The EMBO Journal vol.15 no.15 pp.3813-3822, 1996

The ABC transporter proteins Pati and Pat2 arerequired for import of long-chain fatty acids intoperoxisomes of Saccharomyces cerevisiae

Ewald H.Hettema,Carlo W.T.van Roermund1, Ben Distel,Marlene van den Berg, Cristina Vilela2,Claudina Rodrigues-Pousada2,Ronald J.A.Wanders' andHenk FTabak3Department of Biochemistry, 'Department of Pediatrics, AcademicMedical Centre, Meibergdreef 15, AZ Amsterdam, The Netherlandsand 2Laboratorio de Genetica Molecular. Instituto Gulbenkian deCiencia. Rua da Quinta Grande 6, Oeiras Codex 2781, Portugal

3Corresponding author

Peroxisomes of Saccharomyces cerevisiae are theexclusive site of fatty acid ,-oxidation. We have foundthat fatty acids reach the peroxisomal matrix via twoindependent pathways. The subcellular site of fattyacid activation varies with chain length of the substrateand dictates the pathway of substrate entry into peroxi-somes. Medium-chain fatty acids are activated insideperoxisomes by the acyl-CoA synthetase Faa2p. Onthe other hand, long-chain fatty acids are importedfrom the cytosolic pool of activated long-chain fattyacids via Patlp and Pat2p, peroxisomal membraneproteins belonging to the ATP binding cassette trans-porter superfamily. Patlp and Pat2p are the firstexamples of membrane proteins involved in metabolitetransport across the peroxisomal membrane.Keywords: acyl-CoA synthetase/f-oxidation/fatty acidtransport/peroxisome/yeast

IntroductionPeroxisomes comprise a subcellular compartment ofeukaryotic cells indispensable for mammalian metabolism.The enzymes they contain are involved in several essentialanabolic and catabolic pathways, which are often initiatedor completed elsewhere in the cell (Van den Boschet al., 1992). However, exchange of metabolites betweenperoxisomes and cytosol is hindered by the impermeabilityof the peroxisomal membrane for small molecules (VanRoermund et al., 1995). By analogy with other organelles(Walker and Runswick, 1993), specific transport proteinsfor metabolites have to be present in the peroxisomalmembrane in order to overcome this permeability barrier.In this way, the exchange of metabolites between peroxi-some and cytoplasm can be regulated in response tocellular demands.

In mammals, peroxisomes are involved specifically inthe 3-oxidative chain-shortening of a variety of fatty acidsubstrates including very-long-chain fatty acids (VLCFAs:>C22). After a few cycles of 1-oxidation in peroxisomes,VLCFAs are 1-oxidized to completion in mitochondria.

Prior to n-oxidation, fatty acids need to be activated tothe corresponding acyl-CoA esters. In mammalian cells,long-chain fatty acids (LCFAs) are activated outsideperoxisomes. The site of VLCFA activation is still amatter of debate (Lazo et al., 1990; Lageweg et al., 1991).

Deficiency of one or more peroxisomal functions inman can result in lethal disorders. One of these diseases,X-linked adrenoleukodystrophy (X-ALD), is characterizedby the accumulation of VLCFAs in serum due to adecreased peroxisomal VLCFA 1-oxidation capacity(reviewed by Wanders et al., 1992). The affected gene(X-ALD) was identified by positional cloning and wasshown to encode a peroxisomal ATP binding cassette(ABC) protein (Mosser et al., 1993, 1994). Members ofthis protein superfamily constitute membrane proteinsinvolved in transport of various compounds across mem-branes, ranging from ions to proteins (Higgins, 1992). Byinference, the ALD protein might be involved in VLCFAtransport across the peroxisomal membrane, althoughalternative functions have also been proposed (Valle andGartner, 1993).We have chosen Saccharomyces cerevisiae as a model

system to study transport of fatty acids across the peroxi-somal membrane and their metabolism inside peroxisomesfor a number of reasons. Firstly, in contrast to the situationin mammalian cells, peroxisomes in yeast cells are theonly organelles in which ,-oxidation of fatty acids takesplace (Kunau et al., 1988). This obviates the technicalcomplications due to the existence of iso-enzymes in othercellular compartments. Secondly, during the course of theyeast genome sequencing project, Rodrigues-Pousada andco-workers discovered a gene encoding a member of theABC protein superfamily with a high similarity to theALD protein, here called peroxisomal ABC transporterprotein, Patlp (Bossier et al., 1994). Thirdly, a secondperoxisomal ALD-like protein (Pxalp) has been identifiedrecently (Shani et al., 1995), here named Pat2p. Fourthly,four genes encoding acyl-CoA synthetases (FAAI-FAA4)and their corresponding enzymatic properties have beenreported (Johnson et al., 1994; Knoll et al., 1994). More-over, the contributions of the various proteins involved infatty acid activation and uptake into peroxisomes can becharacterized easily by introduction of targeted deletionsinto the yeast genome. Since S.cerevisiae can use fattyacids as a carbon source, mutants disturbed in fattyacid 1-oxidation can be identified easily by their growthcharacteristics on fatty acid media.We report the existence of two independent pathways

for fatty acid transport across the peroxisomal membrane:one for activated LCFAs which is dependent on theperoxisomal ABC transporters Patlp and Pat2p and onefor medium-chain fatty acids (MCFAs) which is dependenton the peroxisomal acyl-CoA synthetase Faa2p.

