Peroxisomal protein phosphatase PP2A-B' theta interacts with and piggybacks SINA-like 10 E3 ligase into peroxisomes.

Protein phosphatase 2A (PP2A) is targeted to the plant peroxisome via a C-terminal SSL sequence on its regulatory B' theta (θ) subunit. To date the substrates of peroxisomal PP2A are unknown but are thought to be recruited by the regulatory B'θ subunit. Employing yeast two hybrid screening, we have identified Arabidopsis E3 ligase SINA-like 10 as a B'θ binding partner. The E3 ligase SINA-like 10 was found to harbor the PP2A B'-binding Short Linear interaction Motif or SLiM, LxxIxE. This interaction was further verified both in vitro and in vivo using direct pulldown assays and bimolecular fluorescence complementation. Utilizing peroxisomal targeted and a cytosolic version of B'θ (lacking its C-terminal peroxisomal targeting sequence SSL>) bimolecular fluorescence complementation suggests an interaction to occur in the cytosol followed by piggybacking E3 ligase SINA-like 10 into peroxisomes. These results identify a first peroxisomal PP2A interactor, which also obtains a PP2A B'-binding SLiM.

Affinity-based profiling of endogenous phosphoprotein phosphatases by mass spectrometry.

Phosphoprotein phosphatases (PPPs) execute >90% of serine/threonine dephosphorylation in cells and tissues. While the role of PPPs in cell biology and diseases such as cancer, cardiac hypertrophy and Alzheimer's disease is well established, the molecular mechanisms governing and governed by PPPs still await discovery. Here we describe a chemical proteomic strategy, phosphatase inhibitor beads and mass spectrometry (PIB-MS), that enables the identification and quantification of PPPs and their posttranslational modifications in as little as 12 h. Using a specific but nonselective PPP inhibitor immobilized on beads, PIB-MS enables the efficient affinity-capture, identification and quantification of endogenous PPPs and associated proteins ('PPPome') from cells and tissues. PIB-MS captures functional, endogenous PPP subunit interactions and enables discovery of new binding partners. It performs PPP enrichment without exogenous expression of tagged proteins or specific antibodies. Because PPPs are among the most conserved proteins across evolution, PIB-MS can be employed in any cell line, tissue or organism.