Protocol

Protein Phosphatase Protocols

Volume 365 of the series Methods in Molecular Biology pp 235-246

Proteins Interacting With Saccharomyces cerevisiae Type 1 Protein Phosphatase Catalytic Subunit Identified by Single-Step Affinity Purification and Mass Spectrometry

  • Edmund P. WalshAffiliated withUniversity of Newcastle
  • , Douglas J. LamontAffiliated withSchool of Life Sciences, University of Dundee
  • , Kenneth A. BeattieAffiliated withSchool of Life Sciences, University of Dundee
  • , Michael J. R. StarkAffiliated withSchool of Life Sciences, University of Dundee

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Abstract

The catalytic subunit of type 1 protein phosphatase (PP1C) interacts with a large number of polypeptides in eukaryotic cells from yeast to man and these regulatory subunits can both modulate the activity of PP1C and target it to different subcellular locations. Thus, PP1 is really a family of protein phosphatases that share a common catalytic subunit, and identifying and characterizing the PP1-associated proteins is therefore critical to understanding the cellular roles of PP1 and its ability to dephosphorylate specific substrates. Here we describe methods for affinity isolation of PP1C-containing protein complexes in the yeast Saccharomyces cerevisiae and the identification of the associated polypeptides by mass spectrometry. The basic method we describe could be easily adapted to study PP1C-associated proteins in other lower or higher eukaryotes and for characterizing the protein-protein interactions of other protein phosphatases in yeast.

Key Words

Protein-protein interactions Glc7p yeast PP1 affinity isolation protein-A affinity tag mass spectrometry