KatayaProject

A role for PP2A in energy metabolism

Numerous peroxisome-targeted protein phosphatases and kinases have been identified, establishing a framework of key players that control peroxisomal phosphorylation.

Peroxisomes are well known for their importance in fatty acid β-oxidation (a catabolic process by which fatty acid molecules are broken down through successive removal of two carbons) in plants, yeast and mammals. In plants and yeast the process can only take place in peroxisomes whereas in mammals mitochondria are involved. Both peroxisomal and mitochondrial β-oxidation leads to chain shortening of acyl-CoA esters to yield chain-shortened acyl-CoA and acetyl-CoA or enoyl-CoA depending on the substrates (Kaur et al., 2009; Poirier et al., 2006).

In oilseed plants, fatty acid β-oxidation is especially important to supply energy after seed germination. This energy pathway involves fatty acid β-oxidation, glyoxylate cycle, and gluconeogenesis. Triacylglycerides are hydrolysed to fatty acid, which are transferred into peroxisomes where they are β-oxidized to give acetyl-CoA. Acetyl-CoA is processed by the glyoxylate cycle in peroxisomes and gives succinate that is transferred to mitochondria. In mitochondria, succinate is converted to malate that is transported to cytosol and used for sucrose production (Fig 1). The physiological role of β-oxidation in plants also includes production of the phytohormones jasmonic acid and indole-3-acetic acid (IAA) (Kaur et al., 2009; Poirier et al., 2006).

Protein phosphatase 2A (PP2A) is a heterotrimeric complex comprising a catalytic (C), scaffolding (A), and regulatory (B) subunits (Fig.1). In Arabidopsis, 17 regulatory subunits are present, which can lead to 255 possible PP2A holoenzyme combinations with the five catalytic and three scaffolding subunits (Farkas et al., 2007). The regulatory subunits are essential for substrate specificity and localization of the complex and are classified into B, B’, and B’’ non-related families in higher plants (Fig. 1). In Arabidopsis, the close homologs B’η, B’θ, B’γ, and B’ζ were further classified into a subfamily of B’ called B’η. One of our interesting recent finding is that a protein phosphatase 2A complex is imported into plant peroxisomes (Kataya et al., 2015). The involvement of such a complex in regulation of fatty acid metabolism was confirmed by reverse genetics. Plants knocked out with respect to the B'θ subunit of PP2A were impaired in fatty acids mobilization after germination (Kataya et al., 2015a) underpinning the involvement of PP2A in upholding fatty acid degradation. We also investigated if the other subfamily members would be involved in similar processes, where T-DNA insertion lines for B’ζ, and B’η were analyzed by a sugar independence assay. Interestingly, seedlings of the b’ζ mutant showed seedling growth retardation in sucrose-free medium (Kataya et al., 2015c). The PP2A-B’ζ holoenzyme may 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 B’θ that we would like to investigate further.

Recent identification of four additional peroxisomal protein phosphatases

Plant peroxisomes are fragile and difficult to purify and study in vitro. Hence many low-abundant and stress-induced proteins remained unidentified due to this experimental limitation. Using our knowledge from the newly developed bioinformatic methods (Lingner, Kataya et al., 2011), we searched Arabidopsis thaliana for phosphatase-related proteins with a putative PTS (Table 1). From this screen, seven proteins were selected as potential candidates harboring either PTS1 or PTS2. We investigated the functionality of their PTSs and found four leading to peroxisomal targeting. The PTS1 (SAL>) of MAP kinase 1 (MKP1) was found to be a novel PTS1. The full-length MKP1 remained in the cytosol when transiently expressed in Arabidopsis mesophyll protoplasts, but was also detected in peroxisomes when different biotic and abiotic stresses were applied (Kataya et al., 2015). Two PP2C family members (POL-like phosphatases 2 and 3 (PLL2 and PLL3)) were also 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., unpublished, in progress). 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 peroxismal β-oxidation (Kataya et 2016).