue and regulates the breakdown of triacylglycerol in LDs via its phosphorylation. In this study, we used a yeast two-hybrid system to examine the potential function of perilipin. We found direct interaction between perilipin and CGI-58, a deficiency of which correlated with the pathogenesis of Chanarin-Dorfman syndrome (CDS). Endogenous CGI-58 was distributed predominantly on the surface of LDs in differentiated 3T3-L1 cells, and its expression increased during adipocyte differentiation. Overexpressed CGI-58 tagged with GFP gathered at the surface of LDs and colocalized with perilipin. This interaction seems physiologically important because CGI-58 mutants carrying an amino acid substitution identical to that found in CDS lost the ability to be recruited to LDs. These mutations significantly weakened the binding of CGI-58 with perilipin, indicating that the loss of this interaction is involved in the etiology of CDS. Furthermore, we identified CGI-58 as a binding partner of ADRP, another PAT domain protein expressed ubiquitously, by yeast two-hybrid assay. GFP-CGI-58 expressed in non-differentiated 3T3-L1 or CHO-K1 cells was colocalized with ADRP, and the CGI-58 mutants were not recruited to LDs carrying ADRP, indicating that CGI-58 may also cooperate with ADRP.

Using a monoclonal antibody that recognizes a nuclear matrix protein, we selected a cDNA clone from a lambdagt11 human placenta cDNA library. This cDNA encoded a 939-amino acid protein designated nuclear matrix protein NXP-2. Northern blot analysis indicated that NXP-2 was expressed in various tissues at different levels. Forcibly expressed green fluorescent protein-tagged NXP-2 as well as endogenous NXP-2 was localized in the nucleus and distributed to the nuclear matrix. NXP-2 was released from the nuclear matrix when RNase A was included in the buffer for nuclear matrix preparation. Mapping of functional domains was carried out using green fluorescent protein-tagged truncated mutants of NXP-2. The region of amino acids 326-353 was responsible for nuclear matrix binding and contained a cluster of hydrophobic amino acids that was similar to the nuclear matrix targeting signal of acute myeloleukemia protein. The central region (amino acids 500-591) was demonstrated to be required for RNA binding by Northwestern analysis, although NXP-2 lacked a known RNA binding motif. The region of amino acid residues 682-876 was predicted to have a coiled-coil structure. The RNA-binding, nuclear matrix-binding, and coiled-coil domains are structurally separated, suggesting that NXP-2 plays important roles in diverse nuclear functions, including RNA metabolism and maintenance of nuclear architecture.

Editor—Peroxisome biogenesis disorders (PBDs) are lethal genetic diseases characterised by a number of peroxisomal metabolic abnormalities, including the oxidation of very long chain fatty acids (VLCFAs), biosynthesis of bile acids and plasmalogen, and detoxification of H2O2. Peroxisomal matrix proteins are synthesised on free polyribosomes and directed to the organelle by cis acting peroxisome targeting signals (PTSs). PTS1 is a C-terminal tripeptide Ser-Lys-Leu (SKL) sequence and later the consensus sequence was broadened to (S/A/C/K/N)-(K/R/H/Q/N/S)-L, based on subsequent studies. Acyl-CoA oxidase (AOX) has SKL and D bifunctional protein has AKL.1-4 PTS2 is an N-terminal cleavable peptide (-R/KLX5Q/HL) that resides in peroxisomal 3-ketoacyl CoA thiolase (PT), alkyl-dihydroxyacetonephosphate synthase, and phytanoyl-CoA hydroxylase.5-9 PBDs are genetically classified into at least 12 complementation groups (CGs) and each CG contains various clinical phenotypes, for example, Zellweger syndrome (ZS), neonatal adrenoleucodystrophy (NALD), and infantile Refsum disease (IRD).10 11 ZS patients have severe neurological defects, liver dysfunction, and renal cysts and die before 1 year of age. NALD patients have symptoms similar to ZS patients, but they survive a little longer, and IRD patients show milder abnormalities in the central nervous system and survive even longer. We identified the restoration of peroxisome biogenesis in a temperature sensitive (TS) manner in fibroblasts from milder forms of PBDs, that is, all IRD patients and some NALD patients belonging to groups CG-A (CG8), CG-C (CG4), CG-E (CG1), CG-F (CG10), CG-H, and CG6.12-15 In these cells, peroxisomes were formed at 30°C and biochemical activities of peroxisomes, including the oxidation of VLCFAs and dihydroxyacetonephosphate acyltransferase (DHAP-AT), and the import of peroxisomal enzymes, were also restored.16 However, virtually no peroxisomes were formed in ZS cells at 30°C and import of peroxisomal enzymes did not improve.16 Here, we elucidate temperature dependent import and processing …

Peroxisome proliferator-activated receptor (PPAR) constitutes a subfamily among a large group of ligand-activated transcription factors, the nuclear receptor superfamily. We studied the effects of ligand on the intracellular behaviors of PPARalpha. Although nuclear localization of PPARalpha was not affected by a selective ligand, Wy14643, we observed that exogenously expressed PPARalpha was rapidly degraded in HeLa cells, and the ligand significantly stabilized the protein. The stability of PPARalpha was also improved by coexpression of the heterodimer partner retinoid X receptor (RXR) alpha, and further stabilization was not observed with the ligand. These results indicate that PPARalpha is stabilized through heterodimerization with RXR, and the excess protein unpaired with RXR is rapidly turned over, if not bound by an appropriate ligand. These observations on PPARalpha are in sharp contrast to the ligand-stimulated degradation reported on PPARgamma. The ligand-dependent stabilization would have physiological significance when the synthesis of PPARalpha is elevated exceeding the available level of RXR.

Peroxisome biogenesis disorders (PBD), including Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease, are a group of genetically heterogeneous autosomal-recessive diseases caused by mutations in PEX genes that encode peroxins, proteins required for peroxisome biogenesis. Zellweger syndrome patients present the most severe phenotype, whereas neonatal adrenoleukodystrophy patients are intermediate and infantile Refsum disease patients have the mildest features. PEX6 is a causative gene for PBD of complementation group C (CG-C) and encodes the peroxin Pex6p, one of the ATPases associated with diverse cellular activities and a member of the same family of proteins as Pex1p, a causative protein for PBD of CG-E (CG1). Here, we identified the temperature sensitivity of peroxisomes in the fibroblasts of a patient with neonatal adrenoleukodystrophy in CG-C. Peroxisomes were morphologically and biochemically formed at 30 degrees C but not at 37 degrees C. This patient was homozygous for a missense mutation, T-->C at nucleotide 170 resulting in a change from leucine to proline at amino acid 57 (L57P) in Pex6p. CG-C cell mutants (ZP92) in the Chinese hamster ovary transfected with L57P in HsPEX6 revealed the same temperature-sensitive phenotype. However, PEX1-deficient Chinese hamster ovary cell mutants (ZP101) transfected with L111P in PEX1, the counterpart to L57P in PEX6, showed no temperature sensitivity. In addition, ZP92 transfected with G708D in PEX6, the counterpart to the temperature-sensitive mutation G843D in PEX1, revealed no temperature-sensitive phenotype. These results indicate that L57P in Pex6p is a temperature-sensitive mutation causing the milder phenotype in a patient with PBD in CG-C. They also indicate that the amino acid residues responsible for temperature sensitivity do not seem to be conserved between Pex6p and Pex1p

