Fig. 7. Functional domain analysis of Lte1. (A) A schematic representation of the domain structure of Lte1 is shown. The N-terminal region contains a GEFN motif, whereas the Cdc25-related GEF domain resides in the extreme C-terminus. Mutants were either expressed from an integrated allele driven by the GAL1 promoter (GAL1) or from an integrated allele driven by the LTE1 promoter (endogenous). Complementation was scored as the ability to rescue the growth defect of lte1 mutant (SY144) by serial dilution spot analysis after 7 days incubation at 14°C. Lte1 mutants were tested for complementation with or without GFP/HA3 tag with roughly similar outcome. +, cortical localisation; -, cytoplasmic/none localisation; +/-, partial cortical localisation; ND, not determined. (B) Representative cells of Lte1 mutants expressed either from GAL1 promoter or at endogenous level; Lte1C984GFP (SY149); Lte1N801GFP (SY150); wild-type Lte1GFP (SY148); Lte1N157GFP (SY146); Lte1C1334GFP (SY147); wild-type Lte1GFP (SY145). (C) Equal amounts of GST-Lte1N984 protein or GST were bound to glutathione sepharose beads and incubated with MBP-Lte1 (1-500). Bound protein was eluted with glutathione and analysed by SDS-PAGE (10% gels) and immunostaining with anti-MBP antibody.

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