Supplemental Table 1: Known proteins of plant peroxisomes identified in isolated leaf peroxisomes from Arabidopsis.

Supplemental Table 1: Known proteins of plant peroxisomes identified in isolated leaf peroxisomes from Arabidopsis. Acronym Protein Gene locus Size (aa; MW) pi Approach of protein identification PTS1/2 Gelbased Shotgun Photorespiration GOX1 Glycolate oxidase isoform 1 At3g1441 367; 40.3.0 yes yes PRL> GOX2 Glycolate oxidase isoform 2 At3g14420 367; 40.3.1 yes yes ARL> SGT Serine-glyoxylate aminotransferase At2g13360 401; 44.2 7.8 yes yes SRI> HPR Hydroxypyruvate reductase At1g68010 386; 42.2 7.2 yes yes SKL> GGT1 Glutamate-glyoxylate aminotransferase isoform 1 At1g23310 481; 3.3 6. yes yes SKM GGT2 Glutamate-glyoxylate aminotransferase isoform 2 At1g7080 481; 3.4 6.6 yes yes SRM pmdh1 Perox. malate dehydrogenase isoform 1 At2g22780 34; 37. 7. yes yes RIx HL pmdh2 Perox. malate dehydrogenase isoform 2 Atg0660 34; 37.4 8.0 yes yes RIx HL ROS metabolism CAT1 Catalase isoform 1 At1g20630 42; 6.8 7.4 n.d. yes (mpts) CAT2 Catalase isoform 2 At4g300 42; 6. 7.1 yes yes (mpts) CAT3 Catalase isoform 3 At1g20620 42; 6.7 7.6 yes yes (mpts) APX3 Perox. ascorbate peroxidase isoform 3 At4g3000 287; 31.6 7.0 yes yes mpts MDAR1 Monodehydroascorbate reductase isoform 1 At3g2880 434; 46. 6.4 yes n.d. AKI> CSD3 Cu/Zn superoxide dismutase Atg18100 164; 16. 7.6 yes yes AKL> Core fatty acid β-oxidation LACS7 Long-chain acyl-coa synthetase isoform 7 Atg27600 771; 77.1 6.8 yes n.s. 2 SKL> ACX4 Acyl-CoA oxidase isoform 4 At3g1840 436; 47.6 8.3 yes yes SRL> AIM1 Abnormal fluorescence meristem 1 At4g2010 721; 77..3 yes yes SKL> MFP2 Multifunctional protein 2 At3g06860 72; 78.8.2 n.d. yes SRL> PED1/KAT2 Peroxisome Defective 1 At2g3310 462; 48.6 8.6 yes yes RQx HL PKT1/KAT1 Thiolase isoform 1 At1g04710 443; 46.6 8.3 n.d. yes RQx HL PKT2/KAT Thiolase isoform 2 Atg48880 47; 43.2 7.0 yes yes RQx HL Glyoxylate cycle CSY3 Citrate synthase 3 At2g4270 0; 6.2 7.7 yes yes RAx HL ASP3 Aspartate aminotransferase Atg1120 44; 4.0.3 n.d. yes RIx HL Auxiliary β-oxidation enzymes DCI Δ 3, -Δ 2,4 - dienoyl-coa isomerase Atg43280 278; 2. 7.6 yes n.d. AKL> ECH2 Enoyl-CoA hydratase 2 At1g7610 30; 34.1 7.0 yes n.s. 2 SSL> SCP-2 Sterol carrier protein isoform 2 Atg4280 123; 13.6.2 yes yes SKL> ACH2 Acyl-CoA thioesterase At1g01710 427; 48.2 7.2 yes n.d. SKL> AAE7/ACN1 Acyl-activating enzyme isoform 7 At3g1610 6; 63.0 8.4 n.d. yes SRL> JA biosynthesis OPR3 Oxophytodienoic acid isoform 3 At2g0600 31; 42.7 7.7 yes yes SRL> 4CLP1 4-Coumarate-CoA ligase-like protein 1 At4g0160 44;. 8.6 n.d. yes SKM> OPCL1 OPC-8:0 ligase 1 At1g2010 46;.4 8. yes n.d. SKL>

