In vivo subcellular targeting analysis validates a novel peroxisome targeting signal type 2 and the peroxisomal localization of two proteins with putative functions in defense in Arabidopsis

Abstract

Plant peroxisomes are essential organelles that house diverse metabolic activities. To understand the full spectrum of peroxisomal functions, we recently performed a proteomic analysis of Arabidopsis leaf peroxisomes followed by in vivo subcellular targeting validation of some novel proteins. Here, we continue with the targeting analysis and demonstrate that the nonapeptide RVx₅HF, which is present near the N terminus of the newly identified peroxisomal protein, HIT3 (histidine triad family protein 3), is a functional peroxisome targeting signal type 2 (PTS2). In addition, we have confirmed the peroxisomal localization of UP6 (unknown protein 6) and GLX1 (glyoxalase 1 homolog), two proteins with possible roles in stress responses and glutathione-dependent detoxification, respectively. These data, together with results from our previous analysis of the peroxisomal proteome, reinforce the notion that peroxisomes are involved in various stress responses and suggest glutathione as a major antioxidant in plant peroxisomes.

Figure 1

Validation of a new PTS2 peptide and the peroxisomal localization of two novel proteins. Confocal (A and B) or epifluorescent (C) microscopic images were obtained from leaf epidermal cells of 4-week-old tobacco (Nicotiana tabacum) plants co-expressing the indicated YFP fusion and the CFP-PTS1 peroxisomal marker proteins. YFP fusions are shown in green, and the CFP-PTS1-labeled peroxisomes are in red (A and B) or blue (C). Scale bars = 10 µm.

Figure 2

A proposed model for the action of glyoxalase I and II in detoxification. See text for a description of the pathway. GSH, reduced glutathione (γ-L-glutamyl-L-cysteinyl-glycine).