Identification of two novel type 1 peroxisomal targeting signals in Arabidopsis thaliana

Abstract

Peroxisomes lack their own genetic material and must therefore import proteins encoded by genes in the nucleus. Amino acids within these proteins serve as targeting signals: they direct the delivery of the proteins to the organelle. The majority of soluble proteins destined for the peroxisomal matrix utilize a type 1 peroxisomal targeting signal (PTS1): a C-terminal tripeptide that follows the pattern small/basic/hydrophobic. We have discovered two new C-terminal tripeptides that target proteins to peroxisomes in Arabidopsis thaliana. The tripeptides PSL and KRR do not fit the major PTS1 consensus but cause green fluorescent protein to accumulate in peroxisomes of stably transformed Arabidopsis. We have identified forty-one proteins in the Arabidopsis genome that also bear these tripeptides at their C-termini and may therefore be peroxisomal.

Keywords: Arabidopsis thaliana; PTS1; Peroxisome; Protein targeting.

Figure 1

GFP-cDNA fusion proteins in Arabidopsis lines CS84743 and CS84812 accumulate in circular structures about 1μm in diameter. Live samples of lines CS84743 (A–C) and CS84812 (D–F) were observed by epifluorescence and laser scanning confocal microscopy. Fluorescent structures were observed in all organs examined, including leaf epidermis (A), cotyledons (B), root hairs (C), etiolated hypocotyl epidermis (D), leaf primordia (E), and root tips (F). Faint GFP fluorescence is sometimes visible in nuclei and in the cortical cytoplasm. Scale bars = 10μm.

Figure 2

Peroxisome-targeted mCherry fluorescent protein colocalizes with GFP-cDNA fusion proteins in Arabidopsis lines CS84743 and CS84812. Rosette leaves of Arabidopsis lines CS84743 (A–C) and CS84812 (D–F) were infiltrated with Agrobacterium containing a peroxisome-targeted mCherry fluorescent protein. Epifluorescence images of leaf epidermis show fluorescence from GFP-cDNA fusions (A,D), peroxisome-targeted mCherry fluorescent protein (B,E), and an overlay of the two fluorophores (C,F). GFP-labeled organelles in cells that were not transformed with the peroxisome-targeted mCherry showed fluorescence only in the GFP and merged channels (arrows). Scale bars= 5μm.