Polarized localization of yeast Pbs2 depends on osmostress, the membrane protein Sho1 and Cdc42

In Saccharomyces cerevisiae cells, high external osmolarity leads to the activation of a p38-related mitogen-activated protein (MAP) kinase though Pbs2. Pbs2 tagged with green fluorescent protein (Pbs2–GFP) is evenly distributed in the cytoplasm but excluded from the nucleus before and after exposure to stress. Here we show that a catalytically inactive form of Pbs2 attains a highly polarised localization during osmostress. This phenomenon depends of the osmosensor Sho1 and on a functional Cdc42 GTPase. Cdc42, but not the actin cytoskeleton, influences Sho1-dependent activation of the MAP kinase. Sho1 itself accumulates at sites of polar growth, but independently of stress conditions and Cdc42. These observations allow us to define the sequence of events that occurs during propogation of osmostress signals.

[1]  A. Rodal,et al.  Functions and functional domains of the GTPase Cdc42p. , 2000, Molecular biology of the cell.

[2]  Douglas I. Johnson Cdc42: An Essential Rho-Type GTPase Controlling Eukaryotic Cell Polarity , 1999, Microbiology and Molecular Biology Reviews.

[3]  L. Bardwell,et al.  Inhibitory and activating functions for MAPK Kss1 in the S. cerevisiae filamentous- growth signalling pathway , 1997, Nature.

[4]  D. Kellogg,et al.  Control of mitotic events by the Cdc42 GTPase, the Clb2 cyclin and a member of the PAK kinase family , 1998, Current Biology.

[5]  I. Herskowitz,et al.  Functional analysis of the interaction between the small GTP binding protein Cdc42 and the Ste20 protein kinase in yeast. , 1996, The EMBO journal.

[6]  Gerald R. Fink,et al.  MAP Kinases with Distinct Inhibitory Functions Impart Signaling Specificity during Yeast Differentiation , 1997, Cell.

[7]  F. Posas,et al.  Signal transduction by MAP kinase cascades in budding yeast. , 1998, Current opinion in microbiology.

[8]  Thomas J. White,et al.  PCR protocols: a guide to methods and applications. , 1990 .

[9]  Navin Pokala,et al.  High Rates of Actin Filament Turnover in Budding Yeast and Roles for Actin in Establishment and Maintenance of Cell Polarity Revealed Using the Actin Inhibitor Latrunculin-A , 1997, The Journal of cell biology.

[10]  F. Posas,et al.  Osmotic activation of the HOG MAPK pathway via Ste11p MAPKKK: scaffold role of Pbs2p MAPKK. , 1997, Science.

[11]  H. Seliger,et al.  PCR protocols — A guide to methods and applications , 1990 .

[12]  G. Ammerer,et al.  Kinase activity-dependent nuclear export opposes stress-induced nuclear accumulation and retention of Hog1 mitogen-activated protein kinase in the budding yeast Saccharomyces cerevisiae. , 1999, Molecular biology of the cell.

[13]  Pamela A. Silver,et al.  Regulated nucleo/cytoplasmic exchange of HOG1 MAPK requires the importin β homologs NMD5 and XPO1 , 1998, The EMBO journal.

[14]  Douglas I. Johnson,et al.  Characterization of the Saccharomyces cerevisiae cdc42‐1ts Allele and New Temperature‐Conditional‐Lethal cdc42 Alleles , 1997, Yeast.

[15]  J. Segall,et al.  Functional characterization of the Cdc42p binding domain of yeast Ste20p protein kinase , 1997, The EMBO journal.

[16]  G. Fink,et al.  Elements of a single MAP kinase cascade in Saccharomyces cerevisiae mediate two developmental programs in the same cell type: mating and invasive growth. , 1994, Genes & development.

[17]  I. Herskowitz,et al.  The Hog1 MAPK prevents cross talk between the HOG and pheromone response MAPK pathways in Saccharomyces cerevisiae. , 1998, Genes & development.

[18]  R. D. Gietz,et al.  New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. , 1988, Gene.

[19]  W. Toone,et al.  Stress‐activated signalling pathways in yeast , 1998, Genes to cells : devoted to molecular & cellular mechanisms.

[20]  B. Haarer,et al.  Polarity and division site specification in yeast. , 1998, Current opinion in microbiology.

[21]  K. Siegers,et al.  Epitope tagging of yeast genes using a PCR‐based strategy: more tags and improved practical routines , 1999, Yeast.

[22]  G L Johnson,et al.  Organization and regulation of mitogen-activated protein kinase signaling pathways. , 1999, Current opinion in cell biology.

[23]  Fred Winston,et al.  Methods in Yeast Genetics: A Laboratory Course Manual , 1990 .

[24]  P. Pryciak,et al.  Membrane recruitment of the kinase cascade scaffold protein Ste5 by the Gbetagamma complex underlies activation of the yeast pheromone response pathway. , 1998, Genes & development.

[25]  S. Reed,et al.  Role for the Rho-family GTPase Cdc42 in yeast mating-pheromone signal pathway , 1995, Nature.

[26]  T. Maeda,et al.  Activation of yeast PBS2 MAPKK by MAPKKKs or by binding of an SH3-containing osmosensor. , 1995, Science.

[27]  G. Fink,et al.  Methods in yeast genetics , 1979 .

[28]  G. Fink,et al.  Ras2 signals via the Cdc42/Ste20/mitogen-activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[29]  R. Davis,et al.  Structural organization of MAP-kinase signaling modules by scaffold proteins in yeast and mammals. , 1998, Trends in biochemical sciences.

[30]  L. Lim,et al.  Pheromone signalling in Saccharomyces cerevisiae requires the small GTP-binding protein Cdc42p and its activator CDC24 , 1995, Molecular and cellular biology.

[31]  Tatsuya Maeda,et al.  A two-component system that regulates an osmosensing MAP kinase cascade in yeast , 1994, Nature.

[32]  F. Cross,et al.  The Role of Cdc42 in Signal Transduction and Mating of the Budding Yeast Saccharomyces cerevisiae * , 1998, The Journal of Biological Chemistry.

[33]  Francesc Posas,et al.  Requirement of STE50 for Osmostress-Induced Activation of the STE11 Mitogen-Activated Protein Kinase Kinase Kinase in the High-Osmolarity Glycerol Response Pathway , 1998, Molecular and Cellular Biology.

[34]  J. Kyriakis,et al.  Making the connection: coupling of stress-activated ERK/MAPK (extracellular-signal-regulated kinase/mitogen-activated protein kinase) core signalling modules to extracellular stimuli and biological responses. , 1999, Biochemical Society symposium.

[35]  A. Hall,et al.  Signaling to Rho GTPases. , 1999, Experimental cell research.