Patients with peroxisome biogenesis disorders (PBD) can be identified by detection of peroxisomes in their fibroblasts, by means of immunocytochemical staining using an anti-catalase antibody. We report here data on three PBD patients with newly identified mutations (del550C and del642G) in the PEX2 gene which encodes a 35-kDa peroxisomal membrane protein containing two membrane-spanning and a C-terminal cysteine-rich region. Some of the fibroblasts from the patient with the del642G mutation contained numerous catalase-containing particles, whereas no fibroblasts containing such particles were found in the patient with the del550C mutation. We confirmed that the del642G mutation caused a partial defect in peroxisome synthesis and import by expression of the mutated PEX2 into PEX2-defective CHO mutant cells. We propose that the two putative membrane-spanning segments in Pex2p are important domains for peroxisome assembly and import and that a defect in one of these domains severely affects PBD patients. Furthermore, a defect in the C-terminal portion of Pex2p exposed to the cytosol containing a RING finger motif caused the mild phenotype, residual enzyme activities, and mosaic detectable peroxisomes in fibroblasts from the patient.

Most mammalian cell strains genetically deficient in peroxisome biogenesis have abnormal membrane structures called ghosts, containing integral peroxisomal membrane protein, PMP70, but lacking the peroxisomal matrix proteins. Upon genetic complementation, these mutants regain the ability of peroxisome biogenesis. It is postulated that, in this process, the ghosts act as the precursors of peroxisomes, but there has been no evidence to support this. In the present study, we investigated this issue by protein microinjection to a mutant Chinese hamster ovary cell line defective of PEX5, encoding a peroxisome-targeting signal receptor. When recombinant Pex5p and green fluorescent protein (GFP) carrying a peroxisome-targeting signal were co-injected into the mutant cells, the GFP fluorescence gathered over time to particulate structures where PMP70 was co-localized. This process was dependent on both Pex5p and the targeting signal, and, most importantly, occurred even in the presence of cycloheximide, a protein synthesis inhibitor. These findings suggest that the ghosts act as acceptors of matrix proteins in the peroxisome recovery process at least in the PEX5 mutant, and support the view that peroxisomes can grow by incorporating newly synthesized matrix proteins.

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 peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD), are autosomal recessive diseases caused by deficiency of peroxisome assembly as well as malfunction of peroxisomes, where >10 genotypes have been reported. ZS patients manifest the most severe clinical and biochemical abnormalities, while those with NALD and IRD show the least severity and the mildest features, respectively. PEX1 is the causative gene for PBDs of complementation group I (CG1), the highest incidence PBD, and encodes the peroxin, Pex1p, a member of the AAA ATPase family. In the present work, we found that peroxisomes were morphologically and biochemically formed at 30 but not 37 degrees C, in the fibroblasts from all CG1 IRD patients examined, whereas almost no peroxisomes were seen in ZS and NALD cells, even at 30 degrees C. A point missense mutation, G843D, was identified in the PEX1 allele of most CG1 IRD patients. The mutant PEX1, termed HsPEX1G843D, gave rise to the same temperature-sensitive phenotype on CG1 CHO cell mutants upon transfection. Collectively, these results demonstrate temperature-sensitive peroxisome assembly to be responsible for the mildness of the clinical features of PEX1 -defective IRD of CG1.

The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy (NALD), are autosomal recessive diseases caused by defects in peroxisome assembly, for which at least 10 complementation groups have been reported. We have isolated a human PEX1 cDNA ( HsPEX1 ) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary (CHO) cell line, ZP107, transformed with peroxisome targeting signal type 1-tagged “enhanced” green fluorescent protein. This cDNA encodes a hydrophilic protein (Pex1p) comprising 1,283 amino acids, with high homology to the AAA-type ATPase family. A stable transformant of ZP107 with HsPEX1 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX1 expression restored peroxisomal protein import in fibroblasts from three patients with ZS and NALD of complementation group I (CG-I), which is the highest-incidence PBD. A CG-I ZS patient (PBDE-04) possessed compound heterozygous, inactivating mutations: a missense point mutation resulting in Leu-664 → Pro and a deletion of the sequence from Gly-634 to His-690 presumably caused by missplicing (splice site mutation). Both PBDE-04 PEX1 cDNAs were defective in peroxisome-restoring activity when expressed in the patient fibroblasts as well as in ZP107 cells. These results demonstrate that PEX1 is the causative gene for CG-I peroxisomal disorders.