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PubMed1998

Peroxisome targeting signal type 1 (PTS1) receptor is involved in import of both PTS1 and PTS2: studies with PEX5-defective CHO cell mutants.

To investigate the mechanisms of peroxisome assembly and the molecular basis of peroxisome assembly disorders, we isolated and characterized a peroxisome-deficient CHO cell mutant, ZP139, which was found to belong to human complementation group II, the same group as that of our earlier mutant, ZP105. These mutants had a phenotypic deficiency in the import of peroxisomal targeting signal type 1 (PTS1) proteins. Amino-terminal extension signal (PTS2)-mediated transport, including that of 3-ketoacyl coenzyme A thiolase, was also defective in ZP105 but not in ZP139. PEX5 cDNA, encoding the PTS1 receptor (PTS1R), was isolated from wild-type CHO-K1 cells. PTS1R's deduced primary sequence comprised 595 amino acids, 7 amino acids less than the human homolog, and contained seven tetratricopeptide repeat (TPR) motifs at the C-terminal region. Chinese hamster PTS1R showed 94, 28, and 24% amino acid identity with PTS1Rs from humans, Pichia pastoris, and Saccharomyces cerevisiae, respectively. A PTS1R isoform (PTS1RL) with 632 amino acid residues was identified in CHO cells; for PTS1R, 37 amino acids were inserted between residues at positions 215 and 216 of a shorter isoform (PTS1RS). Southern blot analysis of CHO cell genomic DNA suggested that these two isoforms are derived from a single gene. Both types of PEX5 complemented impaired import of PTS1 in mutants ZP105 and ZP139. PTS2 import in ZP105 was rescued only by PTS1RL. This finding strongly suggests that PTS1RL is also involved in the transport of PTS2. Mutations in PEX5 were determined by reverse transcription-PCR: a G-to-A transition resulted in one amino acid substitution: Gly298Glu of PTS1RS (G335E of PTS1RL) in ZP105 and Gly485Glu of PTS1RS (G522E of PTS1RL) in ZP139. Both mutations were in the TPR domains (TPR1 and TPR6), suggesting the functional consequence of these domains in protein translocation. The implications of these mutations are discussed.

Amino Acid Sequence↗
PubMed2000

A missense mutation in the RING finger motif of PEX2 protein disturbs the import of peroxisome targeting signal 1 (PTS1)-containing protein but not the PTS2-containing protein.

SK24 and PT54 mutant cells, which are peroxisome-deficient Chinese hamster ovary (CHO) cells isolated using peroxisomal forms of green fluorescent protein (GFP), were found to be defective in the PEX2 gene. The nucleotide sequences of PEX2 cDNA from the mutant cells were determined to identify mutation sites in the mutant cells. The mutation in SK24 cells changed cysteine to tyrosine at amino acid position 258, which is a component of the RING finger (C(3)HC(4)) motif in the carboxyl terminus of the protein. PT54 cells contained a nonsense mutation in the codon for glutamine at position 101, resulting in premature termination. The immunocytochemical analyses revealed distinct phenotypes between mutant cells defective in the PEX2 gene. Both mutant cells exhibited cytosolic mislocalizations on catalase and urate oxidase containing PTS1. On the other hand, on 3-ketoacyl-CoA thiolase containing PTS2, PT54 cells exhibited cytosolic mislocalization, but SK24 cells exhibited peroxisomal localization. When wild-type or mutant-type PEX2 cDNA was transfected into both mutant cells, the stable transformants restored the phenotype in accordance with the transfected cDNA. These observations indicate that an amino acid substitution, cysteine-258 to tyrosine, in the RING finger motif of PEX2 protein, whose function is required for peroxisomal localizations of both PTS1- and PTS2-containing proteins, results in a complete defect in the PTS1 pathway but not in the PTS2 pathway.

Amino Acid Sequence↗
PubMed1999

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

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

ATPases Associated with Diverse Cellular Activitie↗
PubMed2004

Presence of a bacterial-like citrate synthase gene in Tetrahymena thermophila: recent lateral gene transfers (LGT) or multiple gene losses subsequent to a single ancient LGT?

