Figure 2

Experimental validation of Arabidopsis proteins carrying newly predicted non-canonical PTS1 domains 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 Arabidopsis proteins carrying newly predicted non-canonical PTS1 domains. Subcellular targeting was analyzed by fluorescence microscopy after ca. 48 h expression (ca. 18 h RT plus 30 h ca. 10 °C), or ca. 7 d (ca. 18 h RT plus 6 d ca. 10 °C). Cytosolic constructs, for which subcellular targeting data are shown after short-term expression times, were reproducibly confirmed as cytosolic also after long-term expression. Possibly novel aa residues of PTS1 tripeptides are underlined. To document the efficiency of peroxisome targeting, EYFP images of single transformants were not modified for brightness or contrast (A-H). In double transformants, peroxisomes were labeled with DsRed-SKL or PTS2-CFP with cyan fluorescence having been converted to red for image overlay (I-K). In Figure2K the arrows point at six EYFP-labeled peroxisomes (yellow), while two organelles are only fluorescing in red or green, most likely due to quick organelle movement. EYFP alone was included as negative control (A).