FIG. 5.

Vpr induces cell cycle G₂ arrest through direct interaction with Srk1. (A) Coexpression of vpr and srk1 showed no additive effect on Vpr-induced cell elongation. (a) Overexpression of srk1 induces cell cycle G₂ arrest with cell elongation. The wild-type (SP223) yeast strain was transformed with pREP1-srk1(6HisHA) or pREP1-srk1(K153A)(6HisHA) and grown for 27 h in the minimal selective EMM liquid culture either in the presence or in the absence of thiamine to induce gene expression. The average cell length and standard derivation was measured on at least 100 cells by using the OpenLab software and based on three independent experiments. (b) Coexpression of vpr and srk1 showed no additive effect on Vpr-induced cell elongation. The RE076 fission yeast strain, which carries a single integrated copy of F34Ivpr gene (13), was transformed with pREP1-srk1(6HisHA) or pREP1-srk1(K153A)(6HisHA) and grown and assayed as described for panel Aa. Immunoblot analysis was performed to confirm the expression of srk1(6HisHA), srk1(K153A)(6HisHA), and vpr in the SP223 and RE076 strains transformed with pREP1-srk1(6HisHA) or pREP1-srk1(K153A)(6HisHA). The same levels of Vpr or Srk1 proteins were expressed in each of these samples (data not shown). (B) Deletion of the srk1 gene abolished Vpr-induced cell elongation. The srk1⁺ (RE076) and Δsrk1 (SH2) fission yeast strains were grown for 20 h in the minimal selective EMM liquid culture in the presence [vpr(F34I)-off] or in the absence [vpr(F34I)-on] of thiamine to induce vpr gene expression. Cell length was measured as described for Fig. 1A. Immunoblot analysis was performed to confirm the expression of vpr in the srk1⁺ and Δsrk1 strains. The vpr-repressing and vpr-expressing cells were collected and lysed after 20 h of gene induction. Equal amounts of proteins (25 μg) were loaded onto SDS-PAGE gels, and the Vpr was visualized by using anti-Vpr (dilution, 1/500). The same levels of Vpr proteins were expressed in each of these samples (data not shown). (C) Vpr directly interacts with Srk1, as indicated by immunoprecipitation. The RE007 yeast strain was transformed with pREP1-srk1(6HisHA), pREP1-srk1(K153A)(6HisHA), or pREP1-L3(6HisHA) and was grown in the selective liquid culture for 24 h in the presence or in the absence of thiamine to induce gene expression. Srk1 was then immunoprecipitated with anti-HA antibody from cell extracts. The protein L3 was used here as a nonspecific protein control during the immunoprecipitation experiment. G.I., gene induction; IP, immunoprecipitation; TE, total extract. Bar, 10 μm.