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Pheromone Signaling Mechanisms in Yeast: A Prototypical Sex Machine

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Science  26 Nov 2004:
Vol. 306, Issue 5701, pp. 1508-1509
DOI: 10.1126/science.1104568


The actions of many extracellular stimuli are elicited by complexes of cell surface receptors, heterotrimeric guanine nucleotide–binding proteins (G proteins), and mitogen-activated protein (MAP) kinase complexes. Analysis of haploid yeast cells and their response to peptide mating pheromones has produced important advances in our understanding of G protein and MAP kinase signaling mechanisms. Many of the components, their interrelationships, and their regulators were first identified in yeast. Current analysis of the pheromone response pathway (see the Connections Maps at Science's Signal Transduction Knowledge Environment) will benefit from new and powerful genomic, proteomic, and computational approaches that will likely reveal additional general principles that are applicable to more complex organisms.

All cells have the capacity to sense and discriminate among various environmental stimuli and to then generate an appropriate intracellular response. One common mechanism for detecting and transmitting extracellular signals uses cell surface receptors coupled to intracellular heterotrimeric guanine nucleotide–binding proteins (G proteins). Human G protein–coupled receptors (GPCRs) mediate responses to light, odor, taste, hormones, and neurotransmitters. Prominent examples include receptors for epinephrine, histamine, and serotonin. Drugs such as β-adrenergic receptor blockers (which bind epinephrine receptors), antihistamines, and serotonin-reuptake inhibitors are among the most widely prescribed drugs. Even in the simplest eukaryotes, GPCRs regulate fundamental processes such as cell motility, development, and sexual reproduction. Clearly, a detailed understanding of G protein signaling is important for understanding human disease as well as basic cellular physiology.

The budding yeast Saccharomyces cerevisiae has proven indispensable in elucidating the mechanisms of G protein and mitogen-activated protein (MAP) kinase signaling (1). A combination of genetic, biochemical, and molecular biological analysis of the response of haploid yeast cells to their peptide mating pheromones has established basic principles of G protein signaling and regulation. Examples include the identification of monoubiquitination as a signal for receptor endocytosis (2, 3), definitive demonstration of a positive signaling role for G protein βγ subunits (4), the first discovery of three-tiered structure of the MAP kinase module (5), development of the concept of a kinase-scaffold protein (6), and the first demonstration that regulator of G protein signaling (RGS) proteins desensitize the G proteins (7).

The yeast pheromone response begins with GPCRs at the plasma membrane (Fig. 1). Two haploid cell types, known as MATα and MATa, each secrete small peptide pheromones, called α factor and a factor, respectively. The α factor binds to a specific receptor (Ste2) on MATa cells, whereas a factor binds to a distinct receptor (Ste3) on MATα cells. The pheromone receptors activate a G protein heterotrimer consisting of an α subunit (Gpa1) and a βγ subunit dimer (Ste4-Ste18). Upon activation, the G protein subunits dissociate, and the signal is then transmitted and amplified through multiple effector proteins that bind to Gβγ. Cellular responses ultimately include changes in cytoskeletal structure that lead to polarized cell growth, induction of new gene transcription, changes in nuclear architecture, arrest of cell cycle progression in the G1 phase, and finally cell fusion (mating) to form the a/α diploid. Polarized cell growth is required to establish the site for cell fusion (plasmogamy). New gene transcription is required to produce, for example, proteins that mediate cell adhesion. Growth arrest is required to synchronize the cell cycles of the two mating partners. Nuclear changes are required in preparation for nuclear fusion (karyogamy) and the completion of diploid zygote formation.

Fig. 1.

Components of the pheromone response pathway in yeast. Pheromone (α factor in this case) binds to a cell surface receptor (Ste2) that in turn promotes GTP binding to the G protein α subunit (Gpa1). GTP triggers dissociation of Gα from the G protein βγ subunits (Ste4 and Ste18). Free βγ activates a downstream signaling cascade through the guanine nucleotide exchange factor Cdc24, the protein kinase Ste20, and the kinase scaffold protein Ste5. The MAP kinase Fus3 phosphorylates and activates the transcription factor Ste12, resulting in new gene transcription. Additional components modulate signaling, including receptor kinases, GTPase-accelerating proteins (GAPs) (Sst2, Rga1, Rga2, and Bem3), MAP kinase phosphatases (Msg5, Ptp2, and Ptp3), an activator of Ste11 (Ste50), and inhibitors of Ste12 (Dig1 and Dig2). Some components are omitted for clarity.