N- and S- metabolism URI Uricase At2g26230 30; 34. 8.6 yes yes SKL> SOX Sulfite oxidase At3g0110 33; 43.4 8.7 n.d. yes SNL> Chaperones ACD31.2-Px α-crystallin domain containing protein of 31.2 kda At1g06460 28; 31.2.3 yes yes RLx HF Biogenesis proteins PEX11d PEX11d At2g4740 236; 2. 10.0 yes n.d. n.d. PEX14 PEX14 Atg62810 07;.6 6.0 n.d. yes n.d. For the 36 known plant peroxisomal proteins identified in this study, acronym, gene locus, PTS1/2 or internal PTS (mpts, e.g. CAT1-3), polypeptide size of the longest variant (in amino acid residues, aa, and molecular weight, MW), isoelectric point (pi), additional information on predicted targeting signals, and the method used for protein identification (2-D gel based or shotgun approach) are provided. The values for MW and pi were calculated by EXPASY (www.expasy.ch/). When proteins were represented by only a single peptide in the shotgun approach, it is indicated whether this was sufficient (yes 1 ) or not significant (n.s. 2 ) for protein identification. Transmembrane domains (TMD) were predicted according to Lee et al. (2006, localizome.org/) and Krogh et al. (2001, http://www.cbs.dtu.dk/ services/tmhmm/). n.d., not detected; n.s., not significant References: Krogh, A., Larsson, B., von Heijne, G., and Sonnhammer, E. L. L. (2001) Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. J. Mol. Biol. 30: 67-80. Lee, S., Lee, B., Jang, I., Kim, S., and Bhak, J. (2006b). Localizome: a server for identifying transmembrane topologies and TM helices of eukaryotic proteins utilizing domain information. Nucl. Acids. Res.34: W-103.

Supplemental Table 2. MS data of proteins identified. Acronym Gene locus Size (aa; MW) pi Gel-based approach Shotgun approach PMF Sequence Coverage (%) PMF Peptide sequenced MS/MS Peptides Score score a ion score b 4CLP1 At4g0160 44;. 8.6 - - K.DPNTSLVSFLFR.N K.LAIADSDTGDSLTFSQLK.S 4.8 2.7 6PGDH At3g02360 486; 3.6 7.0 4 12 0 WTVQQAAELSVPAPTIESSLDAR 32 AAE Atg16370 2; 60.8 6. 32 88 FSYTYDDLIHR 4 TVGFTEIDVVDPESGR 3 AAE7/ACN1 At3g1610 6; 63.0 8.4 - - K.IPANYTALTPLWFLDR.A R.YTGM*EQLDVIDTQTGKPVPADGK.T K.ETFAGGWFHSGDIAVK.H 4.8 3.80 3.07 ACAT2 Atg48230 403; 41.4 6. 