Downloaded from http://cshperspectives.cshlp.org/ on February 5, 2023 - Published by Cold Spring Harbor Laboratory Press Lipid Transport between the Endoplasmic Reticulum and Mitochondria VidV.FlisandGu¨ntherDaum InstituteofBiochemistry,GrazUniversityofTechnology,A-8010Graz,Austria Correspondence:[email protected] Mitochondria are partially autonomous organelles that depend on the import of certain proteinsandlipidstomaintaincellsurvivalandmembraneformation.Althoughphospha- tidylglycerol,cardiolipin,andphosphatidylethanolaminearesynthesizedbymitochondrial enzymes,phosphatidylcholine,phosphatidylinositol,phosphatidylserine,andsterolsneed tobeimportedfromotherorganelles.Theoriginofmostlipidsimportedintomitochondriais the endoplasmic reticulum, whichrequires interaction of these twosubcellularcompart- ments.Recently,proteincomplexesthatareinvolvedinmembranecontactbetweenendo- plasmicreticulumandmitochondriawereidentified,buttheirroleinlipidtransportisstill unclear. In the present review, we describe components involved in lipid translocation between the endoplasmic reticulum and mitochondria and discuss functional as well as regulatoryaspectsthatareimportantforlipidhomeostasis. Biological membranes are major structural organelles(Futerman2006).Besidestheexport components of all cell types. They protect ofceramides,theERsuppliesalargeportionof the cell from external influences, organize the lipids to other organelles, which cannot pro- interiorindistinctcompartmentsandallowbal- ducetheirownlipidsorhavealimitedcapacity ancedfluxofcomponents.Besidestheirspecific todoso.Organelleinteractionandtransportof proteome,organellesexhibituniquelipidcom- lipids require specific carrier proteins, mem- positions,whichinfluencetheirshape,physical brane contact sites, tethering complexes, and/ properties, and function. Major lipid classes or vesicle flux. These processes are highly im- foundinbiologicalmembranesarephospholip- portant for the maintenance of cell structure ids,sterols,andsphingolipids. and survival but are still a matter of dispute. Themajor“lipidfactory”withinthecellis Mostprominentorganelleinteractionpartners the endoplasmic reticulum (ER). It is able to aretheERandmitochondria.Asubfractionof synthesizethebulkofstructuralphospholipids, the ER named mitochondria-associated mem- sterols,andstoragelipidssuchastriacylglycer- brane (MAM) (Vance 1990) was described to olsandsterylesters(vanMeeretal.2008).Fur- beinvolvedinlipidtranslocationtomitochon- thermore, initial steps of ceramide synthesis dria.MAMispartoftheERnetwork,whichwas occur in the ER providing precursors for the shown to be in contact or close proximity to formation of complex sphingolipids in other the outer mitochondrial membrane (OMM). Editors:SusanFerro-Novick,TomA.Rapoport,andRandySchekman AdditionalPerspectivesonTheEndoplasmicReticulumavailableatwww.cshperspectives.org Copyright#2013ColdSpringHarborLaboratoryPress;allrightsreserved;doi:10.1101/cshperspect.a013235 CitethisarticleasColdSpringHarbPerspectBiol2013;5:a013235 1 Downloaded from http://cshperspectives.cshlp.org/ on February 5, 2023 - Published by Cold Spring Harbor Laboratory Press V.V.FlisandG.Daum ContactsitesbetweenMAMandmitochondria headgroupandlargehydrophobictail,PEisa wereassumedtofacilitateexchangeofcompo- typical nonbilayer-forming phospholipid (see nentsbetweenthetwocompartments.Interest- Fig.1).Thisstructureappearstobeimportant ingly, MAM harbor a numberof lipid synthe- forsomeperipheralandintegralmembranepro- sizing enzymes (Gaigg et al. 1994). Recently, teins(vandenBrink-vanderLaanetal.2004). molecular components governing membrane TogetherwithCL,PEplaysacrucialroleinmain- contactbetweenthetwoorganelleswereidenti- taining mitochondrial morphology. Loss of fied(Dolmanetal.2005;Csorda´setal.2006;de bothPEandCLislethalprobablyduetodefects BritoandScorrano2008;Kornmannetal.2009; inmitochondrialfusion(Joshietal.2012)and/ Friedman et al. 2010; Lavieu et al. 2010), al- orproteindestabilizing(Bogdanovetal.2008b; though the specific role of these components Osmanetal.2009b).LackofPEsynthesizedin inlipidtranslocationisnotyetclear. the IMM cannot be fully compensated by PE importedfromextramitochondrialsites(Birner etal.2001;Bu¨rgermeisteretal.2004;Joshietal. MAJORPHOSPHOLIPIDCLASSESOF 2012). However, PE derived from exogenous MITOCHONDRIA lyso-PE can restoreboth intra-andextramito- Cardiolipin (CL) and/or phosphatidylglycerol