Novel Proteins, Putative Membrane Transporters, and an ... · Novel Proteins, Putative Membrane Transporters, and an Integrated Metabolic Network Are Revealed by Quantitative Proteomic

Novel Proteins, Putative Membrane Transporters, and anIntegrated Metabolic Network Are Revealed byQuantitative Proteomic Analysis of Arabidopsis CellCulture Peroxisomes1[W][OA]

Holger Eubel2, Etienne H. Meyer2, Nicolas L. Taylor2, John D. Bussell2, Nicholas O’Toole,Joshua L. Heazlewood, Ian Castleden, Ian D. Small, Steven M. Smith, and A. Harvey Millar*

Australian Research Council Centre of Excellence in Plant Energy Biology, M316 (H.E., E.H.M., N.L.T., J.D.B.,J.L.H., I.D.S., S.M.S., A.H.M.), and Centre of Excellence for Computational Systems Biology (N.O., I.C., I.D.S.),University of Western Australia, Crawley, Western Australia 6009, Australia

Peroxisomes play key roles in energy metabolism, cell signaling, and plant development. A better understanding of theseimportant functions will be achieved with a more complete definition of the peroxisome proteome. The isolation of peroxisomesand their separation from mitochondria and other major membrane systems have been significant challenges in the Arabidopsis(Arabidopsis thaliana) model system. In this study, we present new data on the Arabidopsis peroxisome proteome obtained usingtwo new technical advances that have not previously been applied to studies of plant peroxisomes. First, we followed densitygradient centrifugation with free-flow electrophoresis to improve the separation of peroxisomes from mitochondria. Second, weused quantitative proteomics to identify proteins enriched in the peroxisome fractions relative to mitochondrial fractions.We provide evidence for peroxisomal localization of 89 proteins, 36 of which have not previously been identified in other analysesof Arabidopsis peroxisomes. Chimeric green fluorescent protein constructs of 35 proteins have been used to confirm their lo-calization in peroxisomes or to identify endoplasmic reticulum contaminants. The distribution of many of these peroxisomalproteins between soluble, membrane-associated, and integral membrane locations has also been determined. This core per-oxisomal proteome from nonphotosynthetic cultured cells contains a proportion of proteins that cannot be predicted to beperoxisomal due to the lack of recognizable peroxisomal targeting sequence 1 (PTS1) or PTS2 signals. Proteins identified are likelyto be components in peroxisome biogenesis, b-oxidation for fatty acid degradation and hormone biosynthesis, photorespiration,and metabolite transport. A considerable number of the proteins found in peroxisomes have no known function, and potentialroles of these proteins in peroxisomal metabolism are discussed. This is aided by a metabolic network analysis that reveals a tightintegration of functions and highlights specific metabolite nodes that most probably represent entry and exit metabolites thatcould require transport across the peroxisomal membrane.

Within the plant cell, energy metabolism is mainlydistributed among three distinct organelles: plastids,mitochondria, and peroxisomes. Although the pro-teomes of both plastids and mitochondria have beeninvestigated extensively, comparatively little system-

atic analysis of the protein content of plant peroxisomeshas been undertaken. The main obstacle for proteomicsof plant peroxisomes is the availability of purifiedorganelles from model plants that are also amenableto mass spectrometry (MS)-based identification bymatching to protein sequence data. Whereas the prep-aration of peroxisomes in sufficient amounts and purityfrom spinach (Spinacia oleracea), cucumber (Cucumissativus), pea (Pisum sativum), and soybean (Glycine max)for proteomic purposes is possible (Schwitzguebel andSiegenthaler, 1984; Corpas et al., 1994; Lopez-Huertaset al., 1999; Arai et al., 2008), the purification of perox-isomes from Arabidopsis (Arabidopsis thaliana) hasproved to be extremely difficult due to the low yieldof intact organelles and contamination with other cellorganelles. This complicates data analysis and com-promises confidence in the subcellular localization ofthe identified proteins. So far, three studies in Arabi-dopsis have been reported, using greening (Fukao et al.,2002) or etiolated (Fukao et al., 2003) cotyledons ormature plant leaves (Reumann et al., 2007), each usingdifferent purification methods. In these studies, 42putatively peroxisomal proteins were identified from

1 This work was supported by grants from the Australian Re-search Council (ARC) through the Centres of Excellence Program(grant no. CE0561495), by the Western Australian State Governmentvia its Centres of Excellence program, and by a University of WesternAustralia Research Grant to J.D.B. H.E., N.L.T., and J.L.H. aresupported as ARC Australian Postdoctoral Fellows, A.H.M. as anARC Australian Professorial Fellow, and S.M.S. as an ARC Feder-ation Fellow.

2 These authors contributed equally to the article.* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:A. Harvey Millar ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

scription.www.plantphysiol.org/cgi/doi/10.1104/pp.108.129999

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cotyledons and 78 from leaves, but the overlap betweenthe sets from both tissues was only 11 proteins.

The protein composition of peroxisomes from differ-ent tissues is likely to vary significantly as the functionof these organelles changes. Therefore, a full under-standing of peroxisomal function requires experimen-tal analysis of these organelles from a variety of plantorgans during different developmental stages. Peroxi-somes in seedlings of oilseed plants such as Arabidop-sis are mainly involved in the breakdown of fatty acidsderived from storage triacylglycerols via b-oxidationduring germination prior to the initiation of photosyn-thesis (Graham and Eastmond, 2002). Most of theacetyl-CoA generated by fatty acid b-oxidation is fedinto the glyoxylate cycle to produce succinate, whichmay then be exported out of the organelles or used as aprecursor for other metabolites and processes such asgluconeogenesis (Eastmond and Graham, 2001). Leafperoxisomes too perform b-oxidation; however, thisusually happens at a lower rate and is also involved inthe production of signaling compounds and hormonessuch as jasmonic acid (JA) and in the conversion ofindole-3-butyric acid (IBA) into indole-3-acetic acid. Amajor role of peroxisomes in leaf tissue is in photores-piration by oxidation of glycolate derived from theoxygenase reaction of Rubisco to make substrates formitochondria and the reduction of Ser to glycerate forthe return of carbon intermediates to the Calvin cycle(Raghavendra et al., 1998). Peroxisomes in senescingtissue are multifunctional organelles involved in thedegradation of cellular constituents, including fattyacids and the remobilization of nitrogen into ureides(Vicentini and Matile, 1993). The transition from oneform to another is mediated by a change in the proteincontent of existing organelles rather than by a degra-dation and de novo synthesis of organelles (Hayashiet al., 2000). Apart from the above-mentioned func-tions, plant peroxisomes are also involved in nitrogenmetabolism in root nodule cells, amino acid and ureidemetabolism, and the degradation of hydrogen peroxideproduced during a number of their catalytic functions(Hayashi and Nishimura, 2006).

The size of the peroxisomal proteome is unknown,but it is probably substantially smaller than the thou-sands of proteins found in the endosymbiont-derivedmitochondria and chloroplasts. Peroxisomes lack ge-netic material and therefore do not require proteinsinvolved in genome replication, transcription, matura-tion of transcripts, or translation. However, due to thediversity of the plant-specific roles of these organelles,the proteome of the plant peroxisome may well belarger than that of its mammalian or fungal counter-parts (Emanuelsson et al., 2003). All peroxisomal pro-teins are imported posttranslationally into the organelleand therefore require some form of targeting recogni-tion sequence or secondary structure.

Matrix proteins can be directed to the peroxisomes byone of two types of peroxisomal targeting signals(PTSs). PTS1 signals consist of three amino acids atthe C terminus of a peroxisomal protein. Although a

considerable amount of variation in the PTS1 sequenceexists, it usually consists of a small amino acid residue,followed by a basic one, and then a hydrophobicresidue, and it is not cleaved off after import. SKL is atypical PTS1 sequence. PTS2 sequences are composedof nine amino acids located at the N terminus ofperoxisomal proteins and are removed after importinto the organelle. RLx5HL and RIx5HL are typical PTS2sequences. Searches for peroxisome targeting signalswithin the protein-coding regions of the Arabidopsisgenome have identified 256 to 280 proteins containingputative PTS signals (Kamada et al., 2003; Reumannet al., 2004). However, not all matrix proteins foundexperimentally in peroxisomes contain these knownPTS signals. Recently, the presence of a novel PTS1 in anenoyl-CoA hydratase involved in the b-oxidation ofcis-unsaturated fatty acids was described (Ser-Ser-Leu;Goepfert et al., 2006), which was recently confirmed bya study of the leaf peroxisomal proteome (Reumannet al., 2007). Other peroxisomal proteins seem to lackconventional PTS sequences at the N or C terminusbut possess internal sequences serving as targeting sig-nals. The most prominent example is catalase, whichpossesses an internal, PTS1-like targeting sequence(Kamigaki et al., 2003) but is not recognized for importby the normal PTS1 mechanism (Oshima et al., 2008).

Peroxisomal membrane proteins (PMPs) do notpossess PTS1 or PTS2 sequences. Instead, they con-tain a stretch of positively charged amino acids that isusually flanked by transmembrane domains. Some-times, this sequence is referred to as a membrane PTS(mPTS). However, it is not as conserved as conven-tional PTS1 and PTS2 sequences, and the definition ofa consensus sequence for membrane targeting ofperoxisomal proteins is difficult (Trelease, 2002). Ingeneral, two import pathways for proteins destinedfor the peroxisomal membrane are discussed. In thefirst model, proteins are synthesized in the cytosoland subsequently directly inserted into the peroxi-somal membrane and are usually said to have a mPTStype 1 (mPTS1). Alternatively, proteins can be syn-thesized on rough endoplasmic reticulum (ER) andinserted cotranslationally into the ER membrane.Vesicles containing these proteins then bud from theER and fuse with the peroxisomal membrane. Inaddition to the mPTS1 sequence, these proteins alsocontain an ER sorting signal, and the combination ofboth the mPTS1 and the ER signal is referred to asmPTS2. The finding that peroxisomal membraneproteins such as ascorbate peroxidase (APX) andthe peroxins PEX10 and PEX16 are transferred tothe peroxisomes via the ER led to the formulation ofthe ‘‘ER semiautonomous peroxisome maturationand replication’’ model (for review, see Mullen andTrelease, 2006). It uses the largely contradictorymodels of autonomous organelles and purely ER-derived peroxisomes and combines them. Accordingto the semiautonomous maturation model, peroxi-somes can be derived by budding from the ER butalso by fission of existing organelles.

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In a bid to better experimentally define the solubleand membrane proteome of peroxisomes in Arabidop-sis, we have combined conventional centrifugation-based organelle isolation techniques with free-flowelectrophoresis (FFE), which has already been success-fully applied to the isolation of other organelles inplants (Bardy et al., 1998; Eubel et al., 2007) and perox-isomes in mammals (Volkl et al., 1997). As this tech-nique employs surface charge rather than the size ordensity of particles as a separating parameter, it repre-sents a true additional dimension in the purificationprocess and can lead to organellar fractions of greaterpurity. Peroxisomal proteins were analyzed by MS andnonperoxisomal proteins were excluded by quantita-tive comparison between highly purified peroxisomalsamples and other cellular fractions. Where needed,organellar localization was also confirmed by chimericfluorescent protein visualization. A metabolic mappingapproach was adopted to gauge the completeness ofthis peroxisomal proteome and, therefore, our under-standing of the biochemical processes taking placewithin it and the biogenesis of this organelle.

RESULTS

FFE Separation of Organelles to PurifyArabidopsis Peroxisomes

An organellar fraction consisting mainly of mitochon-dria and peroxisomes was obtained from disruptedArabidopsis protoplasts using differential centrifuga-tion and density gradient purification. FFE was thenemployed to separate peroxisomes from the bulk ofmitochondrial material. We have demonstrated previ-ously that FFE is able to increase the purity of mito-chondrial isolates by separating them from plastids andperoxisomes according to differences in the surfacecharge of each organelle, yielding mitochondria thathave seven times less contamination (Eubel et al., 2007).Under the conditions applied, mitochondria, peroxi-somes, and other cellular material migrated close to-gether in FFE, but with different degrees of overlap(Eubel et al., 2007). The electrophoretic mobility ofperoxisomes was lower than that of mitochondria at600 V (Fig. 1A). In order to optimize FFE for theisolation of Arabidopsis peroxisomes, a higher voltage(800 V) was applied to increase the separation betweenthe mitochondrial and peroxisomal fraction peaks (Fig.1B). The distribution of mitochondria and peroxisomeswas monitored using immunodetection of marker pro-teins. At 600 V, the mitochondrial fraction markerprotein, HSP70, peaked in fraction 44, while the perox-isomal marker, KAT2, peaked in fraction 48, but thedistributions were overlapping. By expanding the dis-tributions at 800 V, both mitochondrial and peroxi-somal signals moved further toward anodic fractions,revealing two mitochondrial subpopulations with dif-ferent electrophoretic mobilities peaking between frac-tions 21 to 23 and fractions 29 to 31. Meanwhile, thepeak fractions enriched in peroxisomes were nearly 10

fractions away, in fractions 39 to 45, and devoid ofvisible mitochondrial contamination. While this highervoltage did tend to lead to greater losses of KAT2 signalinto the anodic mitochondrial fractions than was ap-parent at 600 V (Fig. 1A), the displacement of the bulk ofmitochondria from peroxisomes was optimal for thepreparation of peroxisomes. Using this approach, per-oxisomal and mitochondrial samples with only mini-mal amounts of contamination were obtained byselecting the central three fractions of each distributionat 800 V to ensure the best compromise between yieldand purity. In order to produce a highly enrichedmitochondrial sample for comparative functional as-says against the peroxisomal fraction, the mitochondriawere then subjected to another round of FFE at 600 V(i.e. repeating the separation in Fig. 1A) to separatethem from contaminating plastids, which migratedinto the left border of the separation chamber andtherefore were not resolved well from the mitochondriaat 800 V (data not shown).

