Analysis of point mutations in the putative GEF domain of Lte1. (A) Complementation assays for rescue of cold sensitivity of the lte1Δ strain SY144 with integrative YIplac128-based plasmids expressing the mutant forms of LTE1 indicated. (B) Subcellular localization of wild-type (WT), K1273E, and F1387E forms of Lte1-GFP expressed in the lte1Δ strain SY144. (C) Cell cycle–dependent phosphorylation of Lte1. The lte1Δ strain SY144 carried the integrative plasmid expressing the 3HA-tagged forms of Lte1 indicated. After cell arrest with α factor (αF), hydroxyurea (HU), or nocodazole (NOCO), Lte1 status was revealed by Western blotting with anti-HA antibody. Short and long exposures of the same blot are presented. Molecular mass is indicated in kilodaltons. (D) In vivo interaction between wild type (wt) and mutant Lte1-3HA with Ras2. After cell arrest with hydroxyurea, proteins were extracted from the strains indicated. Lte1 was immunoprecipitated with anti-HA antibody. Cell extracts (C.E.) and immunoprecipitates (αHA-IP) were analyzed by Western blotting with anti-HA or anti-Ras2 antibodies. Bar, 5 µm.