chondrial PE deficiencies by remodeling pro- (PG) are considered as mitochondria-specific cesses(RiekhofandVoelker2006).Noteworthy, phospholipids (Zinseret al. 1991). Both lipids mitochondria are the major supplier of PE to are synthesized by mitochondria themselves otherorganelles(Voelker1984). (DavidsonandStanacev1971).Theimportance MitochondrialenzymesofCLandPGsyn- ofCLforATP-productionhasbeenshownwith thesis,aswellasthemajorPSdecarboxylase,are differentmembranetypes,e.g.,bacteria,hydro- synthesized on cytosolic ribosomes, imported genosomes, and mitochondria (Mileykovskaya into mitochondria, and assembled into the etal.2005;Schlame2008;Acehanetal.2011). IMM (Minskoff and Greenberg 1997; Jiang Inmitochondria,themajorityofCLislocalized et al. 2000; Nowicki et al. 2005; Nebaueret al. to the inner membrane (IMM) (Zinser et al. 2007; Choi et al. 2012). Only recently werewe 1991), but substantial amounts were also de- abletodemonstrateexperimentallytheinvolve- tected in the OMM (Gebert et al. 2009). With ment of components of the mitochondrial its uncommon, dimeric structure (Fig. 1) CL TOMcomplexandofmitochondrialproteases together with phosphatidylethanolamine (PE) inimportandprocessingofthemitochondrial interacts with many mitochondrial proteins PS decarboxylase Psd1p from the yeast (Hor- (Osmanetal.2011)andstabilizestheirconfor- vathetal.2012). mation(Joshietal.2012).MutantslackingCL Phosphatidylcholine(PC)hasabighydro- andPEbiosynthesisaresyntheticlethalinyeast philic head group and a long hydrophobictail and bacteria (Gohil et al. 2005). In bacteria, (seeFig.1).Itscylindricalshapemakesitaper- bothlipidsareorganizedinmembraneclusters fect component of bilayer membranes and its (Matsumoto et al. 2006). Specific interaction roleasastructuralcomponentisessential(van partnersofCLareproteinsofATPproduction, Meer et al. 2008). Specific functions of PC in mitochondrial transport systems (Bogdanov mitochondria are not well defined. Similar to etal.2008a;SchlameandRen2009),andpro- PC, specific functions of phosphatidylinositol teins requiredformitochondrial structureand (PI) in mitochondria are not known, but its fusion(Joshietal.2012). essentialroleintotalcellularmetabolismmakes Another prominent mitochondrial phos- it directly or indirectly indispensible for the pholipidisPE.Mitochondriaareabletosynthe- maintenance of mitochondria. The bulkhead sizealargeportionofPEbydecarboxylationof groupofPImakesthisphospholipidaspecific phosphatidylserine(PS)(Vialetal.1982;Trotter membranebilayercomponent. etal.1993;Emotoetal.1999;Birneretal.2001; Phosphatidylserine (PS) has also a cylin- Nerlichetal.2007).Withitssmallhydrophilic dricalshapethatallowsintegrationintobilayer 2 CitethisarticleasColdSpringHarbPerspectBiol2013;5:a013235 Downloaded from http://cshperspectives.cshlp.org/ on February 5, 2023 - Published by Cold Spring Harbor Laboratory Press LipidTransportbetweentheERandMitochondria Phosphatidylserine Phosphatidylcholine Phosphatidylinositol Cylindrical CH3 bilayer C–O2 N+H3 CH3 N+ CH3 HO HO OH CH CH2 OH O O CH2 CH2 H *O P O O O O *O P O *O P O O O CH2 CH CH2 O O CH2 CH CH2 CH2 CH CH2 O O O O R1 R2 R1 R2 R1 R2 Conical Conical Cardiolipin Phosphatidylethanolamine nonbilayer nonbilayer + NH3 OH CH2 CH CH2 CH2 O O CH2 *O P O O P O* O O O *O P O CH2 CH CH2 CH2 CH CH2 O O O O O CH2 CH CH2 R1 R2 R2 R1 O O R1 R2 Figure1.Majorphospholipidclassesofmitochondria.Chemicalformulasandgeometricalformsofthemajor mitochondrialphospholipidsareshown.Theshape-structureconceptoflipidscomparestheareaoftheheadgroup withtheareaoftheiracylchains.Ifthecross-sectionareaoftheheadgroupissimilartothatoftheacylchains,lipids haveanoverallcylindricalshapeandhaveastrongtendencyofself-assemblyintobilayerphasesofbiological membranes.AtypicalexampleforsuchgeometryisPC.Ifthecross-sectionareaoftheheadgroupissmallerthan thatoftheacylchains,lipidshaveaconicalshapeandformstructureswithnegativecurvaturesuchashexagonal phase.ExamplesofthistypearePEandCL.WhilePC,PS,andPIexhibitcylindricalshapeandself-organizeinto bilayers,PEandCLwiththeirconicalshapeinducehexagonalphasesanddisturbbilayerarrangement. membranes(seeFig.1).AlthoughPSispresent Vance1997;vanMeer 2008).OMMandIMM in low concentrations in organelles of eukary- arestronglydifferentwithrespecttotheirlipid oticcells(ZinserandDaum1995),itisimpor- equipment. Whereas the IMM is strongly en- tantasaprecursorforthetwomajorphospho- richedinproteinsandcontainsonly20%lipids