Identification of phosphatases that dephosphorylate Tom6 using synthetic trap-peptides

The identification of phosphatases that dephosphorylate specific sites in proteins remains a major challenge, particularly for the major class of serine/threonine-specific phosphatases, which function as holoenzymes. Here, we report the development of synthetic trap-peptides to identify phosphatases that bind to Tom6, a subunit of the mitochondrial translocase of the outer membrane (TOM complex). The TOM complex is regulated by reversible phosphorylation, and while responsible kinases have been identified, the corresponding phosphatases so far remain unknown. Here, the trap-peptides enriched PP2A and PP4 as full holoenzymes from yeast cytosolic-fractions. We observed that their interaction with Tom6 was mediated through their regulatory subunits Psy2reg and Cdc55reg, and that PP2A was able to dephosphorylate Ser16 of Tom6 in vitro. In summary, synthetic trap-peptides facilitate the identification of complete holoenzymes that bind to the target sequence and reveal PP2A as the first TOM phosphatase.