Evolving models for peroxisome biogenesis.

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Published paper

Evolving

models

for

peroxisome

biogenesis

§

Ewald

H

Hettema

1

,

Ralf

Erdmann

2

,

Ida

van

der

Klei

3

and

Marten

Veenhuis

3

Significantprogresshasbeenmadetowardsour

understandingofthemechanismofperoxisomeformation,in particularconcerningsortingofperoxisomalmembrane proteins,matrixproteinimportandorganellemultiplication. Hereweevaluatetheprogressmadeinrecentyears.Wefocus mainlyonprogressmadeinyeasts.Weindicatethegapsinour knowledgeanddiscussconflictingmodels.

Addresses

1_{DepartmentofMolecularBiologyandBiotechnology,Universityof} Sheffield,WesternBank,SheffieldS102TN,UK

2_{SystemBiochie,RuhrUniversitatBochum,Universitatstr.150,} D-44780,Bochum,Germany

3_{MolecularCellBiology,GroningenBiomolecularSciencesand} Biotechnologyinstitute,UniversityofGroningen,11103,9700CC, Groningen,TheNetherlands

Correspondingauthor:Hettema,EwaldH([email protected])

CurrentOpinioninCellBiology2014,29:25–30

ThisreviewcomesfromathemedissueonCellorganelles

EditedbyWilliamAPrinzandDavidKBanfield

0955-0674/$–seefrontmatter,CrownCopyright#2014Published byElsevierLtd.Allrightsreserved.

http://dx.doi.org/10.1016/j.ceb.2014.02.002

Introduction

Peroxisomesareeukaryoticorganellesboundbyasingle membrane.Theirabundanceandfunctionsvarybetween organisms, celltypesandenvironmental conditions.Ina seminal review, Lazarow and Fujiki [1] proposed that peroxisomalmembraneandmatrix proteinsare synthes-izedonfreepolyribosomesandimported posttranslation-ally into preexisting peroxisomes. The endoplasmic reticulum was presumed to synthesise the membrane phospholipidsofperoxisomes.Thus,theywereconsidered autonomousorganelleslikemitochondriaandchloroplasts. LazarowandFujikistatedthatoneoftheimplicationsof thismodelisthatperoxisomesneverformdenovo[1].

Geneticscreens inthelate 80sand 90sidentified many factorsrequiredforimportofperoxisomalmatrixproteins. The growth and division modelwas challenged by the discovery that mutants that appear to lack peroxisomal membranes canformperoxisomesde novo fromtheER upon complementation. Since then the ER has been

central to studies on peroxisome biogenesis. Below we assesstherecentliteratureonperoxisomalmatrixprotein import andmembraneformation.

Posttranslational

import

of

matrix

proteins

Protein import into peroxisomes differs from protein import into most other organelles as (1) peroxisomes import foldedandevenoligomericproteins and(2) per-oxisomalimport receptorscyclebetweenasoluble, free form in the cytosol and a cargo-loaded form at the peroxisomal membrane, which is associated with the translocon, and atthe endof the cycleisubiquitinated andreleased fromthemembraneinanATP-dependent process [2].Peroxisomalmatrixproteinscontainspecific peroxisomal targeting signals (PTS1 or PTS2) that are post-translationally recognized in the cytosol by the import receptorsPex5and Pex7,respectively[3–7,8].

Receptor-cargo complexes are directed to a docking complex at the peroxisomal membrane (see Figure 1). The PTS1 receptor mediates cargo binding as well as associationwiththeimportmachinery,whereasthePTS2 receptorbindscargobutrequiresauxiliaryproteinssuch as PEX5L in mammals and plants, or proteins of the Pex18familyinfungiformembraneassociationandcargo translocation[2].ThecrystalstructureofPex7incomplex with the Pex18 paralog Pex21 and a PTS2 peptide visualizes the cooperative binding mode of the PTS2 pre-importcomplex [8].