lipids,PEandPC.SupplyofPStomitochondria oftotalmass,theOMMisalipidrichmembrane isessentialbecauseinmanycellsthemajorityof (ZinserandDaum1995;Dolisetal.1996;Daum PEisformedbydecarboxylationofPSinmito- andVance1997).AccumulationofCL,PG,and chondria.ThemetabolicconversionofPStoPE also PE in the IMM appears to be related to uponimportofPSintomitochondriaprovides functionsmentionedabove. aconvenientbiochemicalmethodtostudythis importprocess(Voelker1988,1989b,1991,1992, SYNTHESISOF 1993;Achleitneretal.1995,1999). AMINOGLYCEROPHOSPHOLIPIDS Typically, the lipid composition of mito- INVOLVESINTERACTIONOFORGANELLES chondria shows as major components 40%– 44% PC, 27%–34% PE, 1%–3% PS, 5%– The biosynthetic sequence of aminoglycero- 15% PI, and 13%–14% CL depending on the phospholipid formation starts with the syn- cell type (Zinser and Daum 1995; Daum and thesis of PS in the ER (Fig. 2). The highest CitethisarticleasColdSpringHarbPerspectBiol2013;5:a013235 3 Downloaded from http://cshperspectives.cshlp.org/ on February 5, 2023 - Published by Cold Spring Harbor Laboratory Press V.V.FlisandG.Daum A Endoplasmic reticulum Mitochondria Pss1p/Cho1p CDP-DAG PS PS Serine Golgi network Psd2p –CO2 –CO2 Psd1p Eki1p Ect1p Ept1p Etn PE PE ATP CTP DAG SAM Cho2p/Pem1p Dpl1p SL SAM Opi3p/Pem2p SAM Opi3p/Pem2p Cki1p Cct1p Cpt1p Cho PC ATP CTP DAG B Mitochondria Endoplasmic reticulum Ser PSS1 Cho CPT CCT CK PS PC Cho PS DAG CTP ATP Ser PSD –CO2 PSS2 PE3M T× SAM CDP-Cho Etn EPT ECT EK PE PE Etn CDP-Etn DAG CTP ATP C PL PL CDP-Etn/Cho Methylation pathway Decarboxylation pathway Land’s Acyl-CoA:lysophospholipid PLA2 cycle acyltransferase Fatty acid Acyl-CoA Lyso-PL Figure2.Biosyntheticpathwaysofaminoglycerophospholipids.(A)Biosynthesisofaminoglycerophospholipids inyeast.FormationofPSisaccomplishedintheERandcatalyzedinaCDP-DAG-dependentreactionbythePS synthase,Pss1p.DecarboxylationofPSyieldingPEoccursinmitochondria(viaPsd1p)andaGolgi/vacuolar compartment (viaPsd2p).Three-stepmethylationofPEintheERcatalyzedbyCho2p/Pem1pandOpi3p/ Pem2p leadsto formation ofPC with S-adenosine methionine (SAM)as methyldonor. PC andPE can be formedalsobytheCDP-ethanolamineandCDP-cholinebranchesoftheso-calledKennedypathwaymakinguse ofexogenousorendogenouscholine(Cho)andethanolamine(Etn),respectively.SL,sphingolipids.(B)Amino- glycerophospholipidbiosynthesisinmammaliancells.ThemajormechanismofPSproductioninmammalsis baseexchangewithPC(PSS1)andPE(PSS2)assubstrates.AmajorrouteofPEandPCproductionaretheCDP- ethanolamineandCDP-cholinepathways.Inmammaliancells,PEcanalsobeproducedbydecarboxylationof PS.Inhepatocytes,asinglemethyltransferasecatalyzesmethylationofPEtoPC.(C)TheLand’scycledescribesa sequenceofdeacylationandreacylation.PEandPCareconvertedtolyso-PEandlyso-PCandviceversa. 4 CitethisarticleasColdSpringHarbPerspectBiol2013;5:a013235 Downloaded from http://cshperspectives.cshlp.org/ on February 5, 2023 - Published by Cold Spring Harbor Laboratory Press LipidTransportbetweentheERandMitochondria concentration of PS synthesizing enzymes has pathway cannot fully complement for the mi- been detected in the MAM (mitochondria-as- tochondrial requirement of PE in cells deleted sociatedmembrane)(Kuchleretal.1986;Vance of both PSD1 and PSD2 (Trotter and Voelker 1990).Interestingly,mammaliancellsandyeast 1995). Ethanolamine phosphate, which is an havedifferentpathwaystoproducePS.Inmam- intermediate in this biosynthetic sequence, malian cells, two phosphatidylserine synthases canalsobederivedfromsphingolipiddegrada- (PSS-1andPSS-2)producePSbybaseexchange tion providing a link between sphingolipid attheheadgroupofPEorPC(Vance2008)ina and PE metabolism (Mao et al. 1997; Saba Ca2þ-dependentreaction(Fig.2B).Thisrelease etal.1997).Finally,yeastharborsenzymesthat of Ca2þ into the lumen of the ER is energy catalyze acylation of lyso-PE (Riekhof and dependent. In yeast, formation of PS is cata- Voelker2006; Riekhof etal. 2007b; Deng etal. lyzedbythegeneproductofPSS1/CHO1(Letts 2010).ThistypeofreactionnamedLand’scycle etal.1983),whichrequiresCDP-diacylglycerol includes deacylation/reacylation of phospho- (CDP-DAG)andserine(NikawaandYamashita lipids(Lands1958)andappearstobeimportant 1981) as substrates and depends on Mg2þ or forremodelingprocesses.Thereasonfortheex- Mn2þ(Fig.2A).Cellularenergyisrequiredfor istenceofoverlappingpathwaysisstillamatter the formation of CDP-DAG. In plants, both ofdispute,althoughevidencefordistinctpools pathwaysdescribedaboveareactiveforPSpro- of PE has been presented (Bu¨rgermeisteret al. duction(Delhaizeetal.1999;Manoharanetal. 