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Peroxisomal disorders I: biochemistry andgenetics of peroxisome biogenesis disorders

RJA Wanders and HR Waterham

Department of Pediatrics, AcademicMedical Centre, Emma Children’sHospital, University of Amsterdam, andDepartment of Clinical Chemistry,Laboratory Genetic Metabolic Diseases,Amsterdam, The Netherlands

Key words: fatty acids – genetics –inborn errors – peroxisome biogenesis –peroxisomes

Corresponding author: Prof. Dr RonaldJ. A. Wanders, Lab Genetic MetabolicDiseases, F0-224, Academic MedicalCentre, Meibergdreef 9, 1105 AZAmsterdam, The Netherlands.Tel.: þ31 205665958;fax: þ31 206962596;e-mail: [email protected]

Received 7 May 2004, revised andaccepted for publication 22 June 2004

Wanders RJA, Waterham HR. Peroxisomal disorders I: biochemistryand genetics of peroxisome biogenesis disorders.Clin Genet 2004: 67: 107–133. # Blackwell Munksgaard, 2004

The peroxisomal disorders represent a group of genetic diseases inhumans in which there is an impairment in one or more peroxisomalfunctions. The peroxisomal disorders are usually subdivided into twosubgroups including (i) the peroxisome biogenesis disorders (PBDs) and(ii) the single peroxisomal (enzyme-) protein deficiencies. The PBDgroup is comprised of four different disorders including Zellwegersyndrome (ZS), neonatal adrenoleukodystrophy (NALD), infantileRefsum’s disease (IRD), and rhizomelic chondrodysplasia punctata(RCDP). ZS, NALD, and IRD are clearly distinct from RCDP and areusually referred to as the Zellweger spectrum with ZS being the mostsevere and NALD and IRD the less severe disorders. Studies in the late1980s had already shown that the PBD group is geneticallyheterogeneous with at least 12 distinct genetic groups as concluded fromcomplementation studies. Thanks to the much improved knowledgeabout peroxisome biogenesis notably in yeasts and the successfulextrapolation of this knowledge to humans, the genes responsible for allthese complementation groups have been identified making moleculardiagnosis of PBD patients feasible now. It is the purpose of this reviewto describe the current stage of knowledge about the clinical,biochemical, cellular, and molecular aspects of PBDs, and to provideguidelines for the post- and prenatal diagnosis of PBDs. Less progresshas been made with respect to the pathophysiology and therapy ofPBDs. The increasing availability of mouse models for these disorders isa major step forward in this respect.

Zellweger syndrome (ZS) is the prototype of thegroup of peroxisomal disorders and was firstdescribed in the 1960s in two pairs of sibs, show-ing a series of abnormalities including craniofa-cial, hepatological, ocular, and skeletal aberrations.At about the same time, De Duve and coworkersperformed systematic studies in which rat liverhomogenates were subjected to differential anddensity gradient centrifugation. These studies ledto the identification of a new organelle containing anumber of H2O2-generating oxidases and catalasewhich decomposes H2O2 to O2 and H2O. The con-nection between ZS and peroxisomes first becameapparent in 1973whenGoldfischer et al. (1) reportedthe absence of morphologically identifiable perox-isomes in hepatocytes and kidney tubule cells ofZellweger patients. At that time, however, vir-tually nothing was known about peroxisomes

and it took another 10 years before the true sig-nificance of peroxisomes for human physiologystarted to become clear, thanks to two key obser-vations in Zellweger patients. First, Brown et al.(2) discovered distinct abnormalities in the fattyacid profile of plasma from Zellweger patientswith markedly elevated levels of the very-long-chain fatty acids (VLCFAs) C24:0 and C26:0,whereas normal levels were found for the otherfatty acids including long-chain fatty acids likepalmitic, oleic, and linoleic acid. At that time,peroxisomes were already known to contain afatty acid beta-oxidation system, just like mito-chondria, but the function of this system hadremained obscure. The findings by Brown et al.(2) suggested that peroxisomes are the site ofbeta-oxidation of VLCFAs, which was soonestablished experimentally (3). The second major

Clin Genet 2004: 67: 107–133 Copyright # Blackwell Munksgaard 2004

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discovery demonstrating the crucial role of per-oxisomes in humans appeared 1 year later whenHeymans et al. (4) reported the deficiency of plas-malogens, a special type of phospholipids belong-ing to the group of ether-linked phospholipids, intissues from Zellweger patients. Since then, muchhas been learned about the metabolic role ofperoxisomes and many different functions of per-oxisomes have been identified. In addition, manyof the enzymes involved in the different metabolicpathways within peroxisomes have been charac-terized, purified, and their respective cDNAs andgenes cloned. Parallel to this work, the essentialdetails of peroxisome biogenesis have beenworked out and many of the genes, coding forproteins essential for peroxisome biogenesis,have been identified. Thanks to this explosion ofnew information, enormous progress has beenmade with respect to the identification of newperoxisomal disorders followed by resolution ofthe underlying defects. At present, the group ofperoxisomal disorders comprises 17 well-defineddisorders, which are subdivided into two groupsincluding (i) the peroxisome biogenesis disorders(PBDs) and (ii) the single peroxisomal (enzyme-)protein deficiencies. This review is focused on thefirst group of disorders, the PBDs (Table 1), andwe will begin by discussing what is known aboutthe different PBDs.

The peroxisome biogenesis disorders: aclinically and genetically heterogeneous groupof disorders

The PBD group is comprised of four differentdisorders including ZS, neonatal adrenoleuko-dystrophy (NALD), infantile Refsum’s disease(IRD), and rhizomelic chondrodysplasia punc-tata (RCDP). ZS, NALD, and IRD are clearlydistinct from RCDP and are nowadays usuallyreferred to as ‘the Zellweger spectrum’ with ZSbeing the most severe and NALD and IRD lesssevere disorders. ZS is generally considered as theprototype of the PBD group. ZS is dominated by:(i) the typical craniofacial dysmorphism includinga high forehead, large anterior fontanel, hypo-plastic supraorbital ridges, epicanthal folds, anddeformed earlobes, and (ii) profound neurolog-

ical abnormalities. ZS children show severe psy-chomotor retardation, profound hypotonia,neonatal seizures, glaucoma, retinal degenera-tion, and impaired hearing. There is usuallycalcific stippling of the epiphyses and smallrenal cysts. Brain abnormalities in ZS includenot only cortical dysplasia and neuronal heteroto-pia but also regressive changes. There is dysmyelin-ation rather than demyelination. Patients withNALD have hypotonia and seizures, may havepolymicrogyria, progressive white matter disease,and usually die in late infancy. Patients with IRDmay have external features reminiscent of ZS butdo not show disordered neuronal migration andno progressive white matter disease. Their cogni-tive and motor development varies betweensevere global handicaps and moderate learningdisabilities with deafness and visual impairmentdue to retinopathy. Their survival is variable.Most patients with IRD reach childhood andsome even reach adulthood. Clinical distinctionbetween the different PBD phenotypes is not verywell defined. Common to all three are liver dis-ease, variable neurodevelopmental delay, retino-pathy, and perceptive deafness with onset in thefirst months of life.RCDP is clinically quite different from ZS,

NALD, and IRD and characterized by a dis-proportionally short stature primarily affectingthe proximal parts of the extremities, typicalfacial appearance, including a broad nasal bridge,epicanthus, high arched palate, dysplastic externalears, micrognathia, congenital contractures, char-acteristic ocular involvement, dwarfism, and severemental retardation with spasticity. Most RCDPpatients die in the first decade of life.ZS, NALD, IRD, and RCDP have been found

to be genetically heterogeneous as concludedfrom complementation studies as discussed laterin this review. The molecular defects underlyingthese different complementation groups (CGs)have been resolved in recent years. Two differentstrategies have been very rewarding in the iden-tification of these mutant genes, which includes(i) homology probing, making use of the infor-mation from different yeast mutants, and (ii)functional complementation analysis based onthe generation of peroxisome-deficient Chinese

Table 1. The peroxisome biogenesis disorders

Number Disorder Abbreviation Protein involved Gene Chromosome MIM

1 Zellweger syndrome ZS Peroxins PEX-genes Multiple loci 2141002 Neonatal adrenoleukodystrophy NALD Peroxins PEX-genes Multiple loci 2141103 Infantile Refsum’s disease IRD Peroxins PEX-genes Multiple loci 2023704 Rhizomelic chondrodysplasia

punctata type 1RCDP Type 1 Pex7p PEX7 6q21–q22 215100

Wanders and Waterham

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hamster ovary (CHO) cells. We will proceed bydescribing the current stage of knowledge aboutperoxisome biogenesis.

Peroxisome biogenisis: general aspects

Peroxisomal proteins are all encoded by nucleargenes and translated on free polyribosomes asfirst shown for urate oxidase and catalase, twoperoxisomal matrix proteins, by Goldmann andBlobel (5), and Robbi and Lazarow (6), respect-ively. Later studies have shown the same forperoxisomal membrane proteins (PMPs) (7).After synthesis on free polyribosomes, the newlymade peroxisomal proteins are targeted to per-oxisomes and then imported into pre-existingperoxisomes post-translationally, which impliesthat synthesis and import are sequential ratherthan simultaneous processes. In this way, peroxi-somes get bigger which requires recruitment ofphospholipids most likely from the endoplasmicreticulum (ER) to be incorporated into theperoxisomal membrane. Growth of peroxisomesmay continue until a critical size is reached afterwhich peroxisomes divide into two daughter per-

oxisomes that can then undergo the same cycle ofevents (Fig. 1a).The import of peroxisomal matrix and mem-

brane proteins into peroxisomes is a multistepprocess involving recognition of the cargo pro-tein by a receptor in the cytosol, docking of thereceptor–cargo complex at the peroxisomal mem-brane, translocation across the membrane, cargorelease into the organelle, and receptor recycling.Correct targeting of peroxisomal matrix proteinsis achieved via cis-acting sequences present in theprimary peptide sequences, which are called per-oxisomal targeting signals (PTSs). Most matrixproteins are equipped with a PTS type 1 (PTS1),which is a C-terminal serine-lysine-leucine-COOH (SKL) tripeptide, or a conservative vari-ant thereof, like SHL in D-aminoacid oxidase,AKL in sterol carrier protein 2 (SCP2), etc(Table 2). A few matrix proteins are targeted viaa different signal named PTS2, which is a 9-aminoacid sequence located near the N-terminus withthe amino acids in positions 1, 2, 8, and 9 beingmost important. The consensus PTS2 is R/K-L/V/I-XXXXX-H/Q-A/L in which X is any aminoacid. The PTS1 and PTS2 receptors have been

Fig. 1. Original (a) and modified (b)model for peroxisome biogenesis. (a)The original growth-and-divisionmodel proposed by Lazarow andFujiki (139) in which peroxisomeswere thought to be autonomousorganelles, which could not form denovo. (b) The modified model ofLazarow and Fujiki with peroxisomesnow envisaged as semiautonomousorganelles with the capacity to formde novo.

Peroxisomal disorders

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cloned and characterized from different species.The former, Pex5p, is a tetratricopeptide (TPR)repeat protein, whereas the latter, Pex7p, is a WD40repeat protein, to be discussed later.Similar to matrix proteins, PMPs are synthe-

sized on free cytosolic ribosomes and targeted tothe organelle by cis-acting targeting sequences(mPTS). In contrast to the simple PTS1 andPTS2 sequences found in matrix proteins,PMPs are directed to peroxisomes via, asyet, less well-defined targeting signals, to bediscussed later.

Peroxisome biogenesis: de novo formation ofperoxisomes or not?

As discussed above, peroxisome biogenesisresembles that of mitochondria and chloroplasts,which is true although the details are entirelydifferent. Indeed, protein translocation into per-oxisomes differs markedly from that in mitochon-dria which threads unfolded polypeptide chainsthrough a narrow channel, whereas peroxisomescan import folded and homo-oligomeric proteins

(8), hetero-oligomers (9, 10), and even 4–9-nmgold beads (11). The transport of such largecomplexes somewhat resembles protein transportinto the nucleus, but no such thing as a structureresembling the nuclear pore complex has everbeen observed in the peroxisomal membrane.The concept that peroxisomes multiply by

growth and division of pre-existing peroxisomeswould make peroxisomes belong to the group ofautonomousorganelleswithmitochondria,chloro-plasts, and the endoplasmic reticulum as repre-sentatives. This would imply that peroxisomescannot form de novo. Several experimental obser-vations have been done suggesting that peroxi-somes can form de novo, however. One of themain arguments in favor of de novo biogenesisof peroxisomes has been that cells, mutated inPEX3, PEX16, or PEX19, show no peroxisomalmembrane structures (ghosts), whereas reintro-duction of a wild-type copy of the mutant generestores peroxisome formation. These findingshave been interpreted as evidence for de novosynthesis of peroxisomes from some endomem-brane compartment such as the ER (Fig. 1b).

Table 2. List of bona fide peroxisomal (enzyme) proteins from humans and their PTS1 or PTS2 sequences

Peroxisomal function (Enzyme) protein PTS1/PTS2 Targeting sequence

Fatty acid b-oxidation Acyl-CoA oxidase 1 (straight chain) PTS1 –SKLAcyl-CoA oxidase 2 (branched chain) PTS1 –SKLAcyl-CoA oxidase 3 (pristanoyl-CoA) PTS1 –SKLL-bifunctional protein PTS1 –SKLD-bifunctional protein PTS1 –AKL3-ketothiolase (straight chain) PTS2 –RLQVVLGHL3-ketothiolase (branched chain) PTS1 –AKL2-methylacyl-CoA racemase PTS1 (þMTS) –(K)ASLCarnitine acetyltransferase PTS1 –AKLCarnitine octanoyltransferase PTS1 –THLAcyl-CoA thioesterase PTS1 –SKLBile acid-CoA: taurine/glycine conjugating enzyme PTS1 –SQL2,4-dienoylCoA reductase PTS1 –AKLD2,D3-enoylCoA isomerase PTS1 –SKLD3,5, D2,4-dienoylCoA isomerase PTS1 –SKLVery-long-chain acyl-CoA synthetase (VLACS) PTS1 –LKL

Fatty acid a-oxidation PhytanoylCoA hydroxylase PTS2 –RLQIVLGHL2-hydroxyphytanoylCoA lyase PTS1 –(R)SNM

Etherphospholipid biosynthesis Dihydroxyacetonephosphate acyltransferase PTS1 –AKLAlkyldihydroxyacetonephosphate synthase PTS2 –RLVLSGHL

Glyoxylate detoxification Alanine glyoxylate aminotransferase PTS1 –KKLPipecolic acid degradation L-pipecolate oxidase PTS1 –AHLH2O2 metabolism Catalase PTS1 –(K)ANL

Peroxiredoxin V PTS1 –SQLSterol carrier protein 2 PTS1 –AKLD-aspartate oxidase PTS1 –(K)SNLD-amino acid oxidase PTS1 –SHLHydroxyacid oxidase 3 PTS1 –SRLHydroxyacid oxidase 2 PTS1 –SRLHydroxyacid oxidase 1 (glycolate oxidase) PTS1 –SKL

Others 3-hydroxy-3-methyl glutarylCoA lyase PTS1 (þMTS) –CKLMalonylCoA decarboxylase PTS1 (þMTS) –SKLIsocitrate dehydrogenase PTS1 –AKL

PTS1, peroxisome-targeting signal type 1; PTS2, peroxisome-targeting signal type 2; MTS, mitochondrial targeting signal. In theright hand column, the different targeting sequences are shown using the single letter code for the various amino acids.