68 1 6 GIAAQEAGAFTWEIVPVEVSGGR EQQDDYAVQSFER 72 67 ACD31.2 At1g06460 28; 31.2.3 46 70 DNVSAEFMDGILR QASSAQGFFMR 1 22 K.LANLGTVWSPR.S R.SNVAESTHSYVVAIELPGASINDIR.V K.DNVSAEFM*DGILR.I 3.12 4.8 3.7 ACH2 At1g01710 427; 48.2 7.2 2 41 ILQLDPLELNIFR 2 ACX4 At3g1840 436; 47.6 8.3 4 14 3 ELLGGNGILADFLVAK AFCDLEPIYTYEGTYDINTLVTGR 8 30 R.ELLGGNGILADFLVAK.A R.EVTGIASFKPATR.S 4.4 2.6 AIM1 At4g2010 721; 77..3 43 18 AQLGLPELTLGVIPGFGGTQR FSGGFDINVFQQVHK 62 17 R.FSGGFDINVFQQVHK.T K.AQLGLPELTLGVIPGFGGTQR.L K.LGLIDALVPPGDVLSTSR.K R.IVGAHFFSPAHLM*PLLEIVR.S K.LSETYGSFFKPSR.Y 4.23 4.62 2.38 4.77 3.04 APX3 At4g3000 287; 31.6 7.0 6 16 MGLSDKDIVALSGGHTLGR FDNSYFVELLK 140 88 K.FDNSYFVELLK.G K.LSELGFNPNSSAGK.A 3. 2.14 ASP3 Atg1120 44; 4.0.3 - - R.TEEGKPLVLNVVR.K 2.04

K.EYLPIVGLVEFNK.L K.TIYITQPTWGNHPK.I 4.2 3.03 ATF1 At1g21770 111; 12.6 6.4 2 63 FETDDHEAFIEYK IVWNEGR 77 22 BADH At3g48170 03; 4.. 61 21 3 BGL1 At1g2400 28; 60. 7.2 4 18 7 NEGATVLCGGVRPEHLK R.QLFIGGQWTEPVLR.K e) 3.13 CENHNADVAVDFYHR CSPYIPGYGQHCQDGR 8 68 K.VGVQFYHDLIDELLK.N K.HWITFNEPWVFSR.A R.SGYEAYQVSHNLLLSHAYAVDAFR.N K.GSTDYVGM*NYYTSVFAK.E 4.6 3.28. 4.0 BSMDR/ARP At1g4670 62; 67..0 - - K.IIYAGVNASDVNFSSGR.Y d) 4.1 CAT1 At1g20630 42; 6.8 7.4 - - R.GPILLEDYHLLEK.L R.GPGVQTPVIVR.F R.EGNFDLVGNNFPVFFVR.D K.FPDM*VHALKPNPK.S K.SHIQENWR.I K.IWPEDILPLQPVGR.L R.LGPNYLQLPVNAPK.C R.SIWISYWSQADK.S 3.18 2.67 3.26 2.8 2.64 2.61 4.4 4.3 CAT2 At4g300 42; 6. 7.1 6 1 3 EGNFDLVGNNFPVFFIR GFFEVTHDISNLTCADFLR TWPEDILPLQPVGR 86 78 R.GPILLEDYHLVEK.L R.EGNFDLVGNNFPVFFIR.D R.HM*DGSGVNTYM*LINK.A R.VGGTNHSHATQDLYDSIAAGNYPEWK.L K.TWPEDILPLQPVGR.M 4.6 4.18 4.16 4.73 3.0 CAT3 At1g20620 42; 6.7 7.6 61 2 8 EGNFDLVGNNTPVFFIR GFFEVTHDISNLTCADFLR 12 100 K.YRPSSAYNAPFYTTNGGAPVSNNISSLTIGER.G R.GPVLLEDYHLIEK.V R.APGVQTPVIVR.F R.EGNFDLVGNNTPVFFIR.D K.TNIQEYWR.I R.HM*EGFGVHTYTLIAK.S K.VPTPTNSYTGIR.T K.RWVEILSEPR.L R.WVEILSEPR.L R.GIWISYWSQADR.S R.LNVRPS.I 4.76 3. 2.8 3.3 2.7 3.71 2.2 3.04 3.23 3.04 2.42 CSD3 Atg18100 164; 16. 7.6 86 13 0 HAGDLGNILAGSNGVAEILIK HIPLSGQYSILGR 10 1 K.HIPLSGQYSILGR.A R.HAGDLGNILAGSNGVAEILIK.D R.AVALIAGDNNVR.G 2.4 4.07 3.40 CSY3 At2g4270 0; 6.2 7.7 48 17 0 LYPNVDFYSGLIYR LYDPGYLNTAPVR 76 74 K.APTIAAAAYLR.M K.LYPNVDFYSGLIYR.A 3.63 2.7

DCI Atg43280 278; 2. 7.6 4 8 EVDLAIVADLGTLQR LPSIVGYANAMELALTAR 38 17 ECH2 At1g7610 30; 34.1 7.0 44 80 LSGDYNPLHSDPEFAK 31 K.TVLAGYVDIR.G e) 3.12 ECHIa At4g16210 26; 28.8.0 - - R.FGIFPSWGLSQK.L R.EVSLTSM*PLTADVAGK.L 2. 3.23 ECHIb At4g14430 240; 2.8 8.4 3 14 FHPDTIATILSLLEQAK SLYPELCGILGLETR 100 46 R.FHPDTIATILSLLEQAK.S K.FFSNGFDLAWAQTAGSK.T 3.03 3.23 ECHIc At1g620 240; 2. 