X Oxford University Press 3813

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Fig. 1. (A) Patlp is a peroxisomal membrane protein. Biochemicalfractionation of wild-type cells grown on oleate medium.H, homogenate; P, organellar pellet; and S, supernatant. P wasfractionated further by Nycodenz equilibrium density gradientcentrifugation (fractions 1-20). Patlp and thiolase were detected byWestern blot analysis of cell equivalents. SDH, mitochondrial markersuccinate dehydrogenase and 3HAD, peroxisomal matrix marker3-hydroxyacyl-CoA dehydrogenase. Fraction 1 is at the bottom of thegradient. (B) Patlp is associated with peroxisomal membranes. A glassbead hypotonic homogenate (H) was separated into a 100 000 g pellet(P1) and supernatant fraction (SI). P2 is the pellet obtained afterextraction of P1 with 1 M NaCl to allow dissociation of looselymembrane-associated proteins. Triton X- 114 partitioning ofspheroplasts: 1, starting material; 2, detergent-rich phase; 3, detergent-poor phase. Fractions were analysed by Western blot analysis. Thiolasewas used as control for the peroxisomal matrix.

ResultsPatlp is a peroxisomal membrane proteinThe first indication that Patlp has a role in peroxisomefunction came from studies on its subcellular location.Fractionation of a cell homogenate by differential centri-fugation followed by Western blot analysis showed thatPatlp was almost completely recovered in the crudeorganellar pellet fraction (Figure IA). Subsequent analysisof this organellar fraction by equilibrium density centri-fugation showed that Patlp co-fractionated exactly withthe peroxisomal matrix proteins 3-hydroxyacyl-CoA dehy-drogenase (3HAD) and 3-ketoacyl-CoA thiolase (thiolase).The mitochondria and peroxisomes were well separated,as illustrated by the distribution of the marker enzymessuccinate dehydrogenase (SDH) and 3HAD (Figure IA).Furthermore, Patlp equilibrated at a different density ina peroxisome assembly mutant (Apas2J) (not shown).These observations indicate that Patlp is a peroxisomal

protein. The nature of the association of Patlp withperoxisomes was analysed further. Inspection of the pre-dicted amino acid sequence of Patlp suggested the pres-ence of six or seven membrane-spanning regions (Bossieret al., 1994), which is a feature typical of ABC transporterproteins (Higgins, 1992). We therefore tested whetherPatlp is a membrane-associated protein. Oleate-growncells were homogenized by glass beads in hypotonic bufferand frozen in order to disrupt the peroxisomal membraneand to release peroxisomal matrix proteins. The membrane-associated proteins were separated from the solubilizedmatrix proteins by centrifugation at 100 000 g. Asexpected, most of the peroxisomal matrix enzyme thiolasewas released into the supernatant (S 1), although someresidual thiolase was pelleted (Figure iB). Catalaseactivity, comprised of the cytosolic and peroxisomal iso-enzymes, was fully recovered in the supernatant fraction(SI) (not shown). In order to release proteins looselyassociated with membranes, the membrane pellet (P1) wasresuspended in 1 M NaCl and separated into a 100 000 gpellet (P2) and supematant fraction (S2). Patlp was presentin the pellet fraction (P1) (not shown) and was notsolubilized by treatment of the membranes with 1 M NaCl(Figure lB fraction P2). Patlp behaves the same as themitochondrial inner membrane protein Isp42 (not shown).These experiments illustrate that Patlp is strongly associa-ted with peroxisomal membranes. We used an independentapproach to investigate the possible membrane associationof Patlp by studying its behaviour in Triton X-114partitioning experiments. This technique has been provento predict accurately the membrane localization of aprotein (Brusca and Radolf, 1994). A solution of this non-denaturing detergent partitions into a detergent-rich phasecontaining hydrophobic membrane-spanning proteins anda detergent-poor phase containing water-soluble proteins,upon incubation at 37°C. When spheroplasts of oleate-grown cells were incubated with 2% Triton X-114 andincubated at 37°C, Patlp partitioned to the detergent-richphase in contrast to thiolase, which was present in thedetergent-poor aqueous phase (Figure 1B). Isp42, a mito-chondrial inner membrane protein behaved identicallyto Patlp (experiment not shown). The Triton X-114partitioning experiments support the membrane localiz-ation of Patlp. The subcellular location of Patlp wasstudied further by ultrastructural analysis. Unfortunately,the polyclonal antiserum raised against Patlp failed torecognize Patlp in immunogold electron microscopyexperiments. In order to solve this problem, we taggedPatlp at its N-terminus with a haemagglutinin (NH)epitope of influenza virus. The NH-modified PatIp versionwas able to rescue the mutant phenotype (not shown).Peroxisomes were identified in cross-sections of cells bysmall gold particles coupled to anti-thiolase (Figure 2).NH-Patlp was detected by large gold particles coupledto the NH antiserum. We found that the large gold particlesalso labelled the peroxisomes. Most of the label wasobserved within the vicinity of the peroxisomal membrane.This is seen more clearly in single-labelled cells in areaswith free lying peroxisomes (Figure 2B-D). Recently, asecond S.cerevisiae peroxisomal ABC transporter withhigh similarity to human ALDp was identified, Pxalp(Shani et al., 1995), here called Pat2p.