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Based on studies in the yeasts Yarrowia lipoly-tica and Hansenula polymorpha, it has been pro-posed that peroxisomes can be formed from smallpre-peroxisomal vesicles derived from the ER in aprocess dependent on COPI and COPII, two coatproteins involved in vesicle transport processes.Studies in human fibroblasts, however, haveshown that peroxisome biogenesis occurs inde-pendent of COPI and COPII (12, 13). Further-more, studies by South et al. (14) in the yeastSaccharomyces cerevisiae suggest that proteintraffic into the ER is not required to formperoxisomes. This was concluded from studiesin which the protein entry into the ER wasblocked by inactivation of the ER proteintranslocation factor, Sec61p, or its homolog,Ssh1p. These results argue against the ER asthe site of de novo peroxisome formation.Furthermore, studies by Snyder et al. (15) andHazra et al. (16) have provided compellingevidence against the dogma of the absence ofperoxisomal structures in pex3D, pex16D, andpex19D mutants. Indeed, Snyder et al. (15)identified tiny peroxisomal vesicles and tubulesin Pichia pastoris pex19D cells by deconvolu-tion microscopy using an antibody recognizingendogenous Pex3p. In addition, Hazra et al.(16) reported the identification of vesicularand tubular, torpedo-shaped peroxisomalstructures in P. pastoris pex3D cells andcharacterized these by isopyknic and flotationcentrifugation.The jury is still out on the origin of per-

oxisomes, however, as emphasized by severalvery recent studies. Firstly, Geuze et al. (17)recently presented evidence of the involvementof the ER in peroxisome formation in mousedendritic cells using electron microscopy, immuno-cytochemistry, and three-dimensional imagereconstruction of peroxisomes and associated com-partments. Additional support for the formationof peroxisomes from some endomembrane com-partment has also come from studies by Faberet al. (18) who have shown that an N-terminalfragment of Pex3p expressed in H. polymorpha isassociated with vesicular membrane structuresthat also contain Pex14p. Furthermore, thesestructures appeared to have the potential todevelop into functional peroxisomes after intro-duction of full-length PEX3 and arise from thenuclear membrane. In conclusion, it remains to beestablished whether there are indeed two parallelpathways for peroxisome formation, one frompre-existing peroxisomes and a second de novopathway, which allows peroxisome formationfrom some endomembrane compartment such asthe ER.

Peroxisome biogenesis: a closer look

The realization that a simple organism likebaker’s yeast could be used to study peroxisomebiogenesis and resolve the sorting and targetingof peroxisomal proteins to their correct destin-ation, the peroxisome, has had a tremendousimpact and explains for a large part why the pur-suit of genes defect in PBD patients has been sofruitful in the last few years. The key to theapplication of genetics to the elucidation ofthe mechanism of peroxisome biogenesis and theidentification of the proteins involved was theisolation of peroxisome-deficient mutants (pexmutants) from different yeast species and CHOmutants (19). Erdmann et al. (20) were the first todevice a selection screen based on the notion thatin yeast, peroxisomes are essential for growth onoleate. This follows logically from the fact thatin yeast, fatty acids can only be oxidized inperoxisomes whereas in higher eukaryotes beta-oxidation can occur both in peroxisomes andin mitochondria. S. cerevisiae cells were sub-jected to chemical mutagenesis and grown firston glucose agar plates followed by replica plat-ing onto oleate agar plates to select for cells notgrowing on oleate (onu-mutants). Subsequently,cell fractionation studies were performed toeliminate mutants with no abnormalities in per-oxisome biogenesis but a defect in the fatty acidbeta-oxidation system. This approach resultedin a total of 12 different mutants that turnedout to be peroxisome deficient. Similar screenshave been set up for a variety of different yeastspecies including P. pastoris, H. polymorpha, andY. lipolytica. Additional screens and selections,based on other approaches, have also been setup which together has led to the generation of alarge series of peroxisome biogenesis mutants.Subsequent complementation of these mutantsusing yeast genomic libraries has resulted in theidentification of a large number of genes involvedin peroxisome biogenesis. Initially, these newgenes were all given different names even withinthe same species (i.e. PAF, PAS, PEB, PER, andPAY genes). To simplify matters, all of these geneshave been renamed as PEX genes (PEX1, PEX2,PEX, etc) and the products of these genes arecalled peroxins (Pex1p, Pex2p, Pex3p, etc). Theperoxins were agreed to include all proteinsinvolved in peroxisome biogenesis inclusive ofperoxisome matrix protein import, membranebiogenesis, peroxisome proliferation, and peroxi-some inheritance.In the original study of Erdmann et al. (20),

12 different S. cerevisiae mutants were identifiedin which peroxisome biogenesis was impaired.


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One by one the genes mutated in each of theseso-called pas-mutants have been identified, ofwhich the first one was described by Erdmannet al. in 1991 (21). The gene involved (PEX1)codes for a protein belonging to the family oftriple A (AAA) ATPases, which are involved inthe assembly, organization, and disassembly ofprotein complexes (22). The discovery of thefirst peroxisome biogenesis gene in S. cerevisiaewas soon followed by reports from the samegroup describing the second (PEX3) (23) andthird (PEX4) (24) S. cerevisiae PEX genes. Inpex1D, pex3D, and pex4D cells, the import ofPTS1 and PTS2 proteins is impaired, indicatingthat Pex1p, Pex3p, and Pex4p play an essentialrole in the import of matrix proteins. Later stud-ies revealed that these mutants are different if theimport of PMPs is studied. Indeed, pex1D andpex4D cells are still able to assemble their PMPsinto membranes, whereas pex3D cells lack thisproperty. Studies by Hettema et al. (25) in a seriesof 19 S. cerevisiae mutants have shown that theimport of PTS1 and/or PTS2 proteins is impairedin all mutants except one (pex11D), whereas PMPimport is normal in all these mutants except forthe pex3D and pex19D mutants. These data are inline with the notion that Pex3p and Pex19pbelong to a distinct group of peroxins requiredfor the proper localization and stabilization ofPMPs as discussed in the next section. With therecent identification of three PEX genes in theyeast S. cerevisiae, i.e. PEX 30, 31, and 32 (26),the total number of PEX genes now stands at 32(Table 3).The complete set of 32 PEX genes can be sub-

divided into two groups in which group 1 includesthose genes of which orthologs are found amongmost, if not all, peroxisome-containing species,whereas group 2 refers to those PEX genes whichare only found in single organisms. Most of thePEX genes belong to group 1 with orthologs indifferent species. PEX genes belonging to group 2are PEX18 and PEX21, which are only found inS. cerevisiae (27), and PEX20 which is only foundin Y. lipolytica (28) and Neurospora crassa (29).These results indicate that the principal features ofperoxisome biogenesis are similar among differentorganisms but not identical. Table 3 describes thefull list of PEX genes so far identified and theirdistribution among different species as well assome characteristics of the peroxins encoded bythe different PEX genes.So far, 16 different PEX genes have been iden-

tified inhumans.These includeHsPEX1,HsPEX2,HsPEX3, HsPEX5, HsPEX6, HsPEX7,HsPEX10, HsPEX11a, HsPEX11b, HsPEX11g,HsPEX12, HsPEX13, HsPEX14, HsPEX16,

HsPEX19, and HsPEX26. We will proceed bydescribing the proteins encoded by these PEXgenes and their presumed role in peroxisomebiogenesis. Conceptually, the process of peroxisomebiogenesis can be subdivided into distinct stepsincluding (i) peroxisome membrane assembly,(ii) import of matrix proteins, and (iii) peroxisomeproliferation and maintenance. In the next para-graphs, we will describe what is known about thesedifferent steps with particular emphasis on thesituation in humans. We will begin by describingperoxisome membrane biogenesis and the rolesof Pex3p, Pex16p, and Pex19p.

Peroxisome membrane biogenesis and the humanperoxins HsPEX3p, HsPEX16p, and HsPEX19p

The first clue that the mechanism involved inperoxisome membrane biogenesis is fundamen-tally different from the one used to transportperoxisomal matrix proteins across the peroxiso-mal membrane was the discovery by Santos et al.(30) that cells from Zellweger patients containperoxisome membrane structures, called ghosts,which contain PMPs but lack most, if not all, oftheir matrix protein content. Like the peroxiso-mal matrix proteins, PMPs are synthesized onfree polyribosomes and imported into peroxi-somes by a direct cytosol-to-peroxisome mechan-ism. In general, PMPs lack functional PTS1 andPTS2 signals and their import is independent ofthe PTS1- and PTS2-protein import routes. Thisis true for all bona fide integral PMPs (iPMPs),whereas peripheral PMPs, like dihydroxyacetone-phosphate acyltransferase (DHAPAT) and alkyl-DHAP synthase, use the PTS1- and PTS2-proteinimport routes. Multiple studies have attempted todefine the targeting signals in iPMPs. Thesestudies have clearly shown that iPMPs are nottargeted to peroxisomes via carboxy-terminal oramino-terminal extensions as in PTS1 and PTS2proteins. All data show that the targeting infor-mation is actually contained within the polypep-tide chain itself. Although knowledge abouttargeting signals in iPMPs has remained limitedso far, one signal has been identified in bothsingle- and multi-span transmembrane proteins,which is made up of a basic cluster of amino acidsoriented towards the peroxisomal matrix, in frontof a transmembrane span which directly followsthe basic amino acid cluster. Some proteins mayrequire additional targeting information on thecytosolic side of the peroxisomal membrane as inScPex15p (31). There is increasing evidence, how-ever, which suggests that iPMPs are directed toperoxisomes via multiple, distinct targeting sig-nals rather than a single targeting signal. Indeed,


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Table3.Overview

ofthedifferentPEXgenesandcharacteristicsoftheirprotein

products

(peroxins)

Identifiedin

Gene

Hs

Sc

Yl

Nc

Ce

Humangene

locus

Peroxin

characteristics

Subcellular

localization

Interacting

peroxins

References

PEX1

þþ

þþ

þ7q21–q22

AAA–protein

requiredforperoxisomalmatrix

protein

import;interacts

withPex6p

Mainly

cytosolic,

partly

peroxisomal

Pex6p

(21,117,140)

PEX2

þþ

þþ

þ8q21.1

RING

zincfingerprotein

involvedin

matrix

protein

importdownstream

ofreceptordocking

IntegralPMP

Pex10p

(141)

PEX3

þþ

–þ

–6q23–q24

PMPim

port;possible

dockingfactorforPex19p

IntegralPMP

Pex19p

(23,46)

PEX4

–þ

–þ

–E2–ubiquitin

conjugatingenzymerequired

forperoxisomalmatrix

protein

import

PeripheralPMP

Pex22p

(24,142,143)

PEX5

þþ

þþ

þ12p13.3

TPR–protein;receptorforPTS1proteins

Mainly

cytosolic,

partly

peroxisomal

Pex7p,8p,10p,

12p,13p,14p

(49,50,55)

PEX6

þþ

þþ

þ6p21.1

AAA–protein;interacts

withPex1p,ScPex15p,

andHsPex26p;requiredformatrix

protein

import

Mainly

cytosolic,

partly

peroxisomal

Pex1p,(Sc)Pex15p,

(Hs)Pex26p

(84,95)

PEX7

þþ

–þ

–6q21–q22.2

WD–protein;receptorforPTS2proteins

Mainly

cytosolic,

partly

peroxisomal

Pex5pL,13p,14p,

18p,20p,21p

(59,62,63,64)

PEX8

–þ

þþ

–Involvedin

matrix

protein

importdownstream

ofreceptordocking

LuminalPMP

Pex5p,Pex20p

(144,145)

PEX9

––

þ–

–Involvedin

matrix

protein

import;only

identified

inY.lip

olytica

IntegralPMP

(146)

PEX10

þþ

þþ

–1p36.32

RING

zincfingerprotein;requiredformatrix

protein

import;actingdownstream

ofreceptordocking

IntegralPMP

Pex2p,5p,12p,

and19p

(147,148)

PEX11

þþ

þþ

–15q25.2

(a)

1q21.1(b)

19p13.3

(g)

Involvedin

peroxisomedivisionandproliferationand/or

transportofmedium

chain

fattyacids

IntegralPMP

Pex19p

(101,102,104–106)

PEX12

þþ

–þ

þ17q21.1

RING

zincfingerprotein,requiredformatrix

protein

import,actingdownstream

ofreceptordocking

IntegralPMP

Pex5p,10p,

and19p

(149)

PEX13

þþ

–þ

þ2p14–p16

SH3–protein;matrix

protein

import;involvedin

receptordockingwithPex14p

IntegralPMP

Pex5p,7p,14p,

and19p

(150,151,152)

PEX14

þþ

þþ

1p36.22

Initialsiteofreceptordocking

PMP

Pex5p,7p,13p,

17p,and19p

(153)

PEX15

–þ

––

Requiredformatrix

protein

import;membraneanchor

forPex6p;yeastequivalentofhumanPex26p

IntegralPMP

Pex6p

(31,95)

PEX16

þ–

þþ

11p11.11

RequiredforPMPim

port,togetherwith

Pex3pandPex19p

IntegralPMP

Pex19p

(47,154)

PEX17

–þ

––

–Requiredformatrix

protein

import

PeripheralPMP

Pex14p,Pex19p

(42,79,80)

PEX18

–þ

––

RequiredforPTS2protein

importin

S.cerevisiae;

bindsto

ScPex7p

Mainly

cytosolic,

partially

peroxisomal

Pex7p

(27)

PEX19

þþ

þþ

þ1q22

Cytosolic

PMPreceptor

Mainly

cytosolic,

partly

peroxisomal

Pex3p,10p,12p,

13p,14p,16p,17p,

11ap

,11bp

(43,155,156)


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Table3.(continued)

Identifiedin

Gene

Hs

Sc

Yl

Nc

Ce

Humangene

locus

Peroxin

characteristics

Subcellular

localization

Interacting

peroxins

References

PEX20

––

þþ

–RequiredforPTS2protein

importandthiolase

olig

omerizationin

Y.lip

olytica

Mainly

cytosolic,

partly

peroxisomal

(28)

PEX21

–þ

––

–RequiredforPTS2protein

importin

S.cerevisiae;

bindsto

Pex7p

Mainly

cytosolic

Pex7p,13p,14p

(27)

PEX22

–þ

––

–PMPinvolvedin

matrix

protein

import;membrane

anchorforPex4p

IntegralPMP

Pex4p

(157)

PEX23

––

þþ

–PMPinvolvedin

matrix

protein

import

IntegralPMP

(158)

PEX24

––

þ–

–Involvedin

peroxisomeassembly;highsequence

sim

ilarity

toYlPex28pandYlPex29p

IntegralPMP

(160)

PEX25

–þ

––

–Involvedin

regulatingperoxisomenumber,size,and

distributiontogetherwithPex28p,Pex29p,andVps1p

PeripheralPMP

Pex27p

(159,161)

PEX26

þ–

––

–22q11.21

Matrix

protein

import;recruitsPex1p–Pex6pcomplexto

theperoxisomalmembrane

IntegralPMP

Pex6p

(96)

PEX27

–þ

––

–Controls

peroxisomesizeandnumber;extensivesequence

sim

ilarity

toPex11pandPex25p

PeripheralPMP

Pex25p

(159,162)

PEX28

––

þ–

–Involvedin



IntegralPMP

(163)

PEX29

––

þ–

–Involvedin



IntegralPMP

(163)

PEX30

–þ

––

–Involvedin

thecontrolofperoxisomesizeandproliferation,

togetherwithPex28p,29p,31p,and32p

IntegralPMP

Pex28p,29p,

31p,32p

(26)

PEX31

–þ

––

–Involvedin



IntegralPMP

Pex28p,29p,

30p,32p

(26)

PEX32

–þ

––

–Involvedin



IntegralPMP

Pex28p,29p,

30p,31p

(26)

Abbreviationsused:Hs=Homosapiens;Sc=Saccharomycescerevisiae;Yl=Yarrowia

lipolytica;Nc=Neurospora

crassa;Ce=Caenorhabditis

elegans;PMP=Peripheral

MembraneProteins.


