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PubMed2002

Import of assembled PTS1 proteins into peroxisomes of the yeast Hansenula polymorpha: yes and no!

Previously, Waterham et al. [EMBO J. 12 (1993) 4785] reported that cytosolic oligomeric alcohol oxidase (AO) is not incorporated into peroxisomes after reassembly of the organelles in the temperature-sensitive peroxisome-deficient mutant pex1-6(ts) of Hansenula polymorpha shifted to permissive growth conditions. Here, we show that the failure to import assembled AO protein is not exemplary for other folded proteins because both an artificial peroxisomal matrix protein, PTS1-tagged GFP (GFP.SKL), and the endogenous dimeric PTS1 protein dihydroxyacetone synthase (DHAS) were imported under identical conditions. In vitro receptor-ligand binding studies using immobilised H. polymorpha Pex5p and crude extracts of methanol-induced pex1-6(ts) cells, showed that AO octamers did not interact with the recombinant PTS1 receptor, at conditions that allowed binding of folded GFP.SKL and dimeric DHAS. This shows that import of oligomeric proteins is not a universal pathway for peroxisomal matrix proteins.

Alcohol Oxidoreductases↗
PubMed2001

A novel family of longer chain length dolichols present in oleate-induced yeast Saccharomyces cerevisiae.

The typical size of the yeast dolichol family ranges from 14 to 19 isoprene units D((14-19)) with dolichol(16) being the dominating species. Induction of peroxisome proliferation by growing the cells in medium containing oleate as carbon source induces the synthesis of an additional family of longer dolichols D((19-24)) with D(21) being the most prominent. This phenomenon is abolished in the peroxisome biogenesis deficient strain in which the PEX1 gene (encoding Pex1p peroxin) has been disrupted. The total amount of dolichols in pex1Delta cells is lower than in the wild-type cells, as is the amount of phosphatidylcholine. Moreover, the levels of 3-hydroxy-3-methylglutaryl CoA reductase and farnesyl diphosphate synthase, two key enzymes in dolichol biosynthesis, are decreased in the absence of a functional PEX1 gene. The presence of longer dolichols in oleate-induced Saccharomyces cerevisiae cells, the absence of this additional family in peroxisome deficient cells, and a decrease of the total amount of dolichols in these cells indicate the involvement of peroxisomes in the biosynthesis of dolichols in this organism.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1997

Four distinct secretory pathways serve protein secretion, cell surface growth, and peroxisome biogenesis in the yeast Yarrowia lipolytica.

We have identified and characterized mutants of the yeast Yarrowia lipolytica that are deficient in protein secretion, in the ability to undergo dimorphic transition from the yeast to the mycelial form, and in peroxisome biogenesis. Mutations in the SEC238, SRP54, PEX1, PEX2, PEX6, and PEX9 genes affect protein secretion, prevent the exit of the precursor form of alkaline extracellular protease from the endoplasmic reticulum, and compromise peroxisome biogenesis. The mutants sec238A, srp54KO, pex2KO, pex6KO, and pex9KO are also deficient in the dimorphic transition from the yeast to the mycelial form and are affected in the export of only plasma membrane and cell wall-associated proteins specific for the mycelial form. Mutations in the SEC238, SRP54, PEX1, and PEX6 genes prevent or significantly delay the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX5, PEX16, and PEX17 genes, which have previously been shown to be essential for peroxisome biogenesis, affect the export of plasma membrane and cell wall-associated proteins specific for the mycelial form but do not impair exit from the endoplasmic reticulum of either Pex2p and Pex16p or of proteins destined for secretion. Biochemical analyses of these mutants provide evidence for the existence of four distinct secretory pathways that serve to deliver proteins for secretion, plasma membrane and cell wall synthesis during yeast and mycelial modes of growth, and peroxisome biogenesis. At least two of these secretory pathways, which are involved in the export of proteins to the external medium and in the delivery of proteins for assembly of the peroxisomal membrane, diverge at the level of the endoplasmic reticulum.

Animals↗
PubMed2000

The peroxisome biogenesis factors pex4p, pex22p, pex1p, and pex6p act in the terminal steps of peroxisomal matrix protein import.

