A Membrane Binding Domain in the Ste5 Scaffold Synergizes with Gβγ Binding to Control Localization and Signaling in Pheromone Response
ABSTRACT 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.
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ABSTRACT: The yeast mating cell provides a simple paradigm for analyzing mechanisms underlying the generation of surface polarity. Endocytic recycling and slow diffusion on the plasma membrane were shown to facilitate polarized surface distribution of Snc1p (Valdez-Taubas, J., and H.R. Pelham. 2003. Curr. Biol. 13:1636-1640). Here, we found that polarization of Fus1p, a raft-associated type I transmembrane protein involved in cell fusion, does not depend on endocytosis. Instead, Fus1p localization to the tip of the mating projection was determined by its cytosolic domain, which binds to peripheral proteins involved in mating tip polarization. Furthermore, we provide evidence that the lipid bilayer at the mating projection is more condensed than the plasma membrane enclosing the cell body, and that sphingolipids are required for this lipid organization.The Journal of Cell Biology 07/2006; 173(6):861-6. DOI:10.1083/jcb.200602007 · 9.69 Impact Factor
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ABSTRACT: The class 4 P-type ATPases (“flippases”) maintain membrane asymmetry by translocating phosphatidylethanolamine and phosphatidylserine from the outer leaflet to the cytosolic leaflet of the plasma membrane. In Saccharomyces cerevisiae, five related gene products (Dnf1, Dnf2, Dnf3, Drs2, and Neo1) are implicated in flipping of phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine. In MATa cells responding to α-factor, we found that Dnf1, Dnf2, and Dnf3, as well as the flippase-activating protein kinase Fpk1, localize at the projection (“shmoo”) tip where polarized growth is occurring and where Ste5 (the central scaffold protein of the pheromone-initiated MAPK cascade) is recruited. Although viable, a MATa dnf1∆ dnf2∆ dnf3∆ triple mutant exhibited a marked decrease in its ability to respond to α-factor, which we could attribute to pronounced reduction in Ste5 stability resulting from an elevated rate of its Cln2⋅Cdc28-initiated degradation. Similarly, a MATa dnf1∆ dnf3∆ drs2∆ triple mutant also displayed marked reduction in its ability to respond to α-factor, which we could attribute to inefficient recruitment of Ste5 to the plasma membrane due to severe mislocalization of the cellular phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate pools. Thus proper remodeling of plasma membrane aminoglycerolipids and phosphoinositides is necessary for efficient recruitment, stability, and function of the pheromone signaling apparatus.Molecular Biology of the Cell 11/2014; 26(1). DOI:10.1091/mbc.E14-07-1193 · 4.55 Impact Factor
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ABSTRACT: Proteins are the most versatile among the various biological building blocks and a mature field of protein engineering has lead to many industrial and biomedical applications. But the strength of proteins-their versatility, dynamics and interactions-also complicates and hinders systems engineering. Therefore, the design of more sophisticated, multi-component protein systems appears to lag behind, in particular, when compared to the engineering of gene regulatory networks. Yet, synthetic biologists have started to tinker with the information flow through natural signaling networks or integrated protein switches. A successful strategy common to most of these experiments is their focus on modular interactions between protein domains or domains and peptide motifs. Such modular interaction swapping has rewired signaling in yeast, put mammalian cell morphology under the control of light, or increased the flux through a synthetic metabolic pathway. Based on this experience, we outline an engineering framework for the connection of reusable protein interaction devices into self-sufficient circuits. Such a framework should help to 'refacture' protein complexity into well-defined exchangeable devices for predictive engineering. We review the foundations and initial success stories of protein synthetic biology and discuss the challenges and promises on the way from protein- to protein systems design.Nucleic Acids Research 04/2010; 38(8):2663-75. DOI:10.1093/nar/gkq139 · 8.81 Impact Factor