Measurement of catalase activity as a marker forperoxisomes gave an oxygen production rate of 60mmol min21 in pre-FFE organelle pellets (SupplementalTable S1A), which was approximately 20% of totalcellular extract activity. Total catalase activity was ap-proximately 15 mmol min21 in the FFE-purified perox-isome fraction, which is approximately 23% of theactivity in pre-FFE organelle pellets and approximately4% of total cellular extract activity. This degree ofrecovery is similar to that shown in preparations ofleaf peroxisomes from Arabidopsis when followinghydroxypyruvate reductase activity (Reumann et al.,2007). To quantify the difference between mitochon-drial and peroxisome fractions, catalase and succinatedehydrogenase activities were measured as markerenzymes (Supplemental Table S1A). The specific activ-ity of catalase was much higher in the putative perox-isomal fraction than in the pre-FFE or mitochondrialsample, indicating clear enrichment of peroxisomes inthis fraction. At the same time, the specific activity ofsuccinate dehydrogenase in the putative peroxisomalfraction was only approximately 8% of that measuredin the mitochondrial sample.

Coomassie Brilliant Blue-stained SDS gel lanes of apre-FFE organellar sample compared with post-FFEsamples of pooled peroxisomes and mitochondriafractions are shown in Figure 1C. While the bandingpattern of the pre-FFE sample was very similar to thatof the mitochondrial fraction, the putative peroxisomesdisplayed a distinct pattern with very little resem-blance to the other two samples. Therefore, we con-cluded that the pre-FFE fraction contained mostlymitochondria and only a limited proportion of otherorganelles, whereas the putative peroxisomal fractionwas largely free of mitochondrial proteins. Abundantprotein bands present in the mitochondrial and perox-isomal samples were excised from one-dimensional(1D) gels for identification by MS (Fig. 1C, annotationson gel lanes). The primary protein identification foreach spot is summarized in Supplemental Table S1B,

Arabidopsis Peroxisome Proteome

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supporting these as purified mitochondrial and perox-isomal fractions derived from FFE. Subsequent quan-titative proteomic analysis (see below) allowed thequantification of a series of classical peroxisomal andmitochondrial markers between the two fractions. Thisshows an average ratio of 0.14 for mitochondrial pro-teins in the peroxisomal compared with the mitochon-drial samples and approximately 70-fold enrichmentfor peroxisomal proteins in the peroxisomal comparedwith mitochondrial samples (Fig. 1D). In general agree-ment, the succinate dehydrogenase specific activity inthe peroxisomes was one-twelfth of that in the mito-chondrial sample (18 6 1 versus 231 6 45 nmol O2min21 mg21 protein), and the catalase specific activitywas approximately 30-fold higher in the peroxisomesample (13.7 6 1.7 versus 513 6 181 nmol O2 min21

mg21 protein; Supplemental Table S1A). Based on thesespecific activity measurements and protein ratios, weestimated the approximate peroxisome purity as 85%to 90%. This is based on mitochondria being the largestcontaminant in the peroxisome preparations and thismitochondrial contamination being 8% to 14% (basedon Fig. 1D and Supplemental Table S1A). Also, thecatalase specific activity in the mitochondrial fraction isonly 3% of that in the peroxisome fraction, so the at leastapproximately 30-fold increase in catalase and otherperoxisome markers indicates that approximately 90%of the protein in the peroxisome fraction should be ofperoxisomal origin.

Whole Peroxisome Protein Profiling

An in-depth analysis of the proteins in each fractionwas obtained by two-dimensional differential in gelelectrophoresis isoelectric focusing (DIGE 2D IEF/SDS-PAGE) using three independent peroxisomesand mitochondria preparations. Figure 2 shows thefalse-colored spot intensity maps for both organellefractions (top) as well as a superimposition of the twospot maps for one of the three gel sets. The amount ofoverlap between the two samples is low, and twoclearly distinct patterns can be observed. While themitochondrial pattern is focused around pI valuesbetween 5 and 8 and resembles that of a previouslypublished study using the same material (Millar et al.,2001), the distribution of the peroxisomal pattern isheavily skewed toward the basic side of the pI spec-trum. This shift to basic pI can also be observed on 2Dgels of leaf peroxisome proteins (Reumann et al., 2007).Many peroxisome protein spots of high abundancehave very high pI values, and a considerable numberdid not even reach their pI on the pH 3 to 10 nonlinearimmobilized pH gradient strips but migrated right tothe cathode. The basic nature of many peroxisome pro-teins may be related to the typically alkaline nature (pH8–8.5) of the peroxisome lumen (van Roermund et al.,2004). Quantification of mitochondrial and peroxisomal

Figure 1. Quantification of protein content from 1D SDS-PAGE andimmunoblots of every second of the central 30 fractions collected afterFFE separation at 600 V (A) and 800 V (B). Relative protein quantity isdisplayed as the percentage of the fraction with the highest abundance(top); distribution of marker proteins for mitochondria (mtHSP70) andperoxisomes (3-ketoacyl-CoA thiolase; KAT2) is shown below. C, 1DSDS-PAGE of 40 mg of pre-FFE organelle protein sample and pooledprotein fractions of peroxisomes and mitochondria stained withCoomassie Brilliant Blue. Bands indicated on gels (A1–A5 and B1–B4) were in-gel digested and analyzed by MS (Supplemental TableS1B). Molecular masses in kD are shown at left of gel lanes. D,Quantification of classical mitochondrial and peroxisomal markers infractions shown in C by 2D DIGE analysis (Supplemental Table S2).Each protein’s AGI accession number and description are shown along

with the average ratio of the quantitation between peroxisomal andmitochondrial samples (n 5 3, P , 0.05).

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spot intensities from all three gel sets was performedusing the DeCyder software package (GE Healthcare).In total, 1,019 matched spots were found consistentlyacross the three sets. Spots with P # 0.05 and an averageratio of 2 or greater (mitochondria-peroxisomes) weredesignated as mitochondrial, and those with an averageratio of 2 or greater (peroxisomes-mitochondria) weredesignated as peroxisomal. On average, the peroxisome-mitochondria ratio was approximately 37 for selectedputative peroxisomal protein spots, while the averagemitochondria-peroxisome ratio was approximately5 for selected putative mitochondrial protein spots.Spots from a preparative gel were matched withthose on the DIGE gels. Prominent peroxisomal andmitochondrial spots matched to the DIGE gels (Sup-plemental Fig. S1) were excised for tandem massspectrometry (MS/MS) identification. In total, 136unique proteins were identified from this gel (Sup-plemental Table S2).

As a complement to the gel-based approaches,reverse-phase HPLC separation coupled with MS/

MS was used to gain additional depth in the wholeperoxisome proteome. Four 60-min Intelligent Data-Dependent Acquisition (IDDA) runs of the same sam-ple were conducted in series, with peptides identifiedin each run subsequently excluded from MS/MS acqui-sition in the next run. This analysis was performed onthe same peroxisomal sample used for the 2D gelscontaining 8% to 10% mitochondria (high purity) andalso on a second sample containing twice the amountof mitochondrial contamination (21%; low purity) asdeduced from the respiratory assays. In cases in whicha protein was identified in both samples, the compar-ison of (1) the emPAI values (which is the exponen-tially modified ratio of the observed peptides over thenumber of peptides that can theoretically be observed)and (2) the difference in a protein’s MOWSE scorebetween the samples was taken as a semiquantitativeindicator for localization of a protein. Proteins with ahigher emPAI value or a higher MOWSE score in thehigh-purity sample are putative peroxisomal proteins,and those that produced higher values in the low-purity sample are putative contaminants. In cases inwhich a protein was identified in only the high-puritysample, this has been considered to be an indication ofa higher abundance of the protein in that sample. Atotal of 135 proteins were identified in the high-puritysample and 209 in the low-purity sample, with anoverlap of 93 between both fractions (SupplementalTable S4). DIGE and the MS-derived quantitative data(emPAI and MOWSE difference measures) form animportant part of our final assessment of the peroxi-somal proteome (see below). Sequence information forproteins identified by only a single but significantpeptide is given in Supplemental Data Set S2.

Peroxisomal Membrane Protein Profiling

Three suborganellar fractions were prepared fromwhole peroxisomes by repeated freeze/thaw cycles,followed by centrifugation in order to separate solubleand membrane proteins. Isolation of integral mem-brane proteins was achieved by sodium bicarbonatestripping of an aliquot of the membrane proteins. Atotal of 40 mg of protein from the soluble and completemembrane fractions was separated by 1D SDS-PAGE,whereas only 5 mg could be loaded from the integralmembrane fraction due to the limited amount ofmaterial available. The gel reveals distinct differencesin the protein banding patterns between the peroxi-somal subfractions (Fig. 3). Based on the proteinquantitation (data not shown), we estimate that theintegral proteins account for about 5% of the wholemembrane fraction protein content. Spots from thebands indicated in Figure 3 were excised, digested,and analyzed by liquid chromatography (LC)-MS/MS. From 65 gel bands, a total of 94 unique proteinswere identified using this approach (SupplementalTable S3A). Double SDS-PAGE (dSDS-PAGE) separa-tion of 50 mg of the membrane fraction and 5 mg of theintegral membrane fraction was also performed (Sup-

Figure 2. DIGE 2D IEF/SDS-PAGE of peroxisomal (labeled with Cy5;shown in green) and mitochondrial (labeled with Cy3; shown in red)protein composition. Top, Gel images as derived from the Typhoon Trio(GE Healthcare) fluorescence scanner, analyzed with the DeCydersoftware package (GE Healthcare). Bottom, Fluorescent images wereelectronically overlaid using ImageQuant TL software (GE Healthcare).Yellow spots represent proteins of similar abundance in both samples,with green spots showing an increased abundance in the peroxisomalfraction and red spots indicating a higher abundance in the mitochon-drial fraction. Protein spots from identical preparative gels correspond-ing to these fluorescent proteins were excised, digested with trypsin,and unambiguously identified by MS (Supplemental Fig. S1). Thefluorescent ratios of each identified protein spot are shown in Supple-mental Table S2 and used in Table I.





plemental Fig. S2). The dSDS gel system has an in-creased resolution compared with conventional SDS-PAGE because proteins are separated not only by sizebut also, to a limited extent, by their hydrophobicity(Meyer et al., 2008). Therefore, it is well suited for theseparation of membrane proteins. A set of 49 spotsfrom both gels were cut out, and the identities of 68proteins were derived from tandem MS analysis (Sup-plemental Table S3B). Unfortunately, a DIGE approachwas not feasible for quantification of the membraneproteins in these two approaches. This is due to thelimited resolution inherent to SDS-PAGE gels com-pared with IEF/SDS-PAGE, which often leads to theidentification of more than one protein in a band.Therefore, even if the quantitative analysis of a 1DDIGE gel could indicate a difference in protein abun-dance, it would be impossible to determine the proteinresponsible for the change. To complement the 1D and2D SDS gels, a carbonate-stripped membrane sampleof the high-purity peroxisomal fraction was also ana-lyzed by IDDA-MS/MS for the detection of furtherintegral membrane proteins. This analysis resulted inthe identification of 89 proteins (Supplemental TableS5). We also used a phosphopeptide enrichment strat-egy from organelle peptide preparations to identifyphosphopeptides from peroxisome proteins; the onesignificant match was a Ser phosphopeptide for one ofthe membrane proteins, PMP38 (At2g39970), at Ser-155 (Supplemental Data Set S3).

Confirmation of Localization byGFP-Targeting Experiments

Several hundred unique proteins were identified inthe analyses noted above. While the quantitativeproteomics data provided evidence to validate orinvalidate peroxisomal location in many cases, aselection of proteins for which this analysis was notclear was further verified in vivo on the basis of thetransient expression of fluorescent fusion proteins.For this purpose, proteins carrying a GFP5 insertion10 to 13 amino acids from their C terminus, to allowtargeting by N- and C-terminal sequences and theinfluence of the mature protein sequence on targeting(Tian et al., 2004), were compared with red fluores-cent protein (RFP) fused to the 10 C-terminal aminoacids of the PTS1-containing pumpkin (Cucurbitamaxima) malate synthase. Four proteins (At1g54340,At3g12800, At4g05530, and At4g14430) were used ascontrols. These were found by our MS analysis andhad each previously been documented to be in leafperoxisomes by GFP and MS (Reumann et al., 2007;Table I). As our GFP data were completely convergentwith these localizations, a range of additional pro-teins were selected for analysis (Supplemental DataSet S1). This list does not include all of the proteinswith ambiguous localization data; rather, it merelyfocuses on those for which clarification, in our view,would be most beneficial.

Figure 3. SDS-PAGE separation of40 mg of peroxisomal membraneprotein (A), 5 mg of integral mem-brane protein (B), and 40 mg ofsoluble protein (C) fractions. Mo-lecular masses in kD are shown atleft of each lane, and band numbersextracted for in-gel digestion andprotein identification are shown atright of each lane. Proteins identi-fied are shown in Supplemental Ta-ble S3 and summarized in Table I.