2004). 2000;Ronteinetal.2003).Inalltypesofcells, Mammalian cells have the capacity to syn- PS synthesized in the ER is exported to other thesizetheirentirePEthroughthePSDpathway organelles, including mitochondria where it in mitochondria (Voelker and Frazier 1986), servesasasubstrateforPEsynthesis. but in the presenceof ethanolamine the CDP- PE is the second most abundant lipid of ethanolamine pathway can fulfill most of the eukaryotic cells. It can be synthesized by four extramitochondrial requirements for this lipid different pathways: (i) the CDP-ethanolamine (Vance 2008). The Land’s cycle is important pathway(alsonamedKennedypathway)(Kan- for remodeling phospholipids that have been ferandKennedy1964),(ii)decarboxylationof formedbyotherpathways.Asanexample,for- PStoPE,(iii)baseexchangebetweendifferent mation of PE from lyso-PE can be accom- phospholipids, and (iv) acylation of lyso-PE plishedbyLPEAT2(acyl-CoA:lysophosphatidyl- (see Fig. 2). Yeast has two PS decarboxylases ethanolamine acyltransferase 2) in the brain (PSD) with overlapping functions (Trotter et cells. Ethanolamine phospholipids are major al. 1993). Psd1p is localized to mitochondria constituents of the myelin sheath increasing (Trotteretal.1993),andPsd2phasbeenfound the signal transmission speed along the axons inaGolgi/vacuolarcompartment (Trotterand (Caoetal.2008).Inplants,remodelingofPEto Voelker 1995). Psd1p is a component of the lyso-PE was reported to be involved in plant IMM/intermembrane space and produces the growthpromotionandleafsenescence(Cowan majorityoftotalcellularandmitochondrialPE 2009;Hongetal.2009).Intheparasites,Trypa- (Trotteretal.1993;Birneretal.2001). nosoma brucei and Plasmodium berghei, PE is YeastcanalsoproducePEthroughtheCDP- exclusively produced through the CDP-etha- ethanolamine branch of the Kennedy pathway nolaminebranchoftheKennedypathway(Ser- (KennedyandWeiss 1956).TheCDP-ethanol- ricchioandBu¨tikofer2011). amine pathway incorporates externally added PC is the most abundant phospholipid in orendogenousethanolaminethroughstepwise eukaryoticcells(vanMeeretal.2008).Because phosphorylation, activation with CTP, and at- mitochondria lack PC synthesizing enzymes, tachment to diacylglycerol (DAG). In the final thisphospholipidhastobeimportedfromthe step,phosphoethanolamineistransferredfrom ER. PC can be produced via three pathways: CDP-ethanolamine to a DAG acceptor form- (i)theCDP-cholinebranchoftheKennedypath- ing PE. However, PE synthesized through this way(GibelliniandSmith2010),(ii)methylation CitethisarticleasColdSpringHarbPerspectBiol2013;5:a013235 5 Downloaded from http://cshperspectives.cshlp.org/ on February 5, 2023 - Published by Cold Spring Harbor Laboratory Press V.V.FlisandG.Daum ofPE(SundlerandAkesson1975;Lietal.2005), Cds1p,eitherintheER/MAMorinmitochon- and(iii)theLand’scycle,inwhichLPCAT(lyso- dria. Then, conversion of CDP-DAG to phos- phosphocholine acyltransferase) produces PC phatidylglycerolphosphate(PGP)catalyzedby from lyso-PC and fatty acids (Riekhof et al. Pgs1p occurs (Chang et al. 1998a). PGP is de- 2007a; Hishikawa et al. 2008; Shindou et al. phosphorylated to PG in a reaction that is ac- 2009) (see Fig. 2). In most mammalian cells, complished by the PGP phosphatase Gep4p themajorityofPCisformedthroughtheCDP- (Osman et al. 2010). In the final step of CL choline pathway (Kennedy 1956; Gibellini and synthesis,aphosphatidate(PA)moietyistrans- Smith 2010; Hermansson et al. 2011), and ferredfromCDP-DAGtothehydroxylgroupin only inhepatocytes PEmethylation isthepre- the headgroup of PG. Thecleavage of the py- dominantpathway(SundlerandAkesson1975; rophosphategroupprovidesthechemicalener- Li et al. 2005). Reactions of the CDP-cho- gy for the latter reaction. In yeast (Jiang et al. line pathway are similar to the CDP-ethanol- 1997; Chang et al. 1998b; Tuller et al. 1998), aminepathwayandcatalyzedbycholinekinase, Arabidopsis(Katayamaetal.2004;Nowickietal. phosphocholinecytidyltransferase,andcholine 2005),andhumancells(Chenetal.2006;Hout- phosphotransferase.Inthefinalstep,phospho- kooperetal.2006;Luetal.2006),CLsynthase cholineistransferredfromCDP-cholinetoDAG waslocalizedtomitochondria.Importantly,CL formingPC.Inyeast (Boumannetal.2004)as undergoes remodeling processes (Joshi et al. wellasinmammals(HenneberryandMcMaster 2009; Schlame and Ren 2009) with so-called 1999), enzymes of the both branches of the tafazzins(Malhotraetal.2009)involved.Tafaz- Kennedy pathway have overlapping substrate zinsarephospholipidtransacylasesthattransfer specificities. acyl groups from phospholipids, preferentially The second pathway of PC production in PC, to monolysocardiolipin (MLCL). The re- mammalian cells (Van Pilsum and Carlson versereactionofthismodificationisthetransfer 1970) and yeast (Kodaki and Yamashita 1987) ofanacylgroupfromCLtolyso-PC.Thereac- isPEmethylation.Inthissequence,S-adenosyl- tion does not require activation of fatty acids methionine(SAM) serves as methyl donor for butoccursinalysophospholipid–phospholip- threestepsofmethyltransferasereactions.Inthe idcomplexbydeprotonationandnucleophilic firststep,PEismethylatedtomonomethyl-PE attackontheesterbondoftheacyldonor(Xu and then further converted to dimethyl-PE in etal.2006). the second step. The biosynthetic sequence is PA and CDP-DAG are important interme- completedbyathirdstepofmethylationyield- diatesnotonlyforPG/CLsynthesisbutalsofor ing the final product PC. In yeast, the gene the formation of PI and PS (Athenstaedt and products of CHO2/PEM1 and OPI3/PEM2 Daum 1999). In the yeast, PA is synthesized are involved (Kodaki and Yamashita 1987), from glycerol-3-phosphate and/or dihydroxy- whereashepatocytesharboronlyoneN-meth- acetonephosphate(DHAP)intheERandlipid yltransferase, which catalyzes all three steps of dropletsbytwostepsofacylation.Thefirstacet- PE methylation (Sundler and Akesson 1975; ylationreactionwithDHAPasasubstrateleads Ridgwayetal.1989).Thelowereukaryote,Try- to acyl-DHAP, which is then reduced by the panosoma brucei, lacks genes coding for PE 1-acyl-DHAP reductase Ayr1p in an NADPH- N-methyltransferases(Gibellinietal.2009). dependent reaction to lyso-PA. Alternatively, acylation of glycerol-3-phosphate leads to for- mationoflyso-PA.Inafinalacylationstep,lyso- CARDIOLIPINAND PAisconvertedtoPA(Athenstaedtetal.1999a; PHOSPHATIDYLGLYCEROLSYNTHESIS SorgerandDaum2003).PAcanalsobegener- INMITOCHONDRIA ated by phospholipase D (Osman et al. 2011) SynthesisofPGandCLinmitochondriaoccurs in mitochondria. In plants, PA synthesis oc- byamultistepreactionsequence(Schlameetal. curs in plastids, mitochondria, and micro- 1993). First, PA is converted to CDP-DAG by somes. Dephosphorylation of PA is catalyzed 6 CitethisarticleasColdSpringHarbPerspectBiol2013;5:a013235 Downloaded from http://cshperspectives.cshlp.org/ on February 5, 2023 - Published by Cold Spring Harbor Laboratory Press LipidTransportbetweentheERandMitochondria by mammalian lipin and the yeast ortholog cellshavebeenperformed(ButlerandThomp- Pah1p(CarmanandHan2006,2009;Brindley son 1975; Voelker 1985, 1989a,b, 1990, 1991; etal.2009;HarrisandFinck2011),whichwere Vance1990; Achleitneret al. 1995; Shiao et al. recently shown to be pacemakers and switch 1995;Emotoetal.1999;Kugeetal.2001;Schu- pointsinlipidmetabolism.Dephosphorylation macheretal.2002;WuandVoelker2004;Car- ofPAisacrucialstep,becausetheproductofthe rasco et al. 2006; Riekhof and Voelker 2006; dephosphorylation reaction, DAG, becomes Kornmannetal.2009;Nguyenetal.2012;Padi- substrate for triacylglycerol synthases and thus lla-Lo´pezetal.2012;Tamuraetal.2012).These promotes biogenesis of lipid droplets (Adeyo studies showed that in mammalian cells PS etal.2011).PA,whichisnotdephosphorylated, transport from MAM to the OMM requires ispreferentiallyconvertedtoCDP-DAG,which ATP. Further import of PS to the IMM yields isfurtherusedforthesynthesisofphospholip- PE (Voelker and Frazier 1986; Voelker 1990, ids. CDP-DAG synthase activity in yeast, as 1991;Shiaoetal.1995). well as in mammals, was localized to the ER Experiments with isolated organelles pro- and mitochondria (Kuchler et al. 1986; Kelley vided more detailed information about the andCarman1987;Chenetal.2006). processofphospholipidimportintomitochon- dria.Reconstitutionoftransportsystemsusing isolatedmitochondria andmicrosomes/MAM PHOSPHATIDYLINOSITOLSYNTHESIS (Kuchleretal.1986;Simbenietal.1990;Vance PIisproducedintheERandhastobeimported 1990;Achleitneretal.1999;Emotoetal.1999) intomitochondria.PIissynthesizedfromCDP- revealedthatuptakeofPSbymitochondriade- DAGandinositol(Gardockietal.2005).Inthe pended on the PS concentration in the do- yeast,deletionoftheonlyphosphatidylinositol nor membranes (ER, Golgi) (Wu and Voelker synthasegenePIS1islethal(Nikawaetal.1987). 