Table 4.

Effects of overexpressing STE2 alleles on constitutive activation of the pheromome response pathway and expression of cell-surface α-factor binding sites

STE2 allele expressed	FUS1-lacZ expression (% wild type + α-factor)		Receptor expression (α-factor binding sites/cell)
	−α-factor	+α-factor
ste2Δ	0.7	1.0	−
Wild type	0.4	100	34,000
P258L	18	96	18,000
P258D	3.9	97	700
P258Y	54	103	250

The indicated STE2 alleles were expressed from their normal promoters on high-copy plasmids (pRS424 derivatives) in a ste2Δ mfα1Δ mfα2Δ mutant (KBY16) that contained the FUS1-lacZ gene on a plasmid (pSL307). Where indicated, cells were treated 2 h at 30°C with 1 μM synthetic α-factor (+α-factor); β-galactosidase assays were performed to monitor activation of the pheromone response pathway. Data are expressed as a percent of the activity detected with α-factor–treated cells overexpressing the wild-type STE2 gene. Values shown are the average of a least four independent transformants, each assayed in duplicate; standard errors were 5-30% of the values shown. Radioligand binding assays using [³⁵S]α-factor were performed to determine the number of cell-surface ligand-binding sites (B_max) in cells (KBY16) overexpressing various STE2 alleles from high-copy plasmids (pRS424 derivatives). The values shown for each STE2 allele were calculated from nonlinear regressions of data obtained from two independent transformants, each of which was assayed two to four times. Standard errors for these determinations were 5-15%