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Fig. 3. Growth characteristics of wild-type and mutant cells on oleate-containing agar plates. A 10-fold dilution series of a cell suspensionwas applied directly on the surface of oleate plates and incubated at28°C for 2 weeks. Aindh3 cells lack the peroxisomal malatedehydrogenase and grow slowly on oleate (van Roermund et al.,1995).

Fig. 2. Immunogold electron micrographs showing association ofNH-Patlp with peroxisomal membranes. Apatl cells transformed withpNH-PATl were grown in oleate medium. Thiolase (5 nm gold) andNH-Patlp (10 nm gold). (A) Cells labelled with 5 and 10 nm goldparticles. (B). (C) and (D) Cells marked with 10 nm gold particles.The scale bar indicates 0.2 tM.

Patip and Pat2p are required for growth on oleatemediumDeletion of the PAT] or PAT2 gene did not affect growthon media containing either glucose, acetate, glycerol or

the fatty acids laureate (C12:0) or myristate (C14:0) as

carbon source. However, growth on solid media containingeither the LCFAs oleate (C 18:1) or palmitate (C 16:0)was impaired (Figure 3). Despite this growth deficiency,peroxisomal protein import and organelle biogenesis were

not affected (not shown). Since peroxisome assemblymutants are not able to grow on MCFAs (our unpublishedobservations), the observation that deletion of the PAT]or PAT2 gene does not affect growth on MCFAs supportsthe idea that Pat p and Pat2p are not involved in peroxi-some biogenesis. Rather, the specific growth defect onoleate suggests that Pat p and Pat2p are required for aspecific aspect of fatty acid metabolism.

Patlp and Pat2p are involved in fl-oxidation oflong-chain fatty acidsWe have investigated the enzymatic [-oxidation activityof wild-type and Apati and Apat2 cells using radiolabelledfatty acids of varying chain length. The [-oxidation ofMCFAs (octanoate, decanoate and laureate) was normalin intact Apat] and Apat2 mutant cells, while the oxidationof the LCFAs palmitate and oleate was reduced whencompared with wild-type cells (Figure 4). Double mutantslacking both the PAT] gene and the PAT2 gene exhibitedthe same phenotype as the single mutants. Laureate andoleate 3-oxidation activity in detergent lysates preparedfrom wild-type and Apat] cells was indistinguishable(Figure 4). These results illustrate that the activity of the[-oxidation enzymes themselves was unaffected. The

availability of co-factors for [3-oxidation or the ability totransport the [-oxidation products out of the peroxisomewas also unaffected in Apat] and Apat2 cells, as indicatedby the normal growth on and oxidation of MCFAs inintact cells. Rather, these results imply that a transport stepspecific for LCFA [-oxidation is impaired in Apati cells.

Faa2p is a peroxisomal proteinIn S.cerevisiae four genes have been described that encodeacyl-CoA synthetases. The enzymatic characteristics ofthese proteins purified after expression in Escherichia colihave been reported (Johnson et al., 1994; Knoll et al.,1994). Several proteins involved in 3-oxidation (includingPatlp) are induced during growth on oleate. We previouslyhave identified a promoter element important for transcrip-tional induction of genes by oleate or a derivative thereof(Einerhand et al., 1992). Interestingly, the FAA2 genecontains such an element. Northern blot analysis confirmedthat the FAA2 gene is indeed induced during growth onoleate (not shown).

In order to investigate the subcellular location of Faa2p,we tagged the protein at its N-terminus with the NHepitope and expressed NH-Faa2p from an oleate-induciblepromoter. This modified version of Faa2p could function-ally complement a strain in which the FAA2 gene wasdeleted (see below). Fractionation of a homogenate pre-pared from NH-Faa2p-expressing cells grown on oleateshowed that most NH-Faa2p was present in a crudeorganellar pellet (Figure 5A). Subsequent fractionationof the organellar pellet by equilibrium density gradientcentrifugation showed that NH-Faa2p co-fractionated withthe peroxisomal markers 3HAD and thiolase and was wellseparated from mitochondria (Figure 5A).

Cross-sections of NH-Faa2p-expressing cells were ana-lysed by immunogold electron microscopy. Small goldparticles were used to identify thiolase, and large goldparticles were used to visualize NH-Faa2p. Large goldparticles labelled the peroxisomal membrane (Figure 5B).The association of Faa2p with the peroxisomal mem-

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Fig. 4. Two genetically separable pathways for fatty acid ,B-oxidation. Cells grown on oleate medium were incubated with 1-14C-labelled fatty acidsand 5-oxidation rates were measured (see Materials and methods). The ,B-oxidation rates in wild-type cells were taken as reference (100%) and areexpressed as the sum of [14C]CO2 and water-soluble ,B-oxidation products produced. In lysates of Apatl cells, the 5-oxidation rate was normalized tothe rate observed in lysates prepared from wild-type cells.

brane was unexpected, since the amino acid sequence didnot predict the existence of typical hydrophobic regionsin Faa2p. Indeed, the association of Faa2p with theperoxisomal membrane is very weak as the associationwas lost upon hypotonic lysis of peroxisomes (not shown).

