Isolation of Peroxisomes from Mature Arabidopsis Leaves.
The supernatant of the chloroplast sedimentation was loaded on top of a discontinuous Percoll gradient underlaid with fractions of increasing sucrose concentration (A). The Percoll concentrations refer to the concentrations prior to gradient centrifugation. After centrifugation, the Percoll gradient was fractionated from the top to the bottom into 21 2-mL fractions and analyzed for organellar marker enzyme activities, namely HPR (leaf peroxisomes) (B), fumarase (mitochondria), and NADP-dependent GAPDH (chloroplasts) (C). After washing, the peroxisome fractions of eight Percoll gradients were combined and homogenized, and the resuspension was laid on top of a discontinuous sucrose density gradient followed by ultracentrifugation. After fractionation from the top to the bottom into 14 1-mL fractions, the concentrations of protein and sucrose were determined (D). Marker enzyme activities were analyzed as before ([E] and [F]). Note that the scale between the first and second density gradients is increased by a factor of 5 for HPR and decreased for NADP-GAPDH (factor 5) and fumarase (factor 2).

Two-Dimensional Protein Map of Isolated Leaf Peroxisomes from Arabidopsis.
Leaf peroxisomal proteins (200 μg) of a specific HPR activity of 400 nkat HPR/mg protein were first separated according to pI by isoelectric focusing on commercial IPG strips (18 cm, nonlinear, pH 3.0 to 10) followed by SDS-PAGE on 10 to 15% (w/v) acrylamide gradient gels (A). For the sake of clarity, the annotations of the protein spots within the dashed frames are shown magnified in (B) and (C). Proteins were visualized by colloidal Coomassie blue staining. Stained proteins were excised manually and subjected to an automated platform for protein identification (Jahn et al., 2006). Two artificial Coomassie spots caused by dye crystals were eliminated from the figure. Abbreviations not listed in Supplemental Table 1 online or Table 2 are as follows: ACNA, (cytosolic) aconitase; alb., BSA; ATPase β, ATP synthase subunit β; efTU, elongation factor TU; GDC-P, Gly decarboxylase P protein; PSBP, oxygen-evolving enhancer protein 2; RubisCO LSU and SSU, ribulose-1,5-bis-phosphate carboxylase/oxygenase large and small subunits; SHMT, Ser-hydroxymethyltransferase; VDAC, voltage-dependent anion-selective channel.

Domain Structure of Arabidopsis Transthyretin-Like Protein and Its Splice Variants.
The single Arabidopsis TLP (At5g58220) is a bifunctional protein with an N-terminal decarboxylase (COG3195, OHCU decarboxylase) and a C-terminal hydrolase domain (COG2351, 5-HIU hydrolase) synthesized in three splice variants (A). The full-length variant (TLP₃₂₄) carries a predicted PTS2 (RLRIIGGHL, position 182 to 190) in the linker region between the two functional domains. In addition to the peptide mass information obtained (shown in [A] as black bars with numbering of tryptic peptides), mass spectrometric sequencing by MALDI-TOF-MS unambiguously confirmed the presence of the tryptic peptides T17, T19, and T22 (shown in [A] also as sequences, PTS2 amino acids in bold face), thereby indicating the integrity of the linker region of full-length TLP₃₂₄. Except for the tryptic dipeptide T18 (LR) that escaped detection due to its small size, the entire PTS2 nonapeptide was covered by the sequencing analysis, as can be deduced from the fragment ion mass spectrum of T19 (B). By contrast, both shorter splice variants of TLP (TLP₃₁₁ and TLP₂₈₆) lack the internal PTS2 and TLP₂₈₆ additionally the bulky conserved His residue (His₂₀₉ of TLP₃₂₄, corresponding to His₁₄ of TLP/PucM of Bacillus subtilis) that is implicated in determining the entrance diameter of the internal channel (Jung et al., 2006). Thus, the shorter transcriptional variants probably differ in subcellular localization and function from the peroxisomal isoform. See Supplemental Figure 3P online for details of the central amino acid sequence of the three Arabidopsis TLP splice variants and plant homologs. OHCU, 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline.

Verification of Peroxisome Targeting of Novel Peroxisomal Proteins Carrying Predicted Major or Minor PTS1s.
The cDNAs of six novel peroxisomal proteins identified in isolated leaf peroxisomes from Arabidopsis and one pathway-associated protein (6PGL) were fused in frame to the 3′ end of EYFP and transiently expressed in onion epidermal cells upon cell bombardment. Fluorescence microscopy was used to determine whether fusion proteins were targeted to punctate subcellular structures that coincided with peroxisomes labeled with a peroxisome-targeted version of CFP, MDH-CFP (Fulda et al., 2002). In addition, we analyzed the targeting of EYFP extended C-terminally by the putative 10–amino acid residue PTDs of the respective proteins. 6PGL was not yet identified in isolated leaf peroxisomes but analyzed in context of the detection of another peroxisomal isoform of the OPPP, 6PGDH. The following proteins were found to be targeted to peroxisomes by functional PTS1s (see text for full names): IDH (At1g54340, SRL>; A1, A2, 95% overlapping dots; [B]), SDRb (At3g12800, SKL>; [C1] and [C2]; 74% overlapping dots; [D]), HBCDH (At3g15290, PRL>; [E1] and [E2]; quantification difficult; [F]), 6PGL (At5g24400, SKL>; [G1] and [G2]; 95% overlapping dots; [H]), ECHIb (At4g14430, PKL>; [I1] and [I2]; 97% overlapping dots; [J]), and GSTT1 (At5g41210, SKI>; [K1] and [K2]; 95% overlapping dots [L]). The full-length fusion protein of 6PGDH (At3g02360) remained cytosolic (M), although it was shown to contain a functional PTS1 (SKI>; [N1] and [N2]; 91% overlapping dots).

Subcellular Targeting Analysis of Novel Peroxisomal Proteins Carrying Unknown PTSs.
The generation of EYFP fusion proteins and the fluorescence microscopic analyses were performed as described in Figure 4. Subcellular targeting analysis of ECH2 (At1g76150, SSL>; [A1] to [B2]; [A], 71% overlapping dots; [B], 95% overlapping dots), ATF1 (At1g21770, SSI>; [C1] to [D2]; [C], 60% overlapping dots; [D], 59% overlapping dots), and EH3 (At4g02340, ASL>; [E1] to [F2]; [E], 80% overlapping dots; [F], 73% overlapping dots) established SSL>, SSI>, and ASL> as novel functional PTS1 peptides in plants. Although analysis of full-length TLP₃₂₄ (At5g58220) carrying a putative internal PTS2 (RLx₅HL) at least indicated targeting to punctate subcellular structures (G), we could not provide conclusive evidence for peroxisomal localization of EYFP-TLP₃₂₄ by double labeling owing to a strong cytosolic background fluorescence. When the essential His of the predicted internal PTS2 was mutated to Asp (RLx₅HL to RLx₅DL) to generate EYFP-TLPΔPTS2, the punctate fluorescence pattern disappeared (H), enabling us to identify the full-length version of TLP₃₂₄, but not the two shortened splice variants lacking the predicted PTS2 (TLP₃₁₁ and TLP₂₈₆; see Figure 3) as a peroxisomal protein that is targeted to the organelle by an internal PTS2.