Protein–Protein Interactions and Quantitative Phosphoproteomic Analysis Reveal Potential Mitochondrial Substrates of Protein Phosphatase 2A-B’ζ Holoenzyme

Protein phosphatase 2A (PP2A) is a heterotrimeric conserved serine/threonine phosphatase complex that includes catalytic, scaffolding, and regulatory subunits. The 3 A subunits, 17 B subunits, and 5 C subunits that are encoded by the Arabidopsis genome allow 255 possible PP2A holoenzyme combinations. The regulatory subunits are crucial for substrate specificity and PP2A complex localization and are classified into the B, B’, and B” non-related families in land plants. In Arabidopsis, the close homologs B’η, B’θ, B’γ, and B’ζ are further classified into a subfamily of B’ called B’η. Previous studies have suggested that mitochondrial targeted PP2A subunits (B’ζ) play a role in energy metabolism and plant innate immunity. Potentially, the PP2A-B’ζ holoenzyme is involved in the regulation of the mitochondrial succinate/fumarate translocator, and it may affect the enzymes involved in energy metabolism. To investigate this hypothesis, the interactions between PP2A-B’ζ and the enzymes involved in the mitochondrial energy flow were investigated using bimolecular fluorescence complementation in tobacco and onion cells. Interactions were confirmed between the B’ζ subunit and the Krebs cycle proteins succinate/fumarate translocator (mSFC1), malate dehydrogenase (mMDH2), and aconitase (ACO3). Additional putative interacting candidates were deduced by comparing the enriched phosphoproteomes of wild type and B’ζ mutants: the mitochondrial regulator Arabidopsis pentatricopeptide repeat 6 (PPR6) and the two metabolic enzymes phosphoenolpyruvate carboxylase (PPC3) and phosphoenolpyruvate carboxykinase (PCK1). Overall, this study identifies potential PP2A substrates and highlights the role of PP2A in regulating energy metabolism in mitochondria.publishedVersio

Multi-targeted trehalose-6-phosphate phosphatase I harbors a novel peroxisomal targeting signal 1 and is essential for flowering and development

A putative, non-canonical peroxisomal targeting signal type 1 (PTS1) Pro-Arg-Met > was identified in the extreme C-terminus of trehalose-6-phosphate phosphatase (TPP)I. TPP catalyzes the final step of trehalose synthesis, and the enzyme was previously characterized to be nuclear only (Krasensky et al. in Antioxid Redox Signal 21(9):1289–1304, 2014). Here we show that the TPPI C-terminal decapeptide ending with Pro-Arg-Met > or Pro-Lys-Met > can indeed function as a PTS1. Upon transient expression in two plant expression systems, the free C- or N-terminal end led to the full-length TPPI targeting to peroxisomes and plastids, respectively. The nucleus and nucleolus targeting of the full-length TPPI was observed in both cases. The homozygous T-DNA insertion line of TPPI showed a pleiotropic phenotype including smaller leaves, shorter roots, delayed flowering, hypersensitivity to salt, and a sucrose dependent seedling development. Our results identify novel PTS1s, and TPPI as a protein multi-targeted to peroxisomes, plastids, nucleus, and nucleolus. Altogether our findings implicate an essential role for TPPI in development, reproduction, and cell signaling.publishedVersio

Identification of Peroxisome-Targeted Proteins Implicated in Plant Innate Immunity in Arabidopsis thaliana

