Pheromone-induced anisotropy in yeast plasma membrane phosphatidylinositol-4,5-bisphosphate distribution is required for MAPK signaling.
During response of budding yeast to peptide mating pheromone, the cell becomes markedly polarized and MAPK scaffold protein Ste5 localizes to the resulting projection (shmoo tip). We demonstrated before that this recruitment is essential for sustained MAPK signaling and requires interaction of a pleckstrin homology (PH) domain in Ste5 with phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] in the plasma membrane. Using fluorescently tagged high-affinity probes specific for PtdIns(4,5)P(2), we have now found that this phosphoinositide is highly concentrated at the shmoo tip in cells responding to pheromone. Maintenance of this strikingly anisotropic distribution of PtdIns(4,5)P(2), stable tethering of Ste5 at the shmoo tip, downstream MAPK activation, and expression of a mating pathway-specific reporter gene all require continuous function of the plasma membrane-associated PtdIns 4-kinase Stt4 and the plasma membrane-associated PtdIns4P 5-kinase Mss4 (but not the Golgi-associated PtdIns 4-kinase Pik1). Our observations demonstrate that PtdIns(4,5)P(2) is the primary determinant for restricting localization of Ste5 within the plasma membrane and provide direct evidence that an extracellular stimulus-evoked self-reinforcing mechanism generates a spatially enriched pool of PtdIns(4,5)P(2) necessary for the membrane anchoring and function of a signaling complex.
Garrenton
Lindsay S
LS
Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202, USA.
Stefan
Christopher J
CJ
McMurray
Michael A
MA
Emr
Scott D
SD
Thorner
Jeremy
J
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D022081:Cell Surface Extensions / Q000378:metabolism; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D020935:MAP Kinase Signaling System; D010675:Pheromones / Q000494:pharmacology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D011498:Protein Precursors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism / Q000494:pharmacology
D048868:Adaptor Proteins, Signal Transducing; D005456:Fluorescent Dyes; C048632:MF(ALPHA)1 protein, S cerevisiae; D010675:Pheromones; D019269:Phosphatidylinositol 4,5-Diphosphate; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C080321:STE5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins