1887

Abstract

Cell integrity in yeasts is ensured by a rigid cell wall whose synthesis is triggered by a MAP kinase-mediated signal-transduction cascade. Upstream regulatory components of this pathway in involve a single protein kinase C, which is regulated by interaction with the small GTPase Rho1. Here, two genes were isolated which encode these proteins from ( and ). Sequencing showed ORFs which encode proteins of 1161 and 208 amino acids, respectively. The deduced proteins shared 59 and 85 % overall amino acid identities, respectively, with their homologues from . Null mutants in both genes were non-viable, as shown by tetrad analyses of the heterozygous diploid strains. Overexpression of the gene under the control of the promoter severely impaired growth in both and . On the other hand, a similar construct with did not show a pronounced phenotype. Two-hybrid analyses showed interaction between Rho1 and Pkc1 for the proteins and their homologues. A green fluorescent protein (GFP) fusion to the C-terminal end of KlPkc1 located the protein to patches in the growing bud, and at certain stages of the division process also to the bud neck. N-terminal GFP fusions to KlRho1 localized mainly to the cell surface (presumably the cytoplasmic side of the plasma membrane) and to the vacuole, with some indications of traffic from the former to the latter. Thus, KlPkc1 and KlRho1 have been shown to serve vital functions in , to interact in cell integrity signalling and to traffic between the plasma membrane and the vacuole.

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2006-09-01
2019-10-14
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References

  1. Alberts, A. S., Bouquin, N., Johnston, L. H. & Treisman, R. ( 1998; ). Analysis of RhoA-binding proteins reveals an interaction domain conserved in heterotrimeric G protein beta subunits and the yeast response regulator protein Skn7. J Biol Chem 273, 8616–8622.[CrossRef]
    [Google Scholar]
  2. Arvanitidis, A. & Heinisch, J. J. ( 1994; ). Studies on the function of yeast phosphofructokinase subunits by in vitro mutagenesis. J Biol Chem 269, 8911–8918.
    [Google Scholar]
  3. Ayscough, K. R., Eby, J. J., Lila, T., Dewar, H., Kozminski, K. G. & Drubin, D. G. ( 1999; ). Sla1p is a functionally modular component of the yeast cortical actin cytoskeleton required for correct localization of both Rho1-GTPase and Sla2p, a protein with talin homology. Mol Biol Cell 10, 1061–1075.[CrossRef]
    [Google Scholar]
  4. Bar, E. E., Ellicott, A. T. & Stone, D. E. ( 2003; ). Gβγ recruits Rho1 to the site of polarized growth during mating in budding yeast. J Biol Chem 278, 21798–21804.[CrossRef]
    [Google Scholar]
  5. Bartel, P., Chien, C. T., Sternglanz, R. & Fields, S. ( 1993; ). Elimination of false positives that arise in using the two-hybrid system. Biotechniques 14, 920–924.
