Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

The ‘interactome’ of the Knr4/Smi1, a protein implicated in coordinating cell wall synthesis with bud emergence in Saccharomyces cerevisiae

Abstract

The integrity of the Saccharomyces cerevisiae cell wall requires a functional Pkc1–Slt2 MAP kinase pathway that contributes to transient growth arrest, enabling coordination of cell division with cell wall remodelling. How this coordination takes place is still an open question. Recently, we brought evidence that Knr4 protein, whose absence leads to several cell wall defects, may play a role in this function. Here, we show that Knr4 is a monomeric protein that exhibits an aberrant mobility on a SDS-gel electrophoresis and a non-globular structure. Furthermore, Knr4 is an unstable protein that is degraded as cells enter the stationary phase of growth, while its corresponding gene is constitutively expressed. In exponentially growing cells on glucose, Knr4 appeared to be present in a protein complex that migrates with an apparent Mw superior to 250 kDa. Using the TAP–tag methodology, nine potential partners of Knr4 were identified, which could be distributed into three biological processes. A first group consisted of Slt2 and Pil1, two proteins dedicated to cell wall maintenance and biogenesis. The second group comprised four proteins (Bud6, Act1, Cin8 and Jnm1) implicated in the establishment of cell polarity and bud integrity during mitosis. The last group contained four proteins (Asc1, Ubc1, Hsc82 and Gvp36) that probably deal with the stability/degradation of proteins. Deletion analysis revealed that the domain of interaction covered 2/3 of the Knr4 sequence on the N-terminal side. Moreover, the replacement of the two in vivo phosphorylated Ser200 and Ser203 by alanines led to a mutated protein with reduced protein interactions and a weaker complementation ability towards knr4 null mutant phenotypes. These results together with previous data from genome scale two-hybrid and synthetic interaction screens support the notion that Knr4 is a regulatory protein that participates in the coordination of cell wall synthesis with bud emergence, and that this function may be modulated by phosphorylation of this protein.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. Amberg DC, Zahner JE, Mulholland JW, Pringle JR, Botstein D (1997) Aip3p/Bud6p, a yeast actin-interacting protein that is involved in morphogenesis and the selection of bipolar budding sites. Mol Biol Cell 8:729–753

  2. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–54

  3. Brown RS, Lennon JJ (1995) Mass resolution improvement by incorporation of pulsed ion extraction in a matrix-assisted laser desorption/ionization linear time-of-flight mass spectrometer. Anal Chem 67:1998–2003

  4. Dagkessamanskaia A, Martin-Yken H, Basmaji F, Briza P, Francois J (2001) Interaction of Knr4 protein, a protein involved in cell wall synthesis, with tyrosine tRNA synthetase encoded by TYS1 in Saccharomyces cerevisiae. FEMS Microbiol Lett 200:53–58

  5. DeRisi JL, Iyer VR, Brown PO (1997) Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278:680–686

  6. Ficarro SB, McCleland ML, Stukenberg PT, Burke DJ, Ross MM, Shabanowitz J, Hunt DF, White FM (2002) Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat Biotechnol 20:301–305

  7. Fink AL (2005) Natively unfolded proteins. Curr Opin Struct Biol 15:35–41

  8. Fishel BR, Sperry AO, Garrard WT (1993) Yeast calmodulin and a conserved nuclear protein participate in the in vivo binding of a matrix association region. Proc Natl Acad Sci USA 90:5623–5627

  9. Gavin AC, Bosche M, Krause R, Grandi P, Marzioch M, Bauer A, Schultz J, Rick JM, Michon AM, Cruciat CM, Remor M, Hofert C, Schelder M, Brajenovic M, Ruffner H, Merino A, Klein K, Hudak M, Dickson D, Rudi T, Gnau V, Bauch A, Bastuck S, Huhse B, Leutwein C, Heurtier MA, Copley RR, Edelmann A, Querfurth E, Rybin V, Drewes G, Raida M, Bouwmeester T, Bork P, Seraphin B, Kuster B, Neubauer G, Superti-Furga G (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415:141–147

