Whiteway, M., K.L. Clark, E. Leberer, D. Dignard, & D.Y. Thomas (1994) Genetic identification of residues involved in association of a and /3 G-protein subunits. Mol. Cell. Biol. 14: 3223-3229.
1. Discuss the model of the ste4 haploid-lethal mutant selection depicted in Figure 1A. List the following.
(a) The genotype of the strain used for the selection. (Include the plasmid genes.)
(b) The growth conditions used for the identification of the clones carrying haploid-lethal mutations.
(c) The STE4 mutagenesis method.
2. Discuss the significance of obtaining mutations only in the region of Ste4p between residues 126 and 150.
Describe the method used to isolate suppressors of the ste4 haploid-lethal mutations. Be specific about the ste4 mutant allele used for this selection.
4. List the results that indicate that GPA1-E307K is an allele-specific suppressor of the STE4 haploid-lethal mutations. Draw a model of the interaction of Gpalp and Ste4p based on these results. Indicate the location (within the region of interaction or not) of the various mutant alterations in Gpalp and Ste4p identified in this study.
5. The results in Figure 2 and Table 1 indicate that the GPA1 mutation His 4.3 is a silent mutation. That is, it exhibits wild-type-like activity.
(a) Discuss the results that support this conclusion.
(b) What results would you have expected if the GPA1 mutation His 4.3 had interfered with the activity of Gpalp?
6. Table 2 uses two-hybrid analysis to explore the direct interaction of the GPA1 His 4.3 mutant protein with wild-type Ste4p protein and with the Hpl 21.3 mutant protein.
(a) Which results indicate that the interaction between the Gpal His 4.3 mutant protein and Ste4p or Ste4 Hpl 21.3 mutant protein is comparable? How is this consistent with the phenotype analysis reported in Figure 2 and Table 1?
(b) Which results indicate that the Ste4 Hpl 21.3 mutant protein interferes with the interaction with Gpal protein?
(c) Which results indicate that the GPA1 mutation His 4.3 re-establishes the interaction with Ste4 Hpl 21.3 mutant protein?
(d) Which results indicate that the interaction between these mutant proteins is allele specific?
7. Discuss other Gpal mutations in the region of residue 307 that strengthen the hypothesis that this portion of the Ga subunit is involved in the interaction with G(3-y and may play an important role in the selectivity of the interaction of different Go subunits with their specific Gf3y targets.
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