    [Google Scholar]
  6. Berben, G., Dumont, J., Gilliquet, V., Bolle, P. A. & Hilger, F. ( 1991; ). The YDp plasmids: a uniform set of vectors bearing versatile gene disruption cassettes for Saccharomyces cerevisiae. Yeast 7, 475–477.[CrossRef]
    [Google Scholar]
  7. Bianchi, M. M., Santarelli, R. & Frontali, L. ( 1991; ). Plasmid functions involved in the stable propagation of the pKD1 circular plasmid in Kluyveromyces lactis. Curr Genet 19, 155–161.[CrossRef]
    [Google Scholar]
  8. Birnboim, H. C. & Doly, J. ( 1979; ). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7, 1513–1523.[CrossRef]
    [Google Scholar]
  9. Chen, X. J. ( 1996; ). Low- and high-copy-number shuttle vectors for replication in the budding yeast Kluyveromyces lactis. Gene 172, 131–136.[CrossRef]
    [Google Scholar]
  10. Chen, X. J., Wesolowski-Louvel, M. & Fukuhara, H. ( 1992; ). Glucose transport in the yeast Kluyveromyces lactis. II. Transcriptional regulation of the glucose transporter gene RAG1. Mol Gen Genet 233, 97–105.[CrossRef]
    [Google Scholar]
  11. Denis, V. & Cyert, M. S. ( 2005; ). Molecular analysis reveals localization of Saccharomyces cerevisiae protein kinase C to sites of polarized growth and Pkc1 targeting to the nucleus and mitotic spindle. Eukaryot Cell 4, 36–45.[CrossRef]
    [Google Scholar]
  12. Dong, Y., Pruyne, D. & Bretscher, A. ( 2003; ). Formin-dependent actin assembly is regulated by distinct modes of Rho signaling in yeast. J Cell Biol 161, 1081–1092.[CrossRef]
    [Google Scholar]
  13. Drgonova, J., Drgon, T., Tanaka, K., Kollar, R., Chen, G. C., Ford, R. A., Chan, C. S., Takai, Y. & Cabib, E. ( 1996; ). Rho1, a yeast protein at the interface between cell polarization and morphogenesis. Science 272, 277–279.[CrossRef]
    [Google Scholar]
  14. Dujon, B., Sherman, D., Fischer, G. & 64 other authors ( 2004; ). Genome evolution in yeasts. Nature 430, 35–44.[CrossRef]
    [Google Scholar]
  15. Fujiwara, T., Tanaka, K., Mino, A., Kikyo, M., Takahashi, K., Shimizu, K. & Takai, Y. ( 1998; ). Rho1-Bni1-Spa2p interactions: implication in localization of Bni1 at the bud site and regulation of the actin cytoskeleton in Saccharomyces cerevisiae. Mol Biol Cell 9, 1221–1233.[CrossRef]
    [Google Scholar]
  16. Gietz, R. D. & Sugino, A. ( 1988; ). New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74, 527–534.[CrossRef]
    [Google Scholar]
  17. Goffeau, A. ( 2000; ). Four years of post-genomic life with 6,000 yeast genes. FEBS Lett 480, 37–41.[CrossRef]
    [Google Scholar]
  18. Guarente, L. ( 1983; ). Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol 101, 181–191.
    [Google Scholar]
  19. Guo, W., Tamanoi, F. & Novick, P. ( 2001; ). Spatial regulation of the exocyst complex by Rho1 GTPase. Nat Cell Biol 3, 353–360.[CrossRef]
    [Google Scholar]
  20. Hanahan, D., Jessee, J. & Bloom, F. R. ( 1995; ). Techniques for Transformation of E. coli, vol. 1. Oxford: IRL Press.
  21. Heinisch, J. J. ( 1993; ). PFK2, ISP42, ERG2 and RAD14 are located on the right arm of chromosome XIII. Yeast 9, 1103–1105.[CrossRef]
    [Google Scholar]
  22. Heinisch, J. J. ( 2005; ). Baker's yeast as a tool for the development of antifungal kinase inhibitors – targeting protein kinase C and the cell integrity pathway. Biochim Biophys Acta 1754, 171–182.[CrossRef]
    [Google Scholar]
  23. Heinisch, J. J., Lorberg, A., Schmitz, H. P. & Jacoby, J. J. ( 1999; ). The protein kinase C-mediated MAP kinase pathway involved in the maintenance of cellular integrity in Saccharomyces cerevisiae. Mol Microbiol 32, 671–680.[CrossRef]
    [Google Scholar]
  24. Helliwell, S. B., Schmidt, A., Ohya, Y. & Hall, M. N. ( 1998; ). The Rho1 effector Pkc1, but not Bni1, mediates signalling from Tor2 to the actin cytoskeleton. Curr Biol 8, 1211–1214.[CrossRef]
    [Google Scholar]
  25. Hill, J. E., Myers, A. M., Koerner, T. J. & Tzagoloff, A. ( 1986; ). Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast 2, 163–167.[CrossRef]
    [Google Scholar]
  26. Inoue, S. B., Qadota, H., Arisawa, M., Watanabe, T. & Ohya, Y. ( 1999; ). Prenylation of Rho1 is required for activation of yeast 1,3-β-glucan synthase. J Biol Chem 274, 38119–38124.[CrossRef]
    [Google Scholar]
  27. Iwabuchi, K., Li, B., Bartel, P. & Fields, S. ( 1993; ). Use of the two-hybrid system to identify the domain of p53 involved in oligomerization. Oncogene 8, 1693–1696.