  10. Ghaemmaghami S, Huh WK, Bower K, Howson RW, Belle A, Dephoure N, O’Shea EK, Weissman JS (2003) Global analysis of protein expression in yeast. Nature 425:737–741

  11. Gould KL, Ren L, Feoktistova AS, Jennings JL, Link AJ (2004) Tandem affinity purification and identification of protein complex components. Methods 33:239–244

  12. Harrison JC, Bardes ES, Ohya Y, Lew DJ (2001) A role for the Pkc1p/Mpk1p kinase cascade in the morphogenesis checkpoint. Nat Cell Biol 3:417–420

  13. Hauser NC, Vingron M, Scheideler M, Krems B, Hellmuth K, Entian KD, Hoheisel JD (1998) Transcriptional profiling on all open reading frames of Saccharomyces cerevisiae. Yeast 14:1209–1221

  14. Hazbun TR, Malmstrom L, Anderson S, Graczyk BJ, Fox B, Riffle M, Sundin BA, Aranda JD, McDonald WH, Chiu CH, Snydsman BE, Bradley P, Muller EG, Fields S, Baker D, Yates JR III, Davis TN (2003) Assigning function to yeast proteins by integration of technologies. Mol Cell 12:1353–1365

  15. Heinisch JJ, Lorberg A, Schmitz H-P, Jacoby JJ (1999) The protein kinase C-mediated MAP kinase pathway involved in the maintenance of cellular integrity in Saccharomyces cerevisiae. Mol Microbiol 32:671–680

  16. Ho Y, Gruhler A, Heilbut A, Bader GD, Moore L, Adams SL, Millar A, Taylor P, Bennett K, Boutilier K, Yang L, Wolting C, Donaldson I, Schandorff S, Shewnarane J, Vo M, Taggart J, Goudreault M, Muskat B, Alfarano C, Dewar D, Lin Z, Michalickova K, Willems AR, Sassi H, Nielsen PA, Rasmussen KJ, Andersen JR, Johansen LE, Hansen LH, Jespersen H, Podtelejnikov A, Nielsen E, Crawford J, Poulsen V, Sorensen BD, Matthiesen J, Hendrickson RC, Gleeson F, Pawson T, Moran MF, Durocher D, Mann M, Hogue CW, Figeys D, Tyers M (2002) Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415:180–183

  17. Hong Z, Mann P, Brown NH, Tran LE, Shaw KJ, Hare RS, Didomenico B (1994) Cloning and characterization of KNR4, a yeast gene involved in (1,3)-beta-glucan synthesis. Mol Cell Biol 14:1017–1025

  18. Huh WK, Falvo JV, Gerke LC, Carroll AS, Howson RW, Weissman JS, O’Shea EK (2003) Global analysis of protein localization in budding yeast. Nature 425:686–691

  19. Iakoucheva LM, Kimzey AL, Masselon CD, Smith RD, Dunker AK, Ackerman EJ (2001) Aberrant mobility phenomena of the DNA repair protein XPA. Protein Sci 10:1353–1362

  20. Ito T, Chiba T, Yoshida M (2001) Exploring the protein interactome using comprehensive two-hybrid projects. Trends Biotechnol 19:S23–S27

  21. James P, Halladay J, Craig EA (1996) Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144:1425–1436

  22. Jung US, Levin DE (1999) Genome-wide analysis of gene expression regulated by the yeast cell wall integrity signalling pathway. Mol Microbiol 34:1049–1057

  23. Karas M, Hillenkamp F (1988) Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem 60:2299–2301

  24. Kirchrath L, Lorberg A, Schmitz HP, Gengenbacher U, Heinisch JJ (2000) Comparative genetic and physiological studies of the MAP kinase Mpk1p from Kluyveromyces lactis and Saccharomyces cerevisiae. J Mol Biol 300:743–758

