Abstract
Mitogen Activated Protein Kinases (MAPK) play a central role in transducing extra-cellular signals into defined biological responses. These enzymes, conserved in all eukaryotes, exert their function via the phosphorylation of numerous substrates located throughout the cell and by inducing a complex transcriptional program. The partitioning of their activity between the cytoplasm and the nucleus is thus central to their function. Budding yeast serves as a powerful system to understand the regulation of these fundamental biological phenomena. Under vegetative growth, the MAPK Kss1 is enriched in the nucleus of the cells. Stimulation with mating pheromone results in a rapid relocation of the protein in the cytoplasm. Activity of either Fus3 or Kss1 in the mating pathway is sufficient to drive this change in location by disassembling the complex formed between Kss1, Ste12 and Dig1. Artificial enrichment of the MAPK Kss1 in the nucleus in presence of mating pheromone alters the transcriptional response of the cells and induces a cell-cycle arrest in absence of Fus3 and Far1.
Highlights
The stimulus, demonstrate the relocation of Kss[1] out of the nucleus
Nc is the number of cells in the sample. (d) Comparison of the dynamics of Kss[1] nuclear export and of MAPK activity in cells bearing a Kss1-YFP tag and a Ste7DS-SKARSR (Nc = 806). (e) Correlation of Kss[1] nuclear exit and MAPK activity in single cells 5 minutes after the stimulus
Using an automated image analysis platform, the average nuclear and cytoplasmic fluorescences of hundreds of cells were quantified in time-lapse movies[11]
Summary
The stimulus, demonstrate the relocation of Kss[1] out of the nucleus. In all microscopy images the scale bar represents 5 μm (c). At the single-cell level, the nuclear exit of Kss[1] and the activity of the MAPK five minutes after stimulus are well correlated (Fig. 1e). To test the reversibility of this nuclear depletion, cells combining the Fus3-as and the Kss1-as alleles were stimulated with pheromone and MAPK activity was blocked subsequently with NAPP1.
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