Supplemental Table S1: Peroxisomal matrix proteins of Arabidopsis suitable for bioinformatics specification of peroxisome targeting signals (PTS).

Arabidopsis proteins that have been localized to the matrix of plant peroxisomes or represent the orthologs of known peroxisomal proteins were retrieved from the database based on sequence similarity (http://www.ncbi.nlm.nih.gov/BLAST/; non-redundant database; matrix BLOSUM62). It was next investigated if homologous proteins that correspond to the peroxisomal isoforms could unambiguously be identified in the protein database by a maximum degree of sequence similarity. For three proteins (LACS7, SOD, and peroxisomal Hsp70), peroxisomal and non-peroxisomal isoforms could not be distinguished unambiguously and the proteins had to be excluded. For the remaining PTS1- and PTS2-targeted proteins, homologous sequences from various plant species were identified in the protein and EST databases by sequence similarity and selected if they fulfilled specific criteria irrespective of the nature of the targeting peptide. The C-terminal PTS1 tripeptides and the N-terminal PTS2 nonapeptides of these sequences homologous to PTS1- and PTS2-targeted proteins, respectively, were then analyzed for their amino acid sequence. The number of different plant sequences (391 PTS1 and 168 PTS2 sequences) and the number of different targeting peptides is given for each orthologous group.

a The C-terminal domain of homologs of LACS7 could not be distinguished from those corresponding to the PTS2-targeted protein LACS6.

b Peroxisomal SOD shares high sequence similarity with chloroplastic and cytosolic homologs.

c Peroxisomal Hsp70 is closely related to chloroplastic isoforms that carry an unusual potential PTS2 and lack a second M as potential alternative translation start.