Thecargo-loaded receptoristhoughttoassemblewith componentsofthedockingcomplextoformthe translo-con,whichallowstranslocationoffoldedproteinsacross theperoxisomalmembraneintothematrix.Thecurrent opinionisbasedontheconceptofatransientporethat assembles at the peroxisomal membrane and is disas-sembled after import, with its components being recycled forfurther rounds of proteinimport [9]. The majorconstituentsofthedynamicporeforPTS1import arethePTS1receptorandthePMPPex14:this consti-tutes the minimal functional unit for translocation of matrixproteinsinvivo[10],andinelectrophysiological studies it displays features ofa regulated pore[11]. A majorquestioniswhetherPTS1andPTS2proteinsare imported via common or distinct import pores. Also currently debatediswhetherthecargo-loadedreceptor remainsassociatedwiththepore(shuttlehypothesis)or whetheritisreleasedasasolublereceptor-cargocomplex

§_{Thisisanopen-access articledistributedunderthetermsoftheCreativeCommonsAttributionLicense,which permitsunrestricteduse,}

into theperoxisomal matrix(extendedshuttle hypoth-esis).Oncethecargohasreachedtheperoxisomalmatrix, itisreleased fromthereceptor, whichmayrequirethe intraperoxisomal peripheral membrane protein Pex8 [2,12]orPex14[13].Itisunknownhowfoldedproteins are translocatedthroughthepore.Moreover,theexact compositionoftheporeaswellasthedrivingforcefor cargo translocation remained elusive. During or after dissociation of the receptor-cargo complex, the PTS1 receptorismono-ubiquitinatedataconservedcysteine, whichservesasasignalforATP-dependentdislocation ofthereceptorfromthemembranetothecytosol[14].It remainstobeinvestigatedwhyacysteineandnotalysine istheevolutionarilyconservedresidue.Interestingly,in thePTS2-pathway,Pex7buttheauxiliaryPex18family proteinsare mono-ubiquitinated, again at a conserved cysteine[15,16].

Mono-ubiquitination of receptors is performed by the peroxisomal ubiquitin-conjugating enzyme Pex4 [17]. Mono-ubiquitination is thought to prime the receptor

forrecognitionbyacomplexoftheAAA-typeATPases Pex1andPex6,whichfunctionsasdislocasetorelease themodifiedreceptorfromthemembranetothecytosol [18,19].AWP1hasbeenidentifiedasacofactorof mam-malianPex6thatbindsmono-ubiquitinatedPex5andis involvedintheregulationofPex5export[20].Duringor shortly after export, the PTS1 receptor is deubiquiti-nated and thus made available for another round of import[21,22].

Themechanismofhowfoldedproteinsaretranslocated throughtheperoxisomalporeandalso thedrivingforce forthisprocessremainelusive.Thetaggingofasubstrate bymonoubiquitylationorpolyubiquitylationandits sub-sequentrecognitionandATP-dependentremovalfroma membrane by ATPases of the AAA-family of proteins, resembles the mechanism of the membrane release of proteinsforER-associateddegradation(ERAD)[23,24]. AstheATP-dependentreleaseoftheubiquitinated per-oxisomal receptor from the membrane is the energy-requiringstep ofthematrixproteinimportcascade,the

26 Cellorganelles

Figure1

15 5

Ub Ub

ATP

ADP + Pi

4

1 5

5

PTS1

PTS1 PTS1

6

22 2 10 12 14

5

8 PTS1

Matrix Cytosol

5 14 17

14 13

IV

V III

II

I

VI

Current Opinion in Cell Biology

peroxisomalAAAperoxinsmight induceconformational changes of the receptor in an ATP-dependent manner thatallowsreceptorreleaseandcargotranslocation.This linkofreceptorexportandproteinimportisdescribedby theexport-driven importmodel[23].

Different

mechanisms

of

PMP

sorting

Until recently, two classes of PMPs weredistinguished basedontheirPex19dependencefortargetingto peroxi-somes [25].Class 1 PMPscontaintargeting information thatisrecognizedbyPex19,whichbindsnewly synthes-izedPMPsinthecytosolandactsasachaperone/targeting receptor,thus(1)preventingthemfromaggregatingand (2) delivering them to the peroxisomal membrane for dockingonPex3[26–28].Althoughposttranslational tar-getingofPMPstoperoxisomesinvivoandinvitroiswell documented[25,28–31,32],themechanismofinsertion remainsunclear. The centralroleof Pex3andPex19in PMP biogenesis isevident from thephenotype of pex3

andpex19mutants,inwhichperoxisomalmembranesare

undetectable andclass IPMPs areunstableor misloca-lised[33].Onthebasisoftheseobservationsweproposed thatthesemutants lackanyperoxisomalstructures.