6.2 60 11 3 LNPTLLDSLR EVLIHTIGEYESGSSVVR 4 3 EH3 At4g02340 324; 36.7 6.3 2 14 VNALVNTSVVFNPR ALFGDDYYICR 8 66 ESM1 At3g14210 32; 44.1 7.6 42 12 ANPNADASAQQAFVTNVINR SYFFFDGR 88 34 GGT1 At1g23310 481; 3.3 6. 61 21 2 AMVIINPGNPTGQCLSEANIR HYLSLTSGGLGAYSDSR 118 112 K.ALDYDTLNENVK.K K.KIIFTNVGNPHALGQKPLTFPR.Q K.IIFTNVGNPHALGQK.P K.IIFTNVGNPHALGQKPLTFPR.Q K.HYLSLTSGGLGAYSDSR.G R.DGYPSDPELIFLTDGASK.G K.LVLLGDEVYQQNIYQDER.P K.LVLLGDEVYQQNIYQDERPFISSK.K K.KVLM*EM*GSPFSK.E K.VLM*EM*GSPFSK.E K.EVQLVSFHTVSK.G R.GGYFEM*TNLPPR.V R.LPTGALQAAK.Q K.LLEATGISTVPGSGFGQK.E R.TTILPAEDEM*PEIM*DSFK.K R.TTILPAEDEM*PEIM*DSFKK.F K.FNDEFM*TQYDNNFGYSK.M 2. 6.03 4.81 6.08 4.83 4.46 4.4 4.67 3.01 3.30 2. 2.41 3.07 4.0 3.82 2.6.6 GGT2 At1g7080 481; 3.4 6.6 6 18 1 AMVIINPGNPTGQCLSEANIR HYLSLTSGGLGAYSDSR 118 112 K.EVQLISFHTVSK.G R.GGYFEM*TNIPPR.T K.FNDEFM*SQYADNFGYSR.M 3.3 2.41 4.73 GOX1 At3g1441 367; 40.3.0 7 21 0 IAIQAGAAGIIVSNHGAR VPVFLDGGVR 138 7 -.M*EITNVTEYDAIAK.A K.M*VYDYYASGAEDQWTLQENR.N R.ILIDVNKIDM*ATTVLGFK.I K.IDM*ATTVLGFK.I K.ISM*PIM*VAPTAFQK.M K.M*AHPDGEYATAR.A 3.20 6.24. 3.4 4.03 2.

GOX2 At3g14420 367; 40.3.1 64 16 R.AASAAGTIM*TLSSWATSSVEEVASTGPGIR.F R.FFQLYVYK.N IAIQAGAAGIIVSNHGAR 138 R.ILIDVSKIDM*TTTVLGFK.I K.IDM*TTTVLGFK.I K.ISM*PIM*VAPTAM*QK.M K.M*DEANDSGLASYVAGQIDR.T R.TLSWKDVQWLQTITK.L K.DVQWLQTITK.L K.DVQWLQTITKLPILVK.G R.QLDYVPATISALEEVVK.A R.IPVFLDGGVR.R R.IPVFLDGGVRR.G K.ALALGASGIFIGR.P R.PVVFSLAAEGEAGVR.K R.NHITTEWDTPR.P R.NHITTEWDTPRPSAR.L 4.66 3.07 3.11 3.40 4.03.6 4.4 3.61.00 4.74 3.00 2.7 4.63 3.27 2.4 3.37 GOX3 At4g18360 368; 40. 8.3 - - R.ATSAAGTIMTLSSWATCSVEEVASTGPGIR.F d) 2.26 GR At3g24170 4; 3. 6.4 47 12 GVNLHPQTSLTQLTK TNIPSIWAVGDATNR 4 22 GRP7 At2g21660 176; 16.. 4 8 CFVGGLAWATDDR 7 R.ALETAFAQYGDVIDSK.I R.GFGFVTFKDEK.A R.SITVNEAQSR.G R.EGGGGYGGGEGGGYGGSGGGGG.W GRP8 At4g3260 16; 16.6.6 4 6 SGGGGGYSGGGGGGYSGGGGGGYE R 77 R.TFSQFGDVIDSK.I R.VITVNEAQSR.G R.EGGGYGGGDGGSYGGGGGG.W GSTT1 Atg41210 24; 27.6. - - K.SLSTLETFWLK.G K.ATMPHFDETHEILFK.V HBCDH At3g120 24; 31.7 6.6 44 60 TTVCSQDYAGFVVNR 4 n.d. 4.66 2.63 3.4 6. 2.88 3.33.23 3.4 2.14 HMGDH Atg4340 37; 40.7 6. 46 VEPGSNVAIFGLGTVGLAVAEGAK AAVAYEPNKPLVIEDVQVAPPQAGEVR 83 13 n.d. HPR At1g68010 386; 42.2 7.2 71 23 6 TILSVEDIIDLIGDK YGIAVGNTPGVLTETTAELAASLSLAAA R 108 33 K.PVSIEVYNPNGK.Y K.AFSNM*AVGYNNVDVEAANK.Y K.YGIAVGNTPGVLTETTAELAASLSLAAAR.R R.IVEADEFM*R.G R.GGLYEGWLPHLFVGNLLK.G R.M*M*VEGFK.M K.M*NLIYFDLYQSTR.L K.FVTAYGQFLK.A R.EADLISLHPVLDK.T R.GPVIDEAALVEHLK.E R.GPVIDEAALVEHLKENPM*FR.V 3.6 4.7.40 2.70 3.62 2.26.21 2.40 3.16 4.4 3.6