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For four of these proteins (At5g11520, At1g65520,At1g49670, and At5g27520), the GFP localization con-firmed peroxisomal location, and no previous GFP datahave been reported, to our knowledge. Three of themhave recognizable PTS1 and PTS2 sequences, whileAt5g27520 lacks a recognizable PTS and appears to bea six-transmembrane domain carrier family protein(Fig. 4A). Accordingly, the fluorescence of the At5g27520-GFP construct appears ring like, surrounding the matrix-targeted RFP-SRL.

Five targets had been previously reported to localizeGFP to other cell compartments (At3g02360, cytosol[Reumann et al., 2007]; At5g42020, ER [Kim et al., 2001];At4g29130, mitochondria [Damari-Weissler et al.,2007]; At5g58070, vacuole [Jaquinod et al., 2007]; andAt4g16210, unclear [Cutler et al., 2000]). However,these earlier reports had used terminal GFP fusions,which frequently localize differentially depending onthe terminus to which GFP is fused (Simpson et al.,2001). For example, At3g02360 is localized solely to thecytosol when the whole protein is fused to the C ter-minus of enhanced yellow fluorescent protein (Reumannet al., 2007) but is found to be present in peroxisomes byinternal GFP fusion of the whole protein (Fig. 4B). Theother reported locations were confirmed, except forAt5g58070, which did not allow for a confident positiveassignment of location based on the internal fusion(data not shown).

A set of 14 other proteins analyzed by internal GFPfusion to supplement quantitative proteomic data(At1g02930, At1g07920, At1g44575, At1g44820,At2g16060, At1g77120, At2g17265, At3g52190,

At4g00390, At4g29130, At5g19760, At5g43190,At5g43940, and At5g46710) could not be assigned toperoxisomes by GFP and are most likely mitochon-drial, cytosolic, plastidic, nuclear, or vacuolar contam-inants (Supplemental Data Set S1).

Identification of ER Proteins

Within our list of identified proteins, 14 appeared to bemajor ER proteins (Table II). These proteins were con-sidered to be of special interest, as connections betweenthe ER and the peroxisomes in relation to peroxisomalprotein import have been reported previously (Elgersmaet al., 1997; Flynn et al., 2005; Karnik and Trelease, 2005).One of the proteins detected was calreticulin. The dis-tribution of calreticulin across the 800-V separationsused for peroxisomal purification reveals enrichment ofthe antibody signal in the same lanes as the peroxisomes(Fig. 5A), indicating either the comigration of ER com-ponents with peroxisomes during FFE or the presence ofcalreticulin in these organelles. Analysis of the DIGE 2DIEF/SDS-PAGE data revealed that many of the ERproteins reported in Table II, including three isoformsof calreticulin, were enriched in the peroxisome samplerelative to the mitochondrial sample (Fig. 5B), consis-tent with the enrichment in peroxisomal fractions seenwith the calreticulin antibodies (Fig. 5A).

Eleven of the 14 proteins suspected to be ER proteins(At1g08450, At1g09210, At1g21750, At1g56340,At2g47470, At4g15955, At4g24190, At5g28540,At5g42020, At5g60640, and At5g61790) were analyzedby internal GFP fusions. Besides their obvious ERlocation, none of them could be positively localized toperoxisomes (Table II; Supplemental Data Set S1).However, it became apparent from the analysis of thefluorescent images that some sort of interaction be-tween the ER and the peroxisomes might exist. Fre-quently, peroxisomes appear to be heavily embeddedin the ER, with the fluorescence intensity peaking at theER-peroxisome border (e.g. calreticulin, BIP, and pro-tein disulfide isomerase; Fig. 5C), but so far it is unclearwhether this is a result of a higher ER density in thisarea or a higher concentration of the fusion protein.

Assembling the Data to Define a Peroxisomal Proteome

To ensure maximum depth in protein analysis, mul-tiple strategies employing gel and nongel separation ofproteins and peptides were used in our analysis.Quantitative or semiquantitative measurement of theabundance of proteins was obtained using DIGE fluo-rescence measurements from highly purified peroxi-some versus purified mitochondrial samples and theratio of MOWSE scores, or emPAI (Ishihama et al.,2005), in the nongel LC-MS/MS experiments of high-purity peroxisomal versus low-purity peroxisomalsamples. These provided data to discriminate peroxi-somal proteins from potential contaminants. Alto-gether, 250 unique proteins were identified in thecourse of this study. All of them were assessed by a

Figure 4. Fluorescence images of subcellular localization of selectedproteins found in peroxisome preparations by transient expression ofinternal GFP fusions. A, Localization of membrane carrier proteinAt5g27520 in an Arabidopsis cultured cell. B, Localization of6-phosphogluconate dehydrogenase (At3g02360) in an Arabidopsiscultured cell. GFP images of the indicated proteins are overlaid with aRFP peroxisome marker as outlined in ‘‘Materials and Methods.’’





series of factors to define the final proteome set. Theseinclude the quantitative data generated in this study,our and other GFP data, as well as previous reports ofsubcellular localization from other proteome studies.Additionally, all proteins identified from gels and theliquid phase analysis were examined for the presenceor absence of known PTS1 and PTS2 sequences accord-ing to the AraPerox database (Reumann et al., 2004). Inlight of these data, we have classified 89 of theseproteins as of peroxisomal origin (Table I) and 14 asER proteins that copurify with peroxisomes (Table II).

Subfractionation of the organelles to soluble, peripheralmembrane and integral membrane fractions providedsuborganelle location information.

Hydrophobic proteins are notoriously underrep-resented in proteome studies using 2D IEF-SDSapproaches. To determine our success rate in the iden-tification of hydrophobic proteins, we used the ARA-MEMNON database (Schwacke et al., 2003; release 5.0)to predict transmembrane domains in the set of 89peroxisomal proteins; 16 proteins were predicted tohave one or two transmembrane domains, two proteinsmost probably had three to four transmembrane do-mains, and four proteins had more than four trans-membrane domains. The latter group (At2g39970,At4g04470, At4g39850, and At5g27520) mainly con-sisted of the proteins listed as transporters or integralmembrane proteins in Table I.

Developing a Model of Peroxisome Metabolism

In order to test if our proteins form an integrated setof functions and to predict if many peroxisomal en-zymes are missing from our list, we created a meta-bolic network using data from the AraCyc databaseand visualized it with the Cytoscape software package(Version 2.6.0). This was based on the Enzyme Com-mission (EC) number of all identified proteins asannotated in AraCyc, which links Arabidopsis Ge-nome Initiative (AGI) numbers with EC reactions. Atotal of 44 of the proteins in our peroxisome set fromTable I were assigned EC numbers, making a nonre-dundant set of 28 enzyme nodes and 64 metabolites.Focusing on the metabolites that represent the sub-strates, reactants, and products of the network enablesus to see points of contact between the differentfunctional categories of proteins and also to predictthe need for transporters in the peroxisomal mem-brane by analysis of the metabolite end points of thenetwork. In the network, colored nodes (roundedsquares) represent enzymes in different functionalcategories, metabolites are shown as small gray circles,while the reaction is shown as connecting lines be-tween the enzymes and metabolite nodes (Fig. 6).

The result is a structure derived from our proteomediscovery strategy that appears as a well-connectedsingle metabolic entity, showing that the differentmetabolic pathways within this peroxisome proteomeare interlinked and that there are no large gaps createdby lack of identification of critical enzymes in a se-quence. About one-third of the metabolites do notrepresent starting or end points of a pathway but areconsidered to be intermediates of peroxisome metab-olism by this network. The terminal metabolite nodesof the network are potential substrates to be trans-ported in or out across the peroxisomal membrane viatransporters or pores. Many of these metabolites havebeen reported to be transported, to diffuse freelyacross peroxisomal membranes, or to be incapable ofcrossing the peroxisomal membrane, and these arehighlighted in Figure 6.

Figure 5. Enrichment of ER proteins in peroxisome fractions in vitro,and fluorescence-based evidence for association of ER and peroxi-somes in planta. A, Immunoblots of every second of the central 30fractions collected after FFE separation at 800 V showing distribution ofmarker proteins for peroxisomes (3-ketoacyl-CoA thiolase; KAT2) andER (calreticulin). B, Quantification of a range of ER proteins inperoxisome fractions by 2D DIGE analysis (Supplemental Table S2).Each protein’s AGI accession number and description are shown alongwith the average ratio of the quantitation between peroxisomal andmitochondrial samples (n 5 3, P , 0.05). C, Fluorescence images ofsubcellular localization of selected ER proteins found in peroxisomepreparations by transient expression of internal GFP fusions: CRT3(At1g08450), BiP-1 (At5g28540), and DIL2 (At2g47070). GFP imagesof the indicated proteins are overlaid with a RFP peroxisome marker asoutlined in ‘‘Materials and Methods.’’

Eubel et al.




Tab

leI.

Nonre

dundan

tlist

of

pro

tein

sfo

und

inper

oxi

som

esaf

ter

rem

ova

lof

conta

min

ants

bas

edon

quan

tifica

tion

The

iden

titi

esof

pro

tein

sw

ere

det

erm

ined

by

MS/

MS;

the

pre

dic

ted

mole

cula

rm

ass

(MW

)of

the

mat

chis

show

nal

ong

wit

hth

ebes

tM

OW

SEsc

ore

(P,

0.0

5w

hen

score

.37),

num

ber

of

pep

tides

and

sequen

ceco

vera

ge(S

eq.

Cov.

),an

dth

enum

ber

of

tim

esth

isid

entifica

tion

was

mad

e(N

o.

Exp.)

acro

ssth

ese

ries

of

exper

imen

tsper

form

ed.

Loca

liza

tion

of

pro

tein

sw

ithin

per

oxi

som

esca

nbe

ded

uce

dfr

om

subfr

acti

onat

ion

oforg

anel

les

(Mem

5m

embra

ne

frac

tion,So

l5

solu

ble

frac

tion,W

hole

5w

hole

org

anel

les)

.Q

uan

tita

tive

asse

ssm

ents

pre

sente

din

det

ailin

Supple

men

talT

able

sS2

,S4,a

nd

S5ar

epre

sente

din

the

DIG

Eco

lum

n(P

5hig

her

inper

oxi

som

esa

mple

,M5

hig

her

inm

itoch

ondri

asa

mple

,P/M

5both

loca

liza

tions

poss

ible

)and

the

MS

Hig

hve

rsus

Low

colu

mn

(MO

WSE

score

inhig

h-p

uri

tyco

mpar

edw

ith

low

-puri

tysa

mple

s,w

ith

the

sam

ple

wit

hth

eorg

anel

le[P

or

M]

wit

hth

egr

eate

rsc

ore

dis

pla

yed).

Sim

ilar

ly,th

ela

rges

tem

PAI

score

bas

edon

the

num

ber

ofp

eptides

mat

ched

inth

ehig

h-p

uri

tyve

rsus

low

-puri

tysa

mple

sis

indic

ated

by

Hig

horLo

win

the

emPA

Icolu

mn,a

nd

indep

enden

tGFP

loca

liza

tion

dat

a(p

rese

nte

das

imag

esin

Supple

men

talD

ata

SetS1

)w

ere

use

dto

des

ignat

elo

cati

on

inth

eG

FPco

lum

n.Fu

rther

colu

mns

indic

ate

the

pre

sence

ofPTS1

or

PTS2

sequen

ces,

pre

dic

ted

num

ber

oftr

ansm

embra

ne

dom

ains

(TM

s),pre

vious

iden

tifica

tions

of

each

pro

tein

inpubli

shed

per

oxi

som

epro

teom

es,an

dth

enew

pro

tein

suniq

ue

toth

isan

alys

is.A

ster

isks

indic

ate

wher

ea

singl

epep

tide

wit

ha

score

above

but

appro

achin

gth

eth

resh

old

was

use

dfo

rid

entifica

tion;

spec

tra

and

det

aile

din

terp

reta

tion

are

pro

vided

inSu

pple

men

tal

Dat

aSe

tS2

.

AG

IN

o.

Des

crip

tion

MW

Score

Pep

tides

Seq.

Cov.

No.

Exp.

1D

SDS

1D

SDS

Sol

1D

SDS

Mem

dSD

S

Mem

2D

IEF/

SDS

LC-M

S

Whole

LC-M

S

Mem

DIG

E

MS

Hig

h

vers

us

Low

emPA

IPTS

TM

sO

ur

GFP

Fuka

o

etal

.

(2002)

Fuka

o

etal

.

(2003)

Reu

man

n

etal

.

