Arabidopsis glutathione reductase 1 is dually targeted to peroxisomes and the cytosol

We recently established a proteome methodology for Arabidopsis leaf peroxisomes and identified more than 90 putative novel proteins of the organelle. These proteins included glutathione reductase isoform 1 (GR1), a major enzyme of the antioxidative defense system that was previously reported to be cytosolic. In this follow-up study, we validated the proteome data by analyzing the in vivo subcellular targeting of GR1 and the function of its C-terminal tripeptide, TNL>, as a putative novel peroxisome targeting signal type 1 (PTS1). The full-length protein was targeted to peroxisomes in onion epidermal cells when fused N-terminally with the reporter protein. The efficiency of peroxisome targeting, however, was weak upon expression from a strong promoter, consistent with the idea that the enzyme is dually targeted to peroxisomes and the cytosol in vivo. The reporter protein that was extended C-terminally by 10 amino acid residues of GR1 was directed to peroxisomes, characterizing TNL> as a novel PTS1. The data thus identify plant peroxisomal GR at the molecular level in the first plant species and complete the plant peroxisomal ascorbate-glutathione cycle. Moreover, GR1 is the first plant protein that is dually targeted to peroxisomes and the cytosol. The evolutionary origin and regulatory mechanisms of dual targeting are discussed.Key words: ascorbate-glutathione cycle, dual targeting, proteome analyses, reactive oxygen species, targeting signalsMassive amounts of hydrogen peroxide (H₂O₂) are produced during photosynthesis in peroxisomes by glycolate oxidase activity as part of the photorespiratory cycle.¹ Next to catalase, the ascorbate-glutathione cycle is the secondary scavenging system for H₂O₂ detoxification.^2–4 The cycle comprises four enzymes, ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR) and NADPH-dependent glutathione reductase (GR). GR plays a major physiological role in maintaining and regenerating reduced glutathione in response to biotic and abiotic stresses in plants.⁵ Jiminez et al. (1997) provided biochemical evidence for the presence of the antioxidants ascorbate and glutathione and the enzymes of the ascorbate-glutathione cycle in pea peroxisomes.^6–8 While Arabidopsis APX3, MDAR1 and MDAR4 have been characterized as peroxisomal isoforms,^9–11 the molecular identity of plant peroxisomal GR and DHAR have not been determined in any plant species to date.⁵ Arabidopsis encodes two GR and five DHAR isoforms that are either shown to be or predicted to be cytosolic, mitochondrial or plastidic.¹² We recently identified specific isoforms of GR (GR1, At3g24170) and DHAR (DHAR1, At1g19570) as being peroxisome-associated by proteome analysis of Arabidopsis leaf peroxisomes.^13,14 Both isoforms were previously reported to be or predicted to be cytosolic.¹⁵Arabidopsis GR1 terminates with TNL>, which is related to functional plant PTS1 tripeptides such as SNL> and ANL>.^16,17 Threonine (T), however, has not yet been described as an allowed residue at position −3 of PTS1s in any plant peroxisomal protein.¹⁶ Analysis of homologous plant proteins and expressed sequence tags (ESTs) shows that TNL> is generally highly conserved in putative plant GR1 orthologs (Fig. 1). A few other sequences terminate with related tripeptides, such TSL>, TTL>, NNL> and TKL>. Only a single EST (Picrorhiza kurrooa) carries the canonical PTS1, SKI> (Fig. 1). The data provide only weak additional support for peroxisome targeting of plant GR1 orthologs. However, GR homologs from green algae (chlorophyta) carry canonical PTS1 tripeptides, such as SKL> (Chlamydomonas, Volvox) and AKM> (Micromonas, Fig. 1, Suppl. Fig. 1).Open in a separate windowFigure 1Analysis of PTS1 conservation in plant GR1 homologs. Sequences of full-length protein (FLP) plant GR1 homologs or ESTs (“EST”) were identified by BLAST and phylogenetic analysis, aligned by ClustalX, and conserved residues were shaded by Genedoc. In addition to spermatophyta, homologs from bryophyta and chlorophyta were analyzed for PTS1 conservation. For a phylogenetic analysis of the full-length proteins, see also Supplementary Figure 1. The species abbreviations are as follows: Aa, Artemisia annua; At, Arabidopsis thaliana; Bn, Brassica napus; Br, Brassica rapa; Ci, Cichorium intybus; Cr, Chlamydomonas reinhardtii; Cs, Cynara scolymus; Fv, Fragaria vesca; Ha, Helianthus annuus; Msp, Micromonas sp. RCC 299; Mt, Medicago truncatula; Nt, Nicotiana tabacum; Os, Oryza sativa; Pk, Picrorhiza kurrooa; Ppat, Physcomitrella patens subsp. patens; Ps, Pisum sativum; Ptri, Populus trichocarpa; Rc, Ricinus communis; Rs, Raphanus sativus; Tp, Trifolium pratense; Tpus, Triphysaria pusilla; Vc, Volvox carteri f. nagariensis; Vv, Vitis vinifera; Zm, Zea mays.