R.VGLDVFEEEPFM*K.P R.VGLDVFEEEPFM*KPGLADTK.N K.NAIVVPHIASASK.W R.EGM*ATLAALNVLGR.V K.GYPIWHDPNR.V K.ALGLPVSK.L 2. 2.34 2.80 2.66 2.1 2.4 IDH At1g4340 416; 47.2 7.6 1 6 LIFPFLELDIK YFDLGLPNR 40 1 LACS7 Atg27600 771; 77.1 6.8 37 13 4 YEHLGQLCNDPR LIVVVGGADEHLPSLPR 32 23 K.AVTLVPEPFTLENGLLTPTFK.I e) 2.83 MDAR1 At3g2880 434; 46. 6.4 43 10 METE1 Atg1720 76; 84.4 6.1 42 1 2 YQTLIIATGSTVLR 38 YGAGIGPGVYDIHSPR GGIGVIQIDEAALR 38 34 MFP2 At3g06860 72; 78.8.2 41 13 n.d. n.d. R.FSGGFDISGFGEM*QK.G R.ISAPAAQLGLPELQLGVIPGFGGTQR.L K.ALEM*ILTSKPVK.A R.IVGAHFFSPAHIM*PLLEIVR.T 3.2 3.13 2.23 4.77 MIF At3g1660 112; 12.2 8.8 60 72 ELIATVGSILHTHFSIHPTR 103 NBP At4g1666 146; 16.8.1 - - R.HYLVIPK.E K.EHIPTVNDLQR.R 2.08 2.11 NS/ECHId At1g600 337; 37.1 7.3 48 1 8 LNVLDLQVQIR 7 K.VGSFDAGYGSSIM*SR.L K.M*GLINTVVPLEDLEK.E 3.84 3.14 OASS A1 At4g14880 322; 33.8. 72 13 6 YLSTVLFDATR LFVAIFPSFGER 36 1 OPCL1 At1g2010 46;.4 8. 3 20 8 TPIPLPPNPSLDVTTFISSQAHR TGDLCYIDEDGFIFVVDR 68 61 OPR3 At2g0600 31; 42.7 7.7 6 10 7 ALNGVPNAALAEYYAQR AGFDGIEIHGAHGYLIDQFLK 67 27 R.AGFDGIEIHGAHGYLIDQFLK.D K.QVVEGVVSAIGASK.V R.M*AYNGTFM*SSGGFNK.E K.TFYTQDPVVGYTDYPFLAPFSR.L 4.66 3.48 3.84.32 OZI1 At4g00860 80; 8.6.0 - - R.VAGPPVVMNPISR.Q R.VAGPPVVM*NPISR.Q 3.03 2.12 PED1/KAT2 At2g3310 462; 48.6 8.6 70 172 TNLNPSEVGDIVVGTVLAPGSQR GLPVLGVFR 100 70 R.TSLYGDDVVIVAAHR.T R.GNFKDTYPDDLLAPVLR.A K.DTYPDDLLAPVLR.A 3.0 3.0 2.

K.TNLNPSEVGDIVVGTVLAPGSQR.A R.MAAFYAGFPETVAVR.T R.M*AAFYAGFPETVAVR.T K.AGFYDIGIGAGLESMTTNPMAWEGSVNPAVK.K K.GLPVLGVFR.T R.TFAAVGVDPAIMGIGPAVAIPAAVK.A K.INVNGGAMAIGHPLGATGAR.C 4.62 4.63 3.82.88 2.17 6.04.72 PEX11d At2g4740 236; 2. 10.0 32 41 FVNDLHGLISPVPK AELALVVMYLNK YQDEDYR 3 17 14 PEX14 Atg62810 07;.6 6.0 - - R.VSQEMMITK.N K.IYSADQEVYNGSVTTAR.K K.SYMDIMSMIQR.G R.STDFGYETTTAAR.F R.FTANQNETSTMEPAAFQR.Q R.SWVPPQPPPVAMAEAVEAIR.R PKT1/KAT1 At1g04710 443; 46.6 8.3 - - K.DTFPDELLASVLR.A K.TNVNPSEVGDIVVGTVLGPGSQR.A 2.36.04 3.4 4.00 4.0 3.3 2.18 3.3 PKT2/KAT Atg48880 47; 43.2 7.0 6 1 VNVNGGAIAIGHPLGATGAR VAAYFAGFPDSVPVR 0 4 K.DTLPDDLLASVLK.A R.AGYYDIGIGAGVESM*STDHIPGGGFHGSNPR.A K.VNVNGGAIAIGHPLGATGAR.C 2.3 4.0.3 pmdh1 At2g22780 34; 37. 7. 3 12 1 DDLFNINAGIVR CGIDEVYGLGPLNEYER 74 8 K.VAILGAAGGIGQPLAMLM*K.M R.GFLGQPQLEEALTGM*DLVIIPAGVPR.K K.AIVNIISNPVNSTVPIAAEVFK.K K.AIVNIISNPVNSTVPIAAEVFKK.A K.LM*GVTM*LDVVR.A R.IQNGGTEVVEAK.A 3.84 4.60 4.8 4.22 3.1 3.34 pmdh2 Atg0660 34; 37.4 8.0 33 68 TGAEEVYQLGPLNEYER ANTFVAEVLGLDPR 46 37 K.VAILGAAGGIGQSLSLLM*K.M K.QLEDALTGMDLVIIPAGIPR.K K.QLEDALTGM*DLVIIPAGIPR.K K.CCPNAIVNLISNPVNSTVPIAAEVFK.K K.CCPNAIVNLISNPVNSTVPIAAEVFKK.A K.LLGVTTLDVAR.A R.ANTFVAEVLGLDPR.E K.PPSSFTPQEIEYLTNR.I K.AGAGSATLSMAYAAAK.F K.AGAGSATLSM*AYAAAK.F R.TGAEEVYQLGPLNEYER.I K.AKDELAGSIQK.G.3 2.33.00.3.13 3.76 4.30 4.47 3.34 4.44 4.6 3.73 PYK10 At3g0260 24;.7 6. 