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following earlier work by Dyer et al. (32), Wanget al. (33) reported the identification of threediscrete targeting signals in S. cerevisiaePMP47. Furthermore, Jones et al. (34) showedthat PMP34, the human homolog of C. boidiniiPMP47, contains at least two non-overlapping setsof targeting information (amino acids 1–147 and244–307), either of which is sufficient for insertioninto the membrane. This is in contrast to data byHonsho et al. (35) who reported that PMP47 wastargeted to peroxisomes via a different PTS locatedin the region containing amino acids 183–194.Furthermore, Jones et al. (34) also identified twoindependent sets of targeting information in humanPex13p. In addition, Brosius et al. (36) identifiedtwo distinct, non-overlapping peroxisomalmembrane-targeting signals in rat and humanPMP22, one in the amino-terminal and the otherin the carboxy-terminal end of the protein. Takentogether, these results challenge the assumption thatPMPs are targeted to peroxisomes via single PTSsand rather suggest the involvement ofmultiple, non-overlapping targeting regions in iPMPs.Studies in different yeast mutants as well as in

fibroblasts from PBD patients have shown thatghosts are absent in some mutants indicating thatin these mutants, the targeting of both peroxiso-mal matrix proteins and PMPs is deficient. InS. cerevisiae, the pex3 and pex19 mutants turnedout to lack ghost-like structures. The same wasfound for human fibroblasts mutated in eitherthe PEX3 or PEX19 gene. Furthermore, ghostswere also lacking in fibroblasts from patientsmutated in PEX16. Taken together, these resultsindicate that Pex3p, Pex16p, and Pex19p play anessential role in peroxisome membrane biogenesisas described below.

PEX3The PEX3 gene, first cloned in S. cerevisiaeby Hohfeld et al. (23) encodes a 42–52-kDaprotein, firmly anchored in the peroxisomal mem-brane with its C-terminus exposed to the cytosol,whereas opinions differ with respect to theN-terminus being either cytosolic or intraperox-isomal. The human gene was cloned in 1998 byKammerer et al. (37). Pex3p interacts withPex19p via its C-terminal domain. In humancells with defective PEX3, the peroxin Pex14p ismislocalized to mitochondria, whereas the perox-isomal transporters adreno leuko dystrophyprotein (ALDP) and peroxisomal membraneprotein of 70 kDa (PMP70) are absent and lessabundant, respectively. In CHO pex3D cells,Pex12p and Pex13p are absent and Pex14p lessabundant.

PEX19Pex19p is a farnesylated protein first identified byJames et al. (38) in CHO cells. Subsequent studieshave led to the identification of a number of yeasthomologs as well as human PEX19 (39). The pro-tein is hydrophilic and contains a CAAX boxallowing farnesylation of the cysteine. The exactrole of Pex19p farnesylation is not resolved yet,although it may assist in peroxisomal membraneassociation. Indeed, in S. cerevisiae, farnesylationappears to be essential for its function (40), but thisis not true for P. pastoris (15) and in humans (41).Pex19p is predominantly cytosolic, with only asmall amount bound to the peroxisomal mem-brane, and interacts with a large variety of PMPsincluding peroxins: (i) Pex3p, Pex10p, Pex12p,Pex13p, Pex14p, Pex16p, and Pex17p; (ii) proteinsinvolved in peroxisome proliferation (Pex11a andPex11b); (iii) metabolite transporters (PMP34,PMP70, ALDP, and adreno leuko dystrophyrelated protein (ALDR); and (iv) PMPs ofunknown function (PMP22 and PMP24) (15, 40–44). Based on these results, it is suggested thatPex19p may function as a cytosolic PMP receptoranalogous to Pex5p and Pex7p, which are thecytosolic receptors for PTS1 and PTS2 proteins,respectively. Elegant experiments by Sackstederet al. (41) in which Pex19p was directed to thenucleus by fusing it to a nuclear localization signalhave provided convincing evidence in favor of thissuggestion although this view is disputed by others(43, 45, 46).

PEX16In contrast to Pex3p and Pex19p, which arepresent in multiple mammalian and yeast spe-cies, Pex16p is lacking in most species and hasonly been reported in humans and the yeastY. lipolytica (47) in which Pex16p has differentproperties as compared to human Pex16p play-ing no role in membrane assembly. The humanPEX16 gene was identified by Honsho et al.(48) and encodes a 38.6-kDa integral membraneprotein with two putative membrane-spanningdomains and both the N- and C-termini exposedto the cytosol. Its function is unknown. Cellsdefective in PEX16 lack ghosts as assessed byimmunofluorescence microscopy analysis ofPMP70 (48) and a range of other PMPs (12).

Import of peroxisomal matrix proteins

Recognition of PTS1 and PTS2 proteins in thecytosol by the import receptors Pex5p and Pex7pThe realization that peroxisomal proteinsare synthesized on cytosolic polyribosomes and


115

contain specific targeting signals, which directthem to peroxisomes, implied the existence ofreceptors, recognizing the PTS1 and PTS2sequences. The PTS1 receptor (Pex5p) was firstidentified in 1993 in the yeasts P. pastoris (49) andS. cerevisiae (50). Subsequent studies have led tothe identification of orthologs of Pex5p in a rangeof different species including humans (51–53).Pex5p binds PTS1 proteins in the cytosol andcycles between the cytosol and the peroxisome.In most organisms, Pex5p is mainly localized inthe cytosol with only a small fraction being asso-ciated with peroxisomes. Based on these data, amodel has been proposed called the ‘shuttle-model’ in which Pex5p binds its cargo, i.e. a PTSprotein, in the cytosol, after which the receptor–cargo complex docks at the peroxisomal mem-brane followed by dissociation of the complexand transport of the PTS1 protein across themembrane and recycling of the receptor backinto the cytosol (Fig. 2a). In the yeast Y. lipolytica,however, Pex5p is mainly intraperoxisomal (54).Similar observations have been made in otheryeasts including H. polymorpha (55). This duallocalization of Pex5p has led to a revised modelfor protein import into peroxisomes in which thePex5p–PTS1 protein complex is translocatedacross the peroxisomal membrane in toto followedby recycling of the receptor back into the cytosol.Recent work by Dammai and Subramani (56) sug-gests that such a so-called extended shuttle modelmay also apply to the human situation (Fig. 2b).Pex5p belongs to the family of TPR-containing

proteins, which are characterized by highly degen-erate, repetitive sequences of 34 amino acids.TPRs are found as tandem arrays of 3–16 motifs

in a wide variety of proteins involved in manydifferent cellular processes including cell-cycleregulation, chaperone functions, and proteinphosphorylation. The C-terminal half of Pex5pconsists of two clusters each comprising threeTPR domains (TPR 1–3 and TPR 5–7), whichare linked by a hinge region denoted TPR4. TheTPR domains participate in a special foldingstructure that allows the interaction with the PTS1tripeptide that appears to be embraced by allTPR motifs (57, 58). The importance of theTPR domains for recognition of the PTS1 tripep-tide is immediately clear if it is realized that asingle amino acid change (N489K) within thesixth TPR domain abolishes interaction betweenhuman Pex5p and the PTS1 signal and causesNALD, one of the Zellweger spectrum disorders(51). In addition to binding PTS1 proteins, allPex5p proteins bind Pex13p and Pex14p whereasmammalian Pex5p proteins also bind Pex7p asdiscussed later.

Pex7p, the receptor for PTS2 proteins. The iden-tification by Erdmann et al. (20) of an S. cerevisiaemutant with a defect in PTS2-mediated import,but a normal PTS1-import pathway, led Kunauand coworkers to identify the PTS2 receptor(Pex7p) (59) which turned out to be a memberof the WD-40 family of proteins, a family char-acterized by repeats of approximately 40 aminoacid residues, each containing a central Trp-Asp(WD) motif. WD-40 proteins have been implicatedin interactions with TPR-containing proteins andrecent evidence suggests that Pex5p and Pex7pindeed interact, at least in mammals as discussedbelow. After its initial identification in S. cerevisiae

Cytoplasm

Peroxisomalmembrane

Peroxisomalmatrix

Cytoplasm

Peroxisomalmembrane

Peroxisomalmatrix

Receptor

Peroxisomal matrix protein

Original shuttle model Extended shuttle model

Receptor

Fig. 2. Schematic representation of theoriginal and modified shuttle models. (a)The original shuttle model in which thereceptor shuttles between the cytosol,where a PTS1 or PTS2 protein is pickedup, and the cytosolic face of theperoxisomal membrane, where thereceptor–cargo complex docks followedby dissociation of the receptor–cargocomplex and transfer of the cargoprotein across the peroxisomalmembrane and recycling of the receptorback into the cytosol. (b) The modifiedso-called ‘extended shuttle’ model inwhich the receptor–cargo crosses theperoxisomal membrane en blockfollowed by back transport of theempty receptor from the inside ofperoxisomes to the cytosol.


116

(59–61), subsequent Pex7p proteins have beenidentified in other species including mammals(62–64). The subcellular localization of Pex7p isstill controversial due to conflicting results in humanfibroblasts and S. cerevisiae, which show a pre-dominant cytosolic localization in human fibroblastsvs an entirely peroxisomal localization in S. cerevisiaeas concluded by Zhang and Lazarow (60).

PTS2 protein import route in mammals andyeasts: similar game, different players(HsPex5pL, ScPex18p/Pex21p, and YlPex20p/NcPex20p). Despite the many similarities betweenmammals and yeasts with respect to peroxisomebiogenesis, there are also important differences,one being the role of Pex5p. Indeed, in yeasts,Pex5p is only involved in the import of PTS1 pro-teins, whereas in mammals, Pex5p is involved inboth PTS1- and PTS2-protein import. In contrast,Pex7p is involved in PTS2-protein import only,which is true for both mammals and yeasts. Theexclusive role of Pex5p and Pex7p in PTS1- andPTS2-protein import, respectively, in yeasts, isexemplified by the phenotypes of the pex5 andpex7 yeast mutants in which only the import ofPTS1 proteins (pex5-mutant) or PTS2 proteins(pex7-mutant) is impaired. Subsequent studiesrevealed that in human skin fibroblasts andCHO cells, the situation is different with respectto Pex5p. In fact, analysis of human and CHOpex5-mutants revealed two different phenotypes;in some mutants, only the PTS1-protein importpathway was disrupted whereas in other mutants,both the PTS1- and PTS2-protein import path-ways were blocked. This enigma was resolvedwhen Pex5p was found to exist in two forms inmammals: a long form (Pex5pL) and a short form(Pex5pS). Pex5pS and Pex5pL are identical withone important difference, which is the presence ofan additional internal segment of 37 amino acidspositioned between amino acids 214 and 215(human) or 215 and 216 (Chinese hamster). Thedifferential role of Pex5pS and Pex5pL was clearlyshown by Otera and coworkers (65). These authorsshowed that in pex5-deficient CHO cells disturbedin both PTS1- and PTS2-protein import, Pex5pSonly restored the import of PTS1-proteins whereasPex5pL restored both PTS1- and PTS2-import inthe same cells. The same was shown by Bravermanet al. (66) in human pex5-mutants in which Pex5pLrestored both PTS1- and PTS2-protein importwhereas Pex5pS restored PTS1-protein importonly. These data clearly show that Pex5pL playsan essential role in PTS2-protein import. Subse-quent studies have shown that Pex5pL and Pex7pinteract with one another. The region in Pex5pLnecessary for this interaction was mapped using

truncated versions of Pex5pL and includes theamino-terminal amino acids of the Pex5pL-specific37 amino acids insertion, together with amino acidslying outside this region. The S214F mutation inthis region disrupted binding to Pex7p as shownby Otera and coworkers (67) and resulted in aspecific PTS2-protein import defect, while PTS1-protein import was not affected (68). It is importantto stress that the role of Pex5pL in the import ofPTS2 proteins in mammals is independent of its rolein the import of PTS1 proteins. Indeed, when atruncated version of Pex5pL was expressed con-taining only the amino-terminal half of the proteinwithout a TPR motif, complementation of thePTS2-protein import defect was observed in PEX5-deficient mammalian cells (67, 69).Recent studies have shed new light on the

remarkable difference with respect to the roleof Pex5p between yeasts and mammals. Studiesin the yeast S. cerevisiae have shown thatPTS2-protein import is not only dependent onPex7p but also on Pex18p and Pex21p (27),which are not found in humans. In Y. lipolytica(28) and N. crassa (29), PTS2-protein import isdependent upon another protein named Pex20p.It turns out that the amino acid sequence in the37 amino acid internal region of Pex5pL is highlyconserved in S. cerevisiae Pex18p and Pex21p andY. lipolytica and N. crassa Pex20p, suggesting thatthis region, shared between the four proteins, isinvolved in the formation of an import-competentcomplex of Pex7p and PTS2 proteins. The absenceof the conserved peptide motif in fungi is in linewith the fact that Pex5p plays no role in PTS2-protein import, whereas its presence in differentmammals, protozoa, and plants indicates thatPex5p is also involved in PTS2-protein import inthese organisms. Figure 3 depicts the differentPTS2-protein import pathways and the distinctroles of HsPex5pL, ScPex18p and ScPex21p, andYlPex20p/NcPex20p in the different species. Inter-estingly, Pex5p of Caenorhabditis elegans doesnot contain this conserved peptide motif, whichagrees with the complete lack of the PTS2-proteinimport pathway in this organism (70).

Receptor docking and the essential role of humanPex13p and Pex14pThere is general agreement that Pex13p andPex14p form the docking complex where thePTS1- and PTS2-protein import routes converge.Pex13p is a PMP with two membrane-spanningdomains with both the N- and C-terminusexposed to the cytosol. Pex13p belongs to thefamily of SH3 (Src-Homology 3) proteins. TheSH3 domain in Pex13p is located at its C-ter-minus. SH3 domains are small, non-catalytic


117

protein modules capable of protein–protein inter-actions, which participate in diverse intracellularprocesses. These domains consist of 60–70 aminoacids, have a high sequence similarity, and formstructurally similar conformations. Resolution ofthe three-dimensional structure of various SH3domains and their contact sites with peptideligands has revealed that highly conserved arom-atic amino acid residues form a hydrophobiccleft running between two variable loops: Thishydrophobic cleft forms the binding platformfor ligand association, with the two variableloops contributing to ligand recognition andspecificity. Typically, SHR domains recognizeand bind short proline-rich peptides. Theminimal consensus sequence for this peptideis Pro-X-X-Pro (P-X-X-P), where X is anyamino acid, plus an additional basic amino acidlocated C-terminally (class I: P-X-X-P-X-R) orN-terminally (class II: R-X-X-P-X-X-P) of the

P-X-X-P core. The proline-rich peptide segmentadopts a left-handed polyproline-type helix(PP2) that, depending on the class of the ligand,can bind in two orientations with respect to theSH3 domain.Pex14p is the other member of the docking

machinery and is required for the import ofboth PTS1 and PTS2 proteins. Pex14p is tightlyassociated with the peroxisomal membrane eitheras a peripheral membrane protein or an integralmembrane protein. Pex14p interacts with Pex13pbut can also bind directly to Pex5p and Pex7p.Schliebs et al. (71) have shown that the amino-terminal 78 amino acids of human Pex14p areinvolved in binding of Pex5p with a very highaffinity. Multiple binding sites for Pex14p wereshown to be present in the amino-terminus ofPex5p, and subsequent studies showed that thepentapeptide WXXXF/Y repeats were involvedin binding Pex14p in mammals and in plants

Pex5pL

PTS2p Pex7p

PTS2p

PTS2p PTS2p

Pex7p

Pex7p Pex7p

PTS2p Pex7p

Pex18p

Pex21p

Pex14p Pex

13p

+

Pex18p Pex21p

H. sapiensY. lipolyticaN. crassa

Pex20p

Pex20p

S. cerevisiae

Pex5pL

Fig. 3. PTS2-protein import in mammals and yeast: similar game, different players. In several organisms, including humans (Homosapiens) the long form of the PTS1 receptor (Pex5pL) is needed for the import of PTS2 proteins by forming a complex with Pex7p andits cargo, i.e. a PTS2 protein. In Saccharomyces cerevisiae, PTS2-protein import requires the active participation of two helperproteins (Pex18p and Pex21P) whereas in Yarrowia lipolytica and Neurospora crassa, this task is fulfilled by a single protein, i.e.Pex20p.