  25. Klenova EM, Nicolas RH, U S, Carne AF, Lee RE, Lobanenkov VV, Goodwin GH (1997) Molecular weight abnormalities of the CTCF transcription factor: CTCF migrates aberrantly in SDS-PAGE and the size of the expressed protein is affected by the UTRs and sequences within the coding region of the CTCF gene. Nucleic Acids Res 25:466–474

  26. Klis FM (1994) Review: cell wall assembly in yeast. Yeast 10:851–869

  27. Klis FM, Mol P, Hellingwerf K, Brul S (2002) Dynamics of cell wall structure in Saccharomyces cerevisiae. FEMS Microbiol Rev 26:239–256

  28. Korolyev E, Steinberg-Neifach O, Eshel D (2005) Mutations in the yeast kinesin-like Cin8p are alleviated by osmotic support. FEMS Microbiol Lett 244:379–383

  29. Kren A, Mamnun YM, Bauer BE, Schuller C, Wolfger H, Hatzixanthis K, Mollapour M, Gregori C, Piper P, Kuchler K (2003) War1p, a novel transcription factor controlling weak acid stress response in yeast. Mol Cell Biol 23:1775–1785

  30. Kriwacki RW, Hengst L, Tennant L, Reed SI, Wright PE (1996) Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity. Proc Natl Acad Sci USA 93:11504–11509

  31. Lagorce A, Le Berre-Anton V, Aguilar-Uscanga B, Martin-Yken H, Dagkessamanskaia A, Francois J (2002) Involvement of GFA1, which encodes glutamine-fructose-6-phosphate amidotransferase, in the activation of the chitin synthesis pathway in response to cell-wall defects in Saccharomyces cerevisiae. Eur J Biochem 269:1697–1707

  32. Lesage G, Sdicu AM, Menard P, Shapiro J, Hussein S, Bussey H (2004) Analysis of beta-1,3-glucan assembly in Saccharomyces cerevisiae using a synthetic interaction network and altered sensitivity to caspofungin. Genetics 167:35–49

  33. Lesage G, Shapiro J, Specht CA, Sdicu AM, Menard P, Hussein S, Tong AH, Boone C, Bussey H (2005) An interactional network of genes involved in chitin synthesis in Saccharomyces cerevisiae. BMC Genet 6:8

  34. Levin DE (2005) Cell wall integrity signaling in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 69:262–291

  35. Linding R, Schymkowitz J, Rousseau F, Diella F, Serrano L (2004) A comparative study of the relationship between protein structure and beta-aggregation in globular and intrinsically disordered proteins. J Mol Biol 342:345–353

  36. Liu J, Rost B (2003) NORSp: predictions of long regions without regular secondary structure. Nucleic Acids Res 31:3833–3835

  37. Louvet O, Doignon F, Crouzet M (1997) Stable DNA-binding yeast vector allowing high-bait expression for use in the two-hybrid system. Biotechniques 23:816–820

  38. Madden K, Sheu YJ, Baetz K, Andrews B, Snyder M (1997) SBF cell cycle regulator as a target of the yeast PKC-MAP kinase pathway. Science 275:1781–1784

  39. Markovich S, Yekutiel A, Shalit I, Shadkchan Y, Osherov N (2004) Genomic approach to identification of mutations affecting caspofungin susceptibility in Saccharomyces cerevisiae. Antimicrob Agents Chemother 48:3871–3876

  40. Martin H, Dagkessamanskaia A, Satchanska G, Dallies N, Francois J (1999) KNR4, a suppressor of Saccharomyces cerevisiae cwh mutants, is involved in the transcriptional control of chitin synthase genes. Microbiology 145:249–258

  41. Martin H, Rodriguez-Pachon JM, Ruiz C, Nombela C, Molina M (2000) Regulatory mechanisms for modulation of signaling through the cell integrity Slt2-mediated pathway in Saccharomyces cerevisiae. J Biol Chem 275:1511–1519