Class2PMPs,thebest-studiedexampleofwhichisPex3, containtargetingsignalsthatarenotrecognizedbyPex19. Uponde novo formationof peroxisomesbyinductionof Pex3 expression in S. cerevisiae pex3 cells, most data supportamodelwherebyPex3enterstheERandforms preperoxisomes that develop into peroxisomes[34–36]. Its targeting and subsequent sorting signals have been described[37,38],andtheERtransloconhasbeen impli-cated in this process [37]. In vitro budding reactions release vesicles containing Pex3, dependent on Pex19, cytosolandATP[39,40].Buddingoccursindependently of the machinery required for ER exit of secretory proteins.Thesevesiclesthereforemightrepresent trans-port vesicles that fuse with pre-existing peroxisomes, thereby delivering Pex3 and lipids to peroxisomes (Figure 2).

Figure2

Matrix + Membrane protein import

Fission Fission

Matrix protein import

Pex13 Pex14

ER ER

All PMPs

Pex3, Pex22 (Pex13 Pex14?)

Pex3, Pex22 (Pex13 Pex14?) Pex13/14?

Matrix protein import

Membrane protein import

Pex13/14?

RING-finger PMPs

Vesicle fusion model Growth and division model Reintroduction of peroxisomes

Current Opinion in Cell Biology

AsPex3isnotobservedintheERofWTcells,onehasto proposethatitstransitthrough theERisfast.Thusfar, Pex3hasbeenvisualizedintheERonlyafter overexpres-sion,orbyusingmutantversionsappendedwithlargetags in cellsblocked in peroxisome formation(pex19or pex3

cells).ItisthereforepossiblethatyeastPex3bypassesthe ER in wild type cells and is inserted directly into the peroxisomalmembraneas hasbeen reported forhuman Pex3[41].InlinewiththisisthestudybyKnoopsetal.[42]. TheycoulddetectnoERpoolofPex3afterits reintroduc-tionintopex3cellsandpropose thatH. polymorphaPex3 bypassestheER altogether (see also below). Thisisan issuethatneedsfurtherinvestigation.

Recently,detailedmicrocopystudieschallengedtheview that pex3 and pex19 cells are devoid of peroxisomal structures with the findingthat the H. polymorpha pex3

andpex19cellscontainpre-peroxisomestowhichPex13 and Pex14 localise [42]. This suggests that Pex13 and Pex14 represent a third class of PMPs that also sort independentofPex3andPex19.Pex13-containing mem-branestructureshavebeen found inPichiapastorispex3

cells [43], and in S. cerevisiae pex3 and pex19 cells [44]. Furthermore,mammalianPex13insertsintomembranes independent of Pex19 [45]. Also, Pex13 is required for Pex14sorting[46],underliningthatthesePMPsbehave differentlyfromclassIandIIPMPs.

Peroxisome

multiplication

and

the

contribution

of

the

ER

In wild type yeast cells, peroxisomes receive newly synthesized membrane and matrix proteins and lipids (growth)and multiplybyfission[38,47,48,49,50]. Per-oxisomefissionismediatedbydynamin-relatedproteins andPex11(forrecentreviewsee[51]).Mostperoxisomal membranelipidsaresynthesizedintheER.Theymaybe directlytransferred fromtheER toperoxisomes[59] or reachperoxisomesviavesiculartransport.However,ithas alsobeen proposedthatperoxisomalmembranesderive fromthe ER via budding of vesicles containingPMPs. Tabak and coworkers recently reported that all newly synthesizedS.cerevisiaePMPsinsertintotheER,andthat the Pex13/14 docking complex and the RING-finger complexexit theER indistinct vesicles [44,52].These ‘half importomer’ complexes were proposed to be broughttogetherbyheterotypicvesiclefusiondependent onPex1andPex6,formingapre-peroxisomalmembrane structure able to import matrix proteins. Subsequent fissionwouldproduceperoxisomesoffinalsizeand mem-branecomposition(Figure2).Besidesbeingresponsible for de novo formation of peroxisomes in peroxisome-deficientmutantcells, Tabakand coworkersclaim that thismechanismalsoprovidesa‘continuousstream’ofde novo formed peroxisomes in wild type cells [52,53]. Althoughelegant,thismodelisirreconcilablewithmany oftheindependentstudiesdescribedabove.Thismodel disregards for instance the well established role of

