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. Author manuscript; available in PMC: 2009 Mar 11.
Published in final edited form as: Curr Biol. 2008 Mar 11;18(5):322–330. doi: 10.1016/j.cub.2008.02.005

Figure 1. Pom1 Kinase Interacts with a RhoGAP, Rga4.

Figure 1

(A) Interaction between Pom1 and Rga4 in the yeast two-hybrid assay. Abbreviations: LIM, LIM domain; cc, coiled-coil; GAP, Rho-GAP domain; kinase, Ser/Thr-protein kinase domain. Domain prediction is based on the Pfam database. Numbers indicate positions in amino acid sequences. Interaction between Pom1 and Rga4 in the yeast two-hybrid assay was determined by growth on medium without histidine. “+++,” growth with 8 mM 3-AT; “+,” growth without 3-AT; “–,” no growth even without 3-AT. (B) Pom1 kinase is copurified with Rga4. pom1:myc and pom1:myc rga4:FLAG strains were grown in YES medium and their cell lysate was subjected to anti-FLAG immunoprecipitation, followed by anti-FLAG and anti-myc immunoblotting. (C) Rga4 is a phosphoprotein. Denatured crude lysate, prepared from a rga4:FLAG strain, was incubated with λ protein phophatase (λPP) with and without phosphatase inhibitors, and resolved by SDS-PAGE with reduced bis-acrylamide in the gel. (D) Pom1 affects the phosphorylation state of Rga4. Top, anti-FLAG immunoblotting was performed with the crude lysate of wild-type, Δpom1, Δtea1, and pom1−2 strains expressing Rga4FLAG. Bottom, anti-FLAG immunoblotting of the lysate prepared from the rga4:FLAG Δpom1 strain carrying the pREP1-pom1:myc before (−) and after (+) induction of Pom1myc by thiamine depletion. (E) Pom1 affects the solubility of the Rga4 protein. The supernatant was analyzed by immunoblotting. Lane 1, rga4:FLAG; lane 2, rga4:FLAG Δpom1; lane 3, rga4:FLAG Δpom1 overexpressing Pom1 from the pREP1-pom1:myc plasmid. Anti-Spc1 immunoblotting served as a loading control.