A Cdc24p-Far1p-Gβγ Protein Complex Required for Yeast Orientation during Mating

Oriented cell growth requires the specification of a site for polarized growth and subsequent orientation of the cytoskeleton towards this site. During mating, haploid Saccharomyces cerevisiae cells orient their growth in response to a pheromone gradient overriding an internal landmark for polarized growth, the bud site. This response requires Cdc24p, Far1p, and a heterotrimeric G-protein. Here we show that a two- hybrid interaction between Cdc24p and Gβ requires Far1p but not pheromone-dependent MAP-kinase signaling, indicating Far1p has a role in regulating the association of Cdc24p and Gβ. Binding experiments demonstrate that Cdc24p, Far1p, and Gβ form a complex in which pairwise interactions can occur in the absence of the third protein. Cdc24p localizes to sites of polarized growth suggesting that this complex is localized. In the absence of CDC24-FAR1-mediated chemotropism, a bud site selection protein, Bud1p/Rsr1p, is essential for morphological changes in response to pheromone. These results suggest that formation of a Cdc24p-Far1p-Gβγ complex functions as a landmark for orientation of the cytoskeleton during growth towards an external signal.

[1]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .

[2]  J. Tkacz,et al.  Wall replication in saccharomyces species: use of fluorescein-conjugated concanavalin A to reveal the site of mannan insertion. , 1972, Journal of general microbiology.

[3]  P. Lipke,et al.  Morphogenic effects of alpha-factor on Saccharomyces cerevisiae a cells , 1976, Journal of bacteriology.

[4]  V. Mackay,et al.  Sexual conjugation in yeast. Cell surface changes in response to the action of mating hormones , 1979, The Journal of cell biology.

[5]  R. W. Davis,et al.  Replacement of chromosome segments with altered DNA sequences constructed in vitro. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[6]  R. Schekman,et al.  Localized secretion of acid phosphatase reflects the pattern of cell surface growth in saccharomyces cerevisiae , 1980, The Journal of cell biology.

[7]  J. Pringle,et al.  Roles of the CDC24 gene product in cellular morphogenesis during the Saccharomyces cerevisiae cell cycle , 1981, The Journal of cell biology.

[8]  J. Kilmartin,et al.  Structural rearrangements of tubulin and actin during the cell cycle of the yeast Saccharomyces , 1984, The Journal of cell biology.

[9]  J. Pringle,et al.  Relationship of actin and tubulin distribution to bud growth in wild- type and morphogenetic-mutant Saccharomyces cerevisiae , 1984, The Journal of cell biology.

[10]  Gerald R. Fink,et al.  Methods in Yeast Genetics: A Laboratory Course Manual , 1987 .

[11]  J. Kurjan,et al.  The yeast SCG1 gene: A Gα-like protein implicated in the a- and α-factor response pathway , 1987, Cell.

[12]  K. Arai,et al.  GPA1, a haploid-specific essential gene, encodes a yeast homolog of mammalian G protein which may be involved in mating factor signal transduction , 1987, Cell.

[13]  J. Thorner,et al.  The STE2 gene product is the ligand-binding component of the alpha-factor receptor of Saccharomyces cerevisiae. , 1988, The Journal of biological chemistry.

[14]  N. Baba,et al.  Three-dimensional analysis of morphogenesis induced by mating pheromone alpha factor in Saccharomyces cerevisiae. , 1989, Journal of cell science.

[15]  J. Pringle,et al.  Multicopy suppression of the cdc24 budding defect in yeast by CDC42 and three newly identified genes including the ras-related gene RSR1. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[16]  G. Sprague,,et al.  Pheromones and pheromone receptors are the primary determinants of mating specificity in the yeast Saccharomyces cerevisiae. , 1989, Genetics.

[17]  M. Whiteway,et al.  Overexpression of the STE4 gene leads to mating response in haploid Saccharomyces cerevisiae. , 1990, Molecular and cellular biology.

[18]  I. Herskowitz,et al.  Identification of a gene necessary for cell cycle arrest by a negative growth factor of yeast: FAR1 is an inhibitor of a G1 cyclin, CLN2 , 1990, Cell.

[19]  I. Herskowitz,et al.  Genetic control of bud site selection in yeast by a set of gene products that constitute a morphogenetic pathway , 1991, Cell.

[20]  I. Herskowitz,et al.  Yeast BUD5, encoding a putative GDP-GTP exchange factor, is necessary for bud site selection and interacts with bud formation gene BEM1 , 1991, Cell.