(2007)

New

Acy

l-ac

tiva

ting

enzy

mes

At5

g23050

Ace

tyl-

CoA

synth

etas

e/li

gase

(AA

E17)

79,1

31

68

23

2x

––

––

x–

–H

igh

Hig

hSK

L1

or

2TM

s–

––

–x

At1

g20480

4-C

oum

arat

e-C

oA

liga

se-l

ike

pro

tein

61,3

99

108

49

5–

–x

xx

xx

PH

igh

Hig

hSK

L1

or

2TM

s–

––

–x

At4

g05160

4-C

oum

arat

e-C

oA

liga

se-l

ike

pro

tein

59,8

12

251

712

5x

––

xx

xx

PH

igh

Hig

hSK

M2

TM

s–

––

x

At1

g20560

Ace

tyl-

CoA

synth

etas

e/li

gase

(AA

E1)

61,0

34

194

815

3–

–x

–x

x–

–H

igh

Hig

hSK

LS

––

––

x

At3

g05970

Long-

chai

nfa

tty

acid

-CoA

synth

etas

e

(LA

CS6

)

76,5

55

342

811

5x

––

Xx

xx

PH

igh

Hig

hR

IxH

LS

––

––

x

At3

g16910

Ace

tyl-

CoA

synth

etas

e/li

gase

(AA

E7)

62,9

16

191

67

4x

x–

–x

x–

PH

igh

Hig

hSR

LS

––

–x

At5

g16370

Ace

tyl-

CoA

synth

etas

e/li

gase

(AA

E5)

60,7

00

368

814

2–

––

–x

x–

PH

igh

Hig

hSR

MS

––

–x

At5

g27600

Long-

chai

nfa

tty

acid

-CoA

synth

etas

e

(LA

CS7

)

77,3

04

730

19

22

5x

––

xx

xx

P/M

Hig

hH

igh

RLx

HI

S–

––

x

Core

b-o

xidat

ion

At3

g06860

Fatt

yac

idm

ult

ifunct

ional

pro

tein

(MFP

2)

78,7

90

762

17

25

6x

–x

xx

xx

PH

igh

Sam

eSR

L1

TM

––

–x

At4

g16210

Enoyl

-CoA

hyd

rata

se(E

-CoA

H-2

)28,8

00

665

22

59

5x

–x

xx

x–

–Lo

wSa

me

SKL

1TM

P–

–x

At4

g29010

Fatt

yac

idm

ult

ifunct

ional

pro

tein

(AIM

1)

77,8

09

753

26

33

6x

–x

xx

xx

PH

igh

Sam

eSK

L1

TM

––

–x

At2

g33150

Ace

tyl-

CoA

C-a

cylt

ransf

eras

e

(AC

oA

AT-

2/K

AT2/P

ED1)

48,5

48

918

21

47

6x

–x

xx

xx

PH

igh

Hig

hR

QxH

L2

TM

s–

––

x

At1

g06290

Acy

l-C

oA

oxi

das

e3

(AC

X3)

75,6

29

756

21

25

4x

xx

–x

––

––

–R

AxH

IS

––

––

x

At2

g35690

Acy

l-C

oA

oxi

das

e5

(AC

X5)

74,2

51

216

11

11

1–

–x

––

––

––

Hig

hA

KL

S–

––

–x

At4

g16760

Acy

l-C

oA

oxi

das

e1

(AC

X1)

75,3

29

553

21

22

6x

–x

xx

xx

PH

igh

Hig

hA

RL

S–

––

–x

At1

g04710

Ace

tyl-

CoA

C-a

cylt

ransf

eras

e

(AC

oA

AT1/K

AT1)

46,5

82

165

613

3–

––

x–

xx

–H

igh

Hig

hR

QxH

LS

––

–x

At1

g65520

Del

ta(3

),D

elta

(2)-

enoyl

-CoA

isom

eras

e(E

CI1

)

25,8

91

266

825

4x

x-

–x

x–

PH

igh

Hig

hSK

LS

P–

–x

At3

g51840

Acy

l-C

oA

oxi

das

e4

(AC

X4)

47,5

26

347

820

6x

–x

xx

xx

PH

igh

Hig

hSR

LS

––

–x

At4

g14430

Del

ta(3

),D

elta

(2)-

enoyl

-CoA

isom

eras

e(I

BR

10)

25,7

57

130

313

6x

xx

xx

x–

PH

igh

Hig

hPK

LS

P–

–x

At5

g43280

Enoyl

CoA

hyd

rata

se(E

-CoA

H-5

)29,9

01

174

410

3–

––

xx

–x

P–

–A

KL

S–

––

x

b-O

xidat

ion

of

unsa

tura

ted

subst

rate

s

At1

g36580

2,4

-Die

noyl

-CoA

reduct

ase-

rela

ted

26,5

56

78

818

2–

––

––

xx

–Lo

wH

igh

–S

––

––

x

At2

g07640

2,4

-Die

noyl

-CoA

reduct

ase-

rela

ted

16,8

36

78

719

1–

––

––

x–

–H

igh

––

S–

––

–x

At1

g76150

Enoyl

-CoA

hyd

rata

se2

(EC

H2)

34,0

61

538

15

41

4x

x-

-x

x–

PH

igh

Hig

hSS

LS

––

–x

At3

g12800

Short

-chai

ndeh

ydro

genas

e/re

duct

ase

(SD

R)

fam

ily

pro

tein

31,7

77

537

21

24

6x

xx

xx

x–

PLo

w–

SKL

SP

––

x

At3

g55290

Short

-chai

ndeh

ydro

genas

e/re

duct

ase

(SD

R)

fam

ily

pro

tein

30,1

85

130

313

1–

––

–x

-–

––

–SS

LS

––

–x

At4

g05530

Short

-chai

ndeh

ydro

genas

e/re

duct

ase

(IB

R1)

26,7

48

356

933

5x

–x

xx

x–

PH

igh

Hig

hSR

LS

P–

–x

At5

g42890

Ster

ol

carr

ier

pro

tein

2(S

CP-2

)

fam

ily

pro

tein

13,5

59

70

227

2x

––

––

x–

–H

igh

Sam

eSK

LS

––

–x

(Tab

leco

nti

nues

on

foll

ow

ing

pag

e.)





Tab

leI.

(Conti

nued

from

pre

vious

pag

e.)

AG

IN

o.

Des

crip

tion

MW

Score

Pep

tides

Seq.

Cov.

No.

Exp.

1D

SDS

1D

SDS

Sol

1D

SDS

Mem

dSD

S

Mem

2D

IEF/

SDS

LC-M

S

Whole

LC-M

S

Mem

DIG

E

MS

Hig

h

vers

us

Low

emPA

IPTS

TM

sO

ur

GFP

Fuka

o

etal

.

(2002)

Fuka

o

etal

.

(2003)

Reu

man

n

etal

.

(2007)

New

Oth

erau

xili

ary

b-o

xidat

ion

At4

g04320

Puta

tive

mal

onyl

-CoA

dec

arboxy

lase

58,4

19

45

23

1–

x–

––

––

––

–SR

LS

––

––

x

At4

g02340

Puta

tive

epoxi

de

hyd

rola

se36,6

34

276

821

4x

x–

–x

x–

PH

igh

Sam

e–

S–

––

x

At2

g38180

GD

SLm

oti

fli

pas

e/hyd

rola

se

fam

ily

pro

tein

35,3

83

102

48

2–

––

–x

x–

PH

igh

Sam

eA

RL

S–

––

–x

At3

g15290

3-H

ydro

xy-2

-met

hyl

buty

ryl-

CoA

deh

ydro

genas

e(H

MB

-CoA

DH

-1)

31,6

69

551

16

52

2–

x–

–x

––

P/M

––

PR

LS

––

–x

CoA

pool

regu

lati

on

At4

g00520

Acy

l-C

oA

thio

este

rase

fam

ily

pro

tein

32,1

93

65

211

1–

––

––

x–

–H

igh

Hig

hA

KL

S–

––

–x

At1

g01710

Acy

l-C

oA

thio

este

rase

fam

ily

pro

tein

48,1

25

62

916

2x

––

––

x–

–H

igh

Hig

hSK

LS

––

–x

At3

g61200

Thio

este

rase

fam

ily

pro

tein

20,4

61

50

1*

81

––

––

––

x–

––

SKL

S–

–x

–

At1

g04290

Thio

este

rase

fam

ily

pro

tein

16,8

76

52

1*

71

––

––

––

x–

––

SNL

S–

––

x

Horm

one

bio

synth

esis

/act

ivat

ion

At2

g06050

12-O

xophyt

odie

noat

ere

duct

ase

(OPR

3/D

DE1

)

42,6

64

42

1*

21

–x

––

––

––

––

SRL

S–

––

x

At1

g20510

4-C

oum

arat

e-C

oA

liga

se-l

ike

fam

ily

pro

tein

(OPC

L1)

59,3

37

365

15

25

5x

–x

xx

x–

PH

igh

Hig

hSK

L1

or

2TM

s–

––

x

At3

g06810

Acy

l-C

oA

deh

ydro

genas

e(I

BR

3)

91,6

55

958

21

28

6x

–x

xx

xx

PH

igh

Hig

hSK

LS

––

––

x

Val

cata

boli

sm

At5

g65940

HIB

-CoA

H-6

3-h

ydro

xyis

obuty

ryl-

CoA

hyd

rola

se(C

HY

1)

42,0

47

156

312

4x

x–

––

xx

–H

igh

Hig

hA

KL

S–

––

–x

Car

bon

met

aboli

sm

At3

g14415

Gly

cola

teoxi

das

e(G

OX

/HA

O)

40,2

81

347

818

3x

x–

x–

––

––

Hig

hPR

L1

TM

–x

–x

At3

g14420

Gly

cola

teoxi

das

e(G

OX

/HA

O)

40,3

16

58

34

1–

––

–x

––

P–

–A

RL

1TM

–x

–x

At3

g58750

Cit

rate

synth

ase

2(C

SY2)

56,5

67

1240

28

49

6x

–x

xx

xx

P/M

Hig

hLo

wR

LxH

LS

––

––

x

At1

g23310

Ala

amin

otr

ansf

eras

e(G

GT1)

53,2

67

673

17

35

4x

x–

–x

x–

PH

igh

Hig

hSK

MS

–x

–x

At1

g68010

Hyd

roxy

pyr

uva

tere

duct

ase

(HPR

)42,2

21

448

14

37

2–

x–

–x

––

M–

–SK

LS

–x

–x

At1

g70580

Ala

amin

otr

ansf

eras

e(A

OA

T2)

53,4

10

510

16

41

2–

x–

–x

––

P–

–SR

MS

–x

–x

At5

g09660

NA

Dm

alat

edeh

ydro

genas

e

(PM

DH

2/M

DH

6)

34,9

53

58

26

1–

x–

––

––

––

–R

IxH

LS

–x

–x

At2

g22780

NA

Dm

alat

edeh

ydro

genas

e

(PM

DH

1/M

DH

3)

37,4

42

582

17

48

7x

xx

xx

xx

PH

igh

Hig

hR

IxH

LS

–x

xx

At2

g13360

Ser/

Ala

-gly

oxy

late

amin

otr

ansf

eras

e

(AG

T)

44,1

80

132

58

2–

x–

––

x–

–Lo

wLo

wSR

IS

––

–x

At2

g42790

Cit

rate

synth

ase

3(C

SY3)

56,1

40

870

16

30

7x

xx

xx

xx

P/M

Hig

hH

igh

RLx

HL

S–

––

x

At3

g02360

6-P

hosp

hogl

uco

nat

edeh

ydro

genas

e53,5

44

197

410

3x

x–

––

x–

–H

igh

Hig

hSK

IS

P–

–x

At4

g18360

Gly

cola

teoxi

das

e(G

OX

/HA

O)

40,4

56

700

18

40

6x

xx

xx

x–

PH

igh

Hig

hA

KL

S–

––

x

At5

g11520

Asp

amin

otr

ansf

eras

e3

(ASP

3)

48,9

23

1016

24

52

7x

xx

xx

xx

PLo

wLo

wR

IxH

LS

P–

–x

At1

g54340

NA

DP

isoci

trat

edeh

ydro

genas

e

(ID

HP1/I

CD

H)

47,2

04

624

17

36

4x

x–

–x

x–

PH

igh

Hig

hSR

LS

P–

xx

Nit

roge

nan

dsu

lfur

met

aboli

sm

At1

g65840

Poly

amin

eoxi

das

e4

(PA

O4)

54,8

96

355

10

24

2–

––

–x

x–

–H

igh

Hig

hSR

MS

––

––

x

At2

g42490

Puta

tive

copper

amin

eoxi

das

e86,6

36

474

913

3x

–x

–x

––

P–

–SK

LS

––

––

x

At2

g26230

Puta

tive

uri

case

34,8

59

370

13

30

5x

–x

xx

x–

PH

igh

Hig

hSK

LS

––

–x

At3

g01910

Sulfi

teoxi

das

e(S

OX

)32,6

62

256

616

4x

x–

–x

x–

PH

igh

Hig

hSN

LS

––

–x

Anti

oxi

dan

tdef

ense

At4

g35000

Asc

orb

ate

per

oxi

das

e(A

PX

3)

31,5

52

644

19

44

5x

–x

xx

–x

P/M

––

1TM

–x

xx

At2

g17420

NA

DPH

thio

redoxi

nre

duct

ase

2

(NTR

A)

39,9

89

272

919

3–

x–

–x

x–

PH

igh

Hig

h–

S–

––

–x

At4

g35090

Cat

alas

e2

(CA

T2)

56,8

95

613

20

35

6x

xx

xx

x–

PH

igh

Hig

h–

S–

x–

x

At1

g20620

Cat

alas

e3

(CA

T3)

56,6

60

437

13

20

6x

xx

xx

x–

P/M

Hig

hH

igh

–S

–x

xx

(Tab

leco

nti

nues

on

foll

ow

ing

pag

e.)

Eubel et al.




Tab

leI.

(Continued

from

pre

vious

pag

e.)

AG

IN

o.

Des

crip

tion

MW

Score

Pep

tides

Seq.

Cov.

No.

Exp.

1D

SDS

1D

SDS

Sol

1D

SDS

Mem

dSD

S

Mem

2D

IEF/

SDS

LC-M

S

Whole

LC-M

S

Mem

DIG

E

MS

Hig

h

vers

us

Low

emPA

IPTS

TM

sO

ur

GFP

Fuka

o

etal

.

