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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 1.

Figure 1

Cln2/CDK antagonizes membrane-localized signaling mediated by the Ste5 PM domain
  1. Pheromone response pathway, showing membrane recruitment of Ste5.
  2. Methods for activating membrane-localized signaling. From left to right: α factor (αf) treatment or Gβ overexpression (Whiteway et al., 1990); hyperactive membrane localization of Ste5 via an enhanced PM domain (Winters et al., 2005); membrane targeting of Ste5 via a foreign transmembrane domain (Pryciak and Huntress, 1998); membrane targeting of Ste11 via a prenylation/palmitoylation motif (Winters et al., 2005).
  3. Cln2/CDK inhibition correlates with dependence on the Ste5 PM domain. Pathway-activating components were expressed from the GAL1 promoter and compared for their ability to induce FUS1-lacZ transcription in ste4Δ strains ± PGAL1-CLN2 (n = 4).
  4. Ste20-independent signaling is sensitive to Cln2 inhibition. Wild-type Ste11 (WT) or a Ste20-independent mutant (Ste11-Asp3) was expressed in ste11Δ or ste11Δ ste20Δ strains ± PGAL1-CLN2. FUS1-lacZ induction was measured after α factor treatment (n = 6).
  5. PM domain mutations that disrupt nuclear targeting (NLSm) do not affect Cln2 inhibition. FUS1-lacZ was induced by α factor treatment in ste5Δ ± PGAL1-CLN2 strains expressing Ste5-WT or Ste5-NLSm (n = 9).
  6. Increased Ste5 membrane affinity causes increased resistance to Cln2. Left, Ste5 variants contained PM domain mutations that increase membrane affinity. Right, the native PM domain was replaced with 1 or 2 copies of the PLCδ PH domain. All forms were expressed from the native STE5 promoter in ste5Δ strains ± PGAL1-CLN2, and response to α factor was measured (n = 4-9).

Data in all bar graphs show the mean ± SD.