ReviewPheromone response, mating and cell biology
Introduction
All eukaryotic cells use signal transduction networks to respond in specific ways to external signals from their environment and to coordinate complex cellular changes at the level of metabolism, gene expression, cell division, morphology and cell fate. The process of mating in Saccharomyces cerevisiae is one of the best studied examples of a cellular response to an external signal. Haploid cells mutually stimulate each other by secreting peptide pheromones that bind to cell-surface receptors on cells of opposite cell type. The binding of pheromone to receptor stimulates several cellular responses, including global changes in transcription, arrest of the cell cycle in G1 phase and polarized morphogenesis. These responses allow individual cells to synchronize their cell cycles, elongate and form a projection, then attach to and fuse with a partner cell, yielding a diploid zygote that will ultimately re-enter the mitotic cycle [1], [2]. A receptor–G-protein-coupled mitogen-activated protein kinase (MAPK) cascade mediates all of the responses to pheromone (Fig. 1). The basic elements of a MAPK cascade are three sequentially activated protein kinases, named after the last kinase in the cascade. The mating pathway includes a PAK (p21-activated protein kinase)-type kinase Ste20, a MAPKKK Ste11, a MAPKK Ste7, and two MAPKs Fus3 and Kss1 of which Fus3 is the critical kinase [3], [4], [5. A principal target of the MAPKs is the Ste12 transcription factor, which increases the expression of over 200 genes to mediate the different outputs [6].
Many of the components of the pheromone response pathway are used by three other MAPK cascades (Fig. 2). Ste20 and Ste11 function in the high osmolarity growth pathway that helps cells survive osmotic stress [7] (see also Update). Ste20, Ste11, Ste7, Kss1 and Ste12 are used by both the invasive growth pathway to promote foraging under nutrient deprivation, and by a recently described vegetative growth pathway to promote cell wall integrity in parallel with the protein kinase C pathway [8]. The new Kss1 pathway may be activated by cell wall stress [8] and/or changes in osmolarity, possibly through cell-surface sensors that include Sho1 [9]. This basic MAPK cascade is elaborated on by mating-specific signaling components that include pheromone receptors (Ste2 and Ste3), G-proteins (Gpa1, Ste4, Ste18), a MAPK cascade scaffold (Ste5) and specialized MAPK (Fus3) to promote the pheromone response. Additional pathway specificity comes from the prevention of cross-talk by the MAPKs [4], [7]. Fus3 prevents pheromone-induced activation of the Kss1-dependent pathways at unknown steps [4], while Hog1 prevents osmolarity-induced activation of the Fus3-Kss1 pathways, possibly at the Sho1 step [7].
In this review, I focus on the progress that has been made in defining the regulatory schemes that control mating. I discuss new insights on the localization of key components of signal transduction, and an emerging theme of the underlying nuclear shuttling apparatus that regulates access of cytoplasmic proteins to the plasma membrane for both activation of the MAPK cascade and morphogenesis. In addition, I discuss the progress that has been made in understanding receptor dynamics, a process that is instrumental in setting up asymmetry during morphogenesis and fusion.
Section snippets
Receptor–G-protein coupling
Like other G-protein-coupled receptors, the pheromone receptors have a structural topology of seven transmembrane domains, a third intracellular loop that is involved in G-protein coupling, and a cytoplasmic carboxy-terminal domain that mediates ligand-induced endocytosis and desensitization [10], [11]. Pheromone binding has been thought to drive monomeric receptors into an active complex with the G-protein, leading to exchange of GDP for GTP on Gα, followed by simple dissociation of the Gβγ
A Ste20-induced conformational change may relieve autoinhibition of Ste11
The past couple of years have witnessed important advances in our understanding of how Gβγ activates the downstream MAPK cascade that is headed by the MAPKKK Ste11. This event is of fundamental interest, because Ste11 is the prototype of a large family of poorly understood kinases termed MEKKs (extracellular signal-regulated protein kinase kinases), which function in numerous MAPK cascades. The activation of Ste11 has been thought to involve both direct phosphorylation by the PAK-like kinase
Gβγ recruitment of both Ste20 and the Ste5 scaffold activates Ste11
A second major unresolved issue has been the logistics of how the Gβγ subunits transmit the pheromone signal through Ste20 to Ste11. Ste20 localizes predominantly at sites of new growth at the cell cortex and projection tip through an association with the GTPase Cdc42, however, this interaction is not required for the function of Ste20 in the mating MAPK cascade [10]. Instead, Gβ binds directly to a conserved C-terminal domain in Ste20 that is found in other PAKs [32]. This interaction is
Nuclear shuttling controls access of cytoplasmic Ste5 to the plasma membrane
A critical aspect of the recruitment model is getting Ste5 to Gβ at the right time. Overexpression of Ste5 is sufficient to activate Ste11 fully in the absence of pheromone, suggesting that the access of Ste5 to Gβ must be carefully controlled. Indeed, misactivation of Ste11 has serious consequences for the cell because of its participation in many MAPK cascades, and hyperactivation of Ste11 is known to be lethal.
Recntly, Mahanty et al. [40] have shown that recruitment of Ste5 to Gβ at the
Polarized morphogenesis recapitulates regulatory schemes of the MAPK cascade
Mating cells sense the direction of a pheromone source from a mating partner and undergo polarized growth, forming a projection towards the source. This response is termed chemotropism and is thought to involve the generation of an internal landmark that reflects the axis of the external pheromone signal and overrides internal spatial cues that normally control bud formation [42], [43]. Formation of projections is mediated by the actin cytoskeleton and by many other proteins that normally
Spatial control of the receptor and G-protein
A key element of chemotropism is the asymmetric accumulation of the receptor and G-protein at the internal landmark of the emerging projection tip [42], [52]. Spatial restriction of the receptor and associated G-protein is likely to occur through the septins via Afr1, which binds to Cdc12 and regulates morphogenesis at the receptor step [10], [59], [60. Small differences in receptor occupancy across the cell surface are predicted to be sufficient to generate asymmetry [42], [52]. The site for
Conclusions
The past few years have seen great increases in our understanding of how an external pheromone signal mediates cellular global cellular changes. An emerging theme is the previously unappreciated importance of the underlying cellular apparatus that controls the localization of individual signaling components and the mechanism by which complex multiprotein ‘signalosomes’ are formed through linkages to G-proteins, and stabilized and maintained in response to an external signal. Paramount will be
Update
Recent work by Rait et al. suggests that activation of Ste11 by Ste20 in the high osmolarity growth pathway involves plasma membrane recruitment of Ste20 by Cdc42, and of Pbs2 by Sho1 [67] — an activation mechanism, that resembles that of the pheromone response pathway. A recent study now provides biochemical evidence supporting the existence of endocytosed homo-oligomeric complexes of Ste2, and shows that oligomerization does not involve the carboxyl terminus or cysteine residues of Ste2 [68].
Acknowledgements
I thank the many colleagues who generously provided preprints and reprints for this review and R Arkowitz, C Boone, D Jenness, P Pryciak and M Peter for valuable discussions. This work was funded by National Institutes of Health grant GM46962.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
of special interest
of outstanding interest
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