(2002)

Fuka

o

etal

.

(2003)

Reu

man

n

etal

.

(2007)

New

At1

g20630

Cat

alas

e1

(CA

T1)

56,7

26

885

27

41

6x

xx

xx

x–

PLo

wH

igh

–S

––

–x

At3

g24170

Glu

tath

ione

dis

ulfi

de

reduct

ase

(GR

1)

53,8

37

107

57

1–

x–

––

––

––

––

S–

––

x

At3

g52880

Monodeh

ydro

asco

rbat

e

reduct

ase

1(M

DA

R1)

46,4

58

265

10

22

2–

x–

–x

––

––

––

S–

––

x

At5

g41210

Glu

tath

ione

S-tr

ansf

eras

e(G

STT1)

27,6

36

277

928

4x

–x

xx

––

P–

–SK

IS

––

–x

Red

uci

ng

met

aboli

sm

At3

g56460

Quin

one

reduct

ase-

like

pro

tein

37,2

42

198

621

3x

x–

x–

––

––

–SK

L2

or

3TM

s–

––

–x

At2

g20800

NA

D(P

)Hdeh

ydro

genas

eB

4

(ND

B4)

65,3

31

73

12

14

1–

––

––

x–

–Lo

wH

igh

SSI

S–

––

–x

At1

g49670

Quin

one

oxi

dore

duct

ase

(NA

DPH

:quin

one

reduct

ase)

67,9

01

1433

34

38

6x

–x

xx

xx

PH

igh

Hig

hSR

LS

P–

–x

Tran

sport

er/i

nte

gral

mem

bra

ne

pro

tein

At4

g39850

Per

oxi

som

alA

BC

tran

sport

er

1(P

ED3/C

TS/

PX

A1/P

MP2)

149,4

82

613

17

13

4x

––

x–

xx

–H

igh

Hig

h-

2to

10

TM

s–

––

–x

At4

g04470

Per

oxi

som

alm

embra

ne

pro

tein

(PM

P22)

21,7

05

109

416

4–

–x

x–

xx

–H

igh

Hig

h-

3to

4TM

s–

––

–x

At2

g39970

Per

oxi

som

alm

embra

ne

pro

tein

(PM

P38)

36,1

90

375

10

17

3–

––

x–

xx

–H

igh

Low

-3

to6

TM

s–

––

–x

At5

g27520

Mit

och

ondri

alsu

bst

rate

carr

ier

fam

ily

pro

tein

35,1

37

116

219

3–

–x

––

xx

–H

igh

Hig

h-

3to

6TM

sP

––

–x

Bio

gen/i

mport

/pro

tein

fate

At5

g62810

Per

oxi

som

em

embra

ne

anch

or

pro

tein

(PEX

14/P

ED2)

55,5

61

84

12

3x

–x

–x

––

P–

––

1TM

––

–x

At3

g61070

Per

oxi

som

albio

genes

isfa

ctor

11

fam

ily

pro

tein

(PEX

11E)

25,4

96

545

17

36

2–

–x

––

–x

––

––

1or

2TM

s–

––

–x

At2

g45740

Per

oxi

som

albio

genes

isfa

ctor

11

fam

ily

pro

tein

(PEX

11D

)

25,9

27

239

620

3–

–x

x–

–x

––

––

2TM

s–

––

x

At1

g01820

Per

oxi

som

albio

genes

isfa

ctor

11

fam

ily

pro

tein

(PEX

11C

)

25,8

96

278

925

5x

–x

x–

xx

–H

igh

--

2TM

s–

––

–x

At1

g28320

DEG

15

pro

teas

e76,0

77

44

44

1–

––

––

x-

–H

igh

Hig

hSK

LS

––

––

x

At1

g29260

Per

oxi

som

alta

rget

ing

sign

al

type

2re

cepto

r(P

EX7)

35,4

50

297

10

28

1–

––

–x

––

––

––

S–

––

–x

At1

g47750

Per

oxi

som

albio

genes

isfa

ctor

11

fam

ily

pro

tein

(PEX

11A

)

27,6

60

112

412

3x

––

x–

–x

––

Sam

e–

S–

––

–x

At2

g41790

Pep

tidas

eM

16

fam

ily

pro

tein

/

insu

linas

efa

mil

ypro

tein

110,9

25

67

87

1–

––

––

x–

–Lo

wSa

me

PK

LS

––

––

x

Unkn

ow

n

At1

g50510

Unkn

ow

npro

tein

/indig

oid

ine

synth

ase

Afa

mil

ypro

tein

34,7

78

663

20

56

7x

xx

xx

xx

PH

igh

Hig

hR

IxH

L1

TM

––

––

x

At3

g15000

DA

G8

expre

ssed

pro

tein

42,8

43

37

1*

31

––

––

–x

––

Hig

hSa

me

–S

––

––

x

At1

g35720

Ca2

1-d

epen

den

tm

embra

ne-

bin

din

g

pro

tein

annex

in1

36,1

82

209

618

1–

––

–x

––

M–

––

S–

–x

–

At3

g48170

Bet

aine

aldeh

yde

deh

ydro

genas

e

10A

9(A

LD10A

9)

54,8

83

466

11

21

6x

xx

–x

xx

P/M

Hig

hH

igh

SKL

S–

––

x

At4

g16566

His

tria

dfa

mil

ypro

tein

/HIT

fam

ily

pro

tein

16,7

87

231

840

3–

––

–x

xx

–H

igh

Sam

e–

S–

––

x

At5

g58220

Tran

sthyr

etin

-lik

epro

tein

(TTL)

35,5

42

355

829

3–

x–

–x

x–

PH

igh

Sam

e–

S–

––

x

At1

g34350

Expre

ssed

pro

tein

19,0

81

42

1*

10

1–

––

––

x–

–H

igh

Hig

h–

S–

––

–x

At1

g49350

pfk

B-t

ype

carb

ohyd

rate

kinas

e

fam

ily

pro

tein

40,2

94

39

1*

61

––

––

––

x–

––

SML

S–

––

–x

At5

g10730

Puta

tive

pro

tein

31,0

25

42

1*

31

––

––

x–

––

––

–S

––

––

x





The network shows key features of established per-oxisome biochemistry. It shows citrate synthase andmalate dehydrogenase as key points of contact betweenb-oxidation and organic acid/amino acid metabolismthrough CoA/acetyl-CoA and NAD/NADH pools. Italso shows that antioxidant defense enzymes operate todetoxify hydrogen peroxide produced by a series ofoxidases. The metabolites oxygen, hydrogen peroxide,water, and CoA are the most heavily connected nodes,but removal of these ‘‘currency metabolites’’ (Huss and

Holme, 2007) does not break the highly interconnectedstructure of this metabolic network.

DISCUSSION

Of the 89 peroxisomal proteins identified here, 54have been identified previously by the peroxisomeproteome studies of other tissue types by Fukao et al.(2002, 2003) and Reumann et al. (2007). These proteins

Table II. Nonredundant list of probable ER proteins found in peroxisomal samples but removed on the basis of our GFP data (SupplementalData Set S1) and previous proteomics reports on ER samples

The identities of proteins were determined by MS/MS; the predicted molecular mass (MW) of the match is shown along with the best MOWSE score(P , 0.05 when score . 37), the number of peptides and sequence coverage (Seq. Cov.), and the number of times this identification was made (No.Exp.) across the series of experiments performed. Localization of proteins within peroxisomes can be deduced from subfractionation of organelles(Mem 5 membrane fraction, Sol 5 soluble fraction, Whole 5 whole organelles). Quantitative assessments presented in detail in Supplemental TablesS2, S4, and S5 are presented in the DIGE column (P 5 higher in peroxisome sample, M 5 higher in mitochondria sample, P/M 5 both localizationspossible) and the MS High versus Low column (based on the MOWSE score in high-purity compared with low-purity sample, with the sample with thegreater score displayed in the column). New GFP localization data are shown in Our GFP (presented as images in Supplemental Data Set S1).Literature reports of these proteins in subcellular locations based on GFP data (Cyt, cytosol; Mito, mitochondria; Nuc, nucleus; P-Mem, plasmamembrane; Plast, plastid; Vac, vacuole), MS data, or in protein name annotations are shown in the last three columns; these data were sourced fromthe SUBA database (www.suba.bcs.uwa.edu.au).

AGI No. Description MW Score PeptidesSeq.

Cov.

No.

Exp.

1D

SDS

1D

SDS

Sol

1D

SDS

Mem

dSDS2D

IEF/SDS

LC-MS

Whole

LC-MS

MemDIGE

MS High

versus

Low

Our

GFPGFP MS Annotation

At5g60640 Protein

disulfide

isomerase

(PDIL1-4)

66,316 47 1 3 1 – – – – – x – – Low ER – ER, Mito,

Plast, Vac

–

At5g61790 Calnexin 1

(CNX1)

60,448 151 7 11 2 – – – x – x – – High ER – ER, Mito,

Vac

ER

At5g27330 Unknown

protein

71,967 37 1 1 1 – – – – – – x – – – – ER –

At5g28540 Luminal

binding

protein

(BIP1)

73,600 891 31 39 3 – x – – x x – P Low ER – Nuc, Plast,

Vac

ER

At5g42020 Luminal

binding

protein

(BIP2)

73,500 396 14 16 2 – – – – x x – P Low ER ER ER, Nuc,

Vac

ER

At4g15955 Epoxide

hydrolase-

related

(ATsEH)

19,960 133 5 24 1 – – – – x – – P/M – ER – – –

At4g24190 Heat shock

protein

90 (SHD)

94,146 677 15 20 4 x – x – x x – P Low ER – ER, Nuc,

Plast, Mito

–

At2g47470 Protein

disulfide

isomerase

(PDIL2-1)

39,472 723 16 40 2 – – – – x x – – High ER – ER ER

At1g08450 Calreticulin

3 (CRT3)

49,813 134 6 14 1 – – – – x – – P – ER – P-Mem ER


2 (CRT2)

48,127 318 14 23 1 – – – – x – – P – ER – ER, Mito,

P-Mem

ER

At1g21750 Protein

disulfide

isomerase

(PDIL1-1)

55,567 208 6 13 1 – – – – x – – P – ER – ER, Plast ER


(CRT1))

48,497 199 7 20 3 – – – x x x – P Low ER – ER, Plast,

Mito

ER

At1g77510 Protein

disulfide

isomerase

(PDIL1-2)

56,329 313 12 25 1 – – – – x – – P – – – ER, Plast ER

At2g29960 Peptidyl-

prolyl

cis-trans

isomerase

(CYP5)

21,520 69 5 17 1 – – – – – x – – High – ER ER Cyt

Eubel et al.




participate in all of the classical peroxisomal pathways,such as fatty acid and JA b-oxidation, reactive oxygenspecies detoxification, carbon and nitrogen metabo-lism, and organelle biogenesis (Table I). Interestingly,all of the proteins involved in the peroxisomal steps ofthe photorespiratory pathway have been identifiedhere in cell cultures, even though they are not greenand were grown in the dark for 7 d prior to peroxisomepreparation. A total of 35 proteins are reported here inperoxisomes, to our knowledge, for the first time (TableI). These include a series of isoforms of proteins par-ticipating in well-characterized peroxisomal pathwaysthat are predicted to possess different substrate spec-ificities. Interestingly, these isoforms are often morehydrophobic than their counterparts previously char-acterized by MS, based on comparison of their pre-dicted number of transmembrane domains (Table I).Examples are a member of the 4-coumarate-CoA ligase(4CL)-like proteins (At1g20480), PEX11c, and PEX11e,

indicating that the broadness of the experimental ap-proach helps to identify such proteins. This is alsosupported by the identification of several proteins withunknown function that are reported here, to our knowl-edge, for the first time and that include a number ofputative membrane carrier proteins with four or moretransmembrane domains.

In the following paragraphs, we discuss differentclasses of this newly identified set of 35 proteins; wealso discuss classes of reported peroxisome proteinsnot found here, the close association of peroxisomeswith the ER, and the challenge of defining the widerperoxisome proteome in plants.

Acyl-Activating Enzymes and Acyl-CoA Oxidases

Our data reveal a variety of acyl-activating enzymes(AAEs) in cell culture peroxisomes that provide thepoint of entry for many substrates into the b-oxidation

Figure 6. Visualization of peroxisome metabolic functions based on the proteins identified in this study. Colored nodes (roundedsquares) represent enzymes in different functional categories as shown in the key, metabolites are shown as small gray circles, andreactions are shown as connecting lines between the enzyme and metabolite nodes. The transportability of metabolites across theperoxisome membrane is shown as indicated in the key based on published reports covering plant and nonplant species.





pathway. These proteins are known to possess a certaindegree of substrate specificity. In addition to the two4CL family proteins identified by Reumann et al. (2007;At1g20510 and At4g05160), an additional member ofthis protein family has been identified (At1g20480),which may not be expressed in leaves (Koo et al., 2006).In our study, this protein was identified by five differentapproaches (Table I), strongly suggesting a significantabundance of this protein. At1g20510 and At4g05160are expected to participate in JA biosynthesis, asAt1g20510 preferentially activates OPC 8:0 (Koo et al.,2006) while At4g05160 prefers OPC 6:0. They are alsoreported to accept medium- to long-chain fatty acids assubstrates. These results have been confirmed in acomparison of substrate specificity of 4CL-like proteins(Kienow et al., 2008). None of the tested substratesseemed to suit At1g20480, whose function in the con-text of substrate activation for b-oxidation still remainsunclear. Other new AAEs include At5g16340, whichshares 92% sequence identity with the previouslyfound At5g16370, AAE17 (At5g23050), At1g20560,and LACS6 (At3g05970), a long-chain fatty acid CoAligase (Shockey et al., 2002) targeted to peroxisomes(Fulda et al., 2002). For some of these enzymes, nosubstrate has been identified and their predicted func-tions have only been deduced by amino acid sequencecomparisons. Many of these enzymes with knownfunctions have partially overlapping substrate speci-ficities, according to the literature (Kienow et al., 2008).

