Transcriptional control

The mating MAPK pathway is important for sending signals from the pheromone receptors in the plasma membrane to gene targets in the nucleus, and pheromone signals finally lead to profound changes in the transcriptional program of a cell. Among the products of genes stimulated by pheromone are proteins that activate (e.g. Fus3p) or inhibit (e.g. Msg5p) signaling on the pheromone response pathway (Doi et al., 1994; Elion et al., 1990; Zhan et al., 1997) and proteins needed for cell fusion (e.g. Fus1p), nuclear fusion (e.g. Kar4p) and other mating related functions (Kurihara et al., 1996; McCaffrey et al., 1987; Trueheart et al., 1987). All these genes contain PREs (pheromone response elements) in their 5´regulatory regions, elements which are necessary and sufficient for pheromone regulated transcription (Dolan et al., 1989; Hagen et al., 1991; Kronstad et al., 1987). Ste12p associates with pheromone-inducible promoters as a homomultimer or as a heterodimer with the Mcm1p protein if the PRE is juxtaposed to a distinct target sequence, the P box (Madhani and Fink, 1998). The MAPK cascade mediates pheromone induction of transcription of PRE-containing genes through phosphorylation and activation of at least three nuclear proteins: Dig1p, Dig2p and Ste12p (Cook et al., 1996; Song et al., 1991;

Tedford et al., 1997). Dig1p and Dig2p are related proteins with overlapping and partially redundant functions that act as negative regulators of Ste12p and together efficiently repress the transcription of pheromone responsive genes. In unstimulated cells, Dig1p and Dig2p form a complex containing Fus3p, Kss1p and Ste12p (Cook et al., 1996; Pi et al., 1997; Tedford et al., 1997), thereby preventing transcriptional activation by Ste12p.

Pheromone stimulation increases phosphorylation of Dig1p, Dig2p and Ste12p by Fus3p and Kss1p, resulting in complex dissociation (Elion et al., 1993; Tedford et al., 1997).

Liberated Ste12p is then competent to activate target gene transcription.

1.2.4 Cell cycle regulation

In addition to transcriptional regulation, the mating MAPK cascade is important for mediation of cell cycle arrest in response to pheromone. Pheromone-treated cells arrest in G₁ phase as unbudded cells with a 1N DNA content, and this pheromone-induced cell cycle arrest depends on the CKI (cyclin dependent kinase inhibitor) Far1p (Chang and Herskowitz, 1990; Gartner et al., 1998; Peter et al., 1993; Tyers and Futcher, 1993). Far1p causes cell cycle arrest by inhibiting the activity of G1 cyclin-Cdc28p complexes which are important for G₁/S transition (Jeoung et al., 1998; Peter and Herskowitz, 1994). This function of Far1p is dependent on the MAPK Fus3p, but not on Kss1p, because Far1p is much more efficiently phosphorylated by Fus3p both in vivo and in vitro (Breitkreutz et al., 2001; Peter et al., 1993). The mechanism by which Far1p mediates cell cycle arrest is not well understood (Gartner et al., 1998). Interestingly, Far1p is not only phosphorylated (and thereby activated) by Fus3p, but also by its target G₁ cyclin-Cdc28p, and this phosphorylation seems to trigger ubiquitin-dependent degradation of Far1p (Henchoz et al., 1997; Peter et al., 1993; Tyers and Futcher, 1993). The preferential phosphorylation of Far1p by Fus3p contrasts with the transcriptional targets Ste12p and Dig1p that are phosphorylated by Fus3p and Kss1p to a similar extent. Because far1∆ strains have a severe mating defect, the inability to activate Far1p might partly explain the reduced mating efficiency of fus3∆ strains.

1.2.5 Regulation of cell polarity and morphology

In contrast to the predetermined budding patterns exhibited during vegetative growth, mating cells initiate projection ("shmoo") formation at novel points on the cell surface in response to pheromone signals. Therefore, the mating MAPK cascade must be tightly

coupled to the cytoskeleton. The cytoskeleton-signal transduction relationships during mating have been extensively studied in yeast. In this regard, two proteins have a central function: the small GTPase Cdc42p and Bem1p. The essential GTPase Cdc42p is required to orient the actin cytoskeleton for polarized growth during vegetative growth, for cell division, and to form mating projections (see section 2). Cdc42p interacts with a variety of different proteins that regulate actin cytoskeleton function. Like other small GTPases, Cdc42p can exist in two different conformations depending on the type of guaninnucleotide bound. GTP-bound and GDP-bound forms of Cdc42p exist in a dynamic equilibrium, and regulatory proteins control the cycling between both conformations.

Exchange of GDP for GTP on Cdc42p is triggered by the GEF (guaninnucleotide exchange factor) Cdc24p (Zheng et al., 1994), placing Cdc42p in an activated state. Hydrolysis of the Cdc42p-bound GTP to GDP is predicted to be regulated by the GAPs (GTPase activating proteins) Bem3p and Rga1p (Stevenson et al., 1995; Zheng et al., 1994).

Cdc42p appears to have multiple functions in the mating response. Cdc42p interacts with Ste20p, and this interaction is necessary for proper localization of Ste20p at the shmoo tip.

The Gβ subunit Ste4p interacts not only with Ste5p and Ste20p to activate the MAPK cascade, but also with Cdc24p (Nern and Arkowitz, 1998; Zhao et al., 1995), and mutations in Cdc24p that block interaction with Ste4p also block chemotropic growth.

Thus, the interaction of Ste4p with Cdc24p appears to locally activate Cdc42p and Cdc42p-dependent polarization functions in the vicinity of pheromone-occupied receptors.

Cdc42p is linked to the actin cytoskeleton via the formin homology protein Bni1p (Evangelista et al., 1997; Ozaki-Kuroda et al., 2001), which in turn interacts with the actin monomer-binding protein profilin (Imamura et al., 1997).

Bem1p, like Cdc42p, interacts with a large number of proteins important for the function of the actin cytoskeleton in polarized growth. Proteins found to interact with Bem1p include actin, Ste5p and Ste20p (Leeuw et al., 1995; Lyons et al., 1996). Bem1p colocalizes with Cdc24p and Cdc42p to growth sites, and it is thought to serve as a scaffold that promotes coupling between polarity determinants and Cdc24p-Cdc42p by directly binding both Cdc24p and shmoo-selection proteins (Ayscough and Drubin, 1998;

Bender and Pringle, 1991; Chenevert et al., 1992). Recruitment of Bem1p to sites of polarized growth also depends on the polarity determinant Far1p, which interacts with liberated Gβγ and helps to localize the Cdc24p-Cdc42p module (Butty et al., 1998). Far1p

has a second function as a key regulator of pheromone-induced cell-cycle arrest (Chang and Herskowitz, 1990) (see above).