  42. Martin-Yken H, Dagkessamanskaia A, Basmaji F, Lagorce A, Francois J (2003) The interaction of Slt2 MAP kinase with Knr4 is necessary for signalling through the cell wall integrity pathway in Saccharomyces cerevisiae. Mol Microbiol 49:23–35

  43. Martin-Yken H, Dagkessamanskaia A, Talabi D, Francois J (2002) KNR4 is a member of the PKC1 signalling pathway and genetically interacts with BCK2, a gene involved in cell cycle progression in Saccharomyces cerevisiae. Curr Genet. 41:323–332

  44. von Mering C, Krause R, Snel B, Cornell M, Oliver SG, Fields S, Bork P (2002) Comparative assessment of large-scale data sets of protein–protein interactions. Nature 417:399–403

  45. Millson SH, Truman AW, King V, Prodromou C, Pearl LH, Piper PW (2005) A two-hybrid screen of the yeast proteome for Hsp90 interactors uncovers a novel Hsp90 chaperone requirement in the activity of a stress-activated mitogen-activated protein kinase, Slt2p (Mpk1p). Eukaryot Cell 4:849–860

  46. Page N, Gerard-Vincent M, Menard P, Beaulieu M, Azuma M, Dijkgraaf GJ, Li H, Marcoux J, Nguyen T, Dowse T, Sdicu AM, Bussey H (2003) A Saccharomyces cerevisiae genome-wide mutant screen for altered sensitivity to K1 killer toxin. Genetics 163:875–894

  47. Parrou JL, Enjalbert B, François J (1999) STRE- and cAMP-independent transcriptional induction of Saccharomyces cerevisiae GSY2 encoding glycogen synthase during diauxic growth on glucose. Yeast 15:1471–1484

  48. Pronk JT, Yde SH, Van DJ (1996) Pyruvate metabolism in Saccharomyces cerevisiae. Yeast 12:1607–1633

  49. Pruyne D, Bretscher A (2000) Polarization of cell growth in yeast. J Cell Sci 113:571–585

  50. Puig O, Caspary F, Rigaut G, Rutz B, Bouveret E, Bragado-Nilsson E, Wilm M, Seraphin B (2001) The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24:218–229

  51. Rajavel M, Philip B, Buehrer BM, Errede B, Levin DE (1999) Mid2 is a putative sensor for cell integrity signaling in Saccharomyces cerevisiae. Mol Cell Biol 19:3969–3976

  52. Rigaut G, Shevchenko A, Rutz B, Wilm M, Mann M, Seraphin B (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17:1030–1032

  53. Rose MD, Winston F, Hieter P (1990) Methods in yeast genetics : a laboratory course manual. Cold Spring Harbor Laboratory Press, New York

  54. Seufert W, Jentsch S (1990) Ubiquitin-conjugating enzymes UBC4 and UBC5 mediate selective degradation of short-lived and abnormal proteins. EMBO J 9:543–550

  55. Sheu YJ, Santos B, Fortin N, Costigan C, Snyder M (1998) Spa2p interacts with cell polarity proteins and signaling components involved in yeast cell morphogenesis. Mol Cell Biol 18:4053–4069

  56. Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27

  57. Suzuki M, Igarashi R, Sekiya M, Utsugi T, Morishita S, Yukawa M, Ohya Y (2004) Dynactin is involved in a checkpoint to monitor cell wall synthesis in Saccharomyces cerevisiae. Nat Cell Biol 6:861–871

  58. Takasuka T, Komiyama T, Furuichi Y, Watanabe T (1995) Cell wall synthesis specific cytocidal effect of Hansenula mrakii toxin-1 on Saccharomyces cerevisiae. Cell Mol Biol Res 41:575–581

  59. Tong AH, Evangelista M, Parsons AB, Xu H, Bader GD, Page N, Robinson M, Raghibizadeh S, Hogue CW, Bussey H, Andrews B, Tyers M, Boone C (2001) Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science 294:2364–2368