With regard to the mechanism of fatty acid transportacross the peroxisomal membrane, it was important toestablish whether Faa2p is present on the cytoplasmicor the matrix side of the membrane. The followingexperimental data show that Faa2p is located insideperoxisomes.The localization of Faa2p inside peroxisomes was

demonstrated with protease protection experiments. Inhomogenates of Afaa2 cells expressing NH-Faa2p, theNH epitope remained detectable at increasing trypsinconcentrations. This resistance to degradation was lostwhen membranes were solubilized with 0.3% Triton X-100(Figure 6). Additional experiments revealed that Faa2p isimported via the evolutionarily conserved peroxisometargeting signal type 1 (PTS1)-dependent protein importpathway. Indeed, in cells where the import of PTS-1proteins is blocked (ApasJO cells), NH-Faa2p was mis-located to the cytoplasm. The C-terminal tripeptide ofFaa2p, EKL-COOH, shows a two-out-of-three fit with thePTS-1 consensus sequence. As expected (Gould et al.,1989), the addition of one extra amino acid (lysine; K)to the extreme C-terminus of NH-Faa2p (NH-Faa2p*)abolished import, as revealed by immunogold electronmicroscopy and subcellular fractionation (not shown) andby the acquired sensitivity to trypsin (Figure 6). Inhomogenates of Afaa2 cells expressing cytosolic NH-Faa2p*, the NH epitope was degraded at low trypsinconcentrations in the absence of Triton X-100. The trypsinsensitivity of the mutant version of Faa2p lacking itsPTS-1 is not likely to be due to an aberrant proteinconformation since NH-FAA2p* is enzymatically active(see below). Taken together, our experiments show thatFaa2p is associated with the matrix side of the peroxisomalmembrane.

Faa2p is essential for /3-oxidation of medium-chainfatty acidsWild-type and Afaa2 cells were grown on various fattyacids as carbon source. Diminished growth was observed

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only on laureate (MCFA) and not on plates containingLCFAs such as oleate. Peroxisome assembly was notdisturbed as revealed by electron microscopy (not shown).

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Fig. 6. Faa.2pt is loclated oni thc matr'ix side (f the pet oXiSoImal membt1hl'.lI1CeIS iIItllsti atcd by its resistance to protease deLradation. A\t(o2 cellstransformed with eithierpIpH-FAXA- or1 pNH-FA,-\'\'-were grown oni oIcite miiediulllm. Homogenatcs were inICUhated with an incr,ealsine coinceitraL;tiolontrvpSin in the pIresciecc or1 ahsence of0.3)(' Tr-itoIn X- 100. HaemCIIaeLlutillill-taLeLed FLaap versions aind thiolase were detected hy W'esterin blot analysis.The arro indicates the foil lnth of theseproteios. The'amounSLt oftrIIsin isindicatedI in pg per inCubation.

Ly sates of A/iw2 cells do Inot exhibit any lauicoN1-CoA sylnthetase activity (Figure 7B). wuhereas oleoNl-CoAsvnthetase activity was virtuallV unaffected (inot shown).This illustrates that Faa2p is the mtain if not exclusiVemediumi-clhaini acvl-CoA svnthetase that is expr-essed underour experimllenital coniditions. Indeed. subcellular fractioln-ationi studies reVealed that the laureovi-CoA synthetaseactivity was f-ounid almiiost exclusiVelyN in the crude orcan-ellar pellet (Figur-e 8A) anid co-fi-actionated with peroxi-somn.al im.ar-keri proteins upon equilibrlium density gradientcentrifugLtion (Figul-e 8B).

The f-oxidation capacity of wild-tTpe a.nd AAi(2 cellsthat had been arown oni oleate mediumii was tested forfatty acids of varincm chain length A deficiency becameapparent with fatty acids of shorter- chain length. LCFAsw!ere oxidized at rates that equialled those of wild-typecells (FigrUe 4). These results a.re conisistenit with a defectin the activation of NICFAs destined for f-oxidationi inAdfim2 mutaint cells. NH-Faa2p` and NH-Faa2p expressedin Aftifl2 cells resulted in a somewhat elevated level oflaureoyl-CoA snrthetase activity conmpar-ed with wild-typecells (Figule 7B). 3-Oxidatioin of laureate was restored inAfta(i2 cells expressing NH-Faa2p. The mislocalized butactive NH-Faa'p- could not fully restore the f3-oxidationcapacity of Af/m2 cells. This illustr-ates that the enzymrnaticactivity of Faa2p is required inside peroxisomes forefficient MCFA f3-oxidation (Figui-e 7A).

Location of long-chain fatty acid activationDifferenetial centrifugation of a homnogenate showed that-60U(/c of the oleoyl-CoA synthetase activity was pelletableat 20 000 g (Figure 8A). Whenl the pelletable activitywas analysed furthei by equilibrium density gradientcentrifucation. a bimodal distributioni was founid (Fi LIueSC). However, the fraction of enzyme activity co-micratin c

with peroxisoimies was due to the contribution of Faa2p.Indeed. when the experimiient was repeated with a homo-ceniate denived froIml AfAi2 cells. oleoyl-CoA synthetaseactivity could not be demonstr-ated any more in theperoxisoimial f andctioilaid peak of activitvwas observed at a density somewhat hicher thani thatof mitochoindia (FiLI-e 8D). \We have not studied thesubcellular- location of the oleoyl-CoA synthetase activityin further detail. Fronm these results. it is clear however, thatFaa I p-. Faa3p- or Faa4p-dependent oleoyl-CoA synthetase

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activity is nlot associated with peroxisomes. Deletion ofPATI did nlot influence the subcellular distributioll ofMCFA- or LCFA-CoA svn-thetase activity (not shownl).

