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Ongoing projects:

1- Identifying peroxisomal pro. phosphatases and kinases

2- Identifying peroxisomal phospho-substrates

3- Identifying mitochondrial phosphosubstrates

4- Investigating the functions of newly identified protein phospho-regulating enzumes.

5- Investigate the impact of phosphorylation on peroxisome biogenesis

6- Model for Phospho-Regulation of Triacylglyceride (TAG) Mobilization and Fatty Acid (FA) β-Oxidation.

1- Identifying peroxisomal pro. phosphatases and kinases

To understand regulation of peroxisomal functions by post-translational modification by phosphorylation, it is crucial to identify the key-players involved in this mechanism. The developed bioinformatic analyses combined with phylogenetic analyses are used to identify potential candidates. The signals from the potential candidates to be experimentally validated, and hence the trials to clone the full coding genes to be done. The positive candidates will be further studied.

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2- Identifying peroxisomal phospho-substrates

Protein phosphatase 2A (PP2A) is a heterotrimeric complex comprising a catalytic (C), scaffolding (A), and regulatory (B) subunits. The involvement of such a complex in regulation of fatty acid metabolism was confirmed by reverse genetics. We seek to identify peroxisomal substrates of PP2A by invetigating interactions using BiFC approach, as well as to express and purify the proteins for PPI analyses using Micrscale Thermophoresis (MST) together with other approaches. 

Kataya et al., 2015. Plant Physiology

Kataya et al., 2015. Plant Physiology

3- Identifying mitochondrial phosphosubstrates

Seedlings of the PP2A b’ζ mutant showed mild seedling growth retardation in sucrose-free medium (Kataya et al., 2015c). We suggest PP2A-B’ζ holoenzyme to be involved in the regulation of the mitochondrial succinate/fumarate translocator or affect the enzymes that are involved in the succinate conversion into malate. This may implicate a role for the mitochondrial B’ζ subunit in energy metabolism, perhaps through a synergetic function with PP2A-B’θ that we would like to investigate further. 

4- Investigating the functions of newly identified protein phospho-regulating enzymes.

Two PP2C family members (POL-like phosphatases 2 and 3 (PLL2 and PLL3)) were verified to have a functional PTS1 (SSM>). The full-length cDNAs of PLL2 and PLL3 were amplified from Arabidopsis tissues, and their fusion proteins targeted peroxisomes in two plant expression systems (Kataya et al., 2015c). Another putative protein phosphatase, PAP7, was validated to harbor a functional PTS1 (AHL>). Our reverse genetics studies indicate an impact for PLL3 on seedling germination, possibly by affecting peroxisomal β-oxidation (Kataya et al., 2015c). As an attempt to understand the phospho-regulation of peroxisomal functions, we will investigate the functions of PLL2, PLL3 and PAP7.

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Proxisomal-related assays

Sucrose dependence assay

Auxin precursors conversion assays

5- Investigate the impact of phosphorylation on peroxisome biogenesis

PEX5 and PEX14 phosphorylated residues are studied for a putative regulatory effect in Arabidopsis thaliana. Our MS/MS screen show limited number of phosphorylation for both proteins, which we selected for investigation.

6- Model for Phospho-Regulation of Triacylglyceride (TAG) Mobilization and Fatty Acid (FA) β-Oxidation.

Using functional analyses and deciphering the phosphoproteomes of selected mutants we will investigate further if this model is functional. Additionally, we are working on finding the impact of phospho-residue/s impact on selected β-oxidation mutants, which are related to lipid mobilization.