[21]  J. Pringle,et al.  Use of a screen for synthetic lethal and multicopy suppressee mutants to identify two new genes involved in morphogenesis in Saccharomyces cerevisiae , 1991, Molecular and cellular biology.

[22]  L. Hartwell,et al.  S. cerevisiae α pheromone receptors activate a novel signal transduction pathway for mating partner discrimination , 1991, Cell.

[23]  J. Pringle.  Staining of bud scars and other cell wall chitin with calcofluor. , 1991, Methods in enzymology.

[24]  Michael Snyder,et al.  Specification of sites for polarized growth in Saccharomyces cerevisiae and the influence of external factors on site selection. , 1992, Molecular biology of the cell.

[25]  I. Herskowitz,et al.  A yeast gene (BEM1) necessary for cell polarization whose product contains two SH3 domains , 1992, Nature.

[26]  H. Okamura,et al.  Actin- and tubulin-dependent functions during Saccharomyces cerevisiae mating projection formation. , 1992, Molecular biology of the cell.

[27]  I. Herskowitz,et al.  Phosphorylation of FAR1 in response to alpha-factor: a possible requirement for cell-cycle arrest. , 1992, Molecular biology of the cell.

[28]  J. Thorner,et al.  12 Pheromone Response and Signal Transduction during the Mating Process of Saccharomyces cerevisiae , 1992 .

[29]  Steven N. Hird,et al.  Cell polarity in early C. elegans development. , 1993, Development (Cambridge, England). Supplement.

[30]  J. Segall,et al.  Polarization of yeast cells in spatial gradients of alpha mating factor. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[31]  F. Cross,et al.  Genetic analysis of Cln/Cdc28 regulation of cell morphogenesis in budding yeast. , 1993, The EMBO journal.

[32]  D. Botstein,et al.  Subcellular localization of Cdc42p, a Saccharomyces cerevisiae GTP-binding protein involved in the control of cell polarity. , 1993, Molecular biology of the cell.

[33]  K. Nasmyth,et al.  Yeast G1 cyclins CLN1 and CLN2 and a GAP‐like protein have a role in bud formation. , 1993, The EMBO journal.

[34]  E. Elion,et al.  FUS3 phosphorylates multiple components of the mating signal transduction cascade: evidence for STE12 and FAR1. , 1993, Molecular biology of the cell.

[35]  T. Rabbitts,et al.  The LIM domain: a new structural motif found in zinc-finger-like proteins. , 1994, Trends in genetics : TIG.

[36]  J. Chenevert,et al.  Cell polarization directed by extracellular cues in yeast. , 1994, Molecular biology of the cell.

[37]  R. Cerione,et al.  Interactions between the bud emergence proteins Bem1p and Bem2p and Rho- type GTPases in yeast , 1994, The Journal of cell biology.

[38]  L. Hartwell,et al.  Saccharomyces cerevisiae cells execute a default pathway to select a mate in the absence of pheromone gradients , 1995, The Journal of cell biology.

[39]  I. Herskowitz,et al.  FAR1 is required for oriented polarization of yeast cells in response to mating pheromones , 1995, The Journal of cell biology.

[40]  K. Clark,et al.  Pheromone Response in Yeast: Association of Bem1p with Proteins of the MAP Kinase Cascade and Actin , 1995, Science.

[41]  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.

[42]  Yi Zheng,et al.  Interactions among Proteins Involved in Bud-site Selection and Bud-site Assembly in Saccharomyces cerevisiae (*) , 1995, The Journal of Biological Chemistry.

[43]  J. Chant,et al.  Patterns of bud-site selection in the yeast Saccharomyces cerevisiae , 1995, The Journal of cell biology.

[44]  J. Chant,et al.  Establishment of cell polarity in yeast. , 1995, Cold Spring Harbor symposia on quantitative biology.

[45]  M. Nieto.  Molecular Biology of Axon Guidance , 1996, Neuron.

[46]  E. Elion,et al.  The SH3-domain protein Bem1 coordinates mitogen-activated protein kinase cascade activation with cell cycle control in Saccharomyces cerevisiae , 1996, Molecular and cellular biology.

[47]  D. Drubin,et al.  Origins of Cell Polarity , 1996, Cell.

[48]  J. Chant,et al.  A mechanism of Bud1p GTPase action suggested by mutational analysis and immunolocalization , 1996, Current Biology.