  60. Tong AH, Lesage G, Bader GD, Ding H, Xu H, Xin X, Young J, Berriz GF, Brost RL, Chang M, Chen Y, Cheng X, Chua G, Friesen H, Goldberg DS, Haynes J, Humphries C, He G, Hussein S, Ke L, Krogan N, Li Z, Levinson JN, Lu H, Menard P, Munyana C, Parsons AB, Ryan O, Tonikian R, Roberts T, Sdicu AM, Shapiro J, Sheikh B, Suter B, Wong SL, Zhang LV, Zhu H, Burd CG, Munro S, Sander C, Rine J, Greenblatt J, Peter M, Bretscher A, Bell G, Roth FP, Brown GW, Andrews B, Bussey H, Boone C (2004) Global mapping of the yeast genetic interaction network. Science 303:808–813

  61. Uetz P, Giot L, Cagney G, Mansfield TA, Judson RS, Knight JR, Lockshon D, Narayan V, Srinivasan M, Pochart P, Qureshi-Emili A, Li Y, Godwin B, Conover D, Kalbfleisch T, Vijayadamodar G, Yang M, Johnston M, Fields S, Rothberg JM (2000) A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae. Nature 403:623–627

  62. Vucetic S, Brown CJ, Dunker AK, Obradovic Z (2003) Flavors of protein disorder. Proteins 52:573–584

  63. Watanabe Y, Irie K, Matsumoto K (1995) Yeast RLM1 encodes a serum response factor-like protein that may function downstream of the Mpk1 (Slt2) mitogen-activated protein kinase pathway. Mol Cell Biol 15:5740–5749

  64. Wright PE, Dyson HJ (1999) Intrinsically unstructured proteins: re-assessing the protein structure–function paradigm. J Mol Biol 293:321–331

  65. Zakrzewska A, Boorsma A, Brul S, Hellingwerf KJ, Klis FM (2005) Transcriptional response of Saccharomyces cerevisiae to the plasma membrane-perturbing compound chitosan. Eukaryot Cell 4:703–715

  66. Zhang X, Lester RL, Dickson RC (2004) Pil1p and Lsp1p negatively regulate the 3-phosphoinositide-dependent protein kinase-like kinase Pkh1p and downstream signaling pathways Pkc1p and Ypk1p. J Biol Chem 279:22030–22038

  67. Zhao R, Davey M, Hsu YC, Kaplanek P, Tong A, Parsons AB, Krogan N, Cagney G, Mai D, Greenblatt J, Boone C, Emili A, Houry WA (2005) Navigating the chaperone network: an integrative map of physical and genetic interactions mediated by the hsp90 chaperone. Cell 120:715–727

Download references

Acknowledgments

We thank our colleagues and in particular Dr. Jean Luc Parrou for his help with RT-PCR experiments. We are grateful to M. Crouzet (LBMS, Bordeaux, France), J. Heinisch (University of Osnabrück, Germany ), K. Matsumoto (Nagoya University, Japan) and M. Molina (CSIC, Facultad de Farmacia, Madrid) for kind provision of yeast strains and plasmids. We also thank Miss Faubladier for helping us with the sucrose gradient experiment in Fig. 2. This work was supported in part by grants (QLK3-CT2000-01537) from the European Commission Framework Program and from ‘Fonds de Recherche Hoechst Marion Roussel’ (FRHMR2/9922) to JF. F.B. holds a pre-doctoral grant from Syrian Ministry of Research and Education.

Author information

Correspondence to Jean Francois.

Additional information

Communicated by S. Hohmann

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Basmaji, F., Martin-Yken, H., Durand, F. et al. The ‘interactome’ of the Knr4/Smi1, a protein implicated in coordinating cell wall synthesis with bud emergence in Saccharomyces cerevisiae . Mol Genet Genomics 275, 217–230 (2006). https://doi.org/10.1007/s00438-005-0082-8

Download citation

Keywords

  • KNR4 /SMI1
  • Protein interaction
  • TAP–tag
  • Two-hybrid
  • Cell wall
  • Signalization