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Apat/Afaa2 cells are deficient in fl-oxidation ofMCFAs and LCFAsAs mentioned above, Afaa2 cells lack peroxisomal MCFA-and LCFA-CoA synthetase activity. Nevertheless, Afaa2cells are able to oxidize LCFAs at a normal rate, implyingthat activated LCFAs can be imported efficiently from thecytosol. We hypothesized that Patlp and Pat2p are likelyto function in this transport pathway of activated fattyacids since they are involved in the delivery of LCFAsubstrates for n-oxidation (see above). To test thispossibility, double mutants (Apatl/Afaa2 and Apat2/Afaa2)were constructed and analysed. The double mutants werenot able to grow on medium containing MCFAs or LCFAsas sole carbon source (Figure 3), and 1-oxidation of fattyacids by intact cells was decreased to only 1-4% comparedwith wild-type cells (Figure 4). These results show thatFaa2p functions in a parallel pathway to Patlp and Pat2p,and that no other major pathways exist for the entry ofthese substrates into peroxisomes.

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Fig. 8. Subcellular location of laureoyl-CoA and oleoyl-CoAsynthetase activities. (A) Wild-type and Afaa2 cells grown on oleatemedium were homogenized (H) and fractionated into an organellarpellet (P) and supernatant (S) fraction. The ,B-oxidation capacity ofthese fractions was measured. The organellar pellet fractions werefractionated further by Nycodenz equilibrium density gradientcentrifugation. (B and C) Gradient prepared from wild-type cells.(D) Gradient prepared from AJ.ia2 cells. The enzymatic activities ofC12:0-CoA synthetase, C18:1-CoA synthetase, mitochondrial markersuccinate dehydrogenase (SDH) and peroxisomal matrix marker3-hydroxyacyl-CoA dehydrogenase (3HAD) were determined asdescribed in Materials and methods. The acyl-CoA synthetase activityin each gradient fraction is expressed as a percentage of the sum ofthe acyl-CoA synthetase activity measured throughout the gradient.'M' (mitochondria) and 'P' (peroxisomes) indicate fractions with thehighest SDH and 3HAD activity, respectively.

DiscussionPeroxisomes are the exclusive site of fatty acid ,B-oxidationin S.cerevisiae. Since the peroxisomal membrane isimpermeable to small metabolites (Van Roermund et al.,1995), the question arises as to how fatty acids enterperoxisomes. Here we report that fatty acids reach theperoxisomal matrix via two different pathways, dependingon their chain length. Peroxisome research is hamperedby the technical difficulties of isolating intact organelles.We therefore used 3-oxidation of radiolabelled fatty acidsin intact cells compared with detergent lysates of cells as

a measure of fatty acid transport across the peroxisomalmembrane. We have found that the peroxisomal membraneproteins Patlp and Pat2p are involved in a transport stepspecific for LCFA n-oxidation. Disruption of either PAT]or PAT2 or both genes leads to latency of LCFAf-oxidation. The concept of latency was developed by DeDuve to describe a situation in which an enzyme, whichalthough in principle is catalytically active, shows no

reactivity due to the presence of a substrate-impermeablemembrane functioning as a barrier between substrate andenzyme. We further showed that MCFA oxidation dependsupon the presence of Faa2p, a peroxisomal acyl-CoAsynthetase that activates fatty acids inside peroxisomes.A technical complication in the study of these fatty acidtransport pathways is that they overlap to a certain extent.For instance, the oleoyl-CoA synthetase activity thatcan be measured in purified peroxisomes is due to thecontribution of Faa2p (Figure 8). This is in line with theenzymatic characterization carried out by Knoll et al.(1994). They demonstrated that Faa2p shows a preferencefor laureate, but also displays some reactivity towardsoleate. The contribution of Faa2p in LCFA ,B-oxidation isillustrated by the fact that the residual oleate f-oxidationcapacity in Apatl or Apat2 cells is completely abolished

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Fig. 9. Model for fatty acid entry into peroxisomes. The pathway by which ,B-oxidation substrates enter peroxisomes is dictated by their site ofactivation. Transport of free fatty acids across the peroxisomal membrane is either mediated by spontaneous flip-flop of uncharged fatty acids (A) oris protein facilitated (B). Upon entry into the peroxisomes, the substrates are activated to acyl-CoA esters via the peroxisomal membrane-associatedacyl-CoA synthetase, Faa2p. Activated fatty acids present in the cytoplasm can be used for various processes, including peroxisomal 3-oxidation.Activated fatty acids are translocated across the peroxisomal membrane depending on the peroxisomal ABC transporter proteins Patlp and Pat2p(C). This active transport pathway allows peroxisomes to compete with other cellular processes for cytoplasmic-located substrates.

upon the additional disruption of the FAA2 gene. Althoughour proposal that Patlp and Pat2p are involved in fattyacid transport is based on indirect evidence (the occurrenceof latency in mutants), it is the most simple model toexplain our observations (Figure 9).