118

(67, 72, 73). The number of pentapeptide repeatsdiffers among the different organisms with tworepeats in S. cerevisiae Pex5p, and seven inhuman Pex5pL. Pex14p directly interacts withPex13p via the SH3 domain which involves aclass II P-X-X-P-X-R motif (PPTLPHRDW) inPex14p as shown for S. cerevisiae (74). Binding ofPex5p to the same SH3 domain of Pex13p doesnot occur at the PP-2-binding phase but at anovel interaction site (75). X-ray crystallographyand mass spectrometry data from Dounagamathand coworkers (76, 77) revealed the existence oftwo functionally and structurally independentbinding sites on the SH3 domain of Pex13pfor Pex5p and Pex14p, respectively, with Pex7pbinding at the amino-terminal end of the Pex13p.Although it was initially thought that Pex13p

was the first site of receptor docking, current datasuggest that it is in fact Pex14p, which comes first.This model is supported by data from Oteraet al. (67) and Urquhart et al. (78) who showedthat Pex14p binds to PTS1-loaded Pex5p whereasPex13p only binds to unloaded Pex5p. Oteraet al. (67) proposed that Pex5p bound to a PTS1protein first binds to the Pex13p–Pex14p complexvia interaction with Pex14p after which thePTS1 protein is released from Pex5p followedby dissociation of the Pex13p–Pex14p complex.Subsequently, the unloaded Pex5p is transferredto Pex13p and shuttles back to the cytosol. Thismodel implies that Pex13p and Pex14p formfunctionally distinct subcomplexes, which areboth involved in the import process of peroxi-somal proteins. Taken all data together, Pex14pis indeed the most likely primary dockingprotein. It might well be that other proteins arepart of the docking complex. A good candidate,identified in S. cerevisiae (79) and P. pastoris(42), is Pex17p which behaves as a peripheralmembrane protein tightly bound to the peroxi-somal membrane in S. cerevisiae (79) whereas inP. pastoris, it is an integral membrane proteinwith the carboxyterminus facing the cytosol (42).In S. cerevisiae, Pex17p is thought to be part ofthe docking complex together with Pex14p andPex13p (79), whereas Snyder et al. (42) favored amodel in which Pex17p is also involved in theimport of PMPs. Subsequent studies by Hettemaet al. (25) and Harper et al. (80) showed normalimport of PMPs in both Ppex17pD and Scpex17Dcells, which argues against the model of Snyderet al. (42). Taken together, the bulk of evidencefavors a role of Pex17p in peroxisomal matriximport and not in the import of PMPs. Exceptfrom its identification in S. cerevisiae andP. pastoris, no mammalian Pex17p has beenidentified so far.

Translocation across the peroxisomal membraneand the human peroxins Pex2p, Pex10p, andPex12pThree peroxins belonging to the family of RINGzinc finger proteins, i.e. Pex2p, Pex10p, andPex12p, are thought to be involved in the actualtransport machinery. All three proteins areiPMPs and have a carboxy-terminal RING fingerdomain exposed to the cytosol. Based on thefinding that fibroblasts from PBD patients withmutations in PEX2, PEX10, or PEX12 accumu-lated Pex5p at the level of peroxisomes in contrastto normal fibroblasts, Pex2p, Pex10p, and Pex12pare thought not to be involved in receptor dock-ing but in one of the subsequent steps of proteinimport. Mutant pex2, pex10, or pex12 cells are alldisturbed in the import of peroxisomal matrixproteins while the import of PMPs is not affected.Reguenga et al. (81) have obtained evidence sug-gesting that Pex2p and Pex12p are together ina complex with Pex14p and Pex5p (81). Pex13pis also part of this complex although in non-stoichiometric amounts.Another prediction for the proteins involved in

translocation would be that they should interactdirectly or indirectly either with the cargo to betranslocated or with the receptors for that cargo,Pex5p and/or Pex7p. Pex2p, Pex10p, and Pex12pall contain a C3HC4 zinc-binding domain, orRING finger, a protein module that is thoughtto mediate protein–protein interactions. TheRING finger is essential for the functions ofboth Pex10p and Pex12p, and recent studieshave shown that Pex10p and Pex12p directlyinteract with Pex5p and with each other (82, 83).

Receptor recycling and the role of the humanperoxins Pex1p, Pex6p, and Pex26pThe peroxins Pex1p and Pex6p are members of thelarge family of AAA proteins (ATPases) asso-ciated with a wide range of cellular activities(21, 84). The AAA domain consists of 220–230amino acids and contains two motifs namedWalker A and B, which bind and hydrolyzeATP, respectively (85). The role of Pex1p andPex6p in peroxisome biogenesis has remainedcontroversial with two opposing views. The firstview holds that Pex1p and Pex6p are required forperoxisome biogenesis possibly playing a role insome membrane fusion event involving vesiclesderived from the ER (86). This hypothesis wasstimulated, in part, by the observation that manyof the initially identified members of the AAAATPase family were involved in membrane fusionevents. In line with this postulate, Titorenko et al.(87) showed that in Y. lipolytica, Pex1p and


119

Pex6p stimulate the fusion of pre-peroxisomalvesicles.A complicating factor is that the subcellular loca-

lization of Pex1p and Pex6p is controversial. In ratsand H. polymorpha, Pex1p and Pex6p are asso-ciated with the peroxisomal membrane (88, 89),whereas in P. pastoris and Y. lipolytica, thereseems to be an association with vesicles distinctfrom mature peroxisomes (87, 90, 91). On theother hand, in human cells, both Pex1p and Pex6pare predominantly cytosolic (92, 93), although theseresults were obtained by overexpression. It has beenshown that Pex1p and Pex6p interact with eachother in an ATP-dependent manner (89, 90, 93, 94).The second view holds that Pex1p and Pex6p

are involved in matrix protein transport ratherthan in peroxisome membrane biogenesis. Oneof the arguments is that human cells mutated inPEX1 and PEX6 contain abundant peroxisomalmembrane structures (ghosts) that are larger, notsmaller, than peroxisomes typically present in con-trol fibroblasts. Furthermore, absence of Pex1por Pex6p results in a dramatic instability of Pex5pwith levels falling as low as 1–5% of control. Onthe other hand, Pex5p levels are normal or evenelevated in human cells with inactivating muta-tions in PEX2, PEX10, or PEX12, demonstratingthat Pex5p instability is not a general characteristicof human pex-mutants. A similar reduction inPex5p abundance has been observed in P. pastorispex1- and pex6-mutants (92). Taken together,these results indicate that Pex1p and Pex6p arerequired for the stability of Pex5p and most likelyplay a role in the recycling of the PTS1 receptor.Recent studies have shown that the S. cerevisiae

integral membrane protein Pex15p binds Pex6p inan ATP-dependent manner (95). Interestingly,studies by Matsumoto et al. (96, 97) have led tothe identification of human Pex26p whichanchors both Pex1p and Pex6p to the peroxiso-mal membrane and in fact appears as the humanequivalent of yeast Pex15p as depicted in Fig. 4,which shows the essential features of peroxisomebiogenesis in humans and the presumed role ofPex1p, Pex2p, Pex5p, Pex6p, Pex7p, Pex10p,Pex12p, Pex13p, Pex14p, and Pex26p in theuptake of PTS1 and PTS2 proteins.

Peroxisome proliferation and maintenance

Peroxisomes are markedly dynamic organelles,and the number and matrix enzyme content ofperoxisomes can change dramatically dependingupon environmental conditions and the meta-bolic state. In the yeast S. cerevisiae, for instance,exposure to fatty acids, particularly oleic acid,leads to a large increase in peroxisome abundance

and size. The proliferation of peroxisomes underthese conditions is associated with dramatic changesin gene expression, which requires the transcriptionfactors Pip2 and Oaf1 (98, 99). These two proteinsbind oleate-response elements within transcrip-tional control regions of responsive genes and arerequired for both the transcriptional response tooleic acid and the proliferation of peroxisomes.In rodents, the number, size, and enzyme content

of peroxisomes can be induced dramatically by cer-tain xenobiotics like clofibrate and plasticizers aswell as naturally occurring fatty acids. A key playerin this respect is the peroxisome proliferator--activated receptor-a (PPAR-a), a member of thenuclear receptor super family. Activated PPAR-aforms a heterodimer with a second member of thenuclear receptor super family, the retinoid-X receptor(RXR) together forming an active transcription fac-tor that binds cis-acting elements called peroxisomeproliferator-responsive elements (PPREs). In linewith the important role of PPAR-a in the inductionof peroxisomes in rodents is the fact that PPAR–/–

mice do not show induction of peroxisomes by clofi-brate and other peroxisome proliferators (100).The peroxin Pex11p also plays a major role in

peroxisome proliferation. Indeed, the S. cerevisiaepex11 mutant accumulates only four to five verylarge peroxisomes when incubated in oleic acidcontaining medium, whereas overexpression ofPEX11 led to hyper-proliferation of peroxisomeswith an increased number of peroxisomes ofreduced size (101–103). These data indicate thatPex11 proteins control peroxisome division.According to VanRoermund et al. (104), the defectin peroxisome division and proliferation in pex11Dcells is secondary to the role of Pex11p in perox-isomal beta-oxidation. This conclusion was basedon the finding that pex11D cells display a markedblock in medium chain fatty acid beta-oxidation.Studies by Li and Gould (105) suggest, however,that the defect in medium chain fatty acid beta-oxidation is a secondary phenomenon with Pex11pprimarily controlling peroxisome division.In humans, three distinct genes with high

similarity to yeast PEX11, including PEX11a,PEX11b, andPEX11g, have been identified. Stud-ies by Schrader et al. (106) showed that over-expression of the human PEX11b gene alonewas sufficient to induce peroxisome proliferation,demonstrating that proliferation can occur in theabsence of extracellular stimuli. Furthermore,overexpression of PEX11a also induced peroxi-some proliferation but to a much lower extent.Studies in the mouse, which also has three Pexgenes including Pex11a, Pex11b, and Pex11g,have shown that, although Pex11a expression isinduced by activation of PPAR-a, Pex11a is not


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required for peroxisome proliferation induced byclassical peroxisome proliferators like clofibrate.Interestingly, Pex11a was required for peroxi-some proliferation in response to 4-phenylbuty-rate, which does not act via PPAR-a (107).Studies in the yeast S. cerevisiae by Hoeffner

et al. (108) have shown that the dynamin-likeGTPase Vps1p is required for peroxisomedivision. A mammalian dynamin-like proteinDLP1p, has also been identified (109). Knock-down of DLP1 expression by siRNA causedtubulation of peroxisomes and inhibition of per-oxisome division. In the last few years, a numberof new peroxins have been described, which are allinvolved in regulating peroxisome size, number,and distribution (Table 3). It is clear that muchremains to be learned about the factors involvedin peroxisome proliferation and maintenance.

Laboratory diagnosis of peroxisome biogenesisdisorders

ZS, NALD, and IRD

Although peroxisomes catalyze a variety of dif-ferent metabolic functions, there are three func-tions which are of direct relevance for the

laboratory diagnosis of PBDs. These are: (i)beta-oxidation of fatty acids, including VLCFAsnotably C26:0, pristanic acid, and di- and trihy-droxycholestanoic acid; (ii) biosynthesis of etherphospholipids, notably plasmalogens; and (iii)alpha-oxidation of phytanic acid. In patientsbelonging to the Zellweger spectrum with ZS,NALD, and IRD, all these peroxisomal functionsare deficient, which leads to the accumulationof C26:0, pristanic acid and di- and trihydroxy-cholestanoic acid, the deficiency of plasmalogens,and the accumulation of phytanic acid. It shouldbe added that pristanic acid and phytanic acid aresolely derived from dietary sources and may thusbe completely normal in PBD patients, especiallywhen they are young. In the remaining PBD,i.e. RCDP, in which PEX7 is mutated, peroxiso-mal beta-oxidation is normal, thus explainingthe normal VLCFA profile in these patients.Plasmalogen synthesis and phytanic acid alpha-oxidation, however, are defective in RCDP. As aconsequence, the laboratory diagnosis of PBDsdepends upon the type of PBD, suspected in aparticular patient (see flowcharts of Figs 5 and 6).Plasma VLCFA analysis has turned out to be a

very reliable method for the laboratory diagnosis of

2 12 10 2 12 1014 13 26

6 1

Pex7p

+ +PTS1

PTS1

Pex5pS

Pex5pS

Pex5pSPex

7p

PTS2

Pex7p

PTS2

Pex5pL

Pex5pL+

PTS1-pathwayPTS2-pathway

CYTOSOL

PEROXISOME

PTS2 PTS1

PEROXISOMAL MEMBRANE

Fig. 4. Peroxisome biogenesis in humans and its essential features in which the presumed roles of the different peroxins is shownwithin the framework of the original shuttle model of Fig. 2(a).


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ZS, NALD, and IRD. In a series of>500 Zellwegerspectrum patients ranging from classical ZS to IRDand other mild variants, plasma VLCFAs havealways been found abnormal except for a fewexceptional cases. Indeed, we recently identified aPBD patient with normal VLCFA levels but abnor-mal bile acid intermediates, indicating that thepatient definitely suffered from a peroxisomal dis-order. Subsequent studies led to the identificationof a peroxisome biogenesis defect due to bona fidemutations in the PEX12 gene. These findings indi-cate that great care must be taken with respect tothe laboratory diagnosis of a peroxisomal disorderand that a normal VLCFA profile does not excludea peroxisomal disorder (Fig. 5).It should be added that the finding of an abnor-

mal VLCFA profile in a particular Zellweger

spectrum patient does not necessarily point to aPBD. Indeed, plasma VLCFA may also beabnormal in a number of single peroxisomalenzyme deficiencies, notably D-bifunctional pro-tein deficiency and acyl-CoA oxidase deficiency.Patients suffering from the latter two disordersare clinically indistinguishable from patientssuffering from a Zellweger spectrum disorder.Obviously, plasma VLCFAs are also elevated inX-ALD patients. However, the clinical signs andsymptoms of X-ALD patients are quite differentfrom those of PBD patients.In order to resolve whether the accumulation of

VLCFAs in a particular patient is due to a defectin peroxisome biogenesis or results from anisolated defect in peroxisomal beta-oxidation,fibroblast studies are required. In fibroblasts,

Measure plasma very long chain fatty acids

If abnormal : definite PBD or POD*

PBD POD

D-BP deficiency AOX deficiency

Molecular analysis

Zellweger Spectrum disorder (ZS/NALD/IRD) Clinical suspicion

If normal : No PBD or POD with some exceptions

In case of persistent clinical suspicion : Full analysis of peroxisomal metabolites

notably DHCA/THCA Full analysis ofperoxisomal parameters inplasma and erythrocytes

If normal : Definitely noPBD or POD

If abnormal : PBD or POD

Full work-up of peroxisomal functions in fibroblasts

Complementation analysis

Measure activity of D-bifunctional Protein (DBP) and Acyl-CoA Oxidase (AOX)

Molecular analysis of relevant PEX gene

Fig. 5. Flowchart for the differentialdiagnosis of patients with clinicalsigns and symptoms suggestive of aZellweger spectrum disorder.


