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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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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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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DOI: https://doi.org/10.1038/ncb1201-1051
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