[49]  J. Thorner,et al.  Sst2, a negative regulator of pheromone signaling in the yeast Saccharomyces cerevisiae: expression, localization, and genetic interaction and physical association with Gpa1 (the G-protein alpha subunit) , 1996, Molecular and cellular biology.

[50]  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.

[51]  M. Snyder,et al.  Selection of polarized growth sites in yeast. , 1996, Trends in cell biology.

[52]  E. Craig,et al.  Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. , 1996, Genetics.

[53]  C. Parent,et al.  Molecular genetics of signal transduction in Dictyostelium. , 1996, Annual review of biochemistry.

[54]  H. Dohlman,et al.  Regulation of Membrane and Subunit Interactions by N-Myristoylation of a G Protein α Subunit in Yeast* , 1996, The Journal of Biological Chemistry.

[55]  J. Kurjan,et al.  Analysis of the receptor binding domain of Gpa1p, the G(alpha) subunit involved in the yeast pheromone response pathway , 1997, Molecular and cellular biology.

[56]  J. Kurjan,et al.  Evidence that mating by the Saccharomyces cerevisiae gpa1Val50 mutant occurs through the default mating pathway and a suggestion of a role for ubiquitin-mediated proteolysis. , 1997, Molecular biology of the cell.

[57]  J. Hirschman,et al.  The G beta gamma complex of the yeast pheromone response pathway. Subcellular fractionation and protein-protein interactions. , 1997, The Journal of biological chemistry.

[58]  T. Kimbrough,et al.  Genetic analysis of default mating behavior in Saccharomyces cerevisiae. , 1997, Genetics.

[59]  I. Herskowitz,et al.  Phosphorylation- and ubiquitin-dependent degradation of the cyclin-dependent kinase inhibitor Far1p in budding yeast. , 1997, Genes & development.

[60]  G. Tocchini-Valentini,et al.  Efficient signal transduction by a chimeric yeast–mammalian G protein α subunit Gpa1–Gsα covalently fused to the yeast receptor Ste2 , 1997 .

[61]  S Falkow,et al.  Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans. , 1997, Microbiology.

[62]  I. Herskowitz,et al.  Two active states of the Ras-related Bud1/Rsr1 protein bind to different effectors to determine yeast cell polarity. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[63]  Robert A. Arkowitz,et al.  A Small Conserved Domain in the Yeast Spa2p Is Necessary and Sufficient for Its Polarized Localization , 1997, The Journal of cell biology.

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

[65]  L. Hartwell,et al.  Mating in Saccharomyces cerevisiae: the role of the pheromone signal transduction pathway in the chemotropic response to pheromone. , 1997, Genetics.

[66]  M. Whiteway,et al.  Pheromone signalling and polarized morphogenesis in yeast. , 1997, Current opinion in genetics & development.

[67]  G. Tocchini-Valentini,et al.  Efficient signal transduction by a chimeric yeast-mammalian G protein alpha subunit Gpa1-Gsalpha covalently fused to the yeast receptor Ste2. , 1997, The EMBO journal.

[68]  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.

[69]  J. Rayner,et al.  Identification of the MNN2 and MNN5Mannosyltransferases Required for Forming and Extending the Mannose Branches of the Outer Chain Mannans of Saccharomyces cerevisiae * , 1998, The Journal of Biological Chemistry.

[70]  L. Marsh,et al.  A Role for a Protease in Morphogenic Responses during Yeast Cell Fusion , 1998, The Journal of cell biology.

[71]  Joseph D. Schrag,et al.  Interaction of a G-protein β-subunit with a conserved sequence in Ste20/PAK family protein kinases , 1998, Nature.

[72]  I. Herskowitz,et al.  The role of Far1p in linking the heterotrimeric G protein to polarity establishment proteins during yeast mating. , 1998, Science.

[73]  D. Murphy,et al.  G Protein Signaling Events Are Activated at the Leading Edge of Chemotactic Cells , 1998, Cell.

[74]  David G. Drubin,et al.  A role for the yeast actin cytoskeleton in pheromone receptor clustering and signalling , 1998, Current Biology.

[75]  A. Nern,et al.  A GTP-exchange factor required for cell orientation , 1998, Nature.

[76]  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.

[77]  R. Arkowitz,et al.  Responding to attraction: chemotaxis and chemotropism in Dictyostelium and yeast. , 1999, Trends in cell biology.