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plasmalogen biosynthesis, peroxisomal beta-oxidation, and phytanic acid alpha-oxidation canbe measured reliably. Furthermore, the presenceor absence of peroxisomes can be studied viaimmunofluorescence microscopy analysis usingantibodies against peroxisomal matrix proteinslike catalase. In the majority of patients, suchfibroblast studies lead to an unequivocal diag-nosis allowing discrimination between a PBDand a peroxisomal beta-oxidation disorder at thelevel of D-bifunctional protein or acyl-CoA oxidase(see flowcharts of Fig. 5).With respect to the laboratory diagnosis of

RCDP, analysis of plasmalogens in erythrocytesis also highly reliable. Indeed, in all establishedRCDP patients we have studied through the years(>100),erythrocyteplasmalogenswerealwaysdefi-cient making erythrocyte plasmalogen analysisa highly reliable initial laboratory test indicatingthat if plasmalogens are deficient, a peroxisomalform of RCDP is established. Because RCDPis genetically heterogeneous with three distinctgenetic forms including RCDP type 1, the mostfrequent type of RCDP belonging to the PBDgroup, and the less frequent RCDP types 2 and3 belonging to the group of single peroxisomalenzyme deficiencies, additional studies have tobe done to pinpoint the precise type of RCDP.Resolution between the three types again

requires detailed studies in fibroblasts althoughanalysis of phytanic acid in plasma may also behelpful to discriminate between type 1 and type2/3, respectively. The fact that phytanic acid isderived solely from exogenous sources renders

phytanic acid analysis in plasma, however, anunreliable parameter (110, 111). Indeed, if phyta-nic acid is elevated in a particular RCDP patient,one can be sure of RCDP type 1, which mayprompt direct molecular analysis of the PEX7gene. A normal phytanic acid level, however,may point to RCDP type 2 or type 3, butRCDP type 1 cannot be excluded. In this case,definitive identification of RCDP type 1, 2, or 3requires detailed studies in fibroblasts, whichincludes activity measurements of dihydroxyace-tonephosphate transferase and alkyl-DHAPsynthase, the products of the GNPAT andADHAPS genes, respectively. Figure 6 depictsthe flowchart we use in the daily practice of thelaboratory diagnosis of RCDP.

Molecular basis of the peroxisomal biogenesisdisorders

Due to the potential involvement of many dif-ferent genes, an essential prerequisite for theidentification of the molecular defect in anypatient affected by a peroxisomal biogenesisdisorder is complementation analysis. Comple-mentation analysis is a powerful tool to resolvewhether a particular disorder, or group of dis-orders, is genetically heterogeneous or not. Inpractice, complementation analysis involvesfusion of fibroblasts from two patients, affectedin peroxisome biogenesis, for instance. Fusionwill generate hybrid cells containing nucleifrom the two patients’ fibroblasts. These cellsare called heterokaryons. If the defective genes

Full analysis of peroxisomal functions in fibroblasts

Measure erythrocyte plasmalogens

Rhizomelic chondrodysplasia punctataClinical suspicion

If normal :Definitely not a

peroxisomal formof RCDP

If abnormal :Peroxisomal form of

RCDP, either type 1, 2 or 3Measure phytanic acid

In plasma

If abnormal :RCDP type 1

If normal :RCDP type 2

or 3, or 1

RCDPtype 1

RCDP type 2

RCDP type 3

PEX 7 analysis

GNPAT analysis

ADHAPS analysis

Fig. 6. Flowchart for the differentialdiagnosis of patients with clinical signsand symptoms suggestive of rhizomelicchondrodysplasia punctata.


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in the two patients’ cell lines are different, onewould expect restoration of peroxisome forma-tion, whereas in the other case, when the mutantgenes are identical, no complementation wouldoccur. Tager and coworkers (112, 113) were thefirst to apply complementation analysis to studythe genetic basis of different PBDs. In a firststudy by Brul et al. (113), fibroblasts fromseven PBD patients with phenotypes rangingfrom ZS to NALD, IRD, and RCDP were sub-jected to complementation analysis. These sevenpatients’ cell lines were found to belong to fivedifferent CGs, which immediately suggestedmarked genetic heterogeneity within the PBDgroup. Brul et al. (113) used two different par-ameters to assess complementation, including (i)activity measurement of DHAPAT, a peroxiso-mal enzyme catalyzing the first step in plasma-logen biosynthesis, and (ii) catalase latency. Thelatter method determines whether catalase is per-oxisomal or not. Subsequently, several groupshave also performed complementation analysisusing other methods to assess complementation,including de novo plasmalogen biosynthesis,phytanic acid alpha-oxidation, peroxisomalbeta-oxidation, and immunofluorescence micro-scopy analysis using antibodies raised againstcatalase. The latter method, first applied byYajima et al. (114), allows direct visualizationof peroxisomes in fused cells and is the methodof choice to assess complementation in case ofPBD patients. Because catalase immunofluores-cence is normal in fibroblasts from RCDPpatients, this method can only be applied tocells from Zellweger spectrum patients.A collaborative study between the three main

groups performing complementation analysis has

led to the identification of nine distinct CGs (115).In subsequent years, two additional CGs havebeen identified which brings the total numbernow at 11 or 12, if RCDP is also included (Table 4).With the recent identification of PEX26 as the

defective gene in CG8 by Matsumoto et al. (96,97), the PEX genes underlying each of the CGshave all been identified now. Most CGs includeonly a few patients. One exception to this rule isCG1, with PEX1 as the defective gene, which isby far the largest CG containing more than halfof all Zellweger spectrum patients (116–119). Inour own series of 246 Zellweger spectrum patientsthereby excluding RCDP, 174 patients (59%)were found to belong to CG1 (PEX1) followedby 12% in CG4 (PEX6) and 6% in CG3 (PEX12)(Gootjes et al., unpublished).If all mutant PEX alleles are taken together,

more than 100 mutations have so far beendescribed in literature. In many cases, mutationsare private being restricted to single families only.Most mutations have been described in the PEX1gene (>40). Among these mutations, a fewcommon mutations have been identified. Mostcommon is a missense mutation in exon 15(c.2528G>A) leading to the substitution of theglycine at position 843 of Pex1p by an asparagine(p.G843D) in the second ATP-binding domain.Patients homozygous for this mutation show themild Zellweger spectrum phenotype (NALD/IRD). The frequency of the c.2528G>A(p.G843D) allele ranges from 0.25 to 0.37 in thedifferent cohorts. In our own cohort of PEX1-mutated patients, we found an allele frequency of0.36. Twenty percent of the patients were homo-zygous and 33% were compound heterozygousfor the c.2528G>A allele.

Table 4. Complementation groups in peroxisome biogenesis disorders

Complementation groups Import of peroxisomal matrix proteins

Number Gifu KKI Adam PTS1 PTS2 Import of iPMPs Gene involved Phenotypes

1 A 8 VI – – þ PEX 26 ZS/NALD/IRD2 B 7 (¼5) VII – – þ PEX 10 ZS/NALD/IRD3 C 4 (¼6) III – – þ PEX 6 ZS/NALD/IRD4 D 9 VIII – – – PEX 16 ZS5 E 1 II – – þ PEX 1 ZS/NALD/IRD6 F 10 V – – þ PEX 2 ZS7 G 12 IX – – – PEX 3 NALD8 H 13 X – – þ PEX 13 ZS/NALD9 J 14 XI – – – PEX 19 ZS10 2 IV – –a þ PEX 5 ZS/NALD/IRD11 3 XII – – þ PEX 12 ZS/NALD/IRD12 R 11 I þ – þ PEX 7 RCDP

KKI, ; PTS, peroxisomal targeting signals; iPMP, integral PMPs.aIn a single patient (51), only the PTS1-import pathway was found to be defective with preservation of the PTS2-import pathwaydue to a point mutation in the PEX5 gene causing a N489K amino acid substitution. The mutant Pex5p was unable to sustainPTS1-protein import but was apparently able to form a stable complex with Pex7p and PTS2 proteins allowing a normallyfunctioning PTS2-import pathway.


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The second most common mutation is aT-insertion in exon 13 (c.2097–2098insT), firstdescribed by Maxwell et al. (120) and Collinsand Gould (121), which results in a frame shiftand low steady state PEX1 mRNA levels,presumably caused by nonsense mediated RNAdecay. At the protein level, it leads to truncationof the PEX1 protein within the second AAAdomain, abolishing PEX1 function completely.In its homozygous form, the mutation results inthe severe Zellweger phenotype. In three differentstudies, an allele frequency of around 0.3 hasbeen reported. However, in our own patientcohort, we found an allele frequency of 0.16(Gootjes et al., unpublished). Together, thesetwo mutations account for around 50–60% ofall mutant PEX1 alleles. Interestingly, the muta-tion c.2528G>A leads to a mutant Pex1p, whichallows some residual import of peroxisomalmatrix proteins. The mutation seems to result ina misfolded protein, which is more stable at lowerthan at higher temperatures, which explains themosaic catalase immunofluorescence pattern at37� but a virtually normal pattern at 30� (122,123). The G843D aminoacid substitution mostlikely disrupts the interaction between Pex1pand Pex6p (93, 124).

RCDP

The molecular basis of RCDP type 1 with PEX7as the mutant gene has recently been described intwo large studies describing mutation data in 60(125) and 78 (126) patients with RCDP type 1,respectively. Braverman et al. (125) reported theidentification of a total of 24 mutant PEX7 alleleswhereas Motley et al. (126) described 22 differentmutant alleles. In both studies, one frequentmutation was found (L229X), which leads to atruncated protein with no apparent biologicalfunction. A few additional mutations with lessfrequency have been described in addition to alarge series of mostly private mutations.

Prenatal diagnosis of PBDs

The different PBDs are severe disorders, oftenassociated with early death thus warranting pre-natal diagnosis. Prenatal diagnosis of the differ-ent PBDs requires a full study in the index patientand should not be based on clinical signs andsymptoms only. This is immediately clear if it isrealized that the clinical diagnosis ZS may eitherbe due to a defect in peroxisome biogenesis or adefect in peroxisomal beta-oxidation, notably atthe level of D-bifunctional protein (110). We will

discuss the prenatal diagnosis of the differenttypes of PBDs (ZS/NALD/IRD vs RCDP) below.

ZS/NALD/IRDIf the index patient has been studied in full detail,which includes complementation analysis andmolecular analysis of the PEX gene involved,followed by confirmation of the mutationsfound in DNA from the parents, the preferredprenatal diagnostic method is mutation analysis.In practice, however, the exact molecular defecthas not been determined in every patient. Thismay be due to the fact that fibroblast studiesdid not include complementation analysis thusobstructing identification of the PEX geneinvolved. In such cases, prenatal diagnosis canalso be done in chorionic villous tissue and/orchorionic villous fibroblasts using other methods.Obviously, chorionic villous material, rather thanchorionic villous fibroblasts, is the material ofchoice because of potential problems such asmaternal overgrowth and failure of cells togrow. In principle, a variety of methods can beused for prenatal diagnosis including measure-ment of the activity of: i) DHAPAT, ii) alkyl-DHAP synthase, iii) acyl-CoA oxidase, iv)D-bifunctional protein, and v) immunoblot analysisof peroxisomal enzyme proteins, notably acyl-CoA oxidase, bifunctional protein, and peroxiso-mal thiolase. In our own center, we measure theactivity of DHAPAT and, in addition, performimmunoblot analysis of acyl-CoA oxidase andperoxisomal thiolase. Analysis of acyl-CoA oxi-dase in normal chorionic villous material yieldsimmuno-reactive bands of 70, 50, and 20 kDa,whereas in chorionic villous biopsy material ofaffected fetuses, only the 70-kDa band is observed.In case of peroxisomal thiolase, analysis of nor-mal chorionic villous biopsy material shows asingle band of 41 kDa, whereas in chorionicvillous material of affected fetuses, only the pre-cursor form of peroxisomal thiolase at 44 kDais seen.In fibroblasts of more mildly affected patients,

including patients affected with NALD or IRD,DHAPATismoderatelydeficientandthe immuno-blot profiles of acyl-CoA oxidase and peroxi-somal thiolase are not fully conclusive with the50-kDa and 20-kDa bands of acyl-CoA oxidaseand the 41-kDa band of peroxisomal thiolasepresent, albeit in reduced amounts. For this rea-son, we do not perform any analyzes in chorionicvillous biopsy material, but we prefer other meth-ods in cultured chorionic villous cells, notablyimmunofluorescence microscopy analysis usingspecific antibodies directed against catalase as


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well as measurement of the VLCFA profile byGC/MS and/or C26:0 or pristanic acid beta-oxidation in intact chorionic villous cells. Thebest and easiest procedure is immunofluorescencemicroscopy analysis of catalase, which usuallygives an unambiguous result. In the past 15 years,we have done more than 200 prenatal diagnosesof PBDs with no mistakes.

RCDPAs described above for the prenatal diagnosis ofZS/NALD/IRD, prenatal diagnosis of RCDPshould be done preferably by molecular analysisof the PEX7 gene. Obviously, this requiresdetailed studies in fibroblasts from the indexpatient in order to discriminate between RCDPtype 1, 2, and 3, followed by molecular analysis ofthe PEX7, GNPAT, and ADHAPS gene and con-firmation of the mutations found in the parents.As with the other types of PBD, such detailedstudies often have not been done in fibroblastsfrom particular patients, which implies that insuch cases, prenatal diagnosis should be doneusing enzymatic and/or cell biological methods.In case bona fide abnormalities have been foundin fibroblasts from the index patient, we prefer todo immunoblot analysis of peroxisomal thiolaseplus quantitative determination of plasmalogens.This set of tests usually generates unequivocalresults so that we rarely need to do additionalstudies in cultured chorionic villous cells. Insome cases where studies in fibroblasts from theindex patient have shown only minor abnormal-ities with some 41-kDa thiolase present and onlya partial deficiency of plasmalogens in fibro-blasts, we prefer to do detailed studies in culturedchorionic villous cells with powerful additionalmethods such as de novo plasmalogen biosynth-esis and phytanic acid alpha-oxidation.

Therapy

Treatment options for PBD patients haveremained limited so far. An important problemis that in the severe PBD forms, including ZS andRCDP, abnormalities already develop in utero,which limits potential postnatal treatment. Sup-portive therapies, such as anticonvulsant ther-apy to control seizures, physical and orthopedictherapy, and correction of visual and auditoryimpairment, are important to improve qualityof life. The identification of milder phenotypeswith less pronounced abnormalities and survivalinto the third and even fourth decade of lifehas stimulated attempts to correct the differentbiochemical abnormalities postnatally. Indeed,

efforts have been made to correct the deficiencyof plasmalogens by supplementation of alkylgly-cerol to the diet, to decrease VLCFAs, and espe-cially phytanic acid levels, via dietary regimens,and to reduce the toxicity of the accumulatingbile acid intermediates by supplementing urso-and chenodeoxycholic acid (127). Partial bio-chemical and clinical benefits have been reported,but no definite conclusion can be drawn fromthese studies due to the small number of patientsincluded. In recent years, much interest has cen-tered around docosahexaenoic acid (DHA), apolyunsaturated fatty acid implied in many phys-iological processes, whose levels are markedlyreduced in tissues of Zellweger spectrum patients.Studies by Martinez (128) in 20 PBD patients ofunspecified genotype, but mainly at the mild endof the Zellweger spectrum, have shown improvedliver function, and in addition, improved plasmalevels of peroxisome metabolites and subjectiveimprovement in muscle tone, social contact, andvision. Myelination was claimed to be improvedin more than half of those examined by magneticresonance imaging. These improvements warrantadditional larger scale studies.