2004).AlthoughATPwasrequiredfortransport Furthermore,PIservesasprecursorforcellsig- ofPSinintactandpermeabilizedcells(seebe- naling molecules such as phosphatidylinosi- low),nosuchobservationwasmadeinthere- tolphosphates(PIP),phosphatidylinositolbis- constituted in vitro system. It was argued that phosphates (PIP2) and phosphatidylinositol closeappositionofdonor(ER/MAM)andac- triphosphates (PIP3) and for the biosynthesis ceptor (mitochondria) membranes was suffi- ofGPIanchors(SerricchioandBu¨tikofer2011). cient for transport,butthatATPwasprobably requiredtoprovideappropriateconditionsfor transport of this phospholipid in intact cells. TRANSPORTOFPHOSPHOLIPIDSBETWEEN Experiments making use of the coisolation of THEENDOPLASMICRETICULUMAND MAM with mitochondria suggested that the MITOCHONDRIA contact of these two cellular fractions is fairly AsthebiosyntheticsequenceofPS-PE-PCsyn- tight(Vance1990;Achleitneretal.1999).Alto- thesis occurs in different subcellularcompart- gether,itwasconcludedthatmembranecontact ments (see above), the amounts of intermedi- betweenERandmitochondriaisimportantfor ates and products are indication and measure lipid translocation between these two organ- for transport between organelles. Therefore, elles. interorganelle translocation of aminoglycero- Experimentsperformedwithpermeabilized phospholipidscanbeinvestigatedbyfollowing cellsprovidedausefulalternativetotheexper- the incorporation of serine into PS in the ER, imentsdescribedabove(Limetal.1986;Voelker decarboxylation of PS to PE in the mitochon- 1992;Achleitneretal.1995;Kugeetal.2001;Wu dria, and methylation of PE to PC upon the and Voelker 2001). Permeabilized cells are ob- returnofPEtotheERwithoutisolationofor- tained by mild chemical or mechanical treat- ganelles.Makinguseofthisexperimentalstrat- ment of whole cells, resulting in the internal egy, phospholipid transport studies with in- organelle structures remaining largely intact, tactcells,isolatedorganelles,andpermeabilized while access to the interior is possible for CitethisarticleasColdSpringHarbPerspectBiol2013;5:a013235 7 Downloaded from http://cshperspectives.cshlp.org/ on February 5, 2023 - Published by Cold Spring Harbor Laboratory Press V.V.FlisandG.Daum compoundsthatcannotenteranintactcell.In fectssubstratespecificityoftheubiquitinligase permeabilizedyeastcells(Achleitneretal.1999) (Chandrasekaran et al. 2006; Yan and Xiong [3H]serine is incorporated into PS in the ER. 2010).OnesubstratefortheE3ubiquitinligase Transport of [3H]PS to mitochondria yields isthetranscriptionfactorMet4p,whichisinac- [3H]PE formed by mitochondrial Psd1p. Use tivated upon ubiquitination. It was suggested of psd2Dmutants and depletion of ethanol- thatMet30pmediatesubiquitinationofcertain amine from the medium allows attribution of targetproteinsandmayplayaroleeitherinER- theformed[3H]PEtotransportof[3H]PSfrom mitochondria recognition,inthe inhibition of the ER. Transport of [3H]PE from mitochon- this docking process, or in regulation of tran- driatotheERresultsinformationof[3H]PCas scriptionofafactorinvolvedinlipidtransport. a measure for this transport route (Achleitner Thesecondcomponentidentifiedinthescreen etal.1995,1999;Shiaoetal.1998). was the phosphatidylinositol 4-kinase Stt4p, Theadvantageofpermeabilizedcellsisthat which affects transport of PS to the site of reactions can be manipulated by addition of Psd2p-driven conversion to PE (Trotter et al. chemicals such as divalent ions, energy block- 1998).ThemodeofactionofStt4ponorganelle ers, or cytoskeleton inhibiting reagents (Eilers interactionand/orPStranslocationremainsto et al. 1989) or by removing the cytosol. This beelucidated.ItwassuggestedthattheC2do- feature allowed additional characterization of mainofPsd2p,whichbindsCa2þ,interactswith lipid transport between ER and mitochondria proteinsorlipidsandrecognizesanioniclipids withmammalian(Voelker1990)andyeastcells such as PS and polyphosphoinositides (Choi (Achleitneretal.1995).TransportofPStomi- etal.2005)andmaybethebridgeforPStrans- tochondriawasshowntobedependentonATP location. Finally, the gene product of PDR17/ inmammaliancells(Voelker1989a)butnotin SFH4wasfoundtoaffectPStransporttoPsd2p. yeast(Achleitneretal.1999).However,acertain Reconstitution assays with permeabilized cells amount of ATP must be present in both cell