    [Google Scholar]
  28. Jacoby, J. J., Kirchrath, L., Gengenbacher, U. & Heinisch, J. J. ( 1999; ). Characterization of KLBCK1, encoding a MAP kinase kinase kinase of Kluyveromyces lactis. J Mol Biol 288, 337–352.[CrossRef]
    [Google Scholar]
  29. James, P., Halladay, J. & Craig, E. A. ( 1996; ). Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144, 1425–1436.
    [Google Scholar]
  30. Kaksonen, M., Toret, C. P. & Drubin, D. G. ( 2005; ). A modular design for the clathrin- and actin-mediated endocytosis machinery. Cell 123, 305–320.[CrossRef]
    [Google Scholar]
  31. Kamada, Y., Qadota, H., Python, C. P., Anraku, Y., Ohya, Y. & Levin, D. E. ( 1996; ). Activation of yeast protein kinase C by Rho1 GTPase. J Biol Chem 271, 9193–9196.[CrossRef]
    [Google Scholar]
  32. Kirchrath, L., Lorberg, A., Schmitz, H. P., Gengenbacher, U. & Heinisch, J. J. ( 2000; ). Comparative genetic and physiological studies of the MAP kinase Mpk1p from Kluyveromyces lactis and Saccharomyces cerevisiae. J Mol Biol 300, 743–758.[CrossRef]
    [Google Scholar]
  33. Klebe, R. J., Harriss, J. V., Sharp, Z. D. & Douglas, M. G. ( 1983; ). A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast. Gene 25, 333–341.[CrossRef]
    [Google Scholar]
  34. Kohno, H., Tanaka, K., Mino, A. & 9 other authors ( 1996; ). Bni1 implicated in cytoskeletal control is a putative target of Rho1 small GTP binding protein in Saccharomyces cerevisiae. EMBO J 15, 6060–6068.
    [Google Scholar]
  35. Kono, K., Matsunaga, R., Hirata, A., Suzuki, G., Abe, M. & Ohya, Y. ( 2005; ). Involvement of actin and polarisome in morphological change during spore germination of Saccharomyces cerevisiae. Yeast 22, 129–139.[CrossRef]
    [Google Scholar]
  36. Levin, D. E. ( 2005; ). Cell wall integrity signaling in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 69, 262–291.[CrossRef]
    [Google Scholar]
  37. Li, B. & Fields, S. ( 1993; ). Identification of mutations in p53 that affect its binding to SV40 large T antigen by using the yeast two-hybrid system. FASEB J 7, 957–963.
    [Google Scholar]
  38. Longtine, M. S., McKenzie, A., Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P. & Pringle, J. R. ( 1998; ). Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961.[CrossRef]
    [Google Scholar]
  39. Lorberg, A., Schmitz, H. P., Gengenbacher, U. & Heinisch, J. J. ( 2003; ). KlROM2 encodes an essential GEF homologue in Kluyveromyces lactis. Yeast 20, 611–624.[CrossRef]
    [Google Scholar]
  40. Marelli, M., Smith, J. J., Jung, S. & 10 other authors ( 2004; ). Quantitative mass spectrometry reveals a role for the GTPase Rho1 in actin organization on the peroxisome membrane. J Cell Biol 167, 1099–1112.[CrossRef]
    [Google Scholar]
  41. Nonaka, H., Tanaka, K., Hirano, H., Fujiwara, T., Kohno, H., Umikawa, M., Mino, A. & Takai, Y. ( 1995; ). A downstream target of Rho1 small GTP-binding protein is Pkc1, a homolog of protein kinase C, which leads to activation of the MAP kinase cascade in Saccharomyces cerevisiae. EMBO J 14, 5931–5938.
