In-Depth Proteome Analysis of Arabidopsis Leaf Peroxisomes Combined with in Vivo Subcellular Targeting Verification Indicates Novel Metabolic and Regulatory Functions of Peroxisomes    

Abstract

Peroxisomes are metabolically diverse organelles with essential roles in plant development. The major protein constituents of plant peroxisomes are well characterized, whereas only a few low-abundance and regulatory proteins have been reported to date. We performed an in-depth proteome analysis of Arabidopsis (Arabidopsis thaliana) leaf peroxisomes using onedimensional gel electrophoresis followed by liquid chromatography and tandem mass spectrometry. We detected 65 established plant peroxisomal proteins, 30 proteins whose association with Arabidopsis peroxisomes had been previously demonstrated only by proteomic data, and 55 putative novel proteins of peroxisomes. We subsequently tested the subcellular targeting of yellow fluorescent protein fusions for selected proteins and confirmed the peroxisomal localization for 12 proteins containing predicted peroxisome targeting signals type 1 or 2 (PTS1/2), three proteins carrying PTS-related peptides, and four proteins that lack conventional targeting signals. We thereby established the tripeptides SLM. and SKV. (where . indicates the stop codon) as new PTS1s and the nonapeptide RVx 5 HF as a putative new PTS2. The 19 peroxisomal proteins conclusively identified from this study potentially carry out novel metabolic and regulatory functions of peroxisomes. Thus, this study represents an important step toward defining the complete plant peroxisomal proteome.Surrounded by single membranes, peroxisomes are small, ubiquitous eukaryotic organelles mediating a wide range of oxidative metabolic activities that vary by the species, cell type, and environmental conditions in which the organism lives (Beevers, 1979;Van den Bosch et al., 1992). Plant peroxisomes are essential to physiological processes such as lipid metabolism, photorespiration, and plant hormone biosynthesis and metabolism (Olsen and Harada,

Getting a camel through the eye of a needle: the import of folded proteins by peroxisomes

“…The number of PTS2-targeted proteins ranges from zero in Caenorhabditis elegans, which has lost the PTS2 import pathway altogether (Motley et al, 2000), to an estimated 60 in A. thaliana (Reumann et al, 2009). Interestingly, S. cerevisiae appears to maintain a PTS2 import pathway solely for the transport of 3-ketoacyl thiolase (Grunau et al, 2009).…”

“…50 proteins (Yi et al, 2002;Wiese et al, 2007), whereas plant peroxisomes appear to have approximately double this number (Eubel et al, 2008). However, new functions for peroxisomes are still being discovered by proteomic (Reumann et al, 2007;Wiese et al, 2007;Arai et al, 2008;Eubel et al, 2008;Reumann et al, 2009) and genetic (Lorenz and Fink, 2001;Lipka et al, 2005;Boisson-Dernier et al, 2008) studies.…”

Disrupting Autophagy Restores Peroxisome Function to anArabidopsis lon2Mutant and Reveals a Role for the LON2 Protease in Peroxisomal Matrix Protein Degradation

“…By contrast, mammalian cells expressing a dominant-negative form of rat liver peroxisomal Lon mislocalize catalase to the cytosol (Omi et al, 2008), indicating that peroxisomal LON function somehow promotes matrix protein import. Arabidopsis encodes four LON isoforms; LON2 is the only isoform bearing a canonical PTS1 and localized in peroxisomes (Ostersetzer et al, 2007;Eubel et al, 2008;Reumann et al, 2009). All examined Arabidopsis lon2 alleles are resistant to IBA-induced lateral root formation but respond normally to auxins not requiring b-oxidation Burkhart et al, 2013), suggesting reduced IBA-to-IAA conversion.…”

Cytosolic and Chloroplastic DHARs Cooperate in Oxidative Stress-Driven Activation of the Salicylic Acid Pathway

“…While the encoded proteins may allow DHAR activity in both the chloroplast and the cytosol, some studies point to DHAR1 localization in the peroxisomes (Dixon et al, 2002;Reumann et al, 2009;Grefen et al, 2010;Tang and Yang, 2013). To date, three studies have appeared using single loss-offunction mutants.…”