Figure 4.

Role of CSN subunits in the mating pheromone pathway. (A) Mating efficiency of CSN disruptions. Deletants in csn5 or csn12 show increased mating efficiency by up to 100% when mated with wild-type (WT) cells. Δcsn10 or Δpci8 show little or no effect on mating, while csi1 null is appreciably poorer at mating compared with WT. Error bars are calculated from an average of 5–7 independent mating experiments for each strain. (B) Shmoo formation. Exponentially growing MATa cultures were treated with 1 mM α-factor pheromone. After 1.5 and 3 h, aliquots were fixed and the percentage of cells with shmoo-like projections were counted. Error bars represent the average of triplicate 200-cell samples. Cells deleted for csn5, csn9, csn10 or csn12 respond to pheromone quicker and to a greater extent than WT cells. Deletion of csi1 delays the response to pheromone exposure. (C) Adaptation to pheromone response (halo assay). Cultures of Δsst2 cells containing a vector plasmid (control) or overexpressing individual CSN subunits were plated on selective media and treated with α-factor to the center of the plate. A halo of no-growth encircles the pheromone due to the inability of sst2 cells to exit G₁ arrest. Formation of spontaneous colonies within this halo in Δsst2 culture is at single digits (vector). Overexpression of Csn9, Csn10 or hsCsn1 enhances adaptation, allowing for numerous cells to resume growth within the halo and form colonies. (D) Adaptation to pheromone CSN deletants. As in (C) except that Δsst2 and double deletant strains were plated on complete media. Deletion of csi1 facilitates adaptation to pheromone exposure. Thick growth of Δsst2Δcsi1 occurs at the periphery of the halo, where local concentration of pheromone is lower, and numerous colonies are noticeable throughout the halo. Deletions of other CSN subunits have a mild negative effect on adaptation, decreasing the number of viable colonies compared with the Δsst2 single deletion.