Pex19-dependent posttranslational insertion of newly synthesized PMPs directly into peroxisomes [25– 31,32],the mislocalisation of many S. cerevisiae PMPs tothecytosolinpex3andpex19cells [33],and thatnew peroxisomes are derived from preexisting peroxisomes [47–50]. Furthermore, Pex25 is required for de novo

peroxisomeformation,but mostpex25cells contain per-oxisomes, which suggests the de novo pathway is not essentialforperoxisomemaintenance[54,55]. Addition-ally,thestudiesbyKnoopset al.[42]indicatedthatnot theER, butthePex13/Pex14-containingstructures pre-sentinH.polymorphapex3cellsarethetargetfor reintro-duced Pex3. Although the origin of the Pex13/Pex14 preperoxisomal structures is unclear, their formation is independentofPex3.

TheshapeoftheERappearstoaffectperoxisome multi-plication.Pex30 is aClass 2 PMP[56] thatlocalises to bothperoxisomesandan ERsubdomainfound inclose associationwith peroxisomes[57,58].Ithasbeen pos-tulated that new peroxisomes can form from theseER subdomains. Interestingly, COPI components were found in complex with Pex30, although the functional significanceof this isunclear. The ER reticulons Rtn1, Rtn2 and Ypo1 were identified as core components of Pex30complexes.ERreticulonsareimportantfor main-tainingthemorphologyoftubularER bystabilizingthe stronglycurvedmembranes.Interestingly,incellslacking theERreticulonsorPex30,denovoformationof peroxi-somesisaccelerated[58].Whetherthesemutantsform peroxisomesdenovoallthetimeisnotknown.

A

multi-step

model

for

peroxisome

biogenesis

Thereisoverwhelmingevidenceinfavorofagrowthand division model, whereby the insertion machinery for Class1PMPsiscentral.AsPex3ispresentatsteady-state onperoxisomesinwildtypecells,Pex19boundtonewly synthesizedClass1PMPsmaydockheretodeliverthem. Class 2 PMPs, including Pex3, traffic via the ER and vesiculartransport toperoxisomes.This routemay pro-vide sufficient membrane constituents for continuing growth and division of peroxisomes, although a non-vesicular membrane transfer may contribute as well [59]. In cells forming peroxisomes de novo, Pex3 first insertsin theER and sortsto preperoxisomes, whereit facilitates Class 1 PMP insertion, allowing these mem-branestructurestoimportmatrixproteinsandmatureinto peroxisomes.Pex13andPex14mayfollowthePex3route in cells synthesizing peroxisomes de novo, or they may bypass the ER and insert directly into preperoxisomes thatcanformindependentlyofPex3.WhetherPex13and Pex14followthisrouteinwildtypecellsisanimportant questionand remainsto bedetermined.

Concluding

remarks

The global mechanisms of peroxisome membrane bio-genesisandmatrixproteinimportarebecomingclear,but

manyquestionsremain.Tomentionbut afew:how are ER-insertedPMPssortedawayfromthesecretory path-way? How do PMPs leave the ER, and how are they delivered specifically to peroxisomes?Do Class1PMPs insert spontaneously into peroxisomal membranes after targeting,orisaspecialisedmachineryrequired?Howis peroxisome sizeand numberregulated?Whichproteins makeuptheporesformatrixproteinimport,andhowis matrix proteintranslocationmediated?Newapproaches will need to bedeveloped to shed lightonthese ques-tions.

Acknowledgements

ThisworkwassupportedbytheDeutscheForschungsgemeinschaft (FOR1905), and a Wellcome Trust Senior Research Fellowship in Basic BiomedicalScienceawardedtoEHH,WT084265MA.Theauthorswould liketothankAlisonMotleyfordiscussions.

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and

recommended

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