Mouse models of peroxisome biogenesisdisorders

In recent years, several mouse models have beengenerated in which different genes have beendisrupted, which either code for a peroxin or aperoxisomal enzyme or transport protein. So far,six different Pex gene knockouts have beendescribed (Pex2, 5, 7, 11a, 11b, and 13) (107,129–133). In 1997, the first Pex gene knockoutswere reported by Faust and Hatten (129) andBaes et al. (130). The clinical, biochemical, andcellular phenotypes of the Pex2 and Pex5 homo-zygous knockout mice are remarkably similar.Firstly, both the Pex2(–/–) and Pex5(–/–) miceshow the same metabolic abnormalities as des-cribed in Zellweger patients including elevatedVLCFAs in plasma and tissues, deficient plasma-logens in tissues and erythrocytes, deficient DHAlevels in brain (30–40% decrease) but not in livertissue, and accumulation of the C27 bile acidintermediates di- and trihydroxycholestanoicacid. In addition, marked mitochondrial abnorm-alities were found in various organs of thePex5(–/–) mouse (including liver, proximal kid-ney tubules, adrenal cortex, and heart) and spe-cific cell types (skeletal and smooth muscle cellsand neutrophils) (134). Ultrastructural studiesrevealed the presence of large aggregates of pleo-morphic mitochondria with alterations of the


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mitochondrial outer membrane as well as thecristae. These mitochondrial alterations werequite heterogeneous with normal appearing mito-chondria and severely misshaped mitochondriawithin a single cell. Biochemically, partial defi-ciencies of complex I and V in livers of Pex5(–/–)mice were found amounting to 40% and 65% ofmean control values, respectively. Interestingly,complex IV was much higher in liver ofPex5(–/–) mice(180% of mean control). The sig-nificance of these findings remains to be estab-lished, especially as ATP levels were higher,rather than lower, in Pex5(–/–) livers as com-pared to control livers. Remarkably, the mito-chondria of the Pex2(–/–) mice were described asnormal (129).With respect to the clinical abnormalities in

Pex2(–/–) and Pex5(–/–) mice, mutant pupsshowed intrauterine growth retardation, severehypotonia with failure to eat, and neonataldeath. Most of the affected pups died within24 h. Interestingly, survival was found to dependon the genetic background with embryonic leth-ality in a 129 Svev background and survival for7–10 days in a 129 Svev/Swiss Webster back-ground (135). In the adrenal cortex of Pex2(–/–)mice, abnormal lipid storage was found withcharacteristic lamellar lipid inclusions. In the cen-tral nervous system of newborn mutant mice,there is disordered lamination in the cerebral cor-tex, and an increased cell density in the under-lying white matter, indicating an abnormality ofneuronal migration. Studies in longer survivingPex2 knockout mice showed that neurons thatare delayed in migration at birth eventually popu-late the cortex, but that mislocalization within thecortical laminae occurs. In 1-week-old Pex2(–/–)mice, cerebellar abnormalities were observedincluding reduced size, altered folial patterning,and reduced dendritic arborization of Purkinjecells (135). At birth, no signs of liver fibrosis,renal cysts, calcifications in bone, or facial mal-formation were apparent in the Pex2(–/–) andPex5(–/–) mice, which is different from what isobserved in ZS patients.The Pex5(–/–) mouse was also used for initial

studies on pathophysiologal mechanisms of thedisease. Janssen et al. (136) studied whether thereduced level of DHA in brain of Pex5(–/–) micewas a potential cause of the neuronal migrationdisturbance. Supplementation of pregnant Pex5heterozygous mothers with DHA ethyl esterduring the last 8 days of gestation normalized theDHA content in brain phospholipids with noclinical improvement, however. Indeed, hypoto-nia, growth retardation, and neuronal migrationwere as severe as in untreated mice. Importantly,

because in vivo and in vitro experiments haveshown that glutamatergic neurotransmission viathe N-methyl-D-aspartate (NMDA) receptor,linked to changes in intracellular calcium levels,controls the speed of migration, the potentialinvolvement of NMDA neurotransmission inthe neuronal migration defect of Pex5(–/–) micewas investigated by administering NMDA recep-tor agonists and antagonists to Pex5(–/–)embryos during the migration period (137).Treatment of Pex5(–/–) embryos with NMDAantagonists induced embryonic death whereasNMDA agonists partially reversed the migrationdefect. No changes in NMDA receptor density orglycosylation status were found betweenPex5(–/–) and wild-type brain tissue. A deficit inNMDA signal transduction was demonstrated inneuronal cultures derived from Pex5(–/–) mice bymonitoring calcium influx in response to NMDA.Pex5(–/–) cells were less sensitive to NMDA thanwild-type cells. This effect could be restored bypre-incubation with platelet-activating factor, anetherphospholipid, which requires functional per-oxisomes for its synthesis. Taken together, theseresults suggest that the neuronal migration defectmay result, at least in part, from defectiveNMDA signaling. Maxwell et al. (133) describedanother knockout mouse model in which Pex13was disrupted. The Pex13(–/–) mouse resemblesthe Pex2(–/–) and Pex5(–/–) mice in manyrespects.Recently, Brites et al. (131) described the gen-

eration of a Pex7 knockout mouse as a modelfor RCDP. Homozygous mice are viable anddisplay phenotypic abnormalities, characteristicof RCDP. Pex7(–/–) mice are severely hypotonicat birth and show a marked growth impairment(dwarfism). Mortality in Pex7(–/–) mice is highestin the perinatal period, although some Pex7(–/–)mice survived beyond 18months. Biochemically,Pex7(–/–) mice displayed the same set of abnorm-alities as observed in RCDP type 1 patientsincluding a deficiency of plasmalogens in all tis-sues. Interestingly, neuronal migration was foundto be impaired, although not to the same extent asin Pex2(–/–) and Pex5(–/–) mice. Analysis ofbone ossification in newborn Pex7(–/–) micerevealed a defect in ossification of distal boneelements of the limbs as well as parts of theskull and vertebrae. Interestingly, Rodemer et al.(138) recently described the generation of amouse model in which the gene coding for DHA-PAT (Gnpat), the first enzyme involved in ether-phospholipid biosynthesis, was disrupted. Thephenotype of the Gnpat(–/–) mouse is comparableto that of the Pex7(–/–) mouse, which indicatesthat the pathogenesis of RCDP is predominantly,


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if not exclusively, determined by the inability tosynthesize etherphospholipids.The last two of the Pex knockout mouse

models, generated in recent years, include thePex11a(–/–) and Pex11b(–/–) mice. As describedearlier, Pex11 proteins represent true peroxinsplaying an essential role in peroxisome prolif-eration. Mammals have three PEX11 genes:PEX11a, PEX11b, and PEX11g (105). Pex11a isinducible by peroxisome proliferators, whereasPex11b is expressed constitutively. Pex11a(–/–)mice are phenotypically and metabolically nor-mal. In contrast, mutant mice lacking Pex11bhave a severe clinical phenotype with intrauterinegrowth retardation, hypotonia, and death within24 h, which phenotype strongly resembles that ofmice lacking either Pex2, Pex5, or Pex13.Remarkably, Pex11b(–/–) mice show no peroxi-somal abnormalities. Indeed, plasma VLCFAs aswell as plasmalogen levels were completely nor-mal in contrast to the findings in Pex2(–/–),Pex5(–/–), and Pex13(–/–) mice. These puzzlingresults have been interpreted to imply that neitherVLCFAs nor plasmalogens contribute to theZellweger phenotype, at least in rodents. As faras plasmalogens are concerned, this conclusion ishard to reconcile with the marked phenotype ofthe Gnpat(–/–) mouse, recently generated byRodemer et al. (138).Despite these puzzling data, the increasing

availability of mouse models, particularly thosewith conditional alleles, will allow a better under-standing of pathophysiological mechanisms withthe ultimate perspective of effective treatmentsfor PBD patients.

Acknowledgements

The authors’ work was financially supported by the followinggrants: EU project number QLG1-CT-2001-01277 [mousemodels of peroxisomal diseases (MMPD)]; EU project numberQLG3-CT-2002-00696 [Refsum’s disease: diagnosis, pathology,and treatment (RDDPT)]; NWO project number 901-03-159;NWO project number 916.46.109; NWO project number901-03-097; NWO project number 99008; PBF project number97-0216; PBF project number 99-0220; and Royal DutchAcademy of Sciences. Dr Hans Waterham is a fellow of theRoyal Dutch Academy of Arts and Sciences. The authorsgratefully acknowledge Maddy Festen for excellent prep-aration of the manuscript and Jos Ruiter for preparation ofthe figures.

References

1. Goldfischer S, Moore CL, Johnson AB et al. Peroxisomaland mitochondrial defects in the cerebro-hepato-renalsyndrome. Science 1973: 182: 62–64.

2. Brown FR III, McAdams AJ Cummins JW et al. Cerebro-hepato-renal (Zellweger) syndrome and neonatal adrenoleu-

kodystrophy: similarities in phenotype and accumulation ofvery long chain fatty acids. Johns Hopkins Med J 1982: 151:344–351.

3. Singh I, Moser AE, Goldfischer S, Moser HW. Lignocericacid is oxidized in the peroxisome: implications for theZellweger cerebro-hepato-renal syndrome and adrenoleu-kodystrophy. Proc Natl Acad Sci USA 1984: 81: 4203–4207.

4. Heymans HSA, Schutgens RBH, Tan R, van den Bosch H,Borst P. Severe plasmalogen deficiency in tissues of infantswithout peroxisomes (Zellweger syndrome). Nature 1983:306: 69–70.

5. Goldman BM, Blobel G. Biogenesis of peroxisomes. intra-cellular site of synthesis of catalase and uricase. Proc NatlAcad Sci USA 1978: 75: 5066–5070.

6. Robbi M, Lazarow PB. Synthesis of catalase in two cell-freeprotein-synthesizing systems and in rat liver. Proc NatlAcad Sci USA 1978: 75: 4344–4348.

7. Fujiki Y, Rachubinski RA, Lazarow PB. Synthesis of amajor integral membrane polypeptide of rat liver peroxi-somes on free polysomes. Proc Natl Acad Sci USA 1984: 81:7127–7131.

8. McNew JA, Goodman JM. An oligomeric protein isimported into peroxisomes in vivo. J Cell Biol 1994: 127:1245–1257.

9. Yang X, Purdue PE, Lazarow PB. Eci1p uses a PTS1 toenter peroxisomes: either its own or that of a partner,Dci1p. Eur J Cell Biol 2001: 80: 126–138.

10. TitorenkoVI, Nicaud JM,WangH, ChanH,Rachubinski RA.Acyl-CoA oxidase is imported as a heteropentameric, cofactor-containing complex into peroxisomes of Yarrowia lipolytica.J Cell Biol 2002: 156: 481–494.

11. Walton PA, Hill PE, Subramani S. Import of stably foldedproteins into peroxisomes. Mol Biol Cell 1995: 6: 675–683.

12. South ST, Gould SJ. Peroxisome synthesis in the absence ofpreexisting peroxisomes. J Cell Biol 1999: 144: 255–266.

13. South ST, Sacksteder KA, Li X, Liu Y, Gould SJ. Inhibi-tors of COPI and COPII do not block PEX3-mediatedperoxisome synthesis. J Cell Biol 2000: 149: 1345–1360.

14. South ST, Baumgart E, Gould SJ. Inactivation of the endo-plasmic reticulum protein translocation factor, Sec61p, orits homolog, Ssh1p, does not affect peroxisome biogenesis.Proc Natl Acad Sci USA 2001: 98: 12027–12031.

15. Snyder WB, Faber KN, Wenzel TJ et al. Pex19p interactswith Pex3p and Pex10p and is essential for peroxisomebiogenesis in Pichia pastoris. Mol Biol Cell 1999: 10:1745–1761.

16. Hazra PP, Suriapranata I, Snyder WB, Subramani S. Per-oxisome remnants in pex3delta cells and the requirement ofPex3p for interactions between the peroxisomal dockingand translocation subcomplexes. Traffic 2002: 3: 560–574.

17. Geuze HJ, Murk JL, Stroobants AK et al. Involvement ofthe endoplasmic reticulum in peroxisome formation. MolBiol Cell 2003: 14: 2900–2907.

18. Faber KN, Haan GJ, Baerends RJ, Kram AM, Veenhuis M.Normal peroxisome development from vesicles induced bytruncated Hansenula polymorpha Pex3p. J Biol Chem 2002:277: 1026–11033.

19. Fujiki Y. Peroxisome biogenesis and peroxisome biogenesisdisorders. FEBS Lett 2000: 476: 42–46.

20. Erdmann R, Veenhuis M, Mertens D, Kunau WH. Isol-ation of peroxisome-deficient mutants of Saccharomycescerevisiae. Proc Natl Acad Sci USA 1989: 86: 5419–5423.

21. Erdmann R, Wiebel FF, Flessau A et al. PAS1, a yeast generequired for peroxisome biogenesis, encodes a member of anovel family of putative ATPases. Cell 1991: 64: 499–510.

22. Neuwald AF, Aravind L, Spouge JL, Koonin EV. AAAþ:a class of chaperone-like ATPases associated with the


128

assembly, operation, and disassembly of protein complexes.Genome Res 1999: 9: 27–43.

23. Hohfeld J, Veenhuis M, Kunau WH. PAS3, a Saccharo-myces cerevisiae gene encoding a peroxisomal integral mem-brane protein essential for peroxisome biogenesis. J CellBiol 1991: 114: 1167–1178.

24. Wiebel FF, Kunau WH. The Pas2 protein essential forperoxisome biogenesis is related to ubiquitin-conjugatingenzymes. Nature 1992: 359: 73–76.

25. Hettema EH, Girzalsky W, van den Berg M, Erdmann R,Distel B. Saccharomyces cerevisiae pex3p and pex19p arerequired for proper localization and stability of peroxisomalmembrane proteins. EMBO J 2000: 19: 223–233.

26. Vizeacoumar FJ, Torres-Guzman JC, BouardD, Aitchison JD,Rachubinski RA. Pex30p, Pex31p, and Pex32p form a family ofperoxisomal integral membrane proteins regulating peroxisomesize and number in Saccharomyces cerevisiae. Mol Biol Cell2004: 15: 665–677.

27. Purdue PE, Yang X, Lazarow PB. Pex18p and Pex21p, anovel pair of related peroxins essential for peroxisomaltargeting by the PTS2 pathway. J Cell Biol 1998: 143:1859–1869.

28. Titorenko VI, Smith JJ, Szilard RK, Rachubinski RA.Pex20p of the yeast Yarrowia lipolytica is requiredfor the oligomerization of thiolase in the cytosol andfor its targeting to the peroxisome. J Cell Biol 1998:142: 403–420.

29. Sichting M, Schell-Steven A, Prokisch H, Erdmann R,Rottensteiner H. Pex7p and Pex20p of Neurospora crassafunction together in PTS2-dependent protein import intoperoxisomes. Mol Biol Cell 2003: 14: 810–821.

30. Santos MJ, Imanaka T, Shio H, Lazarow PB. Peroxisomalintegral membrane proteins in control and Zellweger fibro-blasts. J Biol Chem 1988: 263: 10502–10509.

31. Elgersma Y, Kwast L, van den Berg M et al. Overexpressionof Pex15p, a phosphorylated peroxisomal integral mem-brane protein required for peroxisome assembly in S.cere-visiae, causes proliferation of the endoplasmic reticulummembrane. EMBO J 1997: 16: 7326–7341.

32. Dyer JM, McNew JA, Goodman JM. The sorting sequenceof the peroxisomal integral membrane protein PMP47 iscontained within a short hydrophilic loop. J Cell Biol1996: 133: 269–280.

33. Wang X, Unruh MJ, Goodman JM. Discrete targetingsignals direct Pmp47 to oleate-induced peroxisomes inSaccharomyces cerevisiae. J Biol Chem 2001: 276: 10897–10905.

34. Jones JM, Morrell JC, Gould SJ. Multiple distinct targetingsignals in integral peroxisomal membrane proteins. J CellBiol 2001: 153: 1141–1150.