DoktoravhandlingPeroxisomes are subcellular organelles, traditionally known to be involved in processes like photorespiration, fatty acid β-oxidation, and detoxification of reactive oxygen species. Proteome analysis of plant peroxisomes and targeting signal prediction methods are important tools to identify novel peroxisomal proteins. In the present study the accuracy of newly developed methods to predict peroxisome targeting signals type 1 (PTS1) in plant proteins was investigated by in vivo subcellular targeting analyses. Upon application of these prediction methods to the Arabidopsis thaliana genome, 392 gene models were predicted to possess functional PTS1 domains, several proteins of which were validated as peroxisomal and numerous novel PTS1 tripeptides were identified. Furthermore, several detoxification-related enzymes and defense-related Arabidopsis proteins were detected by proteome analyses and PTS1 prediction methods that were potentially targeted to peroxisomes. Two enzymes of the ascorbate-glutathione (ASC-GSH) cycle, glutathione reductase 1 (GR1) and dehydroascorbate reductase 1 (DHAR1), and five glutathione-S transferases (GSTs) had been detected by proteome analysis in leaf peroxisomes. In vivo subcellular localization targeting analyses of the present study verified peroxisomal targeting for GR1 and the protein was found to carry a functional novel PTS1 (TNL>). By contrast, the four GSTs remained cytosolic in the chosen orientation in the back of the reporter protein. New fragmented evidence has been emerging in the literature for an important role of plant peroxisomes in innate immunity. In the present study sixteen defense-related Arabidopsis proteins were experimentally investigated for protein targeting to peroxisomes by in vivo subcellular localization. The proteins of interest included several yet unknown homologs of Arabidopsis NDR1 and tobacco HIN1, the so-called NDR1/HIN1 like (NHL) proteins. In vivo subcellular localization was primarily investigated for three NHL family members (NHL4, NHL6 and NHL25). Peroxisome targeting was verified for NHL4 with strong indications also for NHL6 and NHL25 in being located in peroxisomes. AtIAN12 is a homolog of AIG1/AtIAN8 and had been identified by Arabidopsis leaf peroxisome proteomics. In vivo subcellular localization experiments demonstrated that AtIAN12 protein is targeted to peroxisomes and indicated that the targeting pathway involves posttranslational protein modification by isoprenylation. Taken together, the data indicate for the first time that one NDR1/HIN1 homolog (NHL4) and AtIAN homolog (AtIAN12) are peroxisome associated. Preliminary gene expression analyses indicated that three NHL genes and three AtIAN genes are induced by a bacterial pathogen (Pst DC3000), while NHL6, NHL25, and AtIAN8 are induced by an avirulent Pst DC3000 strain (carrying the effector avrRpt2). Out of the six NHL and AtIAN genes, only NHL6 appeared to be induced in wt Col-0 plants by the bacterial elicitor (flg22), but remained unaffected in Arabidopsis plants carrying a mutation in the flagellin receptor gene FLS2. The data suggested that NHL6 is involved in basal PAMP triggered immunity (PTI). Furthermore, NHL6 transcripts accumulated similarily in both wt plants and npr1 mutant plants after flg22 treatment, which indicates that NHL6 induction is NPR1-independent. Functional studies were initiated through the isolation of homozygous mutants, amiRNA lines and overexpresser lines for selected NHL and AtIAN genes. In homozygous mutants (three nhl mutants and ian11), differences in bacterial proliferation were observed compared to wt plants upon infection with the avirulent bacterium Pst DC3000 (avrRpt2). Overall, the identification of several defense-related proteins in peroxisomes together with preliminary functional data on NHL proteins opens new perspectives to important, multi-layered peroxisome functions in plant innate immunity

Identification of Arabidopsis Protein Kinases That Harbor Functional Type 1 Peroxisomal Targeting Signals

Peroxisomes are eukaryotic specific organelles that perform diverse metabolic functions including fatty acid β-oxidation, reactive species metabolism, photorespiration, and responses to stress. However, the potential regulation of these functions by post-translational modifications, including protein phosphorylation, has had limited study. Recently, we identified and catalogued a large number of peroxisomal phosphorylated proteins, implicating the presence of protein kinases in this organelle. Here, we employed available prediction models coupled with sequence conservation analysis to identify 31 protein kinases from the Arabidopsis kinome (all protein kinases) that contain a putative, non-canonical peroxisomal targeting signal type 1 (PTS1). From this, twelve C-terminal domain-PTS1s were demonstrated to be functional in vivo, targeting enhanced yellow fluorescent protein to peroxisomes, increasing the list of presumptive peroxisomal protein kinases to nineteen. Of the twelve protein kinases with functional PTS1s, we obtained full length clones for eight and demonstrated that seven target to peroxisomes in vivo. Screening homozygous mutants of the presumptive nineteen protein kinases revealed one candidate (GPK1) that harbors a sugar-dependence phenotype, suggesting it is involved in regulating peroxisomal fatty acid β-oxidation. These results present new opportunities for investigating the regulation of peroxisome functions.publishedVersio