Fig. 6. Lte1 is phosphorylated by Cdc28 across the cell cycle. (A) Wild-type cells (SY142) were released from an -factor-induced G1 block into fresh YPDextrose medium at 25°C. Cells were collected at indicated intervals and processed for immunoblotting. The asterisk indicates the position of an unspecific band. (B) Cells containing a non-degradable version of Sic1 under the control of the GAL promoter (SY135) were synchronised in G1 with -factor. Cells were released into either YPSucrose or YPGalactose medium at 30°C. At 15 minute intervals, samples were withdrawn for western blotting to monitor Lte1 phosphorylation and FACS analysis. Cells expressing Lte1 GFP (SY136) were treated in a similar fashion and the effect of Sic1N overproduction on Lte1 localisation scored by microscopy (lower panel). Numbers indicate the percentage of cells with cortical Lte1. (C) Left panel, GST-Cdc28-13 purified from asynchronous yeast cells was used for kinase assays at 25°C and 37°C with either GST-Lte1(300-845) or Histone H1 as substrates. The Cdc28-13 kinase was pre-incubated at 37°C for 30 minutes prior to use in reactions performed at 37°C. Middle panel, GST-Cdc28/Cln2 complexes purified on glutathione sepharose beads from cdc53-1 yeast cells (SY141) arrested at the restrictive temperature were used for kinase assays with either GST-Lte1(300-845) or Histone H1 as substrates. Extract from uninduced cdc53-1 cells was used as a negative control. Right panel, purified GST-Lte1(300-845) and GST-Lte1Cdk(300-845) proteins were used in kinase assays with Cdc28 purified from nocodazole-treated yeast cells. The band of radioactive protein migrated to the same position as the Lte1 protein detected by Coomassie staining (input). (D) Cells expressing Lte1CdkHA3 (SY143) were synchronised in G1 by -factor and released into YPDextrose medium at 25°C. Cells were then treated as in A. (E) Localisation of Lte1GFP in G1 cyclin-depleted cells (SY124) (—Cln2), after 3 hours overexpression of Cdc42-G12V from the GAL promoter (—Cln2+galactose). The number indicates cells with clear cytoplasmic staining. Cells released from the arrest localised Lte1 efficiently at the cortex (+Cln2). Here the number indicates cells with cortical distribution.

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