37 10 GVSQAGVQFYHDLIDELIK 62 R.SGYEAYLVTHNLLISHAEAVEAYR.K d).3 6 RBP At4g1720 360; 38. 8.7 40 8 DGGAAAQAPTPAIGDSAQFPTLGK 81

NNDGPANENGYGGGYR 7 RH11 At3g810 612; 66.0 6.2 3 23 8 SGIATAFFNENNAQLAR VVVAYGGTPIHQQLR 38 20 RH37 At2g4220 633; 67.6 6.6 38 1 VVVAYGGTPINQQLR LAADFLANYIFLAVGR 21 c) 21 SCP-2 Atg4280 123; 13.6.2 4 81 KLGFEEVTYIVDLK IGLVYQINIAPK 86 K.IGLVYQINIAPK.K K.VDATFSFKDDDFVK.V 3.64 4.24 SDRa At4g030 24; 26.8 8. 74 12 VAIVTASTQGIGFGITER VNAVAPGFVPTHFASFITGSSEVR 116 8 R.FGLEGASVVVSSR.K R.VNAVAPGFVPTHFASFITGSSEVR.E 3.3 3.0 SDRb/(DECR At3g12800 28; 31.8.2 60 SLGIQAIGLEGDVR GQVALITGGGSGIGFEISSQFGK 28 8 K.HGASIAIM*GR.R e) 2.3 SDRc At3g0180 266; 2.2 7.1 63 14 VQDILQVSQDEFHR ALYPGADAYASTSAAIHQLVR 4 K.LVM*M*GNEGSLR.S e) 2.63 SDRd At3g20 280; 30.2 8. 61 12 2 GGSVINISSIAGIR IDALINNAGIR 3 14 SGT At2g13360 401; 44.2 7.8 76 17 4 AALDLIFEEGLENIIAR HHLFVPGPVNIPEPVIR 146 113 R.HHLFVPGPVNIPEPVIR.A R.SPAIPALTK.T K.TTSGTPFLFPTTGTGAWESALTNTLSPGDR.I R.M*DEWGVDVALTGSQK.A K.VFFDWNDYLK.F K.LGTYWPYTPSIQLLYGLR.A K.EEWISNTVTAVM*VPPHIDGSEIVR.R R.YNLSLGLGLNK.V 3.72 2.73 3.34.22 3.12 4.0 4.33 3.32 SOX At3g0110 33; 43.4 8.7 - - R.SALVSSYVTPVDLFYK.R K.LADVLELVGIPK.L K.ASITLSQATNPEADVLLAYEM*NGETLNR.D K.AVDSAANVQPENVESVWNLR.G R.GVLNTSWHR.V 4.78 2.3 4.4 3.3 2.40 ST2 At1g0420 1; 16. 7. 22 31 IPPHLLNAGK 41 TGG1 Atg26000 41; 61.1.6 48 17 2 GFIFGVASSAYQVEGGR NWITINQLYTVPTR 3 13 TGG2 Atg280 47; 62.7 7. 3 GINEDGINYYSGLIDGLIAR 3 TLP Atg8220 324; 3.6 6.2 74 14 SGPLMDLVDALNPGTYR SRPPITTHVLDVSR 3

UP1 At1g2410 7; 84.4 4.6 36 18 4 ELSHLLADASEAYEEYHGAVR LEFEAHHNSINQAGSDGVK 88 4 UP2 At3g10 772; 8.0 4.6 36 16 DLVNHPNFDAIIAAGTR FTSLTTSLSELLK 7 66 UP3 At2g31670 263; 28. 6. 0 82 FPGIDQITVGENFSPAR EDLASYAAHPDHVR ISSATAFTHVLHSR 73 6 38 UP4 At1g0340 378; 42.6 8.2 46 13 4 AGGFPEPEIVHYNPK SSLSAEGFDVVYDINGR 4 48 URI At2g26230 30; 34. 8.6 61 13 FKDDVYLPTDEPHGSIEATLSR VLMDTFFGPPETGVYSPSVQR 8 3 K.SGALNLTSGIGGLALLK.T K.FKDDVYLPTDEPHGSIEATLSR.I 3.18 4.3 a) Mascot protein score obtained for the peptide mass fingerprint (PMF). The significance threshold was 60. b) Mascot MS/MS ion scores obtained for the individual peptides sequenced. The significance threshold was 2-32 depending on how many peptides fell within