Citrate synthase is the initial enzyme in the tricarboxylic acid cycle of mitochondria. In plants and fungi, it is the second isozyme in the glyoxylate cycle of peroxisomes (or glyoxysomes), and it is also present in bacteria. Some of the biochemical reactions in the glyoxylate cycle of the ciliated protozoan Tetrahymena pyriformis depend upon mitochondrial enzymes, as T. pyriformis lacks some glyoxysome-specific enzymes. Here we demonstrate a new citrate synthase gene from Tetrahymena thermophila that is different from the mitochondrial counterpart. A potential peroxysome-targeted signal was detected in the N-terminus, suggesting the localization of the enzyme in peroxysomes. Phylogenetic analysis placed the Tetrahymena sequence in a clade consisting of a few sequences from eukaryotes such as cellular slime molds and two land plants, near a green sulfur bacterium and many proteobacteria as a sister group but not in a mitochondrial clade. Southern blot analysis revealed that this type of gene was absent from distantly related ciliates and other species of Tetrahymena except for the closest species, T. mallaccensis. The scattered presence of the bacterial-like genes among distantly related eukaryotes suggests three alternative interpretations of acquisition of the novel glyoxysomal citrate synthase gene via lateral gene transfer (LGT). (1). Some eukaryotes independently acquired the gene from a common bacterium or closely related bacteria via LGT. (2). A hypothetical eukaryote once acquired the gene, which was thereafter independently transferred from the eukaryote to other eukaryotes. (3). A single event of LGT (or duplication) occurred in a certain common ancestor of eukaryotes, followed by multiple losses in many eukaryotic lineages during the subsequent evolution. Considering the monophyly of the bacterial-like eukaryotic citrate synthase genes, the first model is somewhat unlikely, even though it is not impossible. The second and third models can rationally explain the present observation, so these models are discussed in some detail.

Amino Acid Motifs↗
PubMed2006

Generalised and conditional inactivation of Pex genes in mice.

During the past 10 years, several Pex genes have been knocked out in the mouse with the purpose to generate models to study the pathogenesis of peroxisome biogenesis disorders and/or to investigate the physiological importance of the Pex proteins. More recently, mice with selective inactivation of a Pex gene in particular cell types were created. The metabolic abnormalities in peroxisome deficient mice paralleled to a large extent those of Zellweger patients. Several but not all of the clinical and histological features reported in patients also occurred in peroxisome deficient mice as for example hypotonia, cortical and cerebellar malformations, endochondral ossification defects, hepatomegaly, liver fibrosis and ultrastructural abnormalities of mitochondria in hepatocytes. Although the molecular origins of the observed pathologies have not yet been resolved, several new insights on the importance of peroxisomes in different tissues have emerged.

Animals↗
PubMed2006

A temperature-sensitive CHO pex1 mutant with a novel mutation in the AAA Walker A1 motif.

We herein isolated a peroxisome-deficient Chinese hamster ovary mutant, ZPEG252, import-defective of peroxisome targeting signal 1 (PTS1)- and PTS2-proteins at 37 degrees C. The impaired protein import was restored at 30 degrees C, indicating a temperature-sensitive phenotype, similar to that of cells derived from patients with milder peroxisome biogenesis disorders such as infantile Refsum disease. PEX1 expression complemented the mutant phenotype of ZPEG252. Reverse transcription-PCR analysis indicated one point mutation at nucleotide residue 1817 changing a codon (GGG) for Gly(606) to a codon (GAG) for Glu(606) in the sequence for the Walker A1 motif of the AAA cassettes. This novel mutant Pex1pG606E was severely affected in binding to Pex6p at 37 degrees C, but not at 30 degrees C. Pex1pG606E was localized to peroxisomes at 30 degrees C, whilst it was discernible in a cytosolic staining pattern at 37 degrees C. Together, our findings demonstrate that Walker A1 motif of Pex1p is essential for Pex1p-Pex6p interaction and Pex1p targeting to peroxisomes.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2004

Candida albicans CTN gene family is induced during macrophage infection: homology, disruption and phenotypic analysis of CTN3 gene.

We have isolated a Candida albicans gene, coding for a putative peroxisomal carnitine acetyl transferase (CTN) protein, which is up-regulated during macrophage infection. In the present study, we describe the disruption of CTN3 gene (previously called CAT3) to gain insight into its potential role during infection. The ability of disrupted Candida mutants to filament was affected by several solid media. Northern blot analysis revealed that CTN3 gene may be involved not only in conditions of cell starvation but also during the process of germination. In agreement with the putative peroxisomal localization of the corresponding protein, we observed a strong glucose repression of CTN3 gene and, on the contrary, high level of transcription by carbon sources that induce the formation of peroxisomal proteins. Furthermore, we showed the existence of two additional C. albicans CTN encoding sequences, which are also induced during macrophage infection.