Uptake of medium-chain fatty acidsWe have shown that Faa2p is the peroxisomal acyl-CoAsynthetase which is associated with the inside of themembrane. In cells lacking Faa2p, 13-oxidation of MCFAs(octanoate, decanoate and laureate) and MCFA-CoAsynthetase activity are completely abolished. These resultsidentify Faa2p as a link in the chain of events leading touptake of MCFAs into peroxisomes. They leave thequestion open as to how MCFAs cross the peroxisomalmembrane itself: by a protein-mediated process or bypassive flip-flopping from the cytoplasmic leaflet of theperoxisomal membrane to the inner leaflet. Model studieswith artificial membranes have shown that free fatty acidscan flip-flop very efficiently from one leaflet of themembrane to the other (Kamp and Hamilton, 1993), butstudies in E.coli and in mammalian cells (COS and 3T3cells) have identified plasma membrane proteins involvedin LCFA uptake (Black, 1991; Schaffer and Lodish, 1994).Therefore, it is equally conceivable that the import of freeMCFAs into yeast peroxisomes is facilitated by proteinsthat remain to be identified (Figure 9).

Peroxisomal ABC transporters are involved intransport of activated LCFAsDeletion of the PAT] or PAT2 gene leads to a partialdeficiency of LCFA 13-oxidation, resulting in diminished

growth and 1-oxidation compared with wild-type cells.Residual growth and 3-oxidation can be explained by thepartial overlap between the two uptake systems and theaforementioned residual activity of Faa2p towards oleate.

Peroxisomes prepared from Afaa2 cells were devoidof LCFA-CoA synthetase activity. Remarkably, the,B-oxidation activity for oleate was normal. Since fattyacids have to be activated to acyl-CoA esters prior to3-oxidation, we suggest that LCFAs are activated in thecytoplasm prior to import into peroxisomes. This proposalis supported by evidence that activated fatty acids canindeed cross the peroxisomal membrane. Partial comple-mentation of Afaa2 cells by expression of the cytosolicbut active version of Faa2p indicates that MCFAs activatedin the cytosol can be used for 1-oxidation to a certainextent (Figure 7). ,B-Oxidation of MCFAs in these cells isdependent upon a functional Patlp, since Apatl/Afaa2cells transformed with pNH-FAA2* are not able to oxidizeMCFAs (compare Figure 7 lane 3 with lane 5).

Double mutants lacking the FAA2 gene and either thePAT] or the PAT2 gene were not able to grow on oroxidize MCFAs or LCFAs at all. Therefore, substrates for,B-oxidation can cross the peroxisomal membrane by twoparallel pathways, one for free fatty acids preferably ofmedium chain length, which is dependent upon Faa2p,and the other for activated fatty acids preferably of longchain length, which is dependent upon Pat proteins. SinceFaa2p is the only acyl-CoA synthetase with a clearpreference for MCFAs and is located inside peroxisomes(Figure 7), we suggest that MCFAs occur as free fattyacids in the cytoplasm and that the MCFA transportprocess is specific for free MCFAs. This is different for

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E.H.Hettema et al.

LCFAs. The cytosolic pool of LCFA-CoA esters is fed bytwo sources: by de novo synthesis of fatty acids and byuptake from the environment. In Ecoli and mammaliancells, uptake of LCFAs is coupled to esterification withCoA (Black et al., 1992; Schaffer and Lodish, 1994).Thus, the cytoplasmic pool of LCFAs may occur mainlyin the activated form. This might explain why peroxisomesrely on an active transport system of ABC transporters torecruit substrates from the cytosolic pool of activated fattyacids in order to compete with other cellular processes(Figure 9).

Co-operation of Patlp and Pat2pABC transporters exist in two different forms (Higgins,1992): as single proteins composed of two similar halvesor as a complex of two homologous half-transporters.PatIp and Pat2p each represent a half-transporter. Deletionof the PAT] gene results in the same growth characteristicsand 3-oxidation deficiency as deletion of the PAT2 gene.When the two mutations are combined within one strain,no increase in the severity of the phenotype was observed.When either the PAT] or PAT2 gene was deleted in Afaa2cells, [3-oxidation and growth on any fatty acid wascompletely abolished, again showing that deletion of thePAT] gene has the same effect as deletion of the PAT2gene. These results illustrate that Pat 1 p and Pat2p functionin the same 3-oxidation pathway. Although our geneticdata do not rule out the possibility that Patlp and Pat2pfunction in series within the same pathway, it is verylikely that the yeast peroxisomal ABC transporter is aheterodimer of the two half-transporters, conforming withthe general architecture of ABC transporters.X-ALD is a neurodegenerative disorder caused by

an abnormality in peroxisomal fatty acid metabolism.Positional cloning pinpointed the defect to a peroxisomalABC transporter (Mosser et al., 1993, 1994). One of theadvantages of the yeast genome sequencing programpointed out by Dujon (1994) is the discovery of geneswith homology to human genes involved in human patho-logies, as 'such yeast genes may offer a powerful experi-mental system to identify their function'. Considering thebiochemical defect of X-ALD patients, an inability tooxidize VLCFAs (Wanders et al., 1992) which stronglyresembles the phenotype we observed in S.cerevisiae upondisrupting the PAT genes, we suggest that the humanX-ALD ABC transporter protein, which is more similarto Patlp and Pat2p than to any other yeast ABC transporter,might be involved in the uptake of activated VLCFAs intohuman peroxisomes.

