Abstract
The eukaryotic, highly conserved serine (Ser)/threonine-specific protein phosphatase 2A (PP2A) functions as a heterotrimeric complex composed of a catalytic (C), scaffolding (A), and regulatory (B) subunit. In Arabidopsis (Arabidopsis thaliana), five, three, and 17 genes encode different C, A, and B subunits, respectively. We previously found that a B subunit, B'θ, localized to peroxisomes due to its C-terminal targeting signal Ser-Ser-leucine. This work shows that PP2A C2, C5, andA2 subunits interact and colocalize with B'θ in peroxisomes. C and A subunits lack peroxisomal targeting signals, and their peroxisomal import depends on B'θ and appears to occur by piggybacking transport. B'θ knockout mutants were impaired in peroxisomal β-oxidation as shown by developmental arrest of seedlings germinated without sucrose, accumulation of eicosenoic acid, and resistance to protoauxins indole-butyric acid and 2,4-dichlorophenoxybutyric acid. All of these observations strongly substantiate that a full PP2A complex is present in peroxisomes and positively affects β-oxidation of fatty acids and protoauxins.
Highlights
Purified peroxisomes were isolated using a percoll gradient followed by a sucrose density gradient (Reumann and Singhal, 2014). In extracts of these peroxisomes, phosphatase 2A (PP2A) catalytic subunit was detected by Western blotting using antibodies specific for either methylated or demethylated C subunit (Fig. 1a, Supplemental Fig. S1a)
Bimolecular fluorescence complementation (BiFC) (Waadt et al, 2008) was used to identify C and A subunits that are interacting with B′θ in peroxisomes
No PP2A subunits had been reported as part of the proteome of isolated peroxisomes when analyzed by mass spectrometry (Fukao et al, 2002; Reumann et al, 2007; Eubel et al, 2008; Reumann et al, 2009; Quan et al, 2013), but sensitive immunological methods used in this work showed the presence of the PP2A catalytic subunits in extracts of highly purified peroxisomes (Fig. 1a)
Summary
When B′θΔSSL::VYCE(R) was co-expressed with C2::VYNE or C5::VYNE fluorescence remained in the cytosol (Fig. 2V, VI) showing that the B′θ PTS1 is necessary to direct the interacting proteins into peroxisomes. The b′θ mutant seedlings have unique phosphorylated proteins involved in TAG metabolism Very recent studies experimentally identified phosphopeptides related to peroxisomal βoxidation, glyoxysomal cycle, and PTS1 import proteins, and showed that several of the proteins involved in β-oxidation are phosphorylated (Fig. 9 and Supplemental Table S6). In an initial attempt to identify peroxisomal targets of PP2A, total protein extracts were prepared from seven days old b′θ-1 mutant and WT Col-0 seedlings grown on sucrose-free medium in short days.
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