Amino Acid Sequence↗
PubMed2003

Entering a new era of research on plant peroxisomes.

Peroxisomes are globular organelles, of approximately 1 microm in diameter, that are found ubiquitously in eukaryotic cells. In higher plants, peroxisomes have been believed to play a pivotal role in three metabolic pathways: lipid breakdown, photorespiration and H2O2-detoxificaton. However, recent progress using Arabidopsis mutants has suggested that peroxisomes have more diverse functions than are known at present. Extensive studies using genetic and post-genomic approaches will renovate our present understanding of the functions of peroxisomes in plants.

Peroxisomal Targeting Signal 2 Receptor↗
PubMed2002

Peroxisomes: surprisingly versatile organelles.

Peroxisome development is a dynamic process that is not yet completely understood. We use the methylotrophic yeast Hansenula polymorpha as model in our studies on peroxisome homeostasis. Cells of this species may contain different types of peroxisomes that differ in protein composition and capacity to incorporate matrix proteins. This protein import machinery is highly flexible and can accommodate unfolded and complex folded proteins.

Alcohol Oxidoreductases↗
PubMed2002

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

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

Animals↗
PubMed1999

Antibodies against pex14p block ATP-independent binding of matrix proteins to peroxisomes in vitro.

The membrane protein Pex14p is a key component of the protein import machinery of peroxisomes. Antibodies raised against human Pex14p recognise a 66 kDa protein in sunflower glyoxysomes (HaPex14p) and immunoprecipitate in vitro-translated Arabidopsis Pex14p (AtPex14p). These antibodies inhibit the ATP-independent binding to sunflower peroxisome membranes of peroxisome targeting signal type (PTS) 1- and PTS2-targeted matrix proteins, but not an integral membrane protein. These results suggest that Pex14p functions before the ATP-dependent step of peroxisome assembly.

Adenosine Triphosphate↗
PubMed2000

Rapid isolation and characterization of CHO mutants deficient in peroxisome biogenesis using the peroxisomal forms of fluorescent proteins.

We isolated and characterized CHO mutants deficient in peroxisome assembly using green fluorescent protein (GFP) and blue fluorescent protein (BFP) as the fluorescent probes to study the molecular mechanism of peroxisome biogenesis. We used stable transformants of CHO cells expressing GFP appending peroxisome targeting signal-1 (PTS1) and/or peroxisome targeting signal-2 (PTS2) as the parent strains for rapid isolation of the mutants. We have obtained six peroxisome-deficient mutants by visual screening of the mislocalizations of the peroxisomal GFPs. Mutual cell fusion experiments indicated that the six mutants isolated were divided into four complementation groups. Several of the mutants obtained possessed defective genes: the PEX2 gene was defective in SK24 and PT54; the PEX5 gene in SK32 and the PEX7 gene in PT13 and PT32. BE41, which belonged to the fourth complementation group, was not determined. When peroxisomal forms of BFP were transiently expressed in mutant cells, the peroxisomal BFPs appending both PTS1 and PTS2 appeared to bypass either the PTS1 or PTS2 pathway for localization in SK32. This observation suggested that other important machinery, in addition to the PTS1 or PTS2 pathway, could be involved in peroxisome biogenesis. Thus, our approach using peroxisomal fluorescent proteins could facilitate the isolation and analysis of peroxisome-deficient CHO mutants and benefit studies on the identification and role of the genes responsible for peroxisome biogenesis.

Animals↗
PubMed1999

Import of proteins into peroxisomes.

Peroxisomes are organelles that confine an important set of enzymes within their single membrane boundaries. In man, a wide variety of genetic disorders is caused by loss of peroxisome function. In the most severe cases, the clinical phenotype indicates that abnormalities begin to appear during embryological development. In less severe cases, the quality of life of adults is affected. Research on yeast model systems has contributed to a better understanding of peroxisome formation and maintenance. This framework of knowledge has made it possible to understand the molecular basis of most of the peroxisome biogenesis disorders. Interestingly, most peroxisome biogenesis disorders are caused by a failure to target peroxisomal proteins to the organellar matrix or membrane, which classifies them as protein targeting diseases. Here we review recent fundamental research on peroxisomal protein targeting and discuss a few burning questions in the field concerning the origin of peroxisomes.

Intracellular Membranes↗
PubMed2001

How are peroxisomes formed? The role of the endoplasmic reticulum and peroxins.