and isolated organelles showed that Pdr17p types (Shiao et al. 1995) because upstream re- mustbepresentontheacceptormembranefor actionsoflipidprecursorformationareenergy transfer of PS to Psd2p (van den Hazel et al. consuming.Itwasalsoshownthatongoingsyn- 1999;Wuetal.2000).Themechanismofaction thesisofPSisnotrequiredfortranslocationto ofPdr17pisstillunknown. mitochondria because preformed PS was effi- Reconstitution of PS synthesis and trans- ciently used as a substrate for mitochondrial portinpermeabilizedmammaliancellsidenti- PSdecarboxylation.Interestingly,whenexoge- fiedtwoothercomponentsthataffectPStrans- nous mitochondria were added to permeabi- port from the ER/MAM to mitochondria lized cells, they failed to serve as acceptors for (Emoto et al. 1999; Kuge et al. 2001). One of PSimport (Voelker1993).Thisresultsuggests these proteins is S100B (NP_006236), an EF- that pre-existing, stable associations between hand domain-Ca2þ-binding protein. It is not mitochondriaandER/MAMplayanimportant known whether this protein participates in roleinthetransportprocess. transport or promotes stability and/or assem- In a yeast genetic screen, Voelker and co- blyofinteractionsofER/MAMandmitochon- workerssearchedforcomponentsthatinfluence dria.Thesecondcomponentwasonlyindirectly the translocation of PSfrom ERtothe sites of identified in the mammalian cell line R41, decarboxylation in mitochondria (via Psd1p) whichhasadefectinPStransportbetweenthe (Schumacher et al. 2002) and Golgi/vacuoles OMMandIMM. (viaPsd2p)(Trotteretal.1998;WuandVoelker Studies with isolated organelles treated 2001, 2004). The first gene product identified with protease (Shiao et al. 1998; Achleitner was Met30p, which influenced the import of et al. 1999) or dinitrophenol, which alters the PS into mitochondria. MET30 encodes a sub- distance between the ER and mitochondria unit of a multicomponent E3 ubiquitin ligase (Hoviusetal.1992),suggestedtheparticipation (Aghajanetal.2010;Ounietal.2010) andaf- of organelle surface proteins in PS transport. 8 CitethisarticleasColdSpringHarbPerspectBiol2013;5:a013235 Downloaded from http://cshperspectives.cshlp.org/ on February 5, 2023 - Published by Cold Spring Harbor Laboratory Press LipidTransportbetweentheERandMitochondria Electron microscopy supported this view by heritance (Frederick et al. 2004, 2008) and demonstrating close contact zones between mostlikelyconnectsERMEStotheactincyto- ER/MAM and mitochondria, which might skeleton (Kornmann et al. 2011; Michel and serveasbridgesforlipidtranslocation(Csorda´s Kornmann 2012). Itwas assumed that Gem1p et al. 2006). Furthermore, the involvement of shuttlesbetweenafreeandERMESboundform acetylated microtubules in ER-mitochondria (seeFig.3),althoughthemolecularmechanism dynamics was demonstrated, a mechanism by ofGem1premains unknown.Physicalinterac- which membrane contact can be established tion ofPsd1pwithERMESorGem1pwasnot and/or maintained (Friedman et al. 2010). demonstrated.Nevertheless,itwasreportedthat Moreover, mitochondrial division sites pro- a gem1Dpsd2Ddpl1Dyeast mutant grown on a ducedbyDnm1pandDrp1pwerefoundtobe nonfermentablecarbonsourcewasdefectivein physically connected with the ER (Friedman its PC synthesis (Kornmann et al. 2011), most etal.2011). likelybyimpairedPEandPStransport.Asimilar ToshedmorelightontheroleofER-mito- growth phenotypewas reported before for the chondriacontact,geneticandsyntheticgenetic gem1Dsingledeletionstraingrownonsynthetic interactionsanalysismainlywiththeyeast,Sac- glycerol media (Frederick et al. 2008). GEM1 charomyces cerevisiae, were performed (Birner also shows strong synthetic lethality with etal.2003;Kornmannetal.2009;Osmanetal. GEP4,encodingaPGPphosphatase(Kornmann 2009a). Such genetic screens were designed et al. 2011). Gem1p is well conserved in the inawaythatdefectsintwoormoreinteracting eukaryotic kingdom, suggesting that Miro gene products become lethal. Recently, Korn- GTPases are general components of ER-mito- mann et al. (2009) identified components of chondriaconnections.Miro-1,themammalian ER-mitochondriacontactinyeastatthemolec- homologofGem1p,interactswithmitofusin1 ularlevel.Theyshowedthatinwild-typecells,a and 2, the human homologs of yeast Fzo1p. proteincomplextetherstheER/MAMandmi- Mfn-2wassuggestedtotethermitochondriato tochondria.Mutationsinthisproteincomplex