    [Google Scholar]
  42. Park, H. & Lennarz, W. J. ( 2000; ). Evidence for interaction of yeast protein kinase C with several subunits of oligosaccharyl transferase. Glycobiology 10, 737–744.[CrossRef]
    [Google Scholar]
  43. Qadota, H., Python, C. P., Inoue, S. B., Arisawa, M., Anraku, Y., Zheng, Y., Watanabe, T., Levin, D. E. & Ohya, Y. ( 1996; ). Identification of yeast Rho1 GTPase as a regulatory subunit of 1,3-beta-glucan synthase. Science 272, 279–281.[CrossRef]
    [Google Scholar]
  44. Queralt, E. & Igual, J. C. ( 2005; ). Functional connection between the Clb5 cyclin, the protein kinase C pathway and the Swi4 transcription factor in Saccharomyces cerevisiae. Genetics 171, 1485–1498.[CrossRef]
    [Google Scholar]
  45. Robzyk, K. & Kassir, Y. ( 1992; ). A simple and highly efficient procedure for rescuing autonomous plasmids from yeast. Nucleic Acids Res 20, 3790.[CrossRef]
    [Google Scholar]
  46. Saka, A., Abe, M., Okano, H. & 7 other authors ( 2001; ). Complementing yeast rho1 mutation groups with distinct functional defects. J Biol Chem 276, 46165–46171.[CrossRef]
    [Google Scholar]
  47. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  48. Scherman, F., Fink, G. R. & Hicks, J. B. ( 1986; ). Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  49. Schmitz, H. P. & Heinisch, J. J. ( 2003; ). Evolution, biochemistry and genetics of protein kinase C in fungi. Curr Genet 43, 245–254.[CrossRef]
    [Google Scholar]
  50. Schmitz, H. P., Lorberg, A. & Heinisch, J. J. ( 2002; ). Regulation of yeast protein kinase C activity by interaction with the small GTPase Rho1 through its amino-terminal HR1 domain. Mol Microbiol 44, 829–840.[CrossRef]
    [Google Scholar]
  51. Schultz, J., Milpetz, F., Bork, P. & Ponting, C. P. ( 1998; ). smart, a simple modular architecture research tool: identification of signaling domains. Proc Natl Acad Sci U S A 95, 5857–5864.[CrossRef]
    [Google Scholar]
  52. Sherman, D., Durrens, P., Beyne, E., Nikolski, M. & Souciet, J. L. ( 2004; ). Genolevures: comparative genomics and molecular evolution of hemiascomycetous yeasts. Nucleic Acids Res 32, 315–318.[CrossRef]
    [Google Scholar]
  53. Valdivia, R. H. & Schekman, R. ( 2003; ). The yeasts Rho1 and Pkc1 regulate the transport of chitin synthase III (Chs3p) from internal stores to the plasma membrane. Proc Natl Acad Sci U S A 100, 10287–10292.[CrossRef]
    [Google Scholar]
  54. Wach, A., Brachat, A., Pohlmann, R. & Philippsen, P. ( 1994; ). New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10, 1793–1808.[CrossRef]
    [Google Scholar]
  55. Wesolowski-Louvel, M., Tanguy-Rougeau, C. & Fukuhara, H. ( 1988; ). A nuclear gene required for the expression of the linear DNA-associated killer system in the yeast Kluyveromyces lactis. Yeast 4, 71–81.[CrossRef]
    [Google Scholar]
  56. Yamochi, W., Tanaka, K., Nonaka, H., Maeda, A., Musha, T. & Takai, Y. ( 1994; ). Growth site localization of Rho1 small GTP-binding protein and its involvement in bud formation in Saccharomyces cerevisiae. J Cell Biol 125, 1077–1093.[CrossRef]
    [Google Scholar]
  57. Zamenhoff, S. ( 1957; ). Preparation and assay of deoxyribonucleic acids from animal tissue. Methods Enzymol 3, 702–704.
    [Google Scholar]
  58. Zenke, F. T., Kapp, L. & Breunig, K. D. ( 1999; ). Regulated phosphorylation of the Gal4p inhibitor Gal80p of Kluyveromyces lactis revealed by mutational analysis. Biol Chem 380, 419–430.
    [Google Scholar]
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