35. Honsho M, Fujiki Y. Topogenesis of peroxisomal mem-brane protein requires a short, positively charged interven-ing-loop sequence and flanking hydrophobic segments:study using human membrane protein PMP34. J BiolChem 2001: 276: 9375–9382.

36. Brosius U, Dehmel T, Gartner J. Two different targetingsignals direct human peroxisomal membrane protein 22 toperoxisomes. J Biol Chem 2002: 277: 774–784.

37. Kammerer S, Holzinger A, Welsch U, Roscher AA. Cloningand characterization of the gene encoding the human per-oxisomal assembly protein Pex3p. FEBS Lett 1998: 429:53–60.

38. James GL, Goldstein JL, Pathak RK, Anderson RG,Brown MS. PxF, a prenylated protein of peroxisomes.J Biol Chem 1994: 269: 14182–14190.

39. Kammerer S, Arnold N, Gutensohn W et al. Genomicorganization and molecular characterization of a gene

encoding HsPXF, a human peroxisomal farnesylated pro-tein. Genomics 1997: 45: 200–210.

40. Gotte K, Girzalsky W, Linkert M et al. Pex19p, a farnesy-lated protein essential for peroxisome biogenesis. Mol CellBiol 1998: 18: 616–628.

41. Sacksteder KA, Jones JM, South ST, Li X, Liu Y, Gould SJ.PEX19 binds multiple peroxisomal membrane proteins,is predominantly cytoplasmic, and is required for per-oxisome membrane synthesis. J Cell Biol 2000: 148: 931–944.

42. Snyder WB, Koller A, Choy AJ et al. Pex17p is required forimport of both peroxisome membrane and lumenal proteinsand interacts with Pex19p and the peroxisome targetingsignal-receptor docking complex in Pichia pastoris. MolBiol Cell 1999: 10: 4005–4019.

43. Snyder WB, Koller A, Choy AJ, Subramani S. The peroxinPex19p interacts with multiple, integral membrane pro-teins at the peroxisomal membrane. J Cell Biol 2000: 149:1171–1178.

44. Soukupova M, Sprenger C, Gorgas K, Kunau WH, Dodt G.Identification and characterization of the human peroxinPEX3. Eur J Cell Biol 1999: 78: 357–374.

45. Fransen M, Wylin T, Brees C, Mannaerts GP, VanVeldhoven PP. Human pex19p binds peroxisomal integralmembrane proteins at regions distinct from their sortingsequences. Mol Cell Biol 2001: 21: 4413–4424.

46. Fang Y, Morrell JC, Jones JM, Gould SJ. PEX3 func-tions as a PEX19 docking factor in the import of class Iperoxisomal membrane proteins. J Cell Biol 2004: 164:863–875.

47. Eitzen GA, Szilard RK, Rachubinski RA. Enlarged peroxi-somes are present in oleic acid-grown Yarrowia lipolyticaoverexpressing the PEX16 gene encoding an intraperoxiso-mal peripheral membrane peroxin. J Cell Biol 1997: 137:1265–1278.

48. Honsho M, Tamura S, Shimozawa N, Suzuki Y, Kondo N,Fujiki Y. Mutation in PEX16 is causal in the peroxisome-deficient Zellweger syndrome of complementation group D.Am J Hum Genet 1998: 63: 1622–1630.

49. McCollum D, Monosov E, Subramani S. The pas8 mutantof Pichia pastoris exhibits the peroxisomal protein importdeficiencies of Zellweger syndrome cells – the PAS8 proteinbinds to the COOH-terminal tripeptide peroxisomal target-ing signal, and is a member of the TPR protein family. J CellBiol 1993: 121: 761–774.

50. Van der Leij I, FranseMM, ElgersmaY, Distel B, TabakHF.PAS10 is a tetratricopeptide-repeat protein that is essentialfor the import of most matrix proteins into peroxisomes ofSaccharomyces cerevisiae [published erratum appears inProc Natl Acad Sci U S A 1995 June 6; 92 (12): 5759]. ProcNatl Acad Sci USA 1993: 90: 11782–11786.

51. Dodt G, Braverman N, Wong C et al. Mutations in thePTS1 receptor gene, PXR1, define complementation group2 of the peroxisome biogenesis disorders. Nat Genet 1995: 9:115–125.

52. Fransen M, Brees C, Baumgart E et al. Identificationand characterization of the putative human peroxisomalC-terminal targeting signal import receptor. J Biol Chem1995: 270: 7731–7736.

53. Wiemer EA, Nuttley WM, Bertolaet BL et al. Human per-oxisomal targeting signal-1 receptor restores peroxisomalprotein import in cells from patients with fatal peroxisomaldisorders. J Cell Biol 1995: 130: 51–65.

54. Szilard RK, Titorenko VI, Veenhuis M, Rachubinski RA.Pay32p of the yeast Yarrowia lipolytica is an intraperoxiso-mal component of the matrix protein translocation machin-ery. J Cell Biol 1995: 131: 1453–1469.


129

55. van der Klei IJ, Hilbrands RE, Swaving GJ et al. TheHansenula polymorpha PER3 gene is essential for theimport of PTS1 proteins into the peroxisomal matrix.J Biol Chem 1995: 270: 17229–17236.

56. Dammai V, Subramani S. The human peroxisomal target-ing signal receptor, Pex5p, is translocated into the peroxi-somal matrix and recycled to the cytosol. Cell 2001: 105:187–196.

57. Gatto GJ Jr, Geisbrecht BV, Gould SJ, Berg JM. Peroxi-somal targeting signal-1 recognition by the TPR domains ofhuman PEX5. Nat Struct Biol 2000: 7: 1091–1095.

58. Klein AT, Barnett P, Bottger G, Konings D, Tabak HF,Distel B. Recognition of peroxisomal targeting signal type 1by the import receptor Pex5p. J Biol Chem 2001: 276:15034–15041.

59. Marzioch M, Erdmann R, Veenhuis M, Kunau WH. PAS7encodes a novel yeast member of the WD-40 protein familyessential for import of 3-oxoacyl-CoA thiolase, a PTS2-containing protein, into peroxisomes. EMBO J 1994: 13:4908–4918.

60. Zhang JW, Lazarow PB. PEB1 (PAS7) in Saccharomycescerevisiae encodes a hydrophilic, intra-peroxisomal proteinthat is a member of the WD repeat family and is essentialfor the import of thiolase into peroxisomes. J Cell Biol 1995:129: 65–80.

61. Zhang JW, Lazarow PB. Peb1p (Pas7p) is an intraperoxi-somal receptor for the NH2-terminal, type 2, peroxisomaltargeting sequence of thiolase: Peb1p itself is targeted toperoxisomes by an NH2-terminal peptide. J Cell Biol1996: 132: 325–334.

62. Braverman N, Steel G, Obie C et al. Human PEX7 encodesthe peroxisomal PTS2 receptor and is responsible forrhizomelic chondrodysplasia punctata. Nat Genet 1997:15: 369–376.

63. Motley AM, Hettema EH, Hogenhout EM et al. Rhizome-lic chondrodysplasia punctata is a peroxisomal proteintargeting disease caused by a non-functional PTS2 receptor.Nat Genet 1997: 15: 377–380.

64. Purdue PE, Zhang JW, Skoneczny M, Lazarow PB. Rhizo-melic chondrodysplasia punctata is caused by deficiency ofhuman PEX7, a homologue of the yeast PTS2 receptor. NatGenet 1997: 15: 381–384.

65. Otera H, Okumoto K, Tateishi K et al. Peroxisome target-ing signal type 1 (PTS1) receptor is involved in import ofboth PTS1 and PTS2: studies with PEX5-defective CHOcell mutants. Mol Cell Biol 1998: 18: 388–399.

66. Braverman N, Dodt G, Gould SJ, Valle D. An isoform ofPex5p, the human PTS1 receptor, is required for the importof PTS2 proteins into peroxisomes. Hum Mol Genet 1998:7: 1195–1205.

67. Otera H, Setoguchi K, Hamasaki M, Kumashiro T,Shimizu N, Fujiki Y. Peroxisomal targeting signalreceptor Pex5p interacts with cargoes and importmachinery components in a spatiotemporally differen-tiated manner: conserved Pex5p WXXXF/Y motifs arecritical for matrix protein import. Mol Cell Biol 2002:22: 1639–1655.

68. Matsumura T, Otera H, Fujiki Y. Disruption of theinteraction of the longer isoform of Pex5p, Pex5pL, withPex7p abolishes peroxisome targeting signal type 2 proteinimport in mammals. Study with a novel Pex5-impairedChinese hamster ovary cell mutant. J Biol Chem 2000:275: 21715–21721.

69. Dodt G, Warren D, Becker E, Rehling P, Gould SJ.Domain mapping of human PEX5 reveals functional andstructural similarities to Saccharomyces cerevisiae Pex18pand Pex21p. J Biol Chem 2001: 276: 41769–41781.

70. Motley AM, Hettema EH, Ketting R, Plasterk R, Tabak HF.Caenorhabditis elegans has a single pathway to target matrixproteins to peroxisomes. EMBO Rep 2000: 1: 40–46.

71. Schliebs W, Saidowsky J, Agianian B, Dodt G, Herberg FW,Kunau WH. Recombinant human peroxisomal targetingsignal receptor PEX5. Structural basis for interaction ofPEX5 with PEX14. J Biol Chem 1999: 274: 5666–5673.

72. Saidowsky J, Dodt G, Kirchberg K et al. The di-aromaticpentapeptide repeats of the human peroxisome importreceptor PEX5 are separate high affinity binding sites forthe peroxisomal membrane protein PEX14. J Biol Chem2001: 276: 34524–34529.

73. Nito K, Hayashi M, Nishimura M. Direct interaction anddetermination of binding domains among peroxisomalimport factors in Arabidopsis thaliana. Plant Cell Physiol2002: 43: 355–366.

74. Girzalsky W, Rehling P, Stein K et al. Involvement ofPex13p in Pex14p localization and peroxisomal targetingsignal 2-dependent protein import into peroxisomes. J CellBiol 1999: 144: 1151–1162.

75. Barnett P, Bottger G, Klein AT, Tabak HF, Distel B. Theperoxisomal membrane protein Pex13p shows a novel modeof SH3 interaction. EMBO J 2000: 19: 6382–6391.

76. Douangamath A, Filipp FV, Klein AT et al. Topography forindependent binding of alpha-helical and PPII-helical ligandsto a peroxisomal SH3 domain. Mol Cell 2002: 10: 1007–1017.

77. Pires JR, Hong X, Brockmann C et al. The ScPex13p SH3domain exposes two distinct binding sites for Pex5p andPex14p. J Mol Biol 2003: 326: 1427–1435.

78. Urquhart AJ, Kennedy D, Gould SJ, Crane DI. Interactionof Pex5p, the type 1 peroxisome targeting signal receptor,with the peroxisomal membrane proteins Pex14p andPex13p. J Biol Chem 2000: 275: 4127–4136.

79. Huhse B, Rehling P, Albertini M, Blank L, Meller K,Kunau WH. Pex17p of Saccharomyces cerevisiae is anovel peroxin and component of the peroxisomal proteintranslocation machinery. J Cell Biol 1998: 140: 49–60.

80. Harper CC, South ST, McCaffery JM, Gould SJ. Peroxi-somal membrane protein import does not require Pex17p.J Biol Chem 2002: 277: 16498–16504.

81. Reguenga C, Oliveira ME, Gouveia AM, Sa-Miranda C,Azevedo JE. Characterization of the mammalian peroxi-somal import machinery: Pex2p, Pex5p, Pex12p, andPex14p are subunits of the same protein assembly. J BiolChem 2001: 276: 29935–29942.

82. Okumoto K, Abe I, Fujiki Y. Molecular anatomy of theperoxin Pex12p: ring finger domain is essential for Pex12pfunction and interacts with the peroxisome-targeting signaltype 1-receptor Pex5p and a ring peroxin, Pex10p. J BiolChem 2000: 275: 25700–25710.

83. Chang CC, Warren DS, Sacksteder KA, Gould SJ. PEX12interacts with PEX5 and PEX10 and acts downstream ofreceptor docking in peroxisomal matrix protein import.J Cell Biol 1999: 147: 761–774.

84. Spong AP, Subramani S. Cloning and characterization ofPAS5: a gene required for peroxisome biogenesis in themethylotrophic yeast Pichia pastoris. J Cell Biol 1993: 123:535–548.

85. Vale RD. AAA, proteins. Lords of the ring. J Cell Biol2000: 150: F13–F19.

86. Titorenko VI, Rachubinski RA. The endoplasmic reticulumplays an essential role in peroxisome biogenesis. TrendsBiochem Sci 1998: 23: 231–233.

87. Titorenko VI, Chan H, Rachubinski RA. Fusion of smallperoxisomal vesicles in vitro reconstructs an early step inthe in vivo multistep peroxisome assembly pathway ofYarrowia lipolytica. J Cell Biol 2000: 148: 29–44.


130

88. Tsukamoto T, Miura S, Nakai T et al. Peroxisome assem-bly factor-2, a putative ATPase cloned by functional com-plementation on a peroxisome-deficient mammalian cellmutant. Nat Genet 1995: 11: 395–401.

89. Kiel JA, Hilbrands RE, van der Klei IJ et al. Hansenulapolymorpha Pex1p and Pex6p are peroxisome-associatedAAA proteins that functionally and physically interact.Yeast 1999: 15: 1059–1078.

90. Faber KN, Heyman JA, Subramani S. Two AAA familyperoxins, PpPex1p and PpPex6p, interact with each otherin an ATP-dependent manner and are associated withdifferent subcellular membranous structures distinct fromperoxisomes. Mol Cell Biol 1998: 18: 936–943.

91. Titorenko VI, Rachubinski RA. Peroxisomal membranefusion requires two AAA family ATPases, Pex1p andPex6p. J Cell Biol 2000: 150: 881–886.

92. Yahraus T, Braverman N, Dodt G et al. The peroxisomebiogenesis disorder group 4 gene, PXAAA1, encodes acytoplasmic ATPase required for stability of the PTS1receptor. EMBO J 1996: 15: 2914–2923.

93. Tamura S, ShimozawaN, Suzuki Y, TsukamotoT, Osumi T,Fujiki Y. A cytoplasmic AAA family peroxin, Pex1p,interacts with Pex6p. Biochem Biophys Res Commun 1998:245: 883–886.

94. Geisbrecht BV, Schulz K, Nau K et al. Preliminary char-acterization of Yor180Cp: identification of a novel peroxi-somal protein of saccharomyces cerevisiae involved in fattyacid metabolism. Biochem Biophys Res Commun 1999:260: 28–34.

95. Birschmann I, Stroobants AK, van Den BM et al. Pex15pof Saccharomyces cerevisiae provides a molecular basis forrecruitment of the AAA peroxin Pex6p to peroxisomalmembranes. Mol Biol Cell 2003: 14: 2226–2236.

96. Matsumoto N, Tamura S, Fujiki Y. The pathogenic per-oxin Pex26p recruits the Pex1p-Pex6p AAA ATPase com-plexes to peroxisomes. Nat Cell Biol 2003: 5: 454–460.

97. MatsumotoN, Tamura S, Furuki S et al. Mutations in novelperoxin gene PEX26 that cause peroxisome-biogenesisdisorders of complementation group 8 provide a geno-type-phenotype correlation. Am J Hum Genet 2003: 73:233–246.

98. Rottensteiner H, Kal AJ, Filipits M et al. Pip2p: a transcrip-tional regulator of peroxisome proliferation in the yeastSaccharomyces cerevisiae. EMBO J 1996: 15: 2924–2934.

99. Karpichev IV, Small GM. Global regulatory functions ofOaf1p and Pip2p (Oaf2p), transcription factors that regu-late genes encoding peroxisomal proteins in Saccharo-myces cerevisiae. Mol Cell Biol 1998: 18: 6560–6570.