Acyl-CoA oxidases (ACXs) perform the next step inb-oxidation after acyl activation, and four of the sixknown isoforms of this enzyme in Arabidopsis havebeen identified in this study (At4g16760, ACX1;At1g06290, ACX3; At3g51840, ACX4; and At2g35690,ACX5). Only one of these was found by MS in pub-lished reports (Table I). Although these enzymes partlyoverlap in their substrate specificity, they appear toprefer different types of substrates, which they feedinto the b-oxidation process. ACX1 preferentially usesmedium- to long-chain fatty acids and seems to be themost important ACX for JA synthesis (Schilmiller et al.,2007). ACX3 and ACX4 prefer medium- or short-chainFAs. All three are reported to be involved in theb-oxidation of IBA (Adham et al., 2005), although todiffering degrees. Using the GENEVESTIGATOR da-tabase (Zimmermann et al., 2004), the expression pro-files of all six ACXs were compared in different plantorgans. ACX6 is expressed most strongly in root tip,ACX1 in sepal, ACX5 in stamen, and ACX2 and ACX3in endosperm but also strongly in seed, imbibed seed,and embryo, which is consistent with their role inmetabolizing lipids in energy metabolism in oilseeds.The transcript for ACX4 is lowly expressed in mosttissues, but we have detected the protein by five dif-ferent approaches, and it has also been found in leafperoxisomes (Reumann et al., 2007). Interestingly,ACX4 is the most divergent protein within the ACXfamily, as it is missing an oxidase domain (Adham et al.,2005). Expression patterns of ACX2, ACX3, and ACX4were found to cluster during development as well as in

their distribution between plant organs in our analysisof GENEVESTIGATOR data (Zimmermann et al.,2004). ACX2 and ACX6 (which may be a pseudogene,as no transcripts have been reported) were not found inthis study. In ACX2 knockout line seeds, long-chainfatty acids accumulate (Pinfield-Wells et al., 2005),indicating together with its expression pattern thatthis protein is mainly involved in the breakdown ofstorage lipids. Why ACX2 appears to be absent or inlow abundance in cell culture, while ACX3 is present, iscurrently unclear.

In contrast to the AAEs and ACXs, the overlap inidentification of all other core enzymes involved inb-oxidation is much higher between studies reportingperoxisome proteomes from different plant tissues.This may indicate that AAEs and ACXs regulate sub-strate flow into the more generic b-oxidation pathway.A comparison of protein abundance of these enzymesfrom different plant organs or different developmen-tal stages will probably shed more light on the differ-ent functions they fulfill in priming substrates forb-oxidation.

Transporters and Integral Membrane Proteins

Four proteins have been identified that fit clearly intothe category of integral membrane proteins: At2g39970(PMP38), At5g27520, At4g39850 (CTS/PXA/PED3),and At4g04470 (PMP22). All four proteins were foundonly in the peroxisomal membrane fractions, are pre-dicted to possess three or more transmembrane do-mains, and lack known PTS1 and PTS2 sequences fortargeting.

PMP38 (At2g39970) contains functional domainssimilar to those found in the mitochondrial carrierfamily (MCF). A homolog of PMP38 in pumpkin hasbeen localized in the peroxisomal membrane by im-munohistochemistry (Fukao et al., 2001); but in Arabi-dopsis, PMP38 has been, most likely erroneously,detected in the vacuole (Jaquinod et al., 2007). PMP38is a clear homolog of the yeast peroxisomal PMP47 thatwas shown many years ago to be a peroxisomal mem-ber of the yeast MCF (McCammon et al., 1990). Thesubstrate for PMP38 in plants is currently not clear, butpossible candidates are ADP/ATP, 2-oxoglutarate/ma-late, phosphate, or tricarboxylates. The yeast equiva-lent is clearly an ADP/ATP transporter but may alsofunction in DpH formation (Lasorsa et al., 2004). Theclosest Arabidopsis homolog to PMP38 is annotated asa plastidic folate transporter (At5g66380) discovered bycomplementation of folate-deficient hamster cells andbacteria. However, knockout of At5g66380 in Arabi-dopsis did not change folate concentration in the plas-tids, suggesting either a different function in plant cellsor the presence of alternative folate transporters inplastids (Bedhomme et al., 2005). The phosphorylationof PMP38 at Ser-155 (Supplemental Data Set S3) mayindicate that it could be regulated by a phosphoryla-tion/dephosphorylation event. Phosphoproteomics ofyeast mitochondria identified phosphorylation of the

Eubel et al.




main ATP/ADP transporter (AAC1) at Ser-155/Thr-156 or Ser-157. Alignment of the two proteins revealedbroad but low-level sequence similarity, as both areMCF members, but the phosphorylation sites are innearly identical locations within the hydrophilic loopbetween the third and fourth transmembrane domainsof both proteins (Supplemental Data Set S3).

The product of At5g27520 is an unknown functionmember of the MCF and is most closely related insequence to mitochondrial ADP/ATP transporters andthe peroxisomal ADP/ATP transporter identified inyeast. The designation of this carrier as a peroxisomalcarrier is strengthened by its identification here in threeseparate experiments (Table I) and the absence of itsidentification in our focused work to identify mito-chondrial carriers of this type from mitochondria iso-lated from this same cell culture material (Millar andHeazlewood, 2003). We also tested At5g27520 for per-oxisomal localization using transient expression of aGFP fusion protein, confirming its peroxisomal loca-tion in vivo (Fig. 4A).

At4g39850 (CTS/PXA/PED3) possesses an ATP-binding cassette domain and is considered a fullATP-binding cassette transporter, containing 12 trans-membrane domains and acting as a monomer. There isstill some controversy as to its exact mechanistic func-tion and its corequirement for acyl-CoA synthetasescompared with the more clear-cut evidence for half-ATP transporters in acyl-CoA entry to peroxisomes inyeast (Visser et al., 2007). However, it appears from arange of genetic studies that CTS has a role in the entryof a broad range of fatty acid substrates into plantperoxisomes. CTS knockout mutants are resistant toIBA or 4-(2,4-dichlorophenoxy)butyric acid, indicat-ing that the transport of both compounds is facilitatedinto peroxisomes by this protein (Theodoulou et al.,2005). Seed lipid metabolism also seemed to be af-fected by the knockout of this protein, suggesting apotential role of CTS in the import of fatty acids intoperoxisomes (Footitt et al., 2002). Additionally, CTSmutants are JA-deficient and therefore most likely havea reduced import rate of 12-oxo-phytodienoic acid, aplastid-derived JA precursor molecule. However, CTSdoes not seem to be the only route by which 12-oxo-phytodienoic acid is transferred into the organelle;alternative routes via different, unidentified trans-porters or diffusion pathways might exist (Theodoulouet al., 2005).

At4g04470 (PMP22) is an integral membrane proteinthat possesses similarities to known mammalian andyeast peroxisomal proteins (Tugal et al., 1999). But de-spite a series of reports showing that its loss of functionis detrimental in mouse and yeast (Zwacka et al., 1994;Trott and Morano, 2004), relatively little is known aboutits biological function. The most likely roles proposedare as a nonselective membrane channel and/or as areactive oxygen species forming protein in the perox-isome membrane (Visser et al., 2007).

There has been a report recently of the molecularidentification and cloning of the peroxisomal channel

protein in the grass Bromus inermis (Wu et al., 2005).Such a channel has been predicted in a range of speciesto allow free movement of compounds of less than 300D across the membrane, but it has not previously beenidentified in any species (Visser et al., 2007). TheArabidopsis homolog of this protein is an OEP16-likeprotein, At4g16160, but we found no evidence for thisprotein in the membranes of our peroxisomes. Re-cently, GFP localization of this OEP16 homolog inArabidopsis indicated that it localizes to the plastid(Murcha et al., 2007), further discounting the likeli-hood that it is a peroxisomal membrane channel.

Overall, if these identified transporters represent thebulk of the transport functions on mature peroxisomes,then the metabolite pools and needs for exchange (Fig.6) could be understood by the following scenario.Mature peroxisomes may have a static population ofcofactors such as CoA and pyridine nucleotides such asNAD1 and NADP1, which explains their impermeabil-ity to these reagents. Import of fatty acids would bemediated by CTS, while the two MCF-type carrierscould provide a broad entry for organic and aminoacids, ATP/ADP, and inorganic phosphate, and somesmall molecules may diffuse. This would be consistentwith the known properties/substrates of other MCFsand the CTS and might provide for many of the knowntransport properties of plant peroxisomes.

PEX Proteins

Four of the five PEX11 family proteins that have apredicted role in peroxisome proliferation (Orth et al.,2007) in Arabidopsis have been found in this study(PEX11a, -c, -d, and -e). Three of these peroxins(PEX11c, -d, and -e) form a distinct group within theArabidopsis PEX11 genes, while PEX11a and PEX11beach represent a group on their own. While overex-pression of PEX11a and PEX11b resulted merely inelongation of peroxisomes, the overexpression ofPEX11c, -d, and -e led to the induction of a completedivision process. PEX11c, -d, and -e were also the onlyperoxins able to complement the yeast pex11 knockoutmutant (Orth et al., 2007). Only PEX11d was found byMS in the previous proteome study by Reumann et al.(2007) in leaf peroxisomes. As cell culture is a rapidlydividing tissue, peroxisomal biogenesis and prolifera-tion may be enhanced, which might explain the relativeabundance of the PEX11, PEX7, and PEX14 proteinsobserved here. PEX11b, which has not been detectedby us, was recently shown to be involved in light-dependent regulation of peroxisome proliferation (Desaiand Hu, 2008) during seedling morphogenesis andtherefore is most likely of low abundance in dark-growncell culture. Controversy persists about the precise mo-lecular role of PEX11, because despite genetic evidencefor roles in biogenesis and organelle proliferation (Orthet al., 2007), specific isoforms of these membrane pro-teins have also been implicated in metabolic functionsupstream of b-oxidation, most likely in fatty acid acti-





vation or fatty acid transport in yeast (Hettema andTabak, 2000; van Roermund et al., 2000).

Nucleotide Pools and Redox Shuttling in Peroxisomes

The relative importance of putative organic acid/amino acid shuttles to move reductant in or out of theperoxisome versus an internal balance of reducing andoxidizing reactions remains unclear. This is in large partdue to the lack of information about the composition ofperoxisomes and their reductive and oxidative catalyticactivities. A series of known NAD(H)- or NADP(H)-dependent peroxisomal enzymes were found (Table I)and are clustered in Figure 6 based on their common useof these peroxisomal nucleotide pools. The NAD1 clus-ter surrounds malate dehydrogenase, and the NADP1

cluster surrounds isocitrate dehydrogenase. In additionto the enzymes of known substrate and products shownin Figure 6, a series of reductases were found in Table Ithat likely link to these pools. For example, we founddienoyl-CoA reductase proteins. In yeast, this is knownto be an NADPH-dependent enzyme and has beenconvincingly shown to be supplied with NADPH byisocitrate dehydrogenase through genetic studies (vanRoermund et al., 1998). Other reductases thought to beinvolved in the b-oxidation of unsaturated substrates(Table I) were also found in leaf peroxisomes (Reumannet al., 2007), but their specificities for nucleotides areunknown. We also found three putative quinone reduc-tases grouped in the reducing metabolism section ofTable I, and one of these was also found in leaf perox-isomes (Reumann et al., 2007). All three have PTS1signals (Table I) and may link soluble nucleotide redoxpools with membrane redox pools of quinone or otherunknown reductants. These identifications broaden thepossibilities for redox pool regulation in peroxisomesfrom the classical malate/oxaloacetate and malate/Aspshuttles proposed, based on metabolic models of perox-isome function (Visser et al., 2007).

Proteins Previously Identified in Peroxisomes That Are

Missing in This Study

Interestingly, of the seven proteins associated withprotection against pathogen attack and herbivoresfound in leaf peroxisomes (Reumann et al., 2007),none was found in our analysis. The lack of these pro-teins in the cell culture peroxisomes is striking, as in allother areas of peroxisomal metabolism, including pho-torespiration, a large overlap could be observed. Leaf-specific expression of the corresponding genes isnot evident in GENEVESTIGATOR microarray data(Zimmermann et al., 2004). One of the putative de-fense proteins (OZI1; At4g00860) has previously beenfound in the analysis of the mitochondrial proteome(Heazlewood et al., 2004) and on blue-native/SDS gelsof Arabidopsis mitochondrial membranes. The latterdata suggest an association of OZI1 with the cyto-chrome c oxidase complex (complex IV) of the plantrespiratory chain (Millar et al., 2004); subsequently, the

protein was named COX-X2. This protein does notpossess a PTS, and targeting prediction programs donot suggest a peroxisomal location, so in our view thedata point toward it being a mitochondrial protein. Theother six putatively defense-related proteins claimed asperoxisomal (Reumann et al., 2007) have previouslybeen identified in an analysis of the leaf vacuole pro-teome (Carter et al., 2004), and most are predicted tohave secretory signals or ER signal peptides. Anothernotable absence from the peroxisomes analyzed hereare the glyoxylate cycle enzymes isocitrate lyase andmalate synthase. This clearly shows that these cellculture organelles cannot be considered to be catalyz-ing a glyoxylate cycle.

