Towards understanding peroxisomal phosphoregulation in Arabidopsis thaliana.

MAIN CONCLUSION: This work identifies new protein phosphatases and phosphatase-related proteins targeting peroxisomes, and raises the question of a novel protein import pathway from ER to peroxisomes involving peroxisomal targeting signal type 1 (PTS1) Plant peroxisomes are essential for several processes, for example lipid metabolism, free radical detoxification, development, and stress-related functions. Although research on peroxisomes has been intensified, reversible phosphorylation as a control mechanism in peroxisomes is barely studied. Therefore, it is crucial to identify all peroxisomal proteins involved in phosphoregulation. We here started with protein phosphatases, and searched the Arabidopsis thaliana genome for phosphatase-related proteins with putative peroxisomal targeting signals (PTS). Five potential peroxisomal candidates were detected, from which four were confirmed to target peroxisomes or have a functional PTS. The highly conserved Ser-Ser-Met> was validated for two protein phosphatase 2C (PP2C) family members (POL like phosphatases, PLL2 and PLL3) as a functional peroxisomal targeting signal type 1 (PTS1). Full-length PLL2 and PLL3 fused with a reporter protein targeted peroxisomes in two plant expression systems. A putative protein phosphatase, purple acid phosphatase 7 (PAP7), was found to be dually targeted to ER and peroxisomes and experiments indicated a possible trafficking to peroxisomes via the ER depending on peroxisomal PTS1. In addition, a protein phosphatase 2A regulator (TIP41) was validated to harbor a functional PTS1 (Ser-Lys-Val>), but the full-length protein targeted cytosol and nucleus. Reverse genetics indicated a role for TIP41 in senescence signaling. Mass spectrometry of whole seedlings and isolated peroxisomes was employed, and identified new putative phosphorylated peroxisomal proteins. Previously, only one protein phosphatase, belonging to the phospho-protein phosphatase (PPP) family, was identified as a peroxisomal protein. The present work implies that members of two other main protein phosphatase families, i.e. PP2C and PAP, are also targeting peroxisomes.

MAP kinase phosphatase 1 harbors a novel PTS1 and is targeted to peroxisomes following stress treatments.

In Arabidopsis thaliana, twenty mitogen-activated protein kinases (MAPKs/MPKs) are regulated by five MAP kinase phosphatases (MKPs). Arabidopsis MKP1 has an important role in biotic, abiotic and genotoxic stresses and has been shown to interact with and negatively regulate specifically MPK3 and MPK6. MKP1 has been reported to have a role in negative regulation of reactive oxygen species (ROS) and salicylic acid (SA) production. As essential organelles involved in production of ROS and SA, peroxisomes could possibly be an important compartment for MKP1 activity, however MKP1 was previously reported to be cytosolic. By screening Arabidopsis protein phosphatases for peroxisomal targeting signal 1 (PTS1), we identified MKP1 as a putative peroxisomal protein. Arabidopsis MKP1 was found to harbor a non-canonical PTS1-like tripeptide (Ser-Ala-Leu>) that is conserved in MKP1 orthologs. We show experimentally that the C-terminal Ser-Ala-Leu> can function as a novel PTS1, and alanine in position -2, adds more relaxation to the plant PTS1 motif. The full-length MKP1 remained in the cytosol when transiently expressed in Arabidopsis mesophyll protoplasts under standard conditions. When different biotic and abiotic stresses were applied to mesophyll protoplasts, the full length protein changed its targeting to unidentified organelle-like structures that subsequently fused with peroxisomes. Our results identify MKP1 as a protein dually targeted to cytosol and peroxisomes. The finding that MKP1 targets peroxisomes by a non-canonical PTS1 under stressful conditions highlights the complexity of peroxisomal targeting mechanism.