the mass tolerance window about the precursor mass. Only the top ranking peptides matching a query for the first time ("bold red hits") are reported. c) RH37 was accepted as identified on the basis of two peptide matches indicating at least significant homology (threshold 21) though not identity (threshold 2). d, e) When proteins were represented by only a single peptide in the shotgun approach, it is indicated whether this was, upon manual inspection, significant ( d) ) or not significant ( e) ) for protein identification. In the shotgun peptides oxidized methionines within are labelled by an asterics (*), and proteolytic peptide cleavage sites with a dot, indicating fully or incomplete tryptic peptides. Isoform-specific peptides of peroxisomal isoforms sharing high sequence similarity are underlined to indicate specific identification of the respective protein isoform. n.d., not detected

Supplemental Table 3. Sequence conservation of SDR domains in peroxisomal SDRs. Nucleotide binding region Predicted Co-factor binding site Active site residues Pattern 1 Pattern 2 Pattern 3 TGxxxGhG hnhxg GxhhxhSSh Yx[AS][ST]K SDRa At4g030 TAxxxGhG NADP (7 to 4) SDRb/DECR At3g12800 TGxxxGhG NADP (pos. 8 to 0) hnhxs SxhhxhTSh YxVTK hdhxg GxhhxhSAT VxAAK SDRc At3g0180 n.d. n.d. hnhxa GxhhxhATh YxSTS SDRd At3g20 TGxxxGhG NADP (pos. 16 to 8) BSMDR/ARP At4g030 TGxxxGhG NAD (pos. 2 to 44) hnhxg GxhhxhSSh YxCSK hdhxa GxhhxhGSh YxASK The sequences of five peroxisomal SDRs were aligned using kalign (Lassmann and Sonnhammer, 200) and the conservation of active site residues involved in co-factor and substrate binding determined as compared to classical SDR motifs (Kallberg et al., 2002). Co-factor prediction was performed by the general Rossmann fold predictor (http://www.ifm.liu.se/bioinfo/services/, Kallberg and Persson, 2006). The amino acid residues of the catalytic triad (Ser, Tyr, Lys) are underlined. Amino acid residues altered in peroxisomal SDRs are shaded gray. References: Kallberg, Y., Oppermann, U., Jornvall, H., and Persson, B. (2002). Short-chain dehydrogenases/reductases (SDRs). Eur. J. Biochem. 26: 440-4417. Kallberg, Y., and Persson, B. (2006). Prediction of coenzyme specificity in dehydrogenases/reductases. A hidden Markov model-based method and its application on complete genomes. FEBS J. 273: 1177-1184. Lassmann, T., and Sonnhammer, E.L.L. (200). Kalign - an accurate and fast multiple sequence alignment algorithm. BMC Bioinformatics 6: 28.

Supplemental Table 4. Cloning, subcloning, and mutagenesis primers. Acronym Gene locus Vector with insert Primer ( to 3 ) 6PGL Atg24400 6PGL in pgemt for: CACCAAGACTGCGGCCGCTATGGCTACGACAAATATAGG rev: TGCTATCTAGAGCGGCCGCTTAGAGCTTGGAAGATGCTGG EYFP-PTD 6PGL in pcat rev: TATATCTAGATTAGAGCTTGGAAGATGCTGGTTTGTCCAGGAACTTGTACAGCTCGTCCATGCC 6PGDH At3g02360 6PGDH in pgemt for: CACCAAGACTGCGGCCGCTATGGCTGTTCAACCTACAAG rev: TGCTATCTAGAGCGGCCGCTCAGATCTTAGATTGTCTTGC EYFP-PTD 6PGDH in pcat rev: TATATCTAGATCAGATCTTAGATTGTCTTGCAATCTTGAACCACTTGTACAGCTCGTCCATGCC ATF1 At1g21770 ATF1 in pcat for1: TTAAATGGCGACGGGGACGGCGACGGAGAAACCGAAGATCGTGTGGAACGAAGGAAGAC for2: GAATAAGATTGCGGCCGCTATGGCGACGGGGACGGCGAC EYFP-PTD ATF1 in pcat rev: TGCTCTAGATCAGATGCTAGACTTGAGG rev: TATATCTAGAtcagatgctagacttgaggttcttggaatcatCCTTGTACAGCTCGTCCATGCC ECH2 At1g7610 ECH2 in pgemt for: AAGATTGCGGCCGCTATGGCGACTAGCGATTCTG ECHIb At4g14430 ECHIb in pgemt rev: TATATCTAGACTAAAGTGACGAAGATAAC for: AAGATTGCGGCCGCTATGTGTACGTTAGAGAAACG rev: TATATCTAGATTAGAGCTTAGGTGTTGCAAAC EYFP-PTD ECHIb in pcat rev: TATATCTAGATTAGAGCTTAGGTGTTGCAAACACTCTAGTCTCCTTGTACAGCTCGTCCATGCC EH3 At4g02340 EH3 in pgemt for: AAGATTGCGGCCGCTATGGAGAAGATAGAGCACAC rev: TATATCTAGACTACAAGGATGCGGTTTCGCG EYFP-PTD EH3 in pcat rev: TATATCTAGACTACAAGGATGCGGTTTCGCGGGTTCTGAATTTCTTGTACAGCTCGTCCATGCC GSTT1 Atg41210 GSTT1 in pgemt for: AAGATTGCGGCCGCTATGATGAAGCTCAAAGTG rev: TATATCTAGATTAGATCTTGGATTGAAGACCCGGTTTAGATAACTTGTACAGCTCGTCCATGCC EYFP-PTD GSTT1 in pcat rev: TATATCTAGATTAGATCTTGGATTGAAGACCCGGTTTAGATAACTTGTACAGCTCGTCCATGCC HBCDH At3g120 HBCDH in pgemt for: AAGATTGCGGCCGCTATGGCGGAGATGAAGAGTG rev: TATAGAGCTCTCAGAGCCGCGGAGACAGC EYFP-PTD HBCDH in pcat rev: TATATCTAGATCAGAGCCGCGGAGACAGCTTCTGTGTAGCTTCCTTGTACAGCTCGTCCATGCC IDH At1g4340 IDH in pgemt for: CACCAAGACTGCGGCCGCTATGGAATTCGAGAAGAT rev: TGCTATCTAGAGCGGCCGCTCACAACCTGGAGTTGTTGCCTA EYFP-PTD IDH in pcat rev: TATATCTAGATCACAACCTGGAGTTGTTGCCTAACAATCTTCTCTTGTACAGCTCGTCCATGCC EYFP-PTD ECH2 in pcat rev: TATATCTAGACTACAAGGATGCGGTTTCGCGGGTTCTGAATTTCTTGTACAGCTCGTCCATGCC

SDRb/DECR At3g1270 SDRb in pgemt for: AAGATTGCGGCCGCTATGGACTCTCCGTTCAAACC rev: TATATCTAGACTACAGCTTGCTGGTTGGG EYFP-PTD SDRb in pcat rev: TATATCTAGACTACAGCTTGCTGGTTGGGAGACCAACAGGCTTCTTGTACAGCTCGTCCATGCC TLP Atg8220 TLP in pgemt for: AAGATTGCGGCCGCTATGGCGATGGAGATCGGAG rev: TATATCTAGACTAGCTCCCACGGTATGTG EYFP-TLP(H/D) for: GATCGTCTGAGAATCATTGGAGGGGATTTGAATGTTGCAGCTGAAGCAAAAG rev: CTTTTGCTTCAGCTGCAACATTCAAATCCCCTCCAATGATTCTCAGACGATC