Lte1-8N constantly binds to Ras and interferes with its activity. (a) Cells expressing Lte1-ProA (MGY277) and Lte1-8N-ProA (MGY446) were arrested in α-factor, HU, or by expression of nondegradable Sic1. Lte1-ProA was also expressed in cla4Δ cells (MGY567) arrested in HU. ProA-tagged proteins were immunoprecipitated and Lte1 (top panels) and Ras2 (bottom panels) were detected in crude extracts (CE) and after Lte1-ProA immunoprecipitation (ProA-IP). Cells expressing untagged Lte1 were used as controls. (b) Localization of Lte1-8N-GFP and Kel1-CFP in cells overexpressing nondegradable Sic1 (MGY399). DAPI was used to stain nuclei. Asterisk indicates actively growing bud; triangle indicates nongrowing bud. Bar, 5 µm. (c) Cells expressing Lte1-GFP (SY148) or Lte1-8N-GFP (MGY449) under GAL1 promoter control were spotted on glucose- or galactose-containing medium (top panel). Cells expressing Lte1-8N-GFP alone or in combination with Ras2 (MGY517), both under GAL1 promoter control, were spotted on glucose- or galactose-containing medium (center panel). Cells expressing Lte1-8N-GFP and Lte1-8N^R1343E-GFP (MGY527) under GAL1 promoter control were spotted on glucose- or galactose-containing medium (bottom panel). (d) Cells expressing Lte1-GFP (○) and Lte1-8N-GFP (•) under GAL1 promoter control were cultivated in YP-sucrose. 2% galactose was added at time 0 and unbudded cells were counted (n > 200 per point) at the indicated times. Error bars represent SD of three experiments.