A membrane binding domain in the ste5 scaffold synergizes with gbetagamma binding to control localization and signaling in pheromone response.
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.
Winters
Matthew J
MJ
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Lamson
Rachel E
RE
Nakanishi
Hideki
H
Neiman
Aaron M
AM
Pryciak
Peter M
PM
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000502:physiology*; D010675:Pheromones / Q000235:genetics / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D010675:Pheromones; D010743:Phospholipids; C080321:STE5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D020962:Heterotrimeric GTP-Binding Proteins