Arabidopsis ortholog / MIPS accession number / Reference / PTS1/2 / Inclusion in this study / C-terminal tripeptides and N-terminal nonapeptides
of detected homologs
PTS1-targeted proteins:
Alanine-glyoxylate aminotransferase (AGT) / At2g13360 / Liepman and Olsen (2001) / SRI> / yes / 27 sequences, 2 different tripeptides:
SRI> (24), SRV> (3)
Glutamate-glyoxylate aminotransferase (GGT1/2) / At1g23310 (GGT1)
At1g70580 (GGT2) / Liepman and Olsen (2003)
Liepman and Olsen (2003);
Igarashi et al. (2003) / SKM>
SRM> / yes
yes / 31 sequences, 6 different tripeptides:
SRM> (16), SRL> (11), SKL> (1), SKM> (1); IYI> (1), QFL> (1)
Glycolate oxidase (GOX) / At3g14420 (GOX1)
At3g14415 (GOX2)
At4g18360 (GOX3)
At3g14130 (HAOX1)
At3g14150 (HAOX2) / All five proteins:
Reumann (2002) (Homology) / ARL>
PRL>
AKL>
SML>
SML> / yes
yes
no
no
no / 64 sequences, 12 different tripeptides:
PRL> (31), SRL> (8); ARL>(7), PKL> (6), PRM (3), SML> (3), SRM> (1), CRL> (1), CRM> (1), ALL> (1), LRL> (1), ISR> (1)
Hydroxyisobutyryl-CoA hydrolase (HIBCH) / At5g65940 (CHY1)
At2g30650
At2g30660 / Zolman et al. (2001)
(Homology)
(Homology) / AKL>
AKL>
AKL> / yes
no
no / 22 sequences, 5 different tripeptides:
AKL> (10), SKL> (7); PKL (3), SRM> (1), SKI> (1)
Hydroxypyruvate reductase (HPR) / At1g68010 / Mano et al. (1997) / SKL> / yes / 21 sequences, 5 different tripeptides:
SKL> (15), SRL> (2); AKL>(2), SKI> (1), FYL> (1)
Isocitrate lyase (ICL) / At3g21720 / (Homology) / SRM> / yes / 26 sequences, 6 different tripeptides:
SRM> (14), ARM> (7), SRI> (2); SRL> (1), CRI> (1), SRV> (1)
Long-chain acyl CoA synthetase 7 (LACS7) / At5g27600 / Fulda et al. (2002);
Hayashi et al. (2002) / SKL> / noa
Malate synthase (MS) / At5g03860 / (Homology) / SRL> / yes / 28 sequences, 9 different tripeptides:
SRL> (10); SKL> (8), CKL> (3), ARL>(2), SRI> (1), SKI> (1), PRL> (1), FRL> (1), SSL> (1)
Multifunctional protein (MFP) / At4g29010 (AIM1)
At3g06860 (MFP2) / Richmond and Bleecker (1999)
Richmond and Bleecker (1999) / SKL>
SRL> / yes / 58 sequences, 9 different tripeptides:
SRL> (22); SRM> (18), ARL>(8), SKL> (4), PRM (2), SRI> (1), ARM> (1), PRL> (1), AKS> (1)
Oxophytodienoate reductase (OPR3) / At2g06050 / Stintzi and Browse (2000); Schaller et al. (2000);
Strassner et al. (2002) / SRL> / yes / 15 sequences, 5 different tripeptides:
SRL> (7); SRM> (3), ARM> (3), ARL>(1), TEP> (1)
Long-chain acyl CoA
oxidase 1 (ACX1) / At4g16760
At2g35690 / Hooks et al. (1999)
(Homology) / ARL>
AKL> / yes
no / 29 sequences / 8 different tripeptides:
SRL> (10); ARL>(8), AKL> (6), CRL> (1), SRV> (1), SRF> (1), FRV> (1), LWQ> (1)
Short-chain acyl CoA oxidase 4 (ACX4) / At3g51840 / Hayashi et al. (1999) / SRL> / yes / 28 sequences, 6 different tripeptides:
SRL> (20); SRM> (3), ARL>(2), SRV> (1), KAL> (1), LKR> (1)
Sulfite oxidase (SOX) / At3g01910 / Eilers et al. (2001)
Nakamura et al. (2002) / SNL> / yes / 19 sequences, 7 different tripeptides:
SNL> (6), ANL> (3), SKM> (3); SKL> (2), SNM> (2), SSM> (2), SAM> (1)
Superoxide dismutase (SOD) / At5g18100 / Kliebenstein et al. (1998) / AKL> / nob
Uricase (Uric) / At2g26230 / (Homology) / SKL> / yes / 23 sequences, 5 different tripeptides:
SKM> (11), SKL> (9), SRM> (1), AKI>(1), SNM> (1)
PTS2-targeted proteins:
Long-chain acyl-CoA
oxidase 2 (ACX2) / At5g65110 / Hooks et al. (1999) / RIx5HL / yes / 11 sequences, 3 different nonapeptides:
RIx5HL (8), RLx5HL (2), RIx5HI (1)
Medium-chain acyl-CoA oxidase 3 (ACX3) / At1g06290
At1g06310 / Froman et al. (2000);
Eastmond et al. (2000)
(Homology) / RAx5HI
RAx5HI / yes
no / 13 sequences, 3 different nonapeptides:
RTx5HL (8), RAx5HL (3), RAx5HI (2)
Aspartate aminotransferase / At5g11520 / Schultz and Coruzzi (1995) / RIx5HL / yes / 19 sequences, 4 different nonapeptides:
RLx5HL (13), RIx5HL (3), RLx5HF (2), RVx5HL (1)
Citrate synthase (CS) / At2g42790
At3g58740
At3g58750 / (Homology)
(Homology)
(Homology) / RLx5HL
RLx5HL
RLx5HL / yes
yes
yes / 22 sequences, 2 different nonapeptides:
RLx5HL (21), RLx5HI (1)
Heat-shock protein 70 (Hsp70) / At4g24280
At5g49910 / Sung et al. (2001) (2nd M) (Homology) (no 2nd M) / RSx5RT
RSx5RT / noc
Long-chain acyl CoA synthetase 6 (LACS6) / At3g05970 / Fulda et al. (2002);
Hayashi et al. (2002) / RIx5HL / yes / 18 sequences, 6 different nonapeptides:
RLx5HL (11), RIx5HL (3), RLx5HI (1), RLx5HF (1), RVx5HL (1), RLx5HV (1)
Malate dehydrogenase (MDH) / At5g09660
At2g22780 / Berkemeyer et al. (1998);
Fukao et al. (2002);
Fukao et al. (2003) / RIx5HL
RIx5HL / yes
yes / 40 sequences, 4 different nonapeptides:
RIx5HL (33), RMx5HL (5), RLx5HL (1), RIx5HI (1)
Thiolase (Thiol) / At2g33150
At1g04710
At5g48880 / Hayashi et al. (1998)
(Homology)
(Homology) / RQx5HL
RQx5HL
RQx5HL / yes
yes
yes / 45 sequences, 1 nonapeptide:
RQx5HL (45)

LITERATURE CITED

Berkemeyer M, Scheibe R, Ocheretina O (1998) A Novel, non-redox-regulated NAD-dependent malate dehydrogenase from chloroplasts of Arabidopsis Thaliana L. J

Biol Chem 273: 27927–27933

Eastmond PJ, Hooks MA, Williams D, Lange P, Bechtold N, Sarrobert C, Nussaume L, Graham IA (2000) Promoter trapping of a novel medium-chain acyl-CoA

oxidase, which is induced transcriptionally during Arabidopsis seed germination. J Biol Chem 275: 34375–34381

Mano S, Hayashi M, Kondo M, Nishimura M (1997) Hydroxypyruvate reductase with a carboxy-terminal targeting signal to microbodies is expressed in Arabidopsis.

Plant Cell Physiol 38: 449–455

Richmond TA, Bleecker AB (1999) A defect in beta-oxidation causes abnormal inflorescence development in Arabidopsis. Plant Cell 11: 1911–1924

Schultz CJ, Coruzzi GM (1995) The aspartate aminotransferase gene family of Arabidopsis encodes isoenzymes localized to three distinct subcellular compartments.

Plant J 7: 61–75

Sung DY, Vierling E, Guy CL (2001) Comprehensive expression profile analysis of the Arabidopsis Hsp70 gene family. Plant Physiol 126: 789–800

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