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Synthetic trap-peptides identify a TOM complex phosphatase - PP2A dephosphorylates Tom6
Laura Scheinost1,2,3,4, Christina Ludwig5,6, Nico Höfflin1,2
1Faculty of Biology, Institute of Biology III, University of Freiburg, Germany.
The FEBS Journal
|September 2, 2025
Summary
Researchers developed synthetic trap-peptides to identify protein phosphatases. This method identified protein phosphatase 2A (PP2A) as the first phosphatase to dephosphorylate Tom6, a key component of the mitochondrial outer membrane complex.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Identifying specific protein phosphatases, especially serine/threonine-specific ones functioning as holoenzymes, is a significant challenge.
- The mitochondrial translocase of the outer membrane (TOM) complex is regulated by phosphorylation, but its phosphatases remain unknown.
- Kinases for the TOM complex are identified, but phosphatases are elusive.
Purpose of the Study:
- To develop synthetic trap-peptides for identifying phosphatases that bind to Tom6.
- To identify the specific phosphatases responsible for dephosphorylating the TOM complex.
Main Methods:
- Development of synthetic trap-peptides targeting Tom6.
- Affinity enrichment of phosphatases from yeast cytosolic fractions using trap-peptides.
- In vitro dephosphorylation assays to confirm phosphatase activity.
Main Results:
- Synthetic trap-peptides successfully enriched phosphoserine/threonine-specific protein phosphatases 2A (PP2A) and 4 (PP4) as holoenzymes.
- The interaction of PP2A and PP4 with Tom6 was mediated by their regulatory subunits, Cdc55reg and Psy2reg, respectively.
- Protein phosphatase 2A (PP2A) was confirmed to dephosphorylate Ser16 of Tom6 in vitro.
Conclusions:
- Synthetic trap-peptides are effective tools for identifying complete holoenzymes that bind to target sequences.
- Protein phosphatase 2A (PP2A) is identified as the first phosphatase acting on the TOM complex.
- This study elucidates a key regulatory mechanism of mitochondrial protein import.
