Chemical-genetic analysis indicates that two-thirds of the PKC1 genetic interaction network comprises “within-pathway” interactions. (A) Example of sorbitol suppression of the hypersensitivity of a null mutant to staurosporine. Wild-type and api2Δ cells were grown in rich medium and then diluted into wells of a 96-well plate containing fresh medium supplemented with staurosporine (range, 0 μg/ml to 50 μg/ml). The cells were incubated either in the presence (+) or in the absence (−) of sorbitol (1 M). (B) Scheme illustrating the functional classification of the PKC1-interacting genes. Fourteen of the genes behave as encoding proteins that act in the Pkc1 pathway (staurosporine sensitivity is suppressed by osmotic stabilization), six of which encode known CWI pathway components (see text). The remaining seven genes behave as encoding proteins that function, at least in part, parallel to Pkc1 (staurosporine sensitivity is not suppressed by osmotic stabilization).

The basal activity of the Rlm1-LacZ reporter construct is altered in the majority of the Pkc1 genetic network. Each of the 21 network mutants, along with the congenic wild-type strain, was transformed with a plasmid containing LacZ expressed under the control of the Slt2-dependent transcription factor Rlm1. The transformed mutants were assayed for β-galactosidase activity. The relative activity was determined in comparison to the activity of wild-type transformed cells. The experiment was performed on multiple different days and in triplicate on each day. Data from each day were scaled to the average data for the wild-type controls on that day. The average relative activities of the mutants across days and the standard deviations are shown for each mutant. The relative activity for wild-type cells is 1.0 by definition.

Ydl203c/Ack1 activates Rlm1-LacZ reporter activity but is not required for stress induction of the reporter. (A) Wild-type cells were cotransformed with the Rlm1-LacZ reporter plasmid and either a vector control or a plasmid expressing YDL203c under a GAL1,10 promoter. The transformed cells were grown in synthetic medium containing galactose and sucrose to induce the expression of YDL203c. The transformed cells were assayed for β-galactosidase activity. The experiment was performed in triplicate. The relative activity was determined relative to the average activity of the vector controls analyzed on the same day. Standard deviations for the replicates are shown for each strain. (B) Wild-type and yld203cΔ cells transformed with the Rlm1-LacZ reporter construct were examined for their ability to activate the expression of the reporter construct. Overnight cell cultures were diluted and treated with or without 150 μg/ml calcofluor white (CW), 200 μg/ml chlorpromazine (CPZ), or 40 μg/ml latrunculin B (LatB) for 3 hours prior to the assay for β-galactosidase activity. The activity was determined relative to that for untreated, wild-type cells, and all analyses were performed in triplicate on the same day. Standard deviations for replicates are shown for each strain.

Epistasis analysis places Ydl203c/Ack1 in the Pkc1 pathway and upstream of Pkc1. (A) ydl203cΔ cells were transformed with plasmids overexpressing ROM2, TUS1, PKC1, MKK1, or SLT2 and a vector control. Transformed cells were grown in synthetic medium, diluted to an OD₆₀₀ of 0.02, and then serially diluted into fresh medium. The dilutions were spotted onto synthetic medium plates containing tunicamycin (Tm; 1.5 μg/ml). The plates were incubated at 25°C for 2 days, and the growth of transformants was compared to that for the vector control. “+” indicates growth better than that for the vector control. (B) Example of suppression of the tunicamycin hypersensitivity of ydl203cΔ cells by overexpression of ROM2. (C) wsc1Δ/wsc1Δ, rom2Δ, tus1Δ, pkc1^ts, slt2Δ/Δ, and rom2Δ tus1Δ cells and their congenic wild-type cells were transformed with a vector control or a plasmid expressing YDL203c under a GAL1,10 promoter. The transformed cells were streaked onto synthetic medium plates containing galactose and sucrose to induce the expression of YDL203c. Plates were incubated at 25°C, and a duplicate set was incubated at 37°C to examine the ability of Ydl203c to suppress the temperature sensitivity of pkc1^ts, slt2Δ/Δ, and rom2Δ tus1Δ cells. Plates containing benomyl (20 μg/ml) were used to assess the ability of YDL203c overexpression to suppress the benomyl hypersensitivity of rom2Δ and rom2Δ tus1Δ cells. Plates containing chlorpromazine (200 μg/ml) were used to examine the ability of YDL203c overexpression to suppress the chlorpromazine hypersensitivity of tus1Δ cells. Plates containing calcofluor white (25 μg/ml) were used to examine the ability of YDL203c overexpression to suppress the calcofluor white hypersensitivity of wsc1Δ/wsc1Δ cells. All plates were incubated at 25°C for 2 days before the growth of the transformants was assessed. “+” indicates growth better than that of the vector control transformants; “−” indicates growth equal to that of the vector control. (D) Example of the ability of YDL203c overexpression to suppress the benomyl hypersensitivity of rom2Δ cells. Wt, wild type.