A Membrane Binding Domain in the Ste5 Scaffold Synergizes with Gβγ Binding to Control Localization and Signaling in Pheromone Response

Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Molecular Cell (Impact Factor: 14.02). 11/2005; 20(1):21-32. DOI: 10.1016/j.molcel.2005.08.020
Source: PubMed


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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    • "Using this system, ePDZb-Ste5DN (lacking a G-protein binding domain) and ePDZb-Ste11 could be recruited to the plasma membrane-localized AsLOV2 protein upon light stimulation, resulting in mating pathway activation (monitored by PFUS1-DsRed) and cellcycle arrest. This result was consistent with a previous study reporting that Ste5 or Ste11 tethered to the plasma membrane robustly activate the mating MAPK pathway (Winters et al., 2005). Thus, this tool opens the possibility for spatial and temporal control of the yeast signalling pathways using light. "
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    ABSTRACT: All living cells respond to external stimuli and execute specific physiological responses through signal transduction pathways. Understanding the mechanisms controlling signalling pathways is important for diagnosing and treating diseases and for reprogramming cells with desired functions. Although many of the signalling components in the budding yeast Saccharomyces cerevisiae have been identified by genetic studies, many features concerning the dynamic control of pathway activity, cross-talk, cell-to-cell variability or robustness against perturbation are still incompletely understood. Comparing the behaviour of engineered and natural signalling pathways offers insight complementary to that achievable with standard genetic and molecular studies. Here, we review studies that aim at a deeper understanding of signalling design principles and generation of novel signalling properties by engineering the yeast mitogen-activated protein kinase (MAPK) pathways. The underlying approaches can be applied to other organisms including mammalian cells and offer opportunities for building synthetic pathways and functionalities useful in medicine and biotechnology.
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    • "After pheromone stimulus, the Ste5 scaffold is rapidly translocated to the plasma membrane by Gβγ [13,14], where it initiates and amplifies mating signalling [15]. Ste5 membrane binding additionally depends on two membrane-binding regions, an N-terminal amphipathic helix and a PH domain [16,17]. Ste5 also binds the Cdc42 GEF Cdc24, which may contribute to its re-localization to the cell cortex [7]. "
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    • "Finally, to test the hypothesis that heterotrimeric G-protein signaling was down-regulated in the initial projection under high α-factor conditions , we quantified the amount of polarized Ste5-GFP in the first projection at 1 and 2 h (Figure 8A). Proper Ste5 membrane localization depends on binding to free Gβγ (Winters et al., 2005). Under uniform 100 nM conditions, most cells at both time points contained a single projection. "
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