ERandbeinginvolvedinmitochondrialmove- causedslowgrowthorwerelethalforthecells, mentalongaxons(deBritoandScorrano2008; butcouldbesuppressedbyanartificialtether- Miskoetal.2010).Mostrecently,however,the ingconstructcalledChiMERA,whichcarrieda roleofERMESandGem1pintheimportofPS GFP molecule. When staining the mitochon- intomitochondriaanditsconversiontoPEwas driawithmito-trackerandexpressingtheChi- challenged(Nguyenetal.2012).Itwasargued MERA, association was visualized confirming that, despite their genetic and physical inter- that ChiMERA acted as a bridge between the action, ERMES and Gem1p function in dis- ER and mitochondria. The authentic complex tinctpathways,andtheabsenceofERMESand wasnamedERMES(ERmitochondriaencoun- Gem1phadonlylittleeffectonPSimportinto tered structures) and contains the OMM pro- mitochondriaandconversiontoPE.Theeffect teins,Mdm10p,Mdm12p,andMdm34p,which on PC formation as described by Kornmann interactwiththeERmembraneanchoredpro- et al. (2009) was regarded as minor. Nguyen teinMmm1p.IntheabsenceofERMES,theCL et al. (2012) argued that changes in the lipid level and the PS/PC conversion rates were de- profileofcellslackingERMESweresideeffects creased,suggestingtheinvolvementofERMES ofdefectsinmitochondrialmorphology. in phospholipids transport between ER and Recently, another mitochondrial complex mitochondria(Fig.3).Moreover,geneticinter- namedMitOS,MICOS,orMINOSwasidenti- actionsbetweenPSD1,GEM1,andtheERMES fied (Harner et al. 2011; Hoppins et al. 2011; complex were shown (Kornmann et al. 2009). vonderMalsburgetal.2011).ThisIMM-asso- TheCa2þMiro(mitochondriarho-like)GTPase ciated complex functions in cristae formation Gem1p is a regulatory component of ERMES andmorphology(Rabletal.2009).Itwaspro- (Kornmann et al. 2011; Stroud et al. 2011), posed (van der Laan et al. 2012) that MINOS maintains mitochondrial morphology and in- forms the central core of a large and complex CitethisarticleasColdSpringHarbPerspectBiol2013;5:a013235 9 Downloaded from http://cshperspectives.cshlp.org/ on February 5, 2023 - Published by Cold Spring Harbor Laboratory Press V.V.FlisandG.Daum MLCL TAZ1 CL CLD1 CL Nonbilayer P-lipids CRD1 Bilayer P-lipids PG PHB1/PHB2 PA CDP-DAG PGP PGS1 GEP4 MDM35 CDS1 PSD1 IMM UPS1/UPS2 YME1 ATP23 PA CDP-DAG PC PE PS UPS2 UPS1 MDM34 MDM10 OMM 2 M1 GEM1 m MD MMM1 10–40 n CDS1 ER/MAM PA CDP-DAG PEM2 PC PEM1PE PS ERMES Figure 3. Componentsinvolvedintranslocationofphospholipidstoandwithinmitochondriaofyeast.The directionsoflipidtranslocationareindicatedbyarrows.WhilePSandPCareimportedintomitochondria,alarge portionPEisexported.UponimportthemajorityofPSisconvertedtoPEbyPsd1p.DecarboxylationofPStoPE inmitochondriaandmethylationofPEtoPCintheERaresufficientforsupplyingcellswithPEandPCeveninthe absenceofexternalethanolamineorcholine.SimilartoPSandPC,PAandCDP-DAGalsohavetobeimported intomitochondriawheretheyarerequiredforCLsynthesis.ThepathwayofCLsynthesisintheIMMstartswith CDP-DAGandiscompletedduringseveralstepsbyamultienzymecascade.AsmallportionofCLisexportedto theOMM.TheERMEScomplexcontaininggeneproductsofMDM10,MDM34,MDM12,andMMM1tethersER andmitochondria.Inmammaliancells,Mfn1/2seemtohaveasimilarfunction.ERMESassociateswiththe GTPaseGEM1,cyclingbetweenERMESboundandfreeform.UPS1/UPS2interactwithMdm35pandregulate themitochondriallevelsofPEandCL.UPS1andUPS2aredegradedbyATP23andYEM1,respectively.Prohibitin ringlikestructuresmadefromtwoproteinsPHB1/2contributetotheformationofPEandCLclustersinthe IMM.Foradetaileddescriptionofbiosyntheticandtranslocationprocessesandcomponentsinvolved,seetext. organizing system named ERMIONE, which et al. 2009a; Tamura et al. 2009, 2012; Potting includes the ERMES complex, the prohibitin et al. 2010). However, direct involvement of ringlike structures, the TOM and TIM com- MINOS in lipid transport and/or assembly plexes, and Mdm31/32 proteins required for into mitochondrial membrane has not been mtDNA maintenance. MINOSwas also found demonstrated. tointeractwiththeproteinVDACandwiththe fusionproteinUgo1(vanderLaanetal.2012). LINKBETWEENCARDIOLIPINFORMATION BothERMESandMINOSaregeneticallylinked ANDINTRAMITOCHONDRIALTRANSPORT to the prohibitin ring complexes of the IMM OFAMINOGLYCEROPHOSPHOLIPIDS that are integrated into the lipid network me- tabolism(Psd1,Ups1/Ups2)(Birneretal.2003; A link between CL and PE assembly in mito- Gohiletal.2005;Kornmannetal.2009;Osman chondrial membranes, PE synthesis by Psd1p, 10 CitethisarticleasColdSpringHarbPerspectBiol2013;5:a013235