Materials and methodsYeast strains and culture conditionsThe yeast strain used in this study was S.cerevisiae BJ1991 (Mata, leiu2,trpl, itra3-251, prbl-1122, pep4-3, gal2). The paslO deletion mutantwas generated in our laboratory (Van der Leij et al., 1993). Yeasttransformants were selected and grown on minimal medium containing0.67% yeast nitrogen base without amino acids (YNB-WO) (DIFCO),2% glucose and amino acids (20-30,ug/ml) as needed. The liquid mediaused for growing cells for nucleic acid isolation, subcellular fractionation,:-oxidation assays, immunogold electron microscopy and enzymaticassays contained 0.5% potassium phosphate buffer, pH 6.0, 0.3% yeastextract, 0.5% peptone, and either 2% glucose, 3% glycerol or 0.1%oleate-2% Tween 40. Before shifting to these media, the cells weregrown on 0.3% glucose medium for at least 24 h. Oleate plates and

laureate plates contained 0.67% yeast nitrogen base without amino acids(YNB-WO) (DIFCO), 0.1% yeast extract (DIFCO), 2% agar, aminoacids as needed and either 0.1% oleate-0.25% Tween 80 or 25 pMlaureate, respectively. For growth on fatty acid plates, cells were grownovernight in 0.3% glucose medium, washed with water and spotteddirectly on the surface of the plates. Plates were incubated at 28°C for2 weeks and photographed.

Cloning proceduresPatlp is encoded by the open reading frame YKL741 (Bossier et al.,1994) later designated as YKL188c (Dujon, 1994). A genomic HioidIIIfragment containing most of the PATI gene was used to generate a genereplacement construct. The major part of the open reading frame(containing 2246 bp) was deleted, by replacing the Nhel-BglII fragmentwith either a URA3 or a LEU2 expression cassette (pEW44 and pEW49,respectively).The 5' part of the FAA2 gene (-390 to 1600) was amplified from

BJ 1991 genomic DNA using the 5' FAA2 primer (5'-AATCTAGAAGT-CCCGGTGTC-3') and the 3' FAA2 primer (5'-AAATCCTTGTCGGCA-TGG-3') oligonucleotides. PCR with Vent DNA polymerase wasperformed as instructed by the manufacturer using an annealing tempera-ture of 51°C. The PCR product was digested with Xbal and PstI andwas cloned in the multiple cloning site of pBluescript pSK+. Part of theopen reading frame was deleted by replacing the Ba)nHI fragment for aLEU2 or URA3 expression cassette (pEW62 and pEW75, respectively).

The PAT2 gene was amplified as described for the FAA2 gene. The5' PAT2 primer (5'-AAGTTTCCATGGAAGAGAAGG-3') and 3' PAT2primer (5'-CCCTGTGTAAGACGGAAAGC-3') were annealed at 55°C.The PCR product was digested with Sall and NsiI and cloned into SalIl-PstI-digested pUCl9 lacking the HinidlIl site. The internal HindIII-BglIIfragment was replaced by the LEU2 gene (pEW78). In order to generategene replacement mutants, plasmids were linearized and transformed tohaploid BJ1991 cells. All gene replacements were verified by Southernblot analysis and confirmed that Patlp, Pat2p and Faa2p are encoded bysingle-copy genes.

For all expression constructs described, the single-copy or multicopycatalase A (CTAI) promotor expression plasmids based upon Ycplac33and Yeplacl81 were used (Gietz and Sugino, 1988; Elgersma et al.,1993). A 15 amino acid long influenza virus haemagglutinin epitope-encoding adaptor was ligated in the SacI-BamHI site of both plasmids.A more detailed description of the NH tag used in this study will bereported elsewhere (Y.Elgersma et al., manuscript in preparation).

For in-frame fusion of the FAA2 gene with the NH epitope, an XbaIrestriction site was introduced in front of the open reading frame. PCRwas performed with Vent DNA polymerase at an annealing temperature of60°C using XbaI FAA2 primer (5'-AATCTAGAAATATGGCCGCTCCA-3') and 3' FAA2 primer (5'-GTATGGATGTGCATAGGG-3'). The XbaI-PstI PCR fragment was introduced in a single-copy CTAI expressionplasmid containing the NH tag, resulting in an open reading frameencoding NH-tagged FAA2 (pEW67). The expression construct encodingNH-FAA2* was derived from pEW67 by deletion of the HinidIllfragment (pEW70). This leaves the open reading frame completely intactbut adds a single codon for lysine, directly followed by a stop codonpresent in the CTA1 terminator.

