Multi-step phosphorylation of Lte1. (a) Cells expressing Lte1-GFP and a degron form of Cla4-HA (MGY320) were cultivated at 23°C in YP-sucrose. Galactose was then added to induce nondegradable Sic1 and part of the culture was transferred to 37°C for 2.5 h. (b) Localization of Lte1-8N-GFP (MGY409) and Lte1-GFP (SY158) in a cla4Δ background. (c) Cells expressing Lte1-ProA or Lte1-8N-ProA in a WT (MGY261 and MGY415) or a cla4Δ background (MGY443 and MGY444) were arrested with HU and interaction between Lte1 and Ras2 was analyzed by Co-IP. (d) Lte1-8N-ProA purified from cla4Δ (MGY444) cells arrested in HU was treated with phosphatase buffer, with λ-phosphatase alone or in combination with phosphatase inhibitors and analyzed by Western blot. (e) Cells expressing Lte1-3HA and degron Cla4 (MGY313) were cultivated at 25°C and arrested with HU for 2 h. Half of the culture was then transferred at 37°C. Both cultures were sampled at 30-min intervals and Lte1-3HA was analyzed by Western blotting.