Relative Dependence of Different Outputs of the Saccharomyces cerevisiae Pheromone Response Pathway on the MAP Kinase Fus3p

Abstract

Fus3p and Kss1p act at the end of a conserved signaling cascade that mediates numerous cellular responses for mating. To determine the role of Fus3p in different outputs, we isolated and characterized a series of partial-function fus3 point mutants for their ability to phosphorylate a substrate (Ste7p), activate Ste12p, undergo G1 arrest, form shmoos, select partners, mate, and recover. All the mutations lie in residues that are conserved among MAP kinases and are predicted to affect either enzyme activity or binding to Ste7p or substrates. The data argue that Fus3p regulates the various outputs assayed through the phosphorylation of multiple substrates. Different levels of Fus3p function are required for individual outputs, with the most function required for shmoo formation, the terminal output. The ability of Fus3p to promote shmoo formation strongly correlates with its ability to promote G1 arrest, suggesting that the two events are coupled. Fus3p promotes recovery through a mechanism that is distinct from its ability to promote G1 arrest and may involve a mechanism that does not require kinase activity. Moreover, catalytically inactive Fus3p inhibits the ability of active Fus3p to activate Ste12p and hastens recovery without blocking G1 arrest or shmoo formation. These results raise the possibility that in the absence of sustained activation of Fus3p, catalytically inactive Fus3p blocks further differentiation by restoring mitotic growth. Finally, suppression analysis argues that Kss1p contributes to the overall pheromone response in a wild-type strain, but that Fus3p is the critical kinase for all of the outputs tested.

Effects of HSP70 chaperones Ssa1 and Ssa2 on Ste5 scaffold and the mating mitogen-activated protein kinase (MAPK) Pathway in Saccharomyces cerevisiae

“…α-factor was added from a concentrated stock (6 mM in methanol) and samples were shaken for 2 hours, pelleted, washed once with ice-cold water and frozen at −80°C. Pellets were thawed and extracts prepared and quantified exactly as described (85). Units of beta-galactosidase were calculated by the following formula: Units = [A 420 x (1.7/0.0045)]/ [time (min) x extract volume (ml) x protein concentration (mg/ml)].…”

“…CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made CYC1prom-STE7-MYC whole cell extracts was concentrated by 40% ammonium precipitation then dissolved in loading buffer prior to immunoblot analysis as described (85). Co-IPs used 250 µg to 2 mg of whole cell protein extracts, 15 µg of 12CA5, 1-2 µg of 9E10, and 30 µl of protein A agarose beads (Sigma).…”

“…Resistance to 5-fluoro-orotic acid and subsequent complementation tests and confirmation of mitochondrial function was done to isolate ura3 mutant derivatives that revert at very low frequency for several ssa mutant combinations to permit introduction of plasmids harboring a URA3 gene for selection. Mating type was determined by a qualitative patch mating assay (85). Strains were grown at room temperature and 30 o C unless noted otherwise.…”

“…The protein concentration of whole cell extracts was determined with the BioRad assay. For detection of Ste7-MYC, 1 mg of CYC1prom-STE7-MYC whole cell extracts was concentrated by 40% ammonium precipitation then dissolved in loading buffer prior to immunoblot analysis as described (85). Co-IPs used 250 µg to 2 mg of whole cell protein extracts, 15 µg of 12CA5, 1-2 µg of 9E10, and 30 µl of protein A agarose beads (Sigma).…”

“…Cells were grown overnight at room temperature to an A 600 of ~0.3, pelleted, and resuspended at an A 600 of 0.3 in 10 ml aliquots of fresh media. a-factor was added from a concentrated stock (6 mM in methanol) and samples were shaken for 2 hours, pelleted, washed once with ice-cold water and frozen at -80 o C. Pellets were thawed and extracts prepared and quantified exactly as described (85). Units of beta-galactosidase were calculated by the following formula: Units = [A 420 x (1.7/0.0045)]/ [time (min)…”