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Fig. 4. Flp1p interacts with and dephosphorylates Cdc25p. (A) GSTp, GST-Flp1p and GST-Flp1CSp proteins were expressed under the control of the nmt1 promoter in a cdc25 ${Delta}$ cdc2-3w strain and purified with glutathione-Sepharose (GSH) beads. An aliquot of each sample was processed for PAGE and stained with Coomassie Blue to visualise proteins. (B) GST-Flp1p and GST-Flp1CSp but not GSTp associate in vivo with Cdc25p. GSTp (negative control, lanes 1), GST-Flp1p (lanes 2) or GST-Flp1CSp (lanes 3) were purified and processed for the detection of Cdc25p from cdc25⁺ (wt) or cdc25 ${Delta}$ cdc2-3w (cdc25 ${Delta}$ ) cells expressing them (20 hours of induction of the nmt1 promoter). A total extract control from nda3-KM311 metaphase-arrested cells is shown for the detection of Cdc25p (lane C). Note that the Cdc25p protein that associates with the GST-Flp1CSp form is hyperphosphorylated as compared with the migration of Cdc25 from metaphase-arrested cells shown in lane C. (C,D) Purified GSTp, GST-Flp1p and GST-Flp1CSp proteins from A were assayed in vitro for their ability to dephosphorylate an artificial substrate, pNPP (C), or Cdc25p immunoprecipitated from nda3-KM311 metaphase-arrested cells (D). Note in D the mobility shift observed when immunoprecipitated Cdc25p is incubated with GST-Flp1p, in comparison with GSTp and GST-Flp1CSp negative controls.

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