Chapter 16. The MAP Kinase Pathway in Yeast Mating

Introduction

Analyze the Data
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Analyze the Data 16-2: The MAP Kinase Pathway in Yeast Mating

Scaffold proteins can segregate different MAP kinase signaling pathways that share common components. In the yeast mating pathway, the MEK Ste7 phosphorylates and activates the Fus3 MAP kinase, whereas Ste7 phosphorylates and activates the Kss1 MAP kinase in the starvation pathway. The mating pathway is activated by mating factor receptor activation of a G protein; Gβγ recruits the Ste5 scaffold protein and the Ste11, Ste7, and Fus3 components of the kinase cascade. Mutation of the Ste5 binding sites for Ste11 and Ste7 disrupts the mating response, clearly demonstrating the importance of Ste5 for tethering the kinases together. Mutation of the Fus3 binding site on Ste5 gives a more complicated response, suggesting that Ste5-Fus3 interaction may involve more than just tethering. This possibility was investigated with yeast proteins expressed as recombinant proteins in bacteria or insect cells and then purified. See Good et al., 2009, Cell 136:1085–1097.

Question

a. A fluorescence quenching assay was used to measure the activity of the Fus3 and Kss1 MAP kinases using a substrate peptide that can be phosphorylated by both kinases. Phosphorylated peptide binds to gallium coupled to fluorescent beads and quenches the fluorescence. The rate of fluorescence quenching (loss of fluorescence) corresponds to the Fus3 or Kss1 kinase activity. Results of Ste7 phosphorylation, and thereby activation of Kss1 and Fus3, in the presence and absence of Ste5 are shown below:

1. Fus3 or Kss1 alone (control)
2. Fus3 or Kss1 + Ste7
3. Fus3 or Kss1 + Ste7 + Ste5

PKq+gRT5EpClY03hO0vm6NjyTNjNuGh2iG0PY8nhgBpn9LkEIVlhgYwjqgABw4g8zHMpSmVFwCY4+DXweBxV218116gpQwnYRLQxP1d0yb+frPXIue0LKGkebptIaSSGJ3M8LkAV55MMyfNBkFHE7q5K0yEmXcVhOtWiHKMP9MCJeUSKE2aQppWzXJw/8PMTJZeQU03Al1yPpG08qLdbr4IwXZ3AYtIa6aWrQncq4F7NVInx3YRVIpqBXGUjS8QKxRg7SlvMFKf2qOLov2oDI/eZPafbkDh3750hg1ctEjs=
Curve 2 in the Kss1 activation assay indicates that Ste7 is active even in the absence of Ste5. The similarity in the Fus3 curve 3 and the Kss1 curves 2 and 3 indicate that Ste7 can phosphorylate Fus3 and Kss1 approximately equivalently. Comparison of curves 2 and 3 for Fus3 and Kss1 indicates that Ste5 is required for Ste7 to activate Fus3 but is not required for Ste7 to activate Kss1. This supports the possibility that Ste5 plays more than a tethering role in the Fus3 pathway.

Question

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As shown above, Ste7 phosphorylates Kss1 even in the absence of Ste5, so Kss1 is a fully active substrate. In contrast, Ste7 phosphorylates Fus3 poorly unless Ste5 is present, indicating that Fus3 alone is a poor substrate for Ste7 but its phosphorylation can be enhanced by Ste5. Mutation or replacement of the Fus3 MAPK insertion loop activates Fus3 as a substrate even in the absence of Ste5. The most logical conclusion is that in addition to tethering Fus3 and Ste7 together, interaction with Ste5 suppresses MAPK insertion loop inhibition of Fus3 phosphorylation. The inability of Fus3 to be phosphorylated by Ste7, except when it is scaffolded together by Ste5, would contribute to the segregation of the mating and starvation pathways.

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