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MAP kinase dynamics in response to pheromones in budding yeast

Nature Cell Biology volume 3, pages 1051–1059 (2001)Cite this article

Abstract

Although scaffolding is a major regulator of mitogen-activated protein kinase (MAPK) pathways, scaffolding proteins are poorly understood. During yeast mating, MAPK Fus3p is phosphorylated by MAPKK Ste7p, which is activated by MAPKKK Ste11p. This MAPK module interacts with the scaffold molecule Ste5p. Here we show that Ste11p and Ste7p were predominantly cytoplasmic proteins, while Ste5p and Fus3p were found in the nucleus and the cytoplasm. Ste5p, Ste7p and Fus3p also localized to tips of mating projections in pheromone-treated cells. Using fluorescence recovery after photobleaching (FRAP), we demonstrate that Fus3p rapidly shuttles between the nucleus and the cytoplasm independently of pheromones, Fus3p phosphorylation and Ste5p. Membrane-bound Ste5p can specifically recruit Fus3p and Ste7p to the cell cortex. Ste5p remains stably bound at the plasma membrane, unlike activated Fus3p, which dissociates from Ste5p and translocates to the nucleus.

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Figure 1: Subcellular localization of components of the pheromone response pathway.
Figure 2: Nuclear FRAP of Fus3p, Ste5p and the Dig proteins.
Figure 3: Fus3p–GFP shuttles between nucleus and cytoplasm in an α-factor-and phosphorylation-independent manner.
Figure 4: Fus3p–GFP transiently binds to Ste5p at the plasma membrane.
Figure 5: Phosphorylation of Fus3p may trigger its dissociation from Ste5p at the plasma membrane.
Figure 6: A model for the dynamic behaviour of Ste5p and Fus3p in response to pheromones.

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Acknowledgements

We thank P. Pryciak, G. Ammerer, M. Tyers, P. Nurse and E. Leberer for providing plasmids, strains and antibodies, and J. Ellenberger for helpful suggestions about FRAP and FLIP. We are grateful to T. Laroche and M. Allegrini for help with microscopy, N. Perrinjaquet for expert technical assistance, members of the group for stimulating discussion, and R. Iggo and P. Gönczy for critical reading of the manuscript. M.P. is supported by the Swiss National Science Foundation, the Swiss Cancer League and a Helmut Horten Incentive Award.

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  1. Volker M. Stucke

    Present address: Max-Planck Institute for Biochemistry, Department of Cell Biology, Am Klopferspitz 18a, D-82152, Martinsried, Germany

  2. Gerda Jorritsma

    Present address: Hanzehogeschool, Zernikeplein 11, 9747 AS, Groningen, the Netherlands

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  1. Swiss Institute for Experimental Cancer Research (ISREC), Chemin des Boveresses 155, Epalinges/VD, 1066, Switzerland

    Frank van Drogen & Matthias Peter

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Figure S1 Nuclear transport of Fus3p–GFP in wild-type cells as assayed by FRAP does not change within 2 h after addition of α-factor. (PDF 241 kb)

Figure S2 Expression of the GFP-tagged proteins does not interfere significantly with pheromone signalling when expressed in wild-type cells.

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van Drogen, F., Stucke, V., Jorritsma, G. et al. MAP kinase dynamics in response to pheromones in budding yeast. Nat Cell Biol 3, 1051–1059 (2001). https://doi.org/10.1038/ncb1201-1051

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