Mating And Signal Transduction Cascade

Yeast cells exist in a- and a-mating types (Chapter 8). Mating of cells of opposite mating types is coordinated by the release of small peptide hormones (pheromones). The a-cells produce a 12-amino acid peptide, the a-pheromone, and responds to the 13-amino acid peptide a-pheromone, produced by a-cells. The a-cells on the other hand bind the a-pheromone produced by the a-cells. Reception of the pheromone signal triggers a series of events that include changes in cell shape and arrest of cell growth. How these events are triggered by the pheromone signal is of general interest because signaling events must occur in unicellular organisms seeking nutrients in the surrounding environment, or in a fungal pathogen searching for an entry point into a host plant, or in a higher organism responding to growth factors, hormones, neurotransmitters and other sensory input.

The pheromone signaling is the most well-characterized eukaryotic signaling pathway. The hunt for genes in this pathway was facilitated by a selection scheme based on the growth arrest of cells in response to pheromone signaling as schematically depicted in Figure 6.7.

a-mutant unable to produce a-pheromone a-mutant unable to produce a-pheromone

a-mutant unable to produce a-pheromone a-mutant unable to produce to a pheromone a-mutant unable to respond to a-pheromone a-mutant unable to respond to a-pheromone

Figure 6.7 Scheme to isolate mutants defective in pheromone signaling by observing growth inhibition of cells in the presence of pheromone. (a) Wild-type yeast cells of opposite mating type show a zone of growth inhibition where the streaks of growing cells cross each other. The diploid cells produced as a result of mating appear as new growth because they are resistant to pheromones. (b) A mutant showing growth inhibition with no effect on the wild type indicates a defect in the production of the pheromone (top), whereas appearance of a zone of growth inhibition around wild type indicates a mutant that does not respond to the pheromone (bottom). (c) Absence of a zone of growth inhibition in the presence of mutant a and a-cells.

a-mutant unable to produce a-pheromone a-mutant unable to produce to a pheromone a-mutant unable to respond to a-pheromone a-mutant unable to respond to a-pheromone

Figure 6.7 Scheme to isolate mutants defective in pheromone signaling by observing growth inhibition of cells in the presence of pheromone. (a) Wild-type yeast cells of opposite mating type show a zone of growth inhibition where the streaks of growing cells cross each other. The diploid cells produced as a result of mating appear as new growth because they are resistant to pheromones. (b) A mutant showing growth inhibition with no effect on the wild type indicates a defect in the production of the pheromone (top), whereas appearance of a zone of growth inhibition around wild type indicates a mutant that does not respond to the pheromone (bottom). (c) Absence of a zone of growth inhibition in the presence of mutant a and a-cells.

Briefly, streaking yeast cells of opposite mating type on an agar plate in the form of a cross (cross-streaking) results in the appearance of a "zone of growth inhibition" in both the strains as a result of pheromone-induced growth arrest. The "zone of growth inhibition" is eventually populated by the growth of diploid cells, arising as a result of mating and which are non-responsive to pheromones (Figure 6.7a). However, if mutant a-cells defective in producing a-pheromone are cross-streaked on a plate with wild-type a-cell, a zone of growth inhibition is observed around the mutant only because a-pheromone produced by a-cells arrests the growth of a-cells (Figure 6.7b, top). Conversely, if mutant a-cells defective in responding to a-pheromone is plated with wild-type a-cells, a zone of growth inhibition is observed only around the a-cells (Figure 6.7b, bottom). Finally, if both mutant a and a-cells are plated together, no zone of growth inhibition is observed (Figure 6.7c).

Using such a simple visual selection procedure, mutants defective in sending or responding to pheromone signals are identified and the genes cloned by functional complementation. The pheromone pathway has been extensively analyzed by biochemical and molecular genetics techniques, providing us with a wealth of knowledge of a eukaryotic signal transduction pathway (Dohlman and Thorner, 2001).

The biochemical cascade in the mating pathway of S. cerevisiae involves coordinated function of many proteins that work harmoniously, akin to a symphony orchestra. The multi-protein signaling complex is schematically shown in Figure 6.8. The receptors are proteins with seven transmembrane segments embedded in the plasma membrane. These receptors are named G-protein-coupled receptors (GPCRs) because they are associated at

Figure 6.8 Mechanism of pheromone signaling in S. cerevisiae. Binding of pheromone to the G-protein coupled pheromone receptor induces expression of pheromone responsive genes by downstream kinases.

the cytoplasmic side to a heterotrimeric protein complex, the G-proteins. The three protein subunits forming the trimeric complex are named a, p and y. The pheromone receptors are activated by pheromone binding at the extracellular face, which induces a conformational change in the receptor resulting in the dissociation of the a subunit from the p-y heterodimer. The p-y complex functions as an adaptor protein to recruit other signaling molecules. Pheromone signaling activates two separate signaling complexes. The first complex, immediately downstream from the adaptor complex, activates CDC42p by exchanging GDP with GTP, catalyzed by CDC24p (GTP cycle). Active CDC42p regulates two separate cellular processes: reorganization of the actin cytoskeleton leading to the appearance of mating projections and activation of the MAP kinase module. The kinase module contains four separate kinases brought in close proximity to each other by binding to the adaptor protein Ste5. A cascade of protein phosphorylation by kinases leads finally to the activation of Ste12 transcription factor. Ste12 translocates into the nucleus and induces transcription of pheromone-responsive genes. These genes regulate mating and nuclear fusion to begin the diploid phase of yeast life cycle. The molecular mechanisms governing pheromone signaling reflects in composition and characteristics the fundamental nature of signaling pathways found in all higher organisms.

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