A major target of the Gβγ-transmitted signal is a MAP kinase cascade. In this route, a MAP kinase kinase kinase (Ste11) phosphorylates and activates a MAP kinase kinase (Ste7). Ste7 phosphorylates and activates two MAP kinases, Kss1 and Fus3. Genetic analysis indicates that Fus3 acts primarily in the mating pathway leading to haploid cell fusion, whereas Kss1 acts primarily in the invasive or “pseudohyphal” growth pathway in nitrogen-starved cells (8, 9). It is likely that Fus3 and Kss1 normally act separately, but either can function in the absence of the other. In haploid yeast cells, a major fraction of the proteins that make up the MAP kinase cascade (Ste11, Ste7, and Fus3) is bound to a scaffold protein called Ste5. Upon activation, Gβγ can bind to Ste5, where it evidently induces a conformational change in the protein (10). In this context, the Ste5-Ste11-Ste7-Fus3 assembly can be regarded as an effector complex. This scaffolded arrangement presumably allows for more efficient signaling and possibly protects against inappropriate cross talk with the other MAP kinases in yeast.

Another Gβγ effector critical for mating is Cdc24, a guanine nucleotide exchange factor for Cdc42 (11). Cdc42 is a member of the Ras superfamily of small guanosine triphosphatases (GTPases) and specifically regulates actin rearrangements that lead to polarized cell growth and morphogenesis. In yeast, Cdc42 is required for budding in dividing cells and for the formation of a projection that protrudes from the cell after pheromone-triggered cell cycle arrest. This mating projection is where many signaling proteins are concentrated and where cell fusion begins. Cdc42 may also regulate transcription and cell division through its binding to the p21-activated protein kinase Ste20 (12).

Analysis of the yeast mating pathway has provided insights into signal desensitization. A property of signal-response systems in general, and of G protein–coupled receptors in particular, is that prolonged stimulation leads to an attenuated response. At a cellular and molecular level, desensitization typically results from feedback inhibition events that serve to dampen further signaling. Receptors are well known as targets of desensitization, but the first insight that G proteins are also subject to desensitization occurred through analysis of SST2 (supersensitive to pheromone) mutations in yeast (7). Genetic disruption of SST2 allows cells to respond to doses of pheromone that are roughly two orders of magnitude lower than those detected by normal cells. SST2 mutants also prevent recovery from pheromone-induced growth arrest, even if the ligand is removed. Homologs of Sst2 exist in higher eukaryotes and were renamed RGS proteins and shown to accelerate G protein GTPase activity, thus leading to subunit reassociation (1).

Yeast will surely continue to be an attractive and appropriate model for the study of G protein and MAP kinase signaling. The yeast pheromone signaling pathway is structurally and functionally similar to hormone and neurotransmitter signaling pathways in mammals; the G protein and kinase components in particular share extensive sequence similarity with their mammalian counterparts. Yeast is unequaled as a tool for genetic analysis. Its ability to grow stably as a haploid is useful for studying gene mutations that are recessive. Homologous recombination in yeast is useful for studying the physiological function of proteins, through gene replacement and gene disruption. Indeed, yeast is the only system in which nearly every open reading frame has been deleted (13), organized into microarrays (14), fused to fluorescent marker proteins (15), affinity-tagged (16), and purified (17). Consequently it is now possible to study gene function, gene transcription, protein localization, and intermolecular associations in a systematic manner and on a genome-wide scale.

Although our understanding of pheromone signaling is very sophisticated, substantial challenges remain. Little is known about how genes that are essential for viability contribute to pheromone responses. Pathway components probably undergo multiple posttranslational modifications, and technologies to identify the chemical nature and location of those modifications remain nascent. Perhaps the most challenging of all will be the establishment of accurate and quantitative models of the pheromone-response pathway (18). A complete understanding of any cellular process requires models that accurately predict behavior in response to cellular perturbations. Achieving this goal will require a full accounting of every signaling component and every enzymatic reaction, both in cellular space and over time. Newly available genomics and proteomics tools, such as the yeast pheromone response pathway Connections Map at Science's Signal Transduction Knowledge Environment (STKE) (19), will facilitate these efforts, but they will also require new and coordinated efforts by biologists, chemists, mathematicians, and computer scientists.

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