Article 13

Choi, K-Y., B. Satterberg, D.M. Lyons, & E.A. Ellion (1994) Ste5 tethers multiple protein kinases in the MAP kinase cascade required for mating in S. cerevisieae. Cell 78: 499-512.

Epistasis analysis, described in Articles 6 and 7 and elsewhere, places Ste5p upstream of the Stell, Ste7, and Fus3 or Kssl kinases in the mating-type phero-mone response pathway. The results were consistent with a linear pathway as shown below.

Ste5 protein-Stell kinase-Ste7 kinase-Fus3/Kissl kinase

Nevertheless, evidence was accumulating that this simple pathway was not the full story. Kranz et al. (1994) found that overexpression of Ste5p suppressed point mutations (single residue alterations) in Fus3 kinase and did so in an allele-specific manner. Such a result strongly indicates that Ste5p and Fus3p directly physically interact and is not consistent with the proposed linear pathway that places Ste5p three steps upstream of Fus3 kinase. Kranz et al. (1994) also demonstrated that Ste5p and Fus3p associate with each other even in the absence of a pheromone and even if a catalytically inactive Fus3p mutant is used.

In view of the large size of Ste5 protein and the absence of any recognizable sequence motifs (other than homology to Farlp, another large protein of unknown multiple functions), the authors of this article propose to test the possibility that Ste5p serves as a 'scaffold protein', that is a protein to which other proteins attach in order to come into physical proximity with one another and thereby increase the efficiency of their functional interactions. This article also explores the relationship between Ste20 kinase and Ste5p. Epistasis analysis of Ste20 kinase places it downstream of Ste4p but its relationship to Ste5p remains unclear. Two-hybrid analysis and coimmunoprecipitation are used as complementary approaches to analyze the relationships among these proteins of a MAP kinase signaling cascade.

1. This article uses a 'lex A-based' two-hybrid system. Define the term 'lex A-based'. Include the following in your answer.

(a) A diagram of the reporter gene.

(b) A diagram of the structure of the lexA bait construction. Indicate the region encoding the DNA-binding domain, the insertion site for the sequence encoding the bait protein, and the structure of the fusion protein product.

(d) A diagram of the structure of the B42 prey construction. Indicate the insertion site for the sequence encoding the prey protein, and the structure of the fusion protein product.

(e) What is bicoid and what role does it play in this analysis?

2. The results in Table 1 are central to the hypothesis of the authors. That is, Ste5 protein is a scaffold protein capable of interacting with all three of the MAP kinases of the mating-type pheromone response signaling pathway. What evidence in Table 1 supports the following conclusions? Be sure to give the results of the control along with the results of the experiment.

(a) Ste5p interacts with Stel lp.

(b) The N-terminal domain of Stellp is required for the interaction with Ste5p.

(c) The C-terminal domain of Ste7p is required for the interaction with Ste5p.

(d) The interaction of Ste5p with Stellp, Ste7p, or Fus3p is not dependent on the genomic copies of FUS3, STE11, or STE7.

(e) Stel lp interacts with Fus3p and the interaction is not dependent on Ste5p or Ste7p.

3. An interaction between Stel lp and Ste7p is suggested in Table 1 but does not hold up under detailed analysis.

(a) Which initial result suggests an interaction between Stellp and Ste7p?

(b) Which result indicates that this interaction between Stellp and Ste7p is indirect and dependent on the genomic copy of STE5 and is mediated by Ste5p?

(c) Draw a diagram of this interaction.

4. The authors conclude that Ste20p does not interact with Ste5p.

(a) List the data for both the experiment and the control that support this conclusion.

(b) Why do you think that the authors do not consider the 33 units or 61 units of activity seen with the Ste5 and Stell constructs, respectively, to be significant?

(c) The authors state, '. . . LexA-Ste20 . . . functions to repress transcription of a GALl-LexAop-LacZ gene with a LexA operator between the GALl UAS and transcriptional initiation site'. Why is this an important control for this experiment?

5. Describe the results in Figure 1 that demonstrate that Stellp, Ste7p, and Fus3p interact with distinct regions of Ste5p.

6. Describe the results in Figure 1 that demonstrate that Ksslp and Fus3p interact with the same or an overlapping region of Ste5p.

7. Describe the experiment that indicates that binding of Fus3p to Ste5p is essential for the activation of Fus3 kinase.

8. Discuss the functional significance of the finding that Ste5p binds to the N-terminal domain of Stell kinase.

REFERENCE

Kranz, J.A., B. Satterberg, & E.A. Elion (1994) The MAP kinase Fus3 associates with and phosphorylates the upstream signaling component Ste5. Genes Dev. 8: 313-327.

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