Peroxisomes are independent organelles found in virtually all eukaryotic cells. Genetic studies have identified more than 20 PEX genes that are required for peroxisome biogenesis. The role of most PEX gene products, peroxins, remains to be determined, but a variety of studies have established that Pex5p binds the type 1 peroxisomal targeting signal and is the import receptor for most newly synthesized peroxisomal matrix proteins. The steady-state abundance of Pex5p is unaffected in most pex mutants of the yeast Pichia pastoris but is severely reduced in pex4 and pex22 mutants and moderately reduced in pex1 and pex6 mutants. We used these subphenotypes to determine the epistatic relationships among several groups of pex mutants. Our results demonstrate that Pex4p acts after the peroxisome membrane synthesis factor Pex3p, the Pex5p docking factors Pex13p and Pex14p, the matrix protein import factors Pex8p, Pex10p, and Pex12p, and two other peroxins, Pex2p and Pex17p. Pex22p and the interacting AAA ATPases Pex1p and Pex6p were also found to act after Pex10p. Furthermore, Pex1p and Pex6p were found to act upstream of Pex4p and Pex22p. These results suggest that Pex1p, Pex4p, Pex6p, and Pex22p act late in peroxisomal matrix protein import, after matrix protein translocation. This hypothesis is supported by the phenotypes of the corresponding mutant strains. As has been shown previously for P. pastoris pex1, pex6, and pex22 mutant cells, we show here that pex4Delta mutant cells contain peroxisomal membrane protein-containing peroxisomes that import residual amounts of peroxisomal matrix proteins.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2005

Shuttling mechanism of peroxisome targeting signal type 1 receptor Pex5: ATP-independent import and ATP-dependent export.

Peroxisomal matrix proteins are posttranslationally imported into peroxisomes with the peroxisome-targeting signal 1 receptor, Pex5. The longer isoform of Pex5, Pex5L, also transports Pex7-PTS2 protein complexes. After unloading the cargoes, Pex5 returns to the cytosol. To address molecular mechanisms underlying Pex5 functions, we constructed a cell-free Pex5 translocation system with a postnuclear supernatant fraction from CHO cell lines. In assays using the wild-type CHO-K1 cell fraction, (35)S-labeled Pex5 was specifically imported into and exported from peroxisomes with multiple rounds. (35)S-Pex5 import was also evident using peroxisomes isolated from rat liver. ATP was not required for (35)S-Pex5 import but was indispensable for export. (35)S-Pex5 was imported neither to peroxisome remnants from RING peroxin-deficient cell mutants nor to those from pex14 cells lacking a Pex5-docking site. In contrast, (35)S-Pex5 was imported into the peroxisome remnants of PEX1-, PEX6-, and PEX26-defective cell mutants, including those from patients with peroxisome biogenesis disorders, from which, however, (35)S-Pex5 was not exported, thereby indicating that Pex1 and Pex6 of the AAA ATPase family and their recruiter, Pex26, were essential for Pex5 export. Moreover, we analyzed the (35)S-Pex5-associated complexes on peroxisomal membranes by blue-native polyacrylamide gel electrophoresis. (35)S-Pex5 was in two distinct, 500- and 800-kDa complexes comprising different sets of peroxins, such as Pex14 and Pex2, implying that Pex5 transited between the subcomplexes. Together, results indicated that Pex5 most likely enters peroxisomes, changes its interacting partners, and then exits using ATP energy.

Adenosine Triphosphate↗
PubMed2000

Peroxisome biogenesis and molecular defects in peroxisome assembly disorders.

Peroxisome assembly in mammals requires more than 14 genes. So far, we have isolated seven complementation groups (CGs) of peroxisome biogenesis-defective Chinese hamster ovary (CHO) cell mutants, Z65, Z24/ZP107, ZP92, ZP105/ZP139, ZP109, ZP110, ZP114. Two peroxin cDNAs, PEX2 and PEX6, were first cloned by genetic phenotype-complementation assay using Z65 and ZP92, respectively, and were shown to be responsible for peroxisome biogenesis disorders (PBD) such as Zellweger syndrome, of CG-F (the same as CG-X in U.S.A.) and CG-C (the same as CG-IV), respectively. Pex2p is a RING zinc finger membrane protein of peroxisomes and Pex6p is a member of the AAA ATPase family. We likewise isolated PEX12 encoding a peroxisomal integral membrane protein in the RING family, by functional complementation of ZP109, demonstrating PEX12 to be responsible for CG-III PBD. We also cloned PEX1 by screening of human liver cDNA library, using ZP107. PEX1 mutation was delineated to be the genetic cause of PBD in the most highest incidence group, CG-E (the same as CG-I). Moreover, we recently found that Pex5p is involved in transport of not only PTS1- but also PTS2-protein, distinct from yeast Pex5p, using PEX5-defective ZP105 and ZP139. Thus, CHO cell mutants defective in peroxisome biogenesis are indeed shown to be very useful for the studies of peroxisome assembly and delineating pathogenic genes in PBD. Furthermore, we have isolated novel CGs of CHO mutants, ZP119 and ZP126.