Recent data from studies of peroxisome assembly and the subcellular sorting of peroxisomal matrix and membrane proteins have led to an expansion of the 'growth and division' and 'endoplasmic reticulum-vesiculation' models of peroxisome biogenesis into a more flexible, unified model. Within this context, we discuss the proposed role for the endoplasmic reticulum in the formation of preperoxisomes and the potential for 15 Arabidopsis peroxin homologs to function in the biogenesis of peroxisomes in plant cells.

Ascorbate Peroxidases↗
PubMed2000

Peroxisomal remnant structures in Hansenula polymorpha Pex5 cells can develop into normal peroxisomes upon induction of the PTS2 protein amine oxidase.

We have analyzed the properties of peroxisomal remnants in Hansenula polymorpha pex5 cells. In such cells PTS1 matrix protein import is fully impaired. In H. polymorpha pex5 cells, grown on ethanol/ammonium sulfate, conditions that repressed the PTS2 protein amine oxidase (AMO), peroxisomal structures were below the limit of detection. In methanol/ammonium sulfate-grown cells, normal peroxisomes are absent, but a few small membranous structures were observed that apparently represented peroxisomal ghosts since they contained Pex14p. These structures were the target of a Pex10p.myc fusion protein that was produced in pex5 cells under the control of the homologous alcohol oxidase promoter (strain pex5::P(AOX).PEX10.MYC). Glycerol/methanol/ammonium sulfate-grown cells of this transformant were placed in fresh glucose/methylamine media, conditions that fully repress the synthesis of the Pex10p.myc fusion protein but induce the synthesis of AMO. Two hours after the shift Pex10p.myc-containing structures were detectable that had accumulated newly synthesized AMO protein and which during further cultivation developed in normal peroxisomes. Concurrently, the remaining portion of these structures was rapidly degraded. These findings indicate that peroxisomal remnants in pex5 cells can develop into peroxisomes. Also, as for normal peroxisomes in H. polymorpha, apparently a minor portion of these structures actually take part in the development of these organelles.

Amine Oxidase (Copper-Containing)↗
PubMedDate not supplied

Peroxisome biogenesis and protein import in plants, animals and yeasts: enigma and variations? (Review).

Peroxisomes are found in virtually all eukaryotic cells, where they play varied but essential metabolic roles, exemplified by the catastrophic effects of mutations that compromise peroxisome biogenesis and function. This review will aim to provide an accessible introduction to peroxisome biogenesis and protein import for the non-specialist, and draws together recent advances in peroxisome protein targeting and import in plants, animals and yeasts, seeking to define common themes and highlight variations. Despite much progress, many aspects of peroxisome biology remain an enigma, and current questions and controversies in the field are highlighted and discussed.

Amino Acid Sequence↗
PubMed2002

The TRIM37 gene encodes a peroxisomal RING-B-box-coiled-coil protein: classification of mulibrey nanism as a new peroxisomal disorder.

Mulibrey nanism is a rare growth disorder of prenatal onset caused by mutations in the TRIM37 gene, which encodes a RING-B-box-coiled-coil protein. The pathogenetic mechanisms of mulibrey nanism are unknown. We have used transiently transfected cells and antibodies raised against the predicted TRIM37 protein to characterize the TRIM37 gene product and to determine its intracellular localization. We show that the human TRIM37 cDNA encodes a peroxisomal protein with an apparent molecular weight of 130 kD. Peroxisomal localization is compromised in mutant protein representing the major Finnish TRIM37 mutation but is retained in the protein representing the minor Finnish mutation. Colocalization of endogenous TRIM37 with peroxisomal markers was observed by double immunofluorescence staining in HepG2 and human intestinal smooth muscle cell lines. In human tissue sections, TRIM37 shows a granular cytoplasmic pattern. Endogenous TRIM37 is not imported into peroxisomes in peroxin 1 (PEX1(-/-)) and peroxin 5 (PEX5(-/-)) mutant fibroblasts but is imported normally in peroxin 7 (PEX7(-/-)) deficient fibroblasts, giving further evidence for a peroxisomal localization of TRIM37. Fibroblasts derived from patients with mulibrey nanism lack C-terminal TRIM37 immunoreactivity but stain normally for both peroxisomal matrix and membrane markers, suggesting apparently normal peroxisome biogenesis in patient fibroblasts. Taken together, this molecular evidence unequivocally indicates that TRIM37 is located in the peroxisomes, and Mulibrey nanism thus can be classified as a new peroxisomal disorder.

ATPases Associated with Diverse Cellular Activitie↗