Role of Kel1 in Lte1 localization and function. (a) Localization of Lte1-GFP and Kel1-CFP in cells (MGY305) overexpressing nondegradable Sic1 (top panels) and in cells treated with HU (bottom panels). DAPI was used to stain nuclei. Asterisk indicates actively growing bud; triangle indicates nongrowing bud. Bar, 5 µm. (b) WT (MGY369) and cla4Δ (MGY423) cells expressing Lte1-3HA were arrested by overexpression of nondegradable Sic1 (WT) or by HU treatment (WT and cla4Δ). Lte1-HA was visualized by Western blotting. (c) Localization of Lte1-GFP in WT (MGY593) and kel1Δ (MGY594) cells. Cells expressing Lte1-GFP from a MET3 promoter and Sic1 from the GAL1 promoter were cultivated in minimal medium without methionine and 2% sucrose as carbon source. α-Factor or galactose (2%) was added and Lte1 was observed after 2 h. DAPI was used to stain nuclei. Bar, 5 µm. (d) WT (MGY261 and MGY281) and kel1Δ (MGY479 and MGY480) cells expressing Lte1, Lte1-ProA, or Lte1-Cdk-ProA were arrested in HU and ProA-tagged proteins were immunoprecipitated. Immunoprecipitated Lte1-ProA and coimmunoprecipitated Ras2 proteins were detected (top and bottom panels, respectively). Relative amounts of Ras bound to each form of Lte1were quantified. (e) lte1Δ slk19Δ (MGY212) and lte1Δ slk19Δ kel1Δ (MGY 503) strains kept alive by a centromeric URA3-based plasmid expressing Lte1 were transformed with integrative plasmids expressing Lte1 and Lte1-Cdk. Serial dilutions of transformants were spotted on rich medium and 5-FOA–containing plates and cultivated at 30°C for 3 d.