Yeast cells arrest in the G1 phase of the cell cycle upon exposure to mating pheromones. As cells commit to a new cycle, G1 CDK activity (Cln/CDK) inhibits signaling through the mating MAPK cascade. Here we show that the target of this inhibition is Ste5, the MAPK cascade scaffold protein. Cln/CDK disrupts Ste5 membrane localization by phosphorylating a cluster of sites that flank a small, basic, membrane-binding motif in Ste5. Effective inhibition of Ste5 signaling requires multiple phosphorylation sites and a substantial accumulation of negative charge, which suggests that Ste5 acts as a sensor for high G1 CDK activity. Thus, Ste5 is an integration point for both external and internal signals. When Ste5 cannot be phosphorylated, pheromone triggers an aberrant arrest of cells outside G1 either in the presence or absence of the CDK-inhibitor protein Far1. These findings define a mechanism and physiological benefit of restricting antiproliferative signaling to G1.

In the Saccharomyces cerevisiae pheromone response pathway, the G␤␥ complex activates downstream responses by an unknown mechanism involving a MAP kinase cascade, the PAK-like kinase Ste20, and a Rho family GTPase, Cdc42. Here we show that G␤␥ must remain membrane-associated after release from G␣ to activate the downstream pathway. We also show that pheromone stimulates translocation of the kinase cascade scaffold protein Ste5 to the cell surface. This recruitment requires G␤␥ function and the G␤␥-binding domain of Ste5, but not the kinases downstream of G␤␥, suggesting that it is mediated by G␤␥ itself. Furthermore, this event has functional significance, as artificial targeting of Ste5 to the plasma membrane, but not intracellular membranes, activates the pathway in the absence of pheromone or G␤␥. Remarkably, although independent of G␤␥, activation by membrane-targeted Ste5 requires Ste20, Cdc42, and Cdc24, indicating that their participation in this pathway does not require them to be activated by G␤␥. Thus, membrane recruitment of Ste5 defines a molecular activity for G␤␥. Moreover, our results suggest that this event promotes kinase cascade activation by delivering the Ste5-associated kinases to the cell surface kinase Ste20, whose function may depend on Cdc42 and Cdc24.[Key Words: Heterotrimeric G protein; MAP kinase; signal transduction; PAK/Ste20 kinase; Rho/Rac/Cdc42 GTPase]Received April 20, 1998; accepted in revised form July 8, 1998.The mating reaction of the yeast Saccharomyces cerevisiae provides a model signal transduction system in which a G-protein-coupled receptor activates a mitogenactivated protein (MAP) kinase cascade (for review, see Sprague and Thorner 1992;Leberer et al. 1997a). Here, two haploid cells of opposite mating types (a and ␣) fuse into a single diploid cell in response to secreted pheromones (a-factor and ␣-factor), which stimulate cell cycle arrest, transcriptional induction of mating-related genes, and morphological changes. These responses are activated through a pathway that begins with a cell surface receptor and its associated heterotrimeric G protein, G␣␤␥, which is composed of Gpa1 (G␣), Ste4 (G␤), and Ste18 (G␥). Downstream of the G protein lies a cascade of protein kinases-Ste11, Ste7, and Fus3-that is related to mammalian MAP kinase cascades, and an associated ''kinase scaffold'' protein Ste5 (for review, see Herskowitz 1995;Madhani and Fink 1998). Finally, targets of this kinase cascade include Far1, which activates cell cycle arrest, and Ste12, a DNA-binding protein responsible for transcriptional induction.The mechanism by which the kinase cascade is activated by the heterotrimeric G protein remains poorly understood. It is clear that the free G␤␥ complex activates downstream responses, after binding of pheromone to the receptor stimulates its dissociation from G␣, as deletion of the gene (GPA1) encoding G␣ mimics pheromone treatment (for review, see Sprague and Thorner 1992). But the identity of the immediate target of G␤␥ and the molecular mechanism used by G␤␥...

Heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) determine tissue and cell polarity in a variety of organisms. In yeast, cells orient polarized growth toward the mating partner along a pheromone gradient by a mechanism that requires Far1p and Cdc24p. Far1p bound Gbetagamma and interacted with polarity establishment proteins, which organize the actin cytoskeleton. Cells containing mutated Far1p unable to bind Gbetagamma or polarity establishment proteins were defective for orienting growth toward their mating partner. In response to pheromones, Far1p moves from the nucleus to the cytoplasm. Thus, Far1p functions as an adaptor that recruits polarity establishment proteins to the site of extracellular signaling marked by Gbetagamma to polarize assembly of the cytoskeleton in a morphogenetic gradient.