Gene Expression Regulation↗
PubMed2000

Oxygen and haem regulate the synthesis of peroxisomal proteins: catalase A, acyl-CoA oxidase and Pex1p in the yeast Saccharomyces cerevisiae; the regulation of these proteins by oxygen is not mediated by haem.

Saccharomyces cerevisiae genes related to respiration are typically controlled by oxygen and haem. Usually the regulation by these factors is co-ordinated; haem is indicated as the oxygen sensor. However, the responsiveness of peroxisome functions to these regulatory factors is poorly understood. The expression of CTA1, POX1 and PEX1 genes encoding the peroxisomal proteins catalase A, acyl-CoA oxidase and Pex1p peroxin respectively was studied under various conditions: in anaerobiosis, in the absence of haem and in respiratory incompetence caused by the lack of a mitochondrial genome (rho(0)). The influence of haem deficiency or rho(0) on peroxisomal morphology was also investigated. Respiratory incompetence has no effect on the expression of CTA1 and POX1, whereas in the absence of haem their expression is markedly decreased. The synthesis of Pex1p is decreased in rho(0) cells and is decreased even more in haem-deficient cells. Nevertheless, peroxisomal morphology in both these types of cell does not differ significantly from the morphology of peroxisomes in wild-type cells. The down-regulating effect of anoxia on the expression of CTA1 and POX1 is even stronger than the effect of haem deficiency and is not reversed by the addition of exogenous haem or the presence of endogenous haem. Moreover, neither of these genes responds to the known haem-controlled transcriptional factor Hap1p. In contrast with the other two genes studied, PEX1 is up-regulated in anaerobiosis. The existence of one or more novel mechanisms of regulation of peroxisomal genes by haem and oxygen, different from those already known in S. cerevisiae, is postulated.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2003

Genetic heterogeneity of peroxisome biogenesis disorders among Japanese patients: evidence for a founder haplotype for the most common PEX10 gene mutation.

We, as the only diagnostic center for peroxisome biogenesis disorders (PBD) in Japan, identified a total of 31 Japanese patients with PBD during the last 20 years. They were 27 patients with Zellweger syndrome (ZS), including two sib cases, three with neonatal adrenoleukodystrophy (NALD) and one with rhizomelic type chondrodysplasia punctata (RCDP). No patient with infantile Refsum disease has been detected. These patients were genetically subdivided into complementation group A (five ZS and one NALD), B (11 ZS), C (four ZS), E (five ZS and two NALD), F (two ZS), and R (one RCDP). They were subjected to mutation analysis of PEX1, PEX2, PEX6, PEX7, and PEX10. All the 11 ZS patients with group-B PBD had a common mutation, i.e., a homozygous 2-base-pair deletion in PEX10. To determine whether this highly frequent mutation is due to a founder effect, we analyzed single nucleotide polymorphisms within PEX10 among patients and Japanese controls. The mutation apparently arose once on an ancestral chromosome in the Japanese population. Based on the value of 24 PBD patients identified during the last 10 years, we estimated the prevalence of PBD in Japan to be approximately one in 500,000 births.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1984

Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins.

Construction of a family of bacterial expression vectors, pEX1-3, is described. These vectors are derived from a cro-lacZ gene fusion plasmid which expresses large quantities of fusion protein under the control of the PR promoter of bacteriophage lambda. A polylinker has been engineered into the 3' end of the lacZ gene in all three translational reading frames, and stop signals for transcription and translation inserted, so that any open reading frame DNA may be expressed as a hybrid beta-galactosidase protein. cDNA fragments cloned in these vectors can be detected with an efficiency of greater than 1 in 3, thus enabling the detection of rare cDNA molecules. In addition, the low solubility of hybrid proteins leads to a rapid isolation procedure allowing antibodies of pre-determined specificity to be made against expressed regions of cloned DNA. We describe the cloning of albumin and complement C9 genes from a human cDNA library using polyclonal and monoclonal antibodies.

Bacteriophage lambda↗
PubMed1986

Mammalian single-stranded DNA binding protein UP I is derived from the hnRNP core protein A1.

