Experimental Validation of Example Sequences by in Vivo Subcellular Targeting Analysis.
Onion epidermal cells were transformed biolistically with EYFP fusion constructs that were C-terminally extended by the C-terminal decapeptides of plant PTS1 proteins serving as example sequences. Subcellular targeting was analyzed by fluorescence microscopy after ~18 h expression at room temperature only ([B], [C], [E] to [G], [J] to [O], [Q], [T], [V], [X], [Z], [Aa], and [Ab]), at an additional 24 h at ~10°C ([A] and [Ac] to [Ag]), or at an additional 5 to 6 d at ~10°C ([D], [H], [I], [P], [R], [S], [U], [W], and [Y]). Cytosolic constructs, for which subcellular targeting data are shown after short-term expression times, were reproducibly confirmed as cytosolic also after long-term expression. Novel amino acid residues of PTS1 tripeptides are underlined. In double transformants, peroxisomes were labeled with CFP, and cyan fluorescence was converted to red for image overlay ([G], [N], [O], [V], [Z], [Aa], and [Ab]). To document the efficiency of peroxisome targeting, EYFP images of single transformants were not modified for brightness or contrast. The sequences that terminated with LNL> and LCR> were included as putative non-PTS1 sequences ([Af] and [Ag]). Comparative subcellular targeting results obtained under different expression conditions are shown in Supplemental Figure 2 online. For sequence details, see Supplemental Tables 1 and 6 online.

Venn Diagram of PWM- and RI-Model Based PTS1 Protein Predictions for Arabidopsis.
The 392 gene models (GM; i.e., transcriptional and translational protein variants) and 320 gene loci (GL; i.e., protein coding genes) are predicted PTS1 proteins by either the PWM or the RI model. Except for three proteins (At1g21770.1, At4g02340.1, and At5g02660.1), the RI model predicted a protein subset of those predicted by the PWM model to be peroxisome-targeted PTS1 proteins. For details on PWM and RI model predictions for the 35,385 Arabidopsis gene models (TAIR10, November, 2010; 27,416 loci), see Supplemental Data Set 2 online. The 392 gene models (320 gene loci) include 109 gene models (79 gene loci) encoding established plant peroxisomal PTS1 proteins, 12 gene models (10 gene loci) associated with plant peroxisomes based on proteomics data only, and 271 gene models (231 gene loci) that had not yet been associated with peroxisomes, indicating that up to 70% of Arabidopsis PTS1 proteins might have remained unidentified up to now.

Categorization of Plant PTS1 Protein Example Sequences and Summary of Experimentally Validated Amino Acid Residues Forming the Plant PTS1 Motif.
The 2562 positive example sequences were split into three data subsets according to the number of sequences with the same C-terminal tripeptide. Data set 1, containing 2458 sequences and 42 different C-terminal tripeptides, each represented by ≥3 sequences, was used for training of the prediction models, while data sets 2 and 3 contained unseen sequences and C-terminal tripeptides and were used for model testing. Tripeptide residues previously reported to be present in plant PTS1 tripeptides are shaded in gray. According to experimental data and PWM predictions, at least two of the seven high-abundance residues of high targeting strength ([SA][KR][LMI]>, boxed; see Supplemental Figure 1B online) must be combined with one low-abundance residue to yield functional plant PTS1 tripeptides (x[KR][LMI]>, [SA]y[LMI]>, and [SA][KR]z>).

Performance Analysis of the PWM and RI Prediction Models on Example PTS1 Protein Sequences.
The x axis indicates the start position of the C-terminal PTS1 domain that was considered for performance analysis and extends to the extreme C termini of the PTS1 proteins. For the definition of sensitivity, specificity, and harmonic mean, see Supplemental Methods online.

Experimental Validation of Arabidopsis Proteins Newly Predicted to Be Located in Peroxisomes by in Vivo Subcellular Targeting Analysis.
Onion epidermal cells were transformed biolistically with EYFP fusion constructs that were either C-terminally extended by the C-terminal decapeptide of representative Arabidopsis proteins (or the 15–amino acid peptide for PK1, P) or fused with Arabidopsis full-length cDNAs. Novel amino acid residues of newly identified functional PTS1 tripeptides (in addition to those identified in Figure 2) are underlined. Subcellular targeting was analyzed by fluorescence microscopy after ~18 h expression at room temperature only ([A] to [C], [F], [H], [I], [K], [M], [R] to [T], [W], and [X]), at an additional 24 h at ~10°C ([D], [E], [G], [J], [N] to [Q], [U], and [V]), or at an additional 5 to 6 d of expression at ~10°C (L). Cytosolic constructs, for which subcellular targeting data are shown after short-term expression times, were reproducibly confirmed as cytosolic also after long-term expression. In double transformants, peroxisomes were labeled with CFP, and cyan fluorescence was converted to red for image overlay ([A], [H], [L], [M], and [Q] to [W]). The predicted PTS1 domains investigated derived from the following proteins: SCL> (UP9), SPL>(1) (FAH), SWL> (RING), KRL> (Tudor), SYM> (SDRc, At3g01980.1/3/4), APN> (SDRc, At3g01980.2), SEL> (SPK1), SRY> (PHD), SIL> (ANK), IKL> (LCAT), LKL> (CPK1), VKL> (CUT1), AHL> (PAP7), and PK1 (SKL>; Ma and Reumann, 2008). The predicted PTS1 tripeptides of the Arabidopsis full-length proteins are the following: CP (SKL>), CHY1H1 and CHY1H2 (both AKL>), SDRc (SYM>), S28FP (SSM>), NUDT19 (SSL>), pxPfkB (SML>), and CUT1 (VKL>). To document the efficiency of peroxisome targeting, EYFP images of single transformants were not modified for brightness or contrast. The Arabidopsis Genome Initiative codes of the Arabidopsis proteins are listed in Supplemental Table 5 online.