Apparently Nonperoxisomal Proteins inPeroxisome Preparations

The vast majority of proteins that apparently arenonperoxisomal in our preparations have a mitochon-drial origin. Given that mitochondria are probably themost abundant organelles present in dark-grown cells,that they migrate very close to the peroxisomes duringFFE, and that they are widely considered the majorcontaminant in yeast and mammalian peroxisomeproteome analyses (Kikuchi et al., 2004; Marelli et al.,2004; Saleem et al., 2006), this is not surprising. Ourongoing investigation of the Arabidopsis mitochon-drial proteome (Millar et al., 2005), facilitated by tech-nical advances in MS and the increase in the purity ofmitochondrial isolations by FFE (Eubel et al., 2007),enables a very precise assignment of mitochondrialproteins in our overall nonredundant protein list. Thesubtraction of mitochondrial proteins is supported inthe vast majority of cases by the quantitative datagenerated in the course of this study, namely throughDIGE and comparative LC-MS/MS. Contaminationsby cellular compartments other than mitochondria aresignificantly harder to confidently exclude. This isespecially true in the case of the ER proteins identifiedhere, such as calreticulin, calnexin, HSP90, SEC12p,and the lumenal chaperones BIP1 and BIP2. While it isbeyond doubt that these proteins are primarily found inthe ER, it is harder to tell whether this group alsorepresents bona fide proteins in peroxisomal prepara-tions or if they are merely contaminating proteins dueto a copurification of ER vesicles with the peroxisomes.Similar dilemmas have been presented a number oftimes in the literature about separation of the ER andperoxisome proteomes in other species (Saleem et al.,2006, and references therein). The resolution to thesequestions may be tightly linked to peroxisomal biogen-esis and the import of peroxisomal proteins, especiallythose inserted into the membrane of these organelles.An involvement of the ER in the import of proteins intoperoxisomes has been proposed for PEX15 in yeast(Elgersma et al., 1997) and for PEX10 and PEX16 inArabidopsis cell suspension cultures (Flynn et al., 2005;Karnik and Trelease, 2005), although PEX10 has alsobeen reported to be targeted to peroxisomes without a

Eubel et al.




passage through the ER (Sparkes et al., 2005). BIP hasbeen found in the same gradient fraction as APX inpumpkin (Nito et al., 2001). The involvement of the ERwith peroxisomal biogenesis and protein import hasbeen reviewed (Mullen and Trelease, 2006), and ricino-somes emerging from the ER were found to contain BiPand PDI protein (Schmid et al., 2001). Our own GFPlocalization studies did not support the presence of theanalyzed ER proteins inside peroxisomes in vivo (TableII; Fig. 4; Supplemental Data Set S1). The presence ofthese proteins in the peroxisomal samples might beexplained by the close interaction of these two com-partments observed in our GFP images (Fig. 5; Supple-mental Data Set S1) and the potential for isolatingperoxisomes tethered to ER vesicles.

On balance, the data are more in favor of contam-ination of the peroxisomes with a small amount of ERmaterial, either due to comigration in FFE (Fig. 5A) ordue to physical association between ER fragments andperoxisomes (Fig. 5C), but probably not due to thepresence of ER proteins within the peroxisome mem-brane or lumen.

The Complexity of Defining the FullPeroxisomal Proteome

Despite substantial efforts of three separate groupsworking on different Arabidopsis tissue types (Fukaoet al., 2002, 2003; Reumann et al., 2007; Table I), less than20% of the proteins with predicted PTS sequences(Reumann et al., 2004) have been experimentally con-firmed in peroxisomal preparations. In the process,each group has found a range of proteins that lack PTSsignals, suggesting that bioinformatics predictions willbe of limited help. The task of experimentally confirm-ing the complete peroxisomal proteome in Arabidopsismay still be some way off. While 11 proteins (photo-respiratory enzymes, catalases, and malate dehydro-genases) have been found in two or three studies(Table I), significant discrepancies exist between thelists of peroxisomal proteins from our analysis andthose from other Arabidopsis tissues. Reumann et al.(2007) claimed 29 proteins as peroxisomal that were notconfirmed in our study; only four have recognizablePTS sequences (At1g06460, At1g21770, At1g60550, andAt5g48880; Supplemental Table S6). Fukao et al. (2002,2003) claim an entirely separate set of 29 proteins thatwere not confirmed in this study (Supplemental TableS6); only one has a recognizable PTS, malate synthase(At5g03860), and most of the others have currentlyunclear functions in peroxisomes. This study claims 35proteins not found in any of the other studies of leaf orcotyledon peroxisomes (Table I); 21 contain PTS1 orPTS2, and most are involved in fatty acid oxidation. Inaddition, six are transmembrane proteins that wouldnot have a clear PTS. More quantitative data andassessment of targeting are necessary to resolve ifeach of these proteins lacking independent confirma-tion are peroxisomal in a range of plant tissue types.

Further advances in peroxisome purification are re-quired so that 10 to 100 mg of peroxisomes can beisolated from Arabidopsis tissues to allow suborganellefractionation, so effective for improving protein iden-tification in other organelles. However, many proteinshave now been found in two or three independentstudies (Table I). This gives a solid foundation fordetailed analysis of peroxisomal function in plants, as itis likely that many of the major metabolic pathways cannow be reconstructed (Fig. 6).

MATERIALS AND METHODS

Cell Culture Maintenance

An Arabidopsis (Arabidopsis thaliana) suspension cell culture has been

maintained and subcultured as stated elsewhere (Millar et al., 2001). Briefly, 20

mL of a 7-d-old cell culture grown in the light was transferred into 100 mL of

fresh medium. Starting material for the peroxisome preparation was grown in

the dark for 7 d, whereas material used for the maintenance of the culture was

incubated in the light for the same period of time.

Organelle Preparation

The preparation of the organelles was based on Eubel et al. (2007), with some

modifications. Approximately 200 g of cells was used for each preparation.

After enzymatic digestion of the cell wall, the resulting protoplasts were

disrupted by four strokes in a Potter-Elvehjem homogenizer. The homogeni-

zation medium contained bovine serum albumin and EDTA as general protease

inhibitors; in addition, Complete (Roche Applied Science) protease inhibitor

cocktail was used according to the manufacturer’s instructions to inhibit Ser,

Cys, and metalloproteases. The homogenate was centrifuged at 3,000g for 5 min

to remove cell debris. The supernatant was spun at 24,000g for 10 min to

concentrate the organelles. However, in order to avoid pelleting of the organ-

elles, the supernatant of the low-speed spin was layered on top of 5 mL of a

Percoll cushion (60% [v/v] Percoll, 10 mM MOPS-KOH, pH 7.2). The organelle-

containing interphase was taken and diluted 1:1 with wash buffer and loaded

onto discontinuous Percoll density gradients consisting of 5 mL of 50% (v/v)

Percoll and 25 mL of 25% (v/v) Percoll (bottom to top) in wash buffer. After

centrifugation, the mitochondrial/peroxisomal band was found between the

50% (v/v) and the 25% (v/v) Percoll phases. This band was extracted, and

the Percoll was removed by three repeated washes in FFE separation buffer. The

first two washes were performed using a 60% (v/v) Suc cushion (60% [v/v] Suc,

10 mM MOPS-KOH, pH 7.2) at the bottom of the tubes, again to prevent

pelleting of the organelles. The third wash was performed without any cushion,

and the organelles were pelleted. After resuspension in a small volume of FFE

separation buffer (2 mL), the organelle suspension was slowly homogenized in

a Potter-Elvehjem homogenizer in preparation for FFE.

FFE buffer composition and conditions were similar to those described

previously (Eubel et al., 2007) with the exception of the use of either 800 Vor 600

V in experiments as indicated. Using the higher voltage, mitochondria and

plastids were deflected to such an extent that the plastids were running into the

anode stabilization medium, whereas the mitochondria just stayed within the

borders of the separation medium. Although a certain amount of mixing

between those two organelles occurred, the distance between the mitochondria

and the peroxisomes increased, which led to a higher level of purity of the

peroxisomal fraction. After visual inspection of the 96-well plate, mitochon-

drial fractions (contaminated with plastids) and peroxisomal fractions were

pooled separately. The mitochondria were then subjected to a second round of

FFE at 600 Vin order to obtain purer material for the subsequent measurements.

Oxygen Electrode Measurements

Catalase activity was measured using a Clark-type oxygen electrode

(Hansatech) as outlined previously (Eubel et al., 2007). Succinate dehydro-

genase measurements were performed using a Clark-type oxygen electrode in

the presence of 10 mM succinate, 500 nmol of ADP, and 100 nmol of ATP. Fifty

micrograms of protein was used for each assay.





1D SDS-PAGE, 2D IEF/SDS-PAGE, DIGE,

and Immunoblotting

These techniques were performed according to Eubel et al. (2007). For the

preparative 2D gels, 300 mg of protein was used. Primary antibodies to

calreticulin (1:1,000; Agrisera), HSP70 (1:2,000; PM003 from Dr. Tom Elton),

and KAT2 (1:5,000; Germain et al., 2001) were visualized with horseradish

peroxidase-conjugated secondary antibodies (1:10,000) and chemiluminescent

detection of the signal.

dSDS-PAGE

Aliquots of membrane and integral membrane proteins extracted from

peroxisome samples were separated according to Meyer et al. (2008) on 10%

(w/v) Tricine-SDS-PAGE containing 6 M urea. After the migration, gel strips

corresponding to each sample were cut and incubated in acidic solution (100

mM Tris, 150 mM HCl, pH , 2). The strips were then loaded on top of a 16%

(w/v) Tricine-SDS-polyacrylamide gel. The gap between the strip and the gel

was filled with a 10% (w/v) acrylamide mixture. After electrophoresis, the

gels were stained with colloidal Coomassie Brilliant Blue.

Phosphopeptide Isolation

The procedure essentially follows the method outlined by Bodenmiller et al.

(2007). A 1-mg organellar protein pellet was solubilized in 100 mL of 8 M urea, 3

mM EDTA, and 20 mM Tris-HCl, pH 8.0. The sample was reduced with 25 mM

DTT for 1 h at room temperature followed by alkylation of thiols with 50 mM

iodoacetamide for 1 h at room temperature. The solution was diluted to

approximately 1 M urea with 20 mM Tris-HCl, pH 8.0, used to hydrate 25 mg of

trypsin (Invitrogen), and incubated at 37�C overnight. Peptides were desalted

on Bio-Select reverse-phase C18 extraction columns (Grace Vydac) and dried in

a vacuum concentrator. Peptides were reconstituted in 400 mL of solution

containing 50% (v/v) acetonitrile, 2.5% (v/v) trifluoroacetic acid (TFA), and 20

mg mL21 2,5-dihydroxybenzoic acid. This solution was added to 5 mg of

Titanosphere TiO 5 mm (GL Science) equilibrated with 50% (v/v) acetonitrile,

2.5% (v/v) TFA, and 20 mg mL21 2,5-dihydroxybenzoic acid. The slurry was

rotated for 30 min at room temperature in a mobicol spin column (MoBiTec),

then washed twice with the 50% (v/v) acetonitrile, 2.5% (v/v) TFA, and 20 mg

mL21 2,5-dihydroxybenzoic acid solution, twice with 50% (v/v) acetonitrile

and 0.1% (v/v) TFA, and twice with a 0.1% (v/v) TFA solution. Phosphopep-

tides were eluted from the Titanosphere chromatographic material using a

freshly prepared 0.3 M NH4OH solution. Eluted phosphopeptides were dried in

a vacuum desiccator and resuspended in 5% (v/v) acetonitrile and 0.1% (v/v)

formic acid prior to analysis. Phosphopeptides were identified in peptide

mixture analysis by 6510 Q-TOF LC-MS/MS (Agilent Technologies) as outlined

below.

MS Analysis of Peptides

Colloidal Coomassie Brilliant Blue-stained protein spots where cut from

gels and destained twice in 10 mM Na2HCO3 with 50% (v/v) acetonitrile.

Samples were dried at 50�C before being rehydrated with 15 mL of digestion

solution (10 mM NH4CO3 with 12.5 mg mL21 trypsin [Invitrogen] and 0.01%

[v/v] TFA) and incubated overnight at 37�C. Peptides produced from tryp-

sination were twice extracted from gel plugs using 15 mL of acetonitrile. The

supernatant was then collected, and plugs were washed twice with 15 mL of

50% (v/v) acetonitrile and 5% (v/v) TFA and combined with the initial

supernatant. The pooled extracts were dried by vacuum centrifugation and

stored at 4�C before being analyzed by MS using an Agilent XCT Ultra IonTrap

(Agilent Technologies) mass spectrometer. The mass spectrometer was fitted

with an electrospray ionization (ESI) source equipped with a low-flow nebu-

lizer in positive mode and controlled by Chemstation (Rev B.01.03; Agilent

Technologies) and MSD Trap Control software Version 6.1 (Bruker Daltonik).

