FIG 5.
Mth1 is a bona fide substrate for PKA. (A) Pph3-Psy2 deficiency does not change the stability of Mth1. Yeast extracts from WT, psy2Δ, and pph3Δ strains expressing Mth1-HA from the endogenous promoter were collected under the conditions indicated and analyzed by immunoblotting using anti-HA antibody. The anti-PSTAIRE antibody immunoblots were used as loading controls. (B) The top panel is an autoradiograph showing that Mth1-4SA, with mutation of four serines (Ser 111, 112, 161, and 358) to alanines abolishes the GST-Mth1 phosphorylation by PKA in vitro. The bottom panel depicts Coomassie staining showing the equal amount of GST-Mth1 and Ser-to-Ala mutant substrates. (C) At the top is an autoradiograph showing that the mutation of four serines also abolishes the phosphorylation of immunoprecipitated Mth1 by PKA. At the bottom is an immunoblot using anti-HA antibody showing the protein level of WT Mth1 and Mth1-4SA. (D) AT the top left, an autoradiograph shows that PKA-phosphorylated Mth1 is dephosphorylated by Pph3-Psy2 complex in vitro. At the bottom left, an immunoblot shows the Mth1 protein levels used in the assay. Quantification of the phosphorylation levels of Mth1 (shown at the left) is depicted on the right side of the panel. (E) MS analysis identified phosphopeptides containing a single phosphoserine at either S111 or S112 in both immunoprecipitated Mth1 and in vitro PKA-phosphorylated Mth1. (F) Immunoblot with anti-HA antibody showing the comparison of the phosphorylation levels of Mth1, Mth1PA, and Mth1-4SA mutant proteins expressed from pMT52 in a grr1-AAA strain after shifting cells from galactose (Gal)- to glucose (Glc)-containing medium. Immunoblotting with anti-PSTAIRE antibody was used as a loading control.