Antibodies induced against mammalian single-stranded DNA binding protein (ssDBP) UP I were shown to be cross-reactive with most of the basic hnRNP core proteins, the main constituents of 40S hnRNP particles. This suggested a structural relationship between both groups of proteins. Using the anti-ssDBP antibodies, a cDNA clone (pRP10) was isolated from a human liver cDNA library in plasmid expression vector pEX1. By DNA sequencing this clone was shown to encode in its 949 bp insert the last 72 carboxy terminal amino acids of the ssDBP UP I. Thereafter, an open reading frame continued for another 124 amino acids followed by a UAA (ochre) stop codon. Direct amino acid sequencing of a V8 protease peptide from hnRNP core protein A1 showed that this peptide contained at its amino terminus the last 11 amino acids of UP I followed by 19 amino acids which are encoded by the open reading frame of cDNA clone pRP10 immediately following the UP I sequence. This proves that ssDBP UP I arises by proteolysis from hnRNP core protein A1. This finding must lead to a re-evaluation of the possible physiological role of UP I and related ssDBPs. The formerly assumed function in DNA replication, although not completely ruled out, should be reconsidered in the light of a possible alternative or complementary function in hnRNA processing where UP I could either be a simple degradation product of core protein A1 (as a consequence of controlling the levels of active A1) or may continue to function as an RNA binding protein which has lost the ability to interact with the other core proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Cloning of the human cDNA for the U1 RNA-associated 70K protein.

Anti-RNP sera were used to isolate a cDNA clone for the largest polypeptide of the U1 snRNP, a protein of mol. wt 70 kd designated 70K, from a human liver cDNA library constructed in the expression vector pEX1. The cro-beta-galactosidase-70K fusion protein reacted with various anti-RNP patient sera, a rabbit anti-70K antiserum, as well as with a monoclonal antibody specific for this protein. The sequences of four 70K peptides were determined and they match parts of the deduced amino acid sequence of the 1.3 kb insert of p70.1 indicating that it is a genuine 70K cDNA. Screening of a new cDNA library constructed from polysomal mRNA of HeLa cells with the p70.1 clone yielded an overlapping clone, FL70K, which was 2.7 kb long and covered the complete coding and 3'-untranslated sequence of the 70K protein in addition to 680 nucleotides upstream of the putative initiation codon, The predicted mol. wt of the encoded protein is approximately 70 kd. Amino acid analysis of the purified HeLa 70K protein yielded values close or identical to those deduced from the nucleotide sequence of the full-length cDNA. The 70K protein is rich in arginine (20%) and acidic amino acids (18%). Extremely hydrophilic regions containing mixed-charge amino acid clusters have been identified at the carboxyl-terminal half of the protein, which may function in RNA binding. A sequence comparison with two recently cloned RNA binding proteins revealed homology with one region in the U1 RNP 70K protein. This domain may also be responsible for RNA binding.

Amino Acid Sequence↗
PubMed1987

Human ribophorins I and II: the primary structure and membrane topology of two highly conserved rough endoplasmic reticulum-specific glycoproteins.

Ribophorins I and II represent proteins that are postulated to be involved in ribosome binding. They are abundant, highly-conserved glycoproteins located exclusively in the membranes of the rough endoplasmic reticulum. As the first step in the further characterization of the structure and function of these proteins, we have isolated and sequenced full-length human cDNA clones encoding ribophorins I and II using probes derived from a human liver expression library cloned into pEX1. The authenticity of the clones was verified by overlaps in the protein sequence of N-terminal and several internal fragments of canine pancreatic ribophorins I and II. The cDNA clones hybridize to mRNA species of 2.5 kb in length, and encode polypeptides of 68.5 and 69.3 kd, respectively. Primary sequence analysis, coupled with biochemical studies on the topology, indicates that both ribophorins are largely luminally disposed, spanning the membrane once and having 150 and 70 amino acid long cytoplasmically disposed C termini, respectively. Both are synthesized as precursors having cleavable signal sequences of 23 (ribophorin I) and 22 (ribophorin II) amino acids. The topology suggested by the primary structure has been confirmed biochemically using proteolytic enzymes and anti-ribophorin antibodies. Proteolysis of intact microsomes with a variety of enzymes resulted in a reduction in the apparent mol. wt of ribophorin I that would correspond to a loss of its 150-amino acid cytoplasmic tail. In the case of ribophorin II, it is completely resistant to such proteolysis which is consistent with its luminal disposition and fairly hydrophobic C terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1998

A cytoplasmic AAA family peroxin, Pex1p, interacts with Pex6p.

Human PEX1 (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 of HsPEX1-transfected CHO-K1 was incubated with in vitro synthesized 35S-labelled Pex6p, an AAA family peroxin. Immunoprecipitation of Pex1p using anti-Pex1p antibody resulted in concomitant recovery of 35S-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.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1997

NVL: a new member of the AAA family of ATPases localized to the nucleus.