100. Lee SS, Pineau T, Drago J et al. Targeted disruption of thealpha isoform of the peroxisome proliferator-activatedreceptor gene in mice results in abolishment of the pleio-tropic effects of peroxisome proliferators. Mol Cell Biol1995: 15: 3012–3022.

101. Erdmann R, Blobel G. Giant peroxisomes in oleic acid-induced Saccharomyces cerevisiae lacking the peroxisomalmembrane protein Pmp27p. J Cell Biol 1995: 128: 509–523.

102. Marshall PA, Krimkevich Y, Lark R, Dyer J, Veenhuis M,Goodman JM. Pmp27 promotes peroxisomal prolifer-ation. J Cell Biol 1995: 129: 345–355.

103. Sakai Y, Marshall PA, Saiganji A et al. The Candidaboidinii peroxisomal membrane protein Pmp30 has a rolein peroxisomal proliferation and is functionally homolo-gous to Pmp27 from Saccharomyces cerevisiae. J Bacteriol1995: 177: 6773–6781.

104. van Roermund CWT, Tabak HF, van den Berg M,Wanders RJA, Hettema EH. Pex11p plays a primary rolein medium-chain fatty acid oxidation, a process that

affects peroxisome number and size in Saccharomycescerevisiae. J Cell Biol 2000: 150: 489–497.

105. Li X, Gould SJ. PEX11 promotes peroxisome divisionindependently of peroxisome metabolism. J Cell Biol2002: 156: 643–651.

106. Schrader M, Reuber BE, Morrell JC et al. Expression ofPEX11beta mediates peroxisome proliferation in theabsence of extracellular stimuli. J Biol Chem 1998: 273:29607–29614.

107. Li X, Baumgart E, Dong GX et al. PEX11alpha is requiredfor peroxisome proliferation in response to 4-phenylbuty-rate but is dispensable for peroxisome proliferator-activated receptor alpha-mediated peroxisome proliferation.Mol Cell Biol 2002: 22: 8226–8240.

108. Hoepfner D, van Den BM, Philippsen P, Tabak HF,Hettema EH. A role for Vps1p, actin, and the Myo2pmotor in peroxisome abundance and inheritance inSaccharomyces cerevisiae. J Cell Biol 2001: 155: 979–990.

109. Li X, Gould SJ. The dynamin-like GTPase DLP1 is essen-tial for peroxisome division and is recruited to peroxisomesin part by PEX11. J Biol Chem 2003: 278: 17012–17020.

110. Wanders RJA, Barth PG, Heymans HAS. Single peroxi-somal enzyme deficiencies. In: The metabolic and molecu-lar bases of inherited disease. (Scriver CR, Beaudet AL,Sly WS, Valle D, eds). New York: McGraw-Hill, 2001:3219–3256.

111. Gould SJ, Raymond GV, Valle D. The peroxisomal bio-genesis disorder. In: The metabolic and molecular bases ofinherited disease. (Scriver CR, Beaudet AL, SlyWS, Valle D,eds). New York: Mc Graw-Hill, 2001: 3181–3217.

112. Wanders RJA, Saelman D, Heymans HAS et al. Geneticrelation between the Zellweger syndrome, infantileRefsum’s disease, and rhizomelic chondrodysplasia punc-tata. N Engl J Med 1986: 314: 787–788.

113. Brul S, Westerveld A, Strijland A et al. Genetic heterogen-eity in the cerebrohepatorenal (Zellweger) syndrome andother inherited disorders with a generalized impairment ofperoxisomal functions. A study using complementationanalysis. J Clin Invest 1988: 81: 1710–1715.

114. Yajima S, Suzuki Y, Shimozawa N et al. Complementa-tion study of peroxisome-deficient disorders by immuno-fluorescence staining and characterization of fused cells.Hum Genet 1992: 88: 491–499.

115. Shimozawa N, Suzuki Y, Orii T, Moser A, Moser HW,Wanders RJA. Standardization of complementationgrouping of peroxisome – deficient disorders and the sec-ond Zellweger patient with peroxisomal assembly factor-1(PAF-1) defect. Am J Hum Genet 1993: 52: 843–844.

116. Moser AB, Rasmussen M, Naidu S et al. Phenotype ofpatients with peroxisomal disorders subdivided into six-teen complementation groups. J Pediatr 1995: 127: 13–22.

117. Reuber BE, Germain-Lee E, Collins CS et al. Mutations inPEX1 are the most common cause of peroxisome biogen-esis disorders. Nat Genet 1997: 17: 445–448.

118. Gartner J, Preuss N, Brosius U, Biermanns M. Mutations inPEX1 in peroxisome biogenesis disorders: G843D and a mildclinical phenotype. J Inherit Metab Dis 1999: 22: 311–313.

119. Wanders RJA, Mooijer PAW, Dekker C, Suzuki Y,Shimozawa N. Disorders of peroxisome biogenesis:complementation analysis shows genetic heterogeneitywith strong overrepresentation of one group (PEX1 defi-ciency). J Inherit Metab Dis 1999: 22: 314–318.

120. Maxwell MA, Nelson PV, Chin SJ, Paton BC, Carey WF,Crane DI. A common PEX1 frameshift mutation inpatients with disorders of peroxisome biogenesis correlateswith the severe Zellweger syndrome phenotype. HumGenet 1999: 105: 38–44.


131

121. Collins CS, Gould SJ. Identification of a common PEX1mutation in Zellweger syndrome. Hum Mutat 1999: 14:45–53.

122. Imamura A, Tsukamoto T, Shimozawa N et al. Temperature-sensitive phenotypes of peroxisome-assembly processes repre-sent the milder forms of human peroxisome-biogenesisdisorders. Am J Hum Genet 1998: 62: 1539–1543.

123. Imamura A, Tamura S, Shimozawa N et al. Temperature-sensitive mutation in PEX1 moderates the phenotypes ofperoxisome deficiency disorders. Hum Mol Genet 1998: 7:2089–2094.

124. Geisbrecht BV, Collins CS, Reuber BE, Gould SJ. Disrup-tion of a PEX1–PEX6 interaction is the most commoncause of the neurologic disorders Zellweger syndrome,neonatal adrenoleukodystrophy, and infantile Refsum dis-ease. Proc Natl Acad Sci USA 1998: 95: 8630–8635.

125. Braverman N, Chen L, Lin P et al. Mutation analysis ofPEX7 in 60 probands with rhizomelic chondrodysplasiapunctata and functional correlations of genotype withphenotype. Hum Mutat 2002: 20: 284–297.

126. Motley AM, Brites P, Gerez L et al. Mutational spectrumin the PEX7 gene and functional analysis of mutant allelesin 78 patients with rhizomelic chondrodysplasia punctatatype 1. Am J Hum Genet 2002: 70: 612–624.

127. Setchell KD, Bragetti P, Zimmer-Nechemias L et al. Oralbile acid treatment and the patient with Zellweger syn-drome. Hepatology 1992: 15: 198–207.

128. Martinez M. Abnormal profiles of polyunsaturated fattyacids in the brain, liver, kidney and retina of patients withperoxisomal disorders. Brain Res 1992: 583: 171–182.

129. Faust PL, Hatten ME. Targeted deletion of the PEX2peroxisome assembly gene in mice provides a model forZellweger syndrome, a human neuronal migration disor-der. J Cell Biol 1997: 139: 1293–1305.

130. Baes M, Gressens P, Baumgart E et al. A mouse model forZellweger syndrome. Nat Genet 1997: 17: 49–57.

131. Brites P, Motley AM, Gressens P et al. Impaired neuronalmigration and endochondral ossification in Pex7 knockoutmice: a model for rhizomelic chondrodysplasia punctata.Hum Mol Genet 2003: 12: 2255–2267.

132. Li X, Baumgart E,Morrell JC, Jimenez-Sanchez G, Valle D,Gould SJ. PEX11 beta deficiency is lethal and impairsneuronal migration but does not abrogate peroxisomefunction. Mol Cell Biol 2002: 22: 4358–4365.

133. Maxwell M, Bjorkman J, Nguyen T et al. Pex13 inactiva-tion in the mouse disrupts peroxisome biogenesis and leadsto a Zellweger syndrome phenotype. Mol Cell Biol 2003:23: 5947–5957.

134. Baumgart E, Vanhorebeek I, Grabenbauer M et al. Mito-chondrial alterations caused by defective peroxisomal bio-genesis in a mouse model for Zellweger syndrome (PEX5knockout mouse). Am J Pathol 2001: 159: 1477–1494.

135. Faust PL, Su HM, Moser A, Moser HW. The peroxisomedeficient PEX2 Zellweger mouse: pathologic and biochem-ical correlates of lipid dysfunction. J Mol Neurosci 2001:16: 289–297.

136. Janssen A, Baes M, Gressens P, Mannaerts GP, Declercq P,Van Veldhoven PP. Docosahexaenoic acid deficit is not amajor pathogenic factor in peroxisome-deficient mice. LabInvest 2000: 80: 31–35.

137. Gressens P, Baes M, Leroux P et al. Neuronal migrationdisorder in Zellweger mice is secondary to glutamate recep-tor dysfunction. Ann Neurol 2000: 48: 336–343.

138. Rodemer C, Thai TP, Brugger B et al. Inactivation of etherlipid biosynthesis causes male infertility, defects in eyedevelopment and optic nerve hypoplasia in mice. HumMol Genet 2003: 12: 1881–1895.

139. Lazarow PB, Fujiki Y. Biogenesis of peroxisomes. AnnuRev Cell Biol 1985: 1: 489–530.

140. Portsteffen H, Beyer A, Becker E et al. Human PEX1 ismutated in complementation group 1 of the peroxisomebiogenesis disorders. Nat Genet 1997: 17: 449–452.

141. Tsukamoto T, Miura S, Fujiki Y. Restoration by a 35Kmembrane protein of peroxisome assembly in a peroxi-some-deficient mammalian cell mutant. Nature 1991: 350:77–81.

142. Collins CS, Kalish JE, Morrell JC, McCaffery JM, Gould SJ.The peroxisome biogenesis factors pex4p, pex22p, pex1p, andpex6p act in the terminal steps of peroxisomal matrix proteinimport. Mol Cell Biol 2000: 20: 7516–7526.

143. van der Klei IJ, Hilbrands RE, Kiel JA, Rasmussen SW,Cregg JM, Veenhuis M. The ubiquitin-conjugating enzymePex4p of Hansenula polymorpha is required for efficientfunctioning of the PTS1 import machinery. EMBO J 1998:17: 3608–3618.

144. Liu H, Tan X, Russell KA, Veenhuis M, Cregg JM. PER3,a gene required for peroxisome biogenesis in Pichia pas-toris, encodes a peroxisomal membrane protein involved inprotein import. J Biol Chem 1995: 270: 10940–10951.

145. Waterham HR, Titorenko VI, Haima P, Cregg JM,Harder W, Veenhuis M. The Hansenula polymorphaPER1 gene is essential for peroxisome biogenesis andencodes a peroxisomal matrix protein with both carboxy-and amino-terminal targeting signals. J Cell Biol 1994: 127:737–749.

146. Eitzen GA, Aitchison JD, Szilard RK, Veenhuis M,Nuttley WM, Rachubinski RA. The Yarrowia lipolyticagene PAY2 encodes a 42-kDa peroxisomal integralmembrane protein essential for matrix protein import andperoxisome enlargement but not for peroxisome mem-brane proliferation. J Biol Chem 1995: 270: 1429–1436.

147. Tan X, Waterham HR, Veenhuis M, Cregg JM. TheHansenula polymorpha PER8 gene encodes a novel peroxi-somal integral membrane protein involved in proliferation.J Cell Biol 1995: 128: 307–319.

148. Kalish JE, Theda C, Morrell JC, Berg JM, Gould SJ.Formation of the peroxisome lumen is abolished by lossof Pichia pastoris Pas7p, a zinc-binding integral membraneprotein of the peroxisome. Mol Cell Biol 1995: 15: 6406–6419.

149. Kalish JE, Keller GA, Morrell JC et al. Characterizationof a novel component of the peroxisomal protein importapparatus using fluorescent peroxisomal proteins. EMBOJ 1996: 15: 3275–3285.

150. Elgersma Y, Kwast L, Klein A et al. The SH3 domain ofthe Saccharomyces cerevisiae peroxisomal membrane pro-tein Pex13p functions as a docking site for Pex5p, a mobilereceptor for the import PTS1 – containing proteins. J CellBiol 1996: 135: 97–109.

151. Gould SJ, Kalish JE,Morrell JC, Bjorkman J, Urquhart AJ,Crane DI. Pex13p is an SH3 protein of the peroxisomemembrane and a docking factor for the predominantlycytoplasmic PTs1 receptor. J Cell Biol 1996: 135: 85–95.

152. Erdmann R, Blobel G. Identification of Pex13p a peroxi-somal membrane receptor for the PTS1 recognition factor.J Cell Biol 1996: 135: 111–121.

153. Albertini M, Rehling P, Erdmann R et al. Pex14p, aperoxisomal membrane protein binding both receptors ofthe two PTS-dependent import pathways. Cell 1997: 89:83–92.

154. Honsho M, Hiroshige T, Fujiki Y. The membrane biogen-esis peroxin Pex16p. Topogenesis and functional roles inperoxisomal membrane assembly. J Biol Chem 2002: 277:44513–44524.


132

155. Jones JM, Morrell JC, Gould SJ. PEX19 is a predom-inantly cytosolic chaperone and import receptor for class1 peroxisomal membrane proteins. J Cell Biol 2004: 164:57–67.

156. Matsuzono Y, Kinoshita N, Tamura S et al. HumanPEX19: cDNA cloning by functional complementation,mutation analysis in a patient with Zellweger syndrome,and potential role in peroxisomal membrane assembly.Proc Natl Acad Sci USA 1999: 96: 2116–2121.

157. Koller A, Snyder WB, Faber KN et al. Pex22p of Pichiapastoris, essential for peroxisomal matrix protein import,anchors the ubiquitin-conjugating enzyme, Pex4p, on theperoxisomal membrane. J Cell Biol 1999: 146: 99–112.

158. Brown TW, Titorenko VI, Rachubinski RA. Mutants ofthe Yarrowia lipolytica PEX23 gene encoding an integralperoxisomal membrane peroxin mislocalize matrix pro-teins and accumulate vesicles containing peroxisomalmatrix and membrane proteins. Mol Biol Cell 2000: 11:141–152.

159. Rottensteiner H, Stein K, Sonnenhol E, Erdmann R.Conserved function of pex11p and the novel pex25p and

pex27p in peroxisome biogenesis. Mol Biol Cell 2003: 14:4316–4328.

160. Tam YY, Rachubinski RA. Yarrowia lipolytica cellsmutant for the PEX24 gene encoding a peroxisomal mem-brane peroxin mislocalize peroxisomal proteins and accu-mulate membrane structures containing both peroxisomalmatrix and membrane proteins. Mol Biol Cell 2002: 13:2681–2691.

161. Smith JJ, Marelli M, Christmas RH et al. Transcriptomeprofiling to identify genes involved in peroxisome assemblyand function. J Cell Biol 2002: 158: 259–271.

162. Tam YY, Torres-Guzman JC, Vizeacoumar FJ et al.Pex11-related proteins in peroxisome dynamics: a role forthe novel peroxin Pex27p in controlling peroxisome sizeand number in Saccharomyces cerevisiae. Mol Biol Cell2003: 14: 4089–4102.

163. Vizeacoumar FJ, Torres-Guzman JC, TamYY, Aitchison JD,Rachubinski RA. YHR150w and YDR479c encodeperoxisomal integral membrane proteins involved in theregulation of peroxisome number, size, and distribution inSaccharomyces cerevisiae. J Cell Biol 2003: 161: 321–332.


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