Peptides were eluted from a self-packed Microsorb (Varian) C18 (5 mm, 100 A)

reverse-phase column (0.5 3 50 mm) using an Agilent Technologies 1100 series

capillary liquid chromatography system at 10 mL min21 using a 9-min aceto-

nitrile gradient (5%260%, v/v) in 0.1% (v/v) formic acid at a regulated

temperature of 50�C. The method used for initial ion detection utilized a mass

range of 200 to 1,400 mass-to-charge ratio (m/z) with scan mode set to standard

(8,100 m/z s21), ion charge control conditions set at 250,000, and three averages

taken per scan. Smart mode parameter settings were employed using a target of

800 m/z, a compound stability factor of 90%, a trap drive level of 80%, and

optimize set to normal. Ions were selected for MS/MS after reaching an

intensity of 25,000 cps, and two precursor ions were selected from the initial MS

scan. MS/MS conditions employed SmartFrag for ion fragmentation, a scan

range of 70 to 2,200 m/z using an average of three scans, the exclusion of singly

charged ions option, and ion charge control conditions set to 200,000 in ultra

scan mode (26,000 m/z s21). Resulting MS/MS spectra were exported from the

DataAnalysis for LC/MSD Trap Version 3.3 (Build 149) software package

(Bruker Daltonik) using default parameters and 1,000 compound maxima for

AutoMS(n) and compound export. The .mgf files generated were then analyzed

as outlined below.

Protein samples were also analyzed with a nongel approach, using peptide

mixture LC-MS/MS analysis. The protein extracts were digested overnight at

37�C with trypsin (10:1), and insoluble components were removed by centrifu-

gation at 20,000g for 5 min. The supernatant was then dried by vacuum

centrifugation and stored at 4�C before being analyzed by MS. Samples were

analyzed on an Agilent 6510 Q-TOF mass spectrometer (Agilent Technologies)

with an HPLC Chip Cube source. The chip consisted of a 40-nL enrichment

column (Zorbax 300SB-C18 5 u) and a 150-mm separation column (Zorbax

300SB-C18 5 u) driven by the Agilent Technologies 1100 series nano/capillary

liquid chromatography system. Both systems were controlled by MassHunter

Workstation Data Acquisition for Q-TOF (Version B.01.02, Build 65.4, Patches

1,2,3,4; Agilent Technologies). Peptides were resuspended in 5% (v/v) aceto-

nitrile and 0.1% (v/v) formic acid and loaded onto the trapping column at 4 mL

min21 in 5% (v/v) acetonitrile and 0.1% (v/v) formic acid with the chip

switched to enrichment and using the capillary pump. After the sample volume

passed through the enrichment column five times, the chip was then switched

to separation and peptides were eluted from the enrichment column and run

through the separation column during a 1-h gradient (15% to 60% [v/v]

acetonitrile) directly into the mass spectrometer. The mass spectrometer was

run in positive ion mode, and MS scans were run over a range of m/z 275 to 1,500

and at four spectra per second. Precursor ions were selected for auto MS/MS at

an absolute threshold of 500 and a relative threshold of 0.01, with a maximum of

three precursors per cycle, and active exclusion set at two spectra and released

after 1 min. Precursor charge-state selection and preference were set to 21 and

then 31, and precursors were selected by charge and then abundance.

Resulting MS/MS spectra were opened in MassHunter Workstation Qualita-

tive Analysis (Version B.01.02, Build 1.2.122.1, Patches 3; Agilent Technologies),

and MS/MS compounds were detected by Find Auto MS/MS using default

settings. The resulting compounds were then exported as mzdata.xml files and

searched as outlined below.

Data Analysis

Output files were analyzed against an in-house Arabidopsis database

comprising ATH1.pep (release 7) from The Arabidopsis Information Resource

and the mitochondrial and plastid protein sets (The Arabidopsis Information

Resource). This sequence database contained a total of 30,700 protein sequences

(12,656,682 residues). Searches from ion trap data were conducted using the

Mascot search engine Version 2.1.04 (Matrix Science) utilizing error tolerances

of 61.2 D for MS and 60.6 D for MS/MS, Max Missed Cleavages set to 1, the

Oxidation (M) and Carboxymethyl (C) variable modifications, and the Instru-

ment set to ESI-TRAP and Peptide charge set at 21 and 31. Results were filtered

using standard scoring, maximum number of hits set to 20, significance

threshold at P , 0.05, and ion score cutoff at 0. Searches from Q-TOF data

were conducted using the Mascot search engine Version 2.1.04 (Matrix Science)

utilizing error tolerances of 6100 ppm for MS and 60.5 D for MS/MS, Max

Missed Cleavages set to 1, the Oxidation (M) and Carboxymethyl (C) variable

modifications, and the Instrument set to ESI-Q-TOF and Peptide charge set at

21 and 31. Results were filtered using MUDPITscoring, maximum number of

hits set to 20, significance threshold at P , 0.05, and ion score cutoff at 0. Protein

matches were only claimed if at least two distinct peptides were detected per

protein, resulting in MOWSE scores typically higher than 70 (P , 0.05

significance level is score . 37).

IDDA

Following an initial run as outlined above in peptide mixture analysis, the

resulting mzdata.xml files were searched as outlined in ‘‘Data Analysis.’’ The

resulting matches were then filtered by an Ion Score setting of 37, and all

peptides with ion scores of 37 or greater were exported from MASCOT along

with their respective peptide charge into a .csv file. This file was then used to

Eubel et al.




construct an exclusion list, based on peptide (m/z) and charge (z). Isolation

width was set to medium approximately 4 m/z, precursor type was set to

Exclude, retention time was set to 0, and Dm/z was set to 100 ppm. This table was

then loaded into the MassHunter Workstation Data Acquisition for Q-TOF

(Version B.01.02, Build 65.4, Patches 1,2,3,4; Agilent Technologies) software,

and then the next sample was run. Following the second run, the new list of

excluded peptides was added to the previous list and the new list was loaded

for the third run. This process was repeated for the fourth run also. Following

acquisition of data from all four runs, the resulting mzdata.xml files were

concatenated into a single mzdata.xml file using mzdataCombinator Version

1.0.4 (University of Western Australia Centre of Excellence for Computational

Systems Biology; http://www.ce4csb.org/software.shtml). The resulting files

were then used to search an in-house Arabidopsis database as outlined above.

GFP Localization Studies

GFP fusions were constructed according to a modification of the fluorescent

tagging of full-length proteins (FTFLP) method (Tian et al., 2004). FTFLP

introduces GFP into genomic clones (including native promoters and termi-

nators) in a stretch of hydrophilic amino acids in a position corresponding to

approximately 10 amino acids from the C terminus. In its original context,

FTFLP is claimed to represent a good first pass for spatial and temporal

localization of proteins for which no other information is available (Tian et al.,

2004; Tzafrir et al., 2004) and is suggested to be a good alternative to

constructing both N- and C-terminal fusions (Goodin et al., 2007). Tian et al.

(2004) present proof of principle for marker proteins targeted to numerous

organelles, including MFP2, which was directed to peroxisomes. A number of

studies have specifically used FTFLP to preserve the N-terminal targeting

signal and the C-terminal retention motifs (Chen et al., 2005; Li et al., 2005;

Drakakaki et al., 2006; Anand et al., 2007; Goh et al., 2007; Blakeslee et al., 2008;

Gallavotti et al., 2008). We adapted the method for high-throughput analysis of

full-length proteins via a transient expression assay. For many of the proteins

examined here, there were either no clear predicted targeting signals or

potentially conflicting N- and C-terminal signals. Thus, GFP was still inserted

10 to 13 amino acids from the C terminus, but this was done using coding

sequence alone and driven by the 35S promoter for transient expression in

onion (Allium cepa) cell epidermal peel and Arabidopsis cell culture. The

position of 10 amino acids from the C terminus largely avoids structural and

functional protein domains and preserves both N- and C-terminal targeting

sequences in full-length proteins (Tian et al., 2004). The aim of this approach

was to avoid masking potential N- or C-terminal targeting signals with GFP

and to reduce the chance that blocking the biologically relevant signal might

allow cryptic, physiologically meaningless signals to take precedence (Simpson

et al., 2001; Tian et al., 2004; Goodin et al., 2007; Pottosin and Schonknecht, 2007).

GFP5 was amplified including adapters as described (Tian et al., 2004). The

portion of the gene corresponding to the N terminus of the protein was

amplified using appropriate primers (primers P1 and P2). Templates for

amplification were cloned cDNAs if available (ordered from the Arabidopsis

Biological Resource Center if available) or total cDNA. Oligonucleotides P3 and

P4, corresponding to the C-terminal 10 to 13 amino acids plus the stop codon of

the protein (i.e. 33–42 nucleotides), were synthesized to constitute a template

for direct inclusion in a second round of PCR. Primers P1 and P4 included

adapters that partially overlapped with attB Gateway cloning sequences, while

primers P2 and P3 had adapters that overlapped with those bounding the GFP

fragment; the adapters were as described by Tian et al. (2004). Three-template

PCR thus included three overlapping templates (the P1-P2 PCR fragment, GFP,

and the P3-P4 oligonucleotides). Three-template PCR products were cloned

into Gateway vector pDONR207 and sequenced to check PCR accuracy. The

GFP constructs were then introduced into a pGREEN vector modified for

Gateway. We used pGREEN0179 containing cauliflower mosaic virus 23 35S

promoter and cauliflower mosaic virus terminator with the Gateway A cassette

inserted between them in the SmaI site of the pGREEN multiple cloning site. For

colocalization studies, pGREEN0049-RFP-SRL (Pracharoenwattana et al., 2005)

was used as a peroxisomal marker. Plasmids were precipitated onto 1-mm gold

particles and biolistically transformed into Arabidopsis cultured cells or onion

epidermal peel as described by Thirkettle-Watts et al. (2003). Fluorescence

images were obtained using an Olympus BX61 epifluorescence microscope

with HQ GFP (U-MGFPHQ) and RFP (U-MRFPHQ) filters and CellR software.

Alternatively, a Leica TCS SP2 AOBS multiphoton confocal microscope was

used with laser excitation of GFP at 488 nm and RFP at 561 nm. Emission

collection was in the ranges of 500 to 550 nm and 570 to 700 nm, respectively.

Confocal images were captured using Leica confocal software.

Network Analysis and Visualization

AraCyc (www.arabidopsis.org/biocyc/; Zhang et al., 2005) is a database of

biochemical pathways for Arabidopsis. Over 90% of the pathways in the

current release of AraCyc (4.5) have been manually curated with experimental

data. Proteins identified in this study were associated with biochemical

reactions in AraCyc release 4.5 by downloading from the Plant Metabolic

Network FTP server (ftp://ftp.plantcyc.org/Pathways/) the tab-delimited

text files ‘‘aracyc_compounds.20080611’’ and ‘‘aracyc_dump.20080611.’’ For

each AGI code in Table I, pathway names, EC numbers, and enzyme names that

have been associated by AraCyc with that AGI code were extracted from

the ‘‘aracyc_dump’’ file. Biochemical reactions and their compounds were

extracted from the lines in the ‘‘aracyc_compounds’’ file, which contained both

the pathway name and the EC number associated with each AGI code. A total of

44 of the AGI numbers in our peroxisome set shown in Table I were assigned EC

numbers in this manner, making a nonredundant set of 28 enzyme nodes and 64

metabolites. It should be noted that multiple distinct reactions, corresponding

to differing substrates, can be assigned to a single EC number in AraCyc. These

often occur when an EC number occurs in the context of separate biochemical

pathways. Therefore, we extracted only those biochemical reactions for which

both the EC number and the pathway name matched those associated with an

AGI code, to avoid incorporating metabolites that may only be involved in

pathways outside of the peroxisome.

After the recovery of these data, the set of unique EC numbers and bio-

chemical reactions was parsed to generate a Simple Interaction Format file to

represent a metabolic network. The Simple Interaction Format file and other

data, such as AGI codes associated with EC numbers, enzyme names, and node

types (enzyme or metabolite), were inputted into the Cytoscape software

(Version 2.6.0; Shannon et al., 2003) for network visualization and analysis.

Network images were exported from Cytoscape as .svg files, imported into

Adobe Illustrator, and modified visually for presentation purposes.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. 2D IEF/SDS-PAGE gels and spots annotated

that were analyzed by MS.

Supplemental Figure S2. dSDS-PAGE gels and spots annotated that were

analyzed by MS.

Supplemental Table S1. A, Catalase and succinate dehydrogenase activ-

ity assays. B, Gel spot identifications by MS of protein bands from

Figure 1C.

Supplemental Table S2. Gel spot identifications by MS of gel spots from

Supplemental Figure S1 and the corresponding DIGE ratios from

Figure 2.

Supplemental Table S3. A, Gel band identifications by MS for Figure 3. B,

Gel spot identifications by MS for Supplemental Figure S2 (dSDS).

Supplemental Table S4. Complex peptide mixture MS analysis by IDDA

of digested whole peroxisomes of two different purities.

Supplemental Table S5. Complex peptide mixture MS analysis by IDDA

of peroxisome membrane sample.

Supplemental Table S6. Proteins reported in peroxisomes by Reumann

et al. (2007) and Fukao et al. (2002, 2003) but not found in Table I.

Supplemental Data Set S1. Images of fluorescent tagging of full-length

proteins, supporting Table I and Table II GFP claims.

Supplemental Data Set S2. Single peptide mass spectral analysis sup-

porting the data in Table I.

Supplemental Data Set S3. Phosphopeptide analysis of PMP38.

Received September 16, 2008; accepted October 10, 2008; published October

17, 2008.

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