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. Author manuscript; available in PMC: 2007 Aug 9.
Published in final edited form as: Cell. 2007 Feb 9;128(3):519–531. doi: 10.1016/j.cell.2006.12.032

Figure 5.

Figure 5

Phosphorylation of Ste5 by Cln2/CDK
  1. Phosphorylation of the Ste5 N-terminus by Cln2/Cdc28 in vitro. Bacterially-expressed GST-Ste51-125 fusions (WT, 8A, and 8E) were phosphorylated by recombinant Cln2-Cdc28. Histone H1 served as a control substrate.
  2. Cln2 expression in vivo alters Ste5 electrophoretic mobility. HA-tagged Ste5 (WT, 8A, and 16E) was immunoprecipitated from the indicated strains after 3 hr galactose induction (to express Cln2), resolved by SDS-PAGE, and analyzed by anti-HA immunoblot.
  3. The Ste5 mobility shift is due to phosphorylation. Ste5-HA3 was immunoprecipitated from ste5Δ ± PGAL1-CLN2 strains, and treated with calf intestinal phosphatase (CIP).
  4. Effects on Ste5 mobility are specific to Cln2. Ste5-HA3 derivatives (WT, 8A, ΔNLS, ΔNLS+8A) were analyzed as in panel B, using various PGAL1-cyclin strains.
  5. Ste5ΔNLS is more fully modified by Cln2. Ste5-HA3 derivatives were analyzed as in panel B.
  6. Ste5ΔNLS modification is elevated after Start and requires Cln1/2. WT and mutant strains (“cln1,2” = cln1 cln2) expressing Ste5-HA3 (ΔNLS or ΔNLS+8A) were incubated for 3 hr at 37°C.
  7. Modification of the Ste5 N-terminus is cell cycle dependent. Cells (cdc15-2 or cdc28-13) harboring Ste5-HA3 (ΔNLS or ΔNLS+8A) were arrested at 37°C for 3 hr, then transferred to 25°C to resume cycling. Samples were collected at 20 min intervals (0-180 min). As cdc15 cells arrest with large buds, emergence of small buds was used to follow cell cycle progression (c.f., Figure S1D).

In panels B-G, Ste5-HA3 derivatives were expressed from the native STE5 promoter.