We report the cloning of NVL, a newly recognized human gene that encodes an approximately 110-kDa nuclear protein designated NVLp (nuclear VCP-like protein), which is a member of a rapidly growing family of ATP-binding proteins recently denoted the AAA family (ATPases associated with diverse cellular activities) (W. H. Kunau et al., 1993, Biochimie 75:209-224). NVL was isolated by degenerate PCR using oligonucleotides corresponding to the yeast PEX1 gene, which is necessary for peroxisomal biogenesis. Two cDNAs, designated NVL.1 and NVL.2, may represent alternatively spliced forms of a single gene that maps to chromosome 1q41-q42.2. NVL has greatest similarity to the VCP subfamily of AAA proteins, is widely expressed, and encodes a nuclear protein with two highly similar ATP-binding domains. We speculate that NVLp is involved in an ATP-dependent nuclear process.

Adenosine Triphosphatases↗
PubMed2004

Non-coordinate expression of peroxisome biogenesis, beta-oxidation and glyoxylate cycle genes in mature Arabidopsis plants.

The expression of three genes that encode proteins involved in peroxisome biogenesis, beta-oxidation and the glyoxylate cycle was studied in Arabidopsis plants by fusing their promoter regions to the reporter gene luciferase. Malate synthase showed an extremely restricted pattern of expression, being detected only in young seedlings and the root tips of older plants. PEX1 and 3-ketoacyl thiolase (PED1) were expressed in roots, mature leaves, stems and flowers. However, only thiolase was up-regulated by starvation. Immunoblotting confirmed that neither malate synthase nor the other unique glyoxylate cycle enzyme isocitrate lyase are expressed in senescent leaves. These results indicate that, in contrast to cucumber, pumpkin and barley, the glyoxylate cycle does not play a role in the recycling of carbon from the turnover of membrane lipids during senescence and starvation in Arabidopsis.

Arabidopsis↗
PubMed2006

Thyroid papillary carcinoma recurring as squamous cell carcinoma: report of a case.

We report a case of local squamous cell carcinoma recurrence of thyroid papillary carcinoma, 4 years after subtotal thyroidectomy, in an 82-year-old woman. The papillary cancer of the right thyroid was histopathologically classified as T2a, N0, M0, Ex1; pT2a, pN1b, pEx1; Stage III. Fine-needle aspiration cytology of the recurrent tumor revealed atypical squamous epithelium-like cells with keratinization. The tumor was judged cytologically to be class III, defined as a suspicious malignancy and, after reoperation, it was diagnosed histopathologically as papillary carcinoma recurrence with extensive squamous metaplasia. The recurrent papillary carcinoma was thought to have changed to a squamous cell carcinoma because most of the tumor was occupied by atypical squamous cells, with a small amount of glandular tissue. The primary tumor was histologically diagnosed as a well-differentiated papillary carcinoma at the initial operation. It contained numerous tall neoplastic cells with eosinophilic granules and pseudostratified nuclei, indicating that it could potentially transform into squamous cell carcinoma. We report this case as an example of how squamous cell carcinoma of the thyroid can develop.

Adenocarcinoma, Follicular↗
PubMed1987

Isolation of cDNA clones corresponding to the Mr = 150,000 subunit of chick type VI collagen.

Type VI collagen is a disulfide-bonded protein with an unusual structure in that the molecule contains three short triple-helical domains and very extended non-collagenous regions. The molecule is a heterotrimer composed in the chick of two polypeptides of similar apparent size in SDS-PAGE (Mr = 140- and 150,000) but different structure, and a third component that is much larger (Mr = 260,000) than the other two chains. We report here on the isolation of several overlapping cDNA clones from a chicken aorta mRNA expression library in the plasmid vector pEX1. Antibodies affinity purified onto the fusion proteins recognized the chick type VI collagen Mr = 150,000 subunit. Northern blots using the cDNA inserts from the above clones revealed a single RNA species of about 4,600 nucleotides sufficient to code for a protein with the size of the Mr = 150,000 subunit.

Animals↗
PubMed1992

Construction of dystrophin fusion proteins to raise targeted antibodies to different epitopes.

For the study of the structure and function relationship of dystrophin, defective in DMD, and for diagnostic purposes it is important to dispose of antibodies against different parts of the protein. We have made five different constructs for the expression of fusion proteins containing parts of the four domains of dystrophin. Two different recombinant expression vectors, pATH2 and pEX1, were used. Rabbits were immunized with the fusion products and several polyclonal antibodies were raised. At a later stage, monoclonal antibodies were also raised to some of the fusion proteins. One polyclonal antibody, named P20 AB, is directed against the region covering amino acid sequence 1749-2248 or the nucleotide sequence 5456-6953 of the mRNA, which corresponds to the major deletion-prone region of the DMD gene. We show the particular value, sensitivity and specificity of the P20 AB in dystrophin analysis.

Antibody Formation↗