In order to tag Patlp with the NH epitope, a BaniHI site was introducedat the 5' end of the open reading frame by PCR with 5' PAT1 primer(5'-CGGGATCCATAATGATCTCAACAGCT-3') and 3' PAT1 primer(5'-GCCCTCTTAGCAGTGC-3'). PCR was performed with an annealingtemperature of 48°C. The PCR fragment was digested with BamnHI andEcoRI and subcloned into the BamHI-EcoRI site of pBluescript pSK+(pEW42). To obtain a full-length PAT] open reading frame, a NheI-KpnI genomic fragment was cloned into pEW42 digested with NheI andKpnI (pEW54). The DNA sequence from the 5' BamHI site to the Nhelsite was analysed to rule out PCR-introduced mutations. Subsequently,the BamHI fragment containing the complete PATI open reading framewas introduced into a multicopy catalase expression plasmid containingthe NH epitope (pNH-PATl).

All single-copy expression plasmids described contained the URA3gene as auxotrophic marker. If needed, the URA3 marker was replacedby the TRPI gene-containing AatII-NarI fragment from Ycplac22or the LEU2 gene-containing fragment from Ycplac 111 (Gietz andSugino. 1988).

Production of Patip polyclonal antiserumPart of Patlp was expressed in Ecoli by cloning the PstI-HinidIIIfragment of the PATI gene (encoding amino acids 625-839) in plasmidpQE-10 (Qiagen). The six histidine residues fused to part of Patlp

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allowed rapid purification by nickel-chelating chromatography underdenaturing conditions as instructed by the manufacturer. The proteinwas purified further by SDS-PAGE, visualized with 0.25 M KCl/l mMdithiothreitol (DTT) and subsequently excised and eluted from the gelin elution buffer (50 mM Tris-HCl pH 8.0, 0.1% SDS, 0.1 mM EDTA,5 mM DTT, 0.15 M NaCI). This protein was used to immunize rabbits.

Protease protection, SDS-PAGE and Western blottingTransformants grown overnight on oleate medium were converted tospheroplasts with Zymolyase lOOT (1 mg/g cells). The spheroplasts werewashed twice in 1.2 M sorbitol, 5 mM 2[N-morpholino]ethane sulfonicacid (MES) pH 5.5, 1 mM EDTA and 1 mM KCI. The washedspheroplasts were lysed by osmotic shock in 0.65 M sorbitol, 5 mMMES pH 5.5, 1 mM EDTA and 1 mM KCI. Intact cells and nuclei wereremoved from the homogenate by two centrifugation steps at 600 gduring 10 min. For protease protection experiments, 100 lt of homogenatewith a protein concentration of 1 mg/ml was added to 100 ,ul of 0.65 Msorbitol, 5 mM MES pH 5.5, 1 mM EDTA and 1 mM KCI containingeither 0, 10, 30 or 100 tg trypsin either in the presence or absence of0.3% Triton X-100. The samples were incubated at room temperaturefor 15 min. The reaction was stopped by the addition of trichloroaceticacid (TCA) (10%). SDS-PAGE was performed according to Sambrooket al. (1989). Proteins were transferred to nitrocellulose using a Bio-Rad Transblot apparatus according to the manufacturer's instructions.Western blots were incubated with rabbit polyclonal antisera raisedagainst 3-ketoacyl-CoA thiolase, Patlp and the haemagglutinin epitope.Antibody complexes were either detected by incubation with goat anti-rabbit Ig-conjugated alkaline phosphatase or peroxidase.

/3-Oxidation measurements,-Oxidation assays in intact cells were done essentially as described(Van Roermund et al., 1995) with the following modifications. Incuba-tions were performed at 28°C and substrates were solubilized inax-cyclodextrin. The substrates used included [1-_4C]octanoate, [1-14C]_decanoate, [1-_4C]laureate, [1-_4C]myristate, [1-_4C]palmitate and[I - 14C]oleate.

3-Oxidation measurements in cell-free extracts were followed byquantification of 14C-labelled 3-oxidation products.

Miscellaneous3-Hydroxyacyl-CoA dehydrogenase activity was measured on a Cobas-Fara centrifugal analyser by following the 3-keto-octanoyl-CoA-depend-ent rate of NADH consumption at 340 nm (Wanders et al., 1990).Succinate dehydrogenase activity was measured according to Munujoset al. (1993). Acyl-CoA synthetase activity was measured essentially asdescribed (Knoll et al., 1994). Protein concentrations were determinedby the bicinchoninic acid method (Smith et al., 1985). Triton X-114partitioning experiments of oleate-grown spheroplasts were performedaccording to Brusca and Radolf (1994).

Subcellular fractionations were performed as described (Van der Leijet al., 1992). Organellar pellets (20 000 g pellets) were used forcontinuous Nycodenz gradients as described (Van Roermund et al.,1995). DNA manipulations were performed as described (Sambrooket al., 1989). Immunogold electron microscopy was performed asdescribed by Distel et al. (1992).

AcknowledgementsWe are grateful to P.Borst, A.Verkley, A.M.Motley, A.ten Asbroek,Y.Elgersma, I.Braakman and L.IJlst for stimulating discussions andsupport. We thank Dr P.van der Sluijs for the NH epitope and NHantiserum and B.Metzig for constructing the NH-PAT1 expressionplasmid. We thank M.Meyer for the Isp42 antiserum. E.H.H. wassupported by The Netherlands Foundation for Scientific Research(Medical Sciences).

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Received oln Janucary, 12, 1996; revised oan April 23, 1996

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