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Activation of mitogen-activated protein (MAP) kinase cascade signaling by yeast mating pheromones involves recruitment of the Ste5 scaffold protein to the plasma membrane by the receptor-activated Gbetagamma dimer. Here, we identify a putative amphipathic alpha-helical domain in Ste5 that binds directly to phospholipid membranes and is required for membrane recruitment by Gbetagamma. Thus, Ste5 signaling requires synergistic Ste5-Gbetagamma and Ste5-membrane interactions, with neither alone being sufficient. Remarkably, the Ste5 membrane binding domain is a dual-function motif that also mediates nuclear import. Separation-of-function mutations show that signaling requires the membrane-targeting activity of this domain, not its nuclear-targeting activity, and heterologous lipid binding domains can substitute for its function. This domain also contains imperfections that reduce membrane affinity, and their elimination results in constitutive signaling, explaining some previous hyperactive Ste5 mutants. Therefore, weak membrane affinity is advantageous, ensuring a normal level of signaling quiescence in the absence of stimulus and imposing a requirement for Gbetagamma binding.

The Saccharomyces cerevisiae kinase Ste20 is a member of the p21-activated kinase (PAK) family with several functions, including pheromone-responsive signal transduction. While PAKs are usually activated by small G proteins and Ste20 binds Cdc42, the role of Cdc42-Ste20 binding has been controversial, largely because Ste20 lacking its entire Cdc42-binding (CRIB) domain retains kinase activity and pheromone response. Here we show that, unlike CRIB deletion, point mutations in the Ste20 CRIB domain that disrupt Cdc42 binding also disrupt pheromone signaling. We also found that Ste20 kinase activity is stimulated by GTP-bound Cdc42 in vivo and this effect is blocked by the CRIB point mutations. Moreover, the Ste20 CRIB and kinase domains bind each other, and mutations that disrupt this interaction cause hyperactive kinase activity and bypass the requirement for Cdc42 binding. These observations demonstrate that the Ste20 CRIB domain is autoinhibitory and that this negative effect is antagonized by Cdc42 to promote Ste20 kinase activity and signaling. Parallel results were observed for filamentation pathway signaling, suggesting that the requirement for Cdc42-Ste20 interaction is not qualitatively different between the mating and filamentation pathways. While necessary for pheromone signaling, the role of the Cdc42-Ste20 interaction does not require regulation by pheromone or the pheromone-activated G␤␥ complex, because the CRIB point mutations also disrupt signaling by activated forms of the kinase cascade scaffold protein Ste5. In total, our observations indicate that Cdc42 converts Ste20 to an active form, while pathway stimuli regulate the ability of this active Ste20 to trigger signaling through a particular pathway.

Summary Background Signaling through mitogen-activated protein kinase (MAPK) cascade pathways can show various input-output behaviors, including either switch-like or graded responses to increasing levels of stimulus. Prior studies suggest that switch-like behavior is promoted by positive feedback loops and nonprocessive phosphorylation reactions, but it is unclear whether graded signaling is a default behavior or if it must be enforced by separate mechanisms. Scaffold proteins have been hypothesized to promote graded behavior. Results Here, we experimentally probe the determinants of graded signaling in the yeast mating MAPK pathway. We find that graded behavior is robust, as it resists perturbation by loss of several negative feedback regulators. However, the pathway becomes switch-like when activated by a crosstalk stimulus that bypasses multiple upstream components. To dissect the contributing factors, we developed a method for gradually varying the signal input at different pathway steps in vivo. Input at the beginning of the kinase cascade produced a sharp, threshold-like response. Surprisingly, the scaffold protein Ste5 increased this threshold behavior when limited to the cytosol. However, signaling remained graded whenever Ste5 was allowed to function at the plasma membrane. Conclusions The results suggest that the MAPK cascade module is inherently ultrasensitive, but is converted to a graded system by the pathway-specific activation mechanism. Scaffold-mediated assembly of signaling complexes at the plasma membrane allows faithful propagation of weak signals, which consequently reduces pathway ultrasensitivity. These properties help shape the input-output properties of the system to fit the physiological context.

Abstract. During conjugation, haploid S. cerevisiae cells find one another by polarizing their growth toward each other along gradients of pheromone (chemotropism). We demonstrate that yeast cells exhibit a second mating behavior: when their receptors are saturated with pheromone, wild-type a cells execute a default pathway and select a mate at random. These matings are less efficient than chemotropic matings, are induced by the same dose of pheromone that induces shmoo formation, and appear to use a site near the incipient bud site for polarization. We show that the SPA2 gene is specifically required for the default pathway: spa2A mutants cannot mate if pheromone concentrations are high and gradients are absent, but can mate if gradients are present, ste2A, sst2A, and farlA mutants are chemotropism-defective and therefore must choose a mate by using a default pathway; consistent with this deduction, these strains require SPA2 to mate. In addition, our results suggest that farl mutants are chemotropism-defective because their mating polarity is fixed at the incipient bud site, suggesting that the FAR1 gene is required for inhibiting the use of the incipient bud site during chemotropic mating. These observations reveal a molecular relationship between the mating and budding polarity pathways.