Together with identification of the peroxisomal isoform of DHAR (DHAR1, At1g19570,14
), all four members of the peroxisomal ascorbate-glutathione cycle have been identified in Arabidopsis at the molecular level. The pronounced conservation of TNL> in higher plant orthologs (, Suppl.
) strongly suggests that most plant GR1 orthologs, even though they are frequently annotated as cytosolic, are generally dually targeted to peroxisomes and the cytosol. Plant GR1 can thus regenerate reduced glutathione in the peroxisome matrix and substantially contribute to H2
detoxification. Moreover, peroxisomal GR1 provides the substrate for several other glutathione-dependent enzymes that have been recently discovered in plant peroxisomes.13,14,19
Glutathione is thereby emerging as a major antioxidant in plant peroxisomes, protecting peroxisomal and cellular enzymes against a range of peroxides, xenobiotics and possibly heavy metals.20
Dual or even multiple subcellular targeting of a single protein to several compartments emerges as a more prevalent phenomenon than previously assumed. Six Arabidopsis proteins are dually targeted to mitochondria and peroxisomes,21,22
and three proteins are dually targeted to plastids and peroxisomes (watermelon Hsp70;23
soybean aspartate aminotransferase;24
). GR1 is thus the first plant protein that is dually targeted to peroxisomes and the cytosol. In the green lineage, most species bear two GR genes as a result of an early gene duplication event. The two genes cluster into a clade of chloroplastic/mitochondrial isoforms with N-terminal extensions and a clade of cytosolic/peroxisomal isoforms terminating with known or PTS1-related tripeptides (Suppl.
). PTS1 conservation analysis in cytosolic/peroxisomal isoforms allows the prediction as to whether the cytosolic or peroxisomal function of GR1 is the most fundamental and most evolutionarily ancient task. As revealed by phylogenetic analysis of full-length protein sequences and cDNAs assembled from ESTs, one of two GR homologs from green algae carries a strong canonical PTS1 tripeptide, such as SKL> (Chlamydomonas, Volvox) and AKM> (Micromonas). The data indicate that the peroxisome-targeting function of GR evolved soon after gene duplication in Viridiplantae and that these GR1 homologs are most likely exclusively peroxisomally targeted in chlorophyta (Suppl.
, see also ). Dual targeting of GR1 to peroxisomes and the cytosol appears to have evolved at a later stage, prior to the divergence of mosses. The GR1 homolog from Physcomitrella carries a PTS1-related tripeptide, LKV>, of weak predicted targeting strength, similar to TNL> in higher plants. Notably, leucine (L) has recently been experimentally verified as another allowed residue at pos. -3 in plant PTS1 tripeptides (Lingner T, Antonicelli GE, Kataya A, Reumann S, unpubl. data).
Figure 3 Model of the evolutionary development of the peroxisomal and cytosolic functions of GR1 in spermatophyta. The canonical nature and high peroxisome targeting strength of PTS1s in GR homologs of chlorophyta (Chlamydomonas, Volvox and Micromonas) indicate (more ...)
The most intriguing question of multiple protein targeting concerns the possible existence and identity of the underlying regulatory mechanism. Two mechanisms that regulate protein distribution between the cytosol and peroxisomes can be envisioned: (1) a rather static mechanism of protein distribution that is largely determined by the PTS strength, i.e., the affinity of the PTS1 domain to its receptor, PEX5p, and (2) a dynamic mechanism of post-transcriptional or post-translational nature that allows adequate responses to changing demands in GR1 compartmentalization. Notably, even in the case of a rather simple regulation of protein distribution by PTS1 strength, the peroxisome import rate by weak PTS1s in vivo depends on the rate of gene expression; low-abundance proteins with weak PTS1s may still be imported quantitatively into peroxisomes, but not high-abundance proteins. Therefore, the low peroxisome import rate of GR1 upon expression from the strong 35S promoter does not allow the conclusion to be made that GR1 is largely cytosolic in vivo. This question needs to be addressed by expressing N-terminally or internally tagged GR1 versions, preferentially in a gr1
knockout background, from the native GR1 promoter. The fact that GR1 was identified in leaf peroxisomes that were isolated from plants grown under standard conditions13,14
supports the idea of a constitutive role of GR1 in peroxisomes.
In light of the strong need and major function of the peroxisomal ascorbateglutathione cycle under sudden conditions of catalase inactivation,25
it is reasonable to postulate that the ratio of GR1 distribution between peroxisomes and the cytosol is dynamic and adjustable depending on H2
overproduction in the matrix by post-transcriptional and/or post-translational mechanisms. Interestingly, pea (and later, Arabidopsis) GR2 was one of the first proteins shown to be dually targeted to two plant cell compartments, mitochondria and plastids.12,26
Dual targeting of GR1/2 thus emerges as a specific property of the enzyme. The regulatory mechanism(s) underlying dual targeting of both GR1 and GR2 are still unknown and remain to be studied. The exclusively cytosolic localization of EYFP-GR1 in tobacco protoplasts () may indicate that peroxisome targeting is prevented in this expression system by PTS1 inaccessibility caused by conformational changes or binding of an accessory protein that regulates enzyme distribution between the cytosol and peroxisomes.