Phosphorylated Lte1 interacts with Ras and allows cortical distribution of Lte1. (a) Cells expressing Lte1-ProA (MGY415) were arrested by overexpression of nondegradable Sic1 or by HU treatment, and ProA-tagged proteins were immunoprecipitated. Lte1 (top panels) and Ras2 (bottom panels) were detected in crude extracts (CE) and after Lte1-ProA immunoprecipitation (ProA-IP). (b) Cells expressing Lte1-3HA and a nondegradable Clb2 under the control of GAL1 promoter (MGY239) were cultivated in YP-sucrose and arrested in G1 with α-factor. The culture was split and 2% glucose or galactose was added while the arrest was maintained. Cells were harvested at the indicated times and Lte1 and Clb2[Δdb] were detected by Western blotting. (c) WT cells expressing Lte1-Cdk-3HA (MGY240) or cla4Δ mutants expressing Lte1-3HA (MGY592) and a nondegradable Clb2 under the control of GAL1 promoter were treated as in b. Lte1 and Clb2[Δdb] were detected by Western blotting. (d) Cells expressing Lte1-GFP from a MET3 promoter and a nondegradable Clb2 under the control of GAL1 promoter (MGY565) were cultivated at 30°C in minimal medium without methionine and 2% sucrose. Cells were arrested in G1 with α-factor and localization of Lte1 was observed after 90 min (time 0). Then the culture was split and 2% glucose or galactose was added while the arrest was maintained. Lte1 localization was observed after 90 min. The percentage of cells with indicated Lte1-GFP distribution is indicated (n > 120). (e) Cells treated as in b were harvested after 60 min of addition of glucose or galactose and Lte1-HA was immunoprecipitated. Co-immunoprecipitated Ras2 and Lte1-HA were detected by Western blotting.