Article 16

Butty, A-C., P..M. Pryciak, L.S. Huang, I. Herskowitz, & M. Peter (1998) The role of Farlp in linking the heterotrimeric G protein to polarity establishment proteins during yeast mating. Science 282: 1511-1516.

In addition to identifying farl-s mutants, Chenevert et al. (1994) isolated the mutant alleles of several other genes involved in polarized morphogenesis during mating (schmoo formation) as well as in vegetative cell division. These included alterations in CDC24, a GDP-GTP exchange factor for Cdc42p, referred to as cdc24-m mutants because they affect mating at permissive temperatures in addition to having a defect in cell division at higher temperatures. Cdc42p is a small GTPase in the same family of proteins as the mammalian Rholp and is involved in organizing the actin cytoskeleton (reviewed in Johnson, 1999; Pruyne & Bretscher, 2000; Takai et al., 2001).

Chenevert et al. (1994) also identified schmooless alleles of BEM1, a gene previously shown to be involved in cell polarization during vegetative budding (Chant et al., 1991; Chenervert et al., 1992). Reports that Beml protein binds to actin and interacts with Cdc42p, Cdc24p, Farlp, Ste5p, and Ste4p suggested the possibility that these proteins form a large complex that is essential for directing the recruitment of the actin cytockeleton to the region of the cell surface exposed to the highest concentration of pheromone (Peterson et al., 1994; Leeuw et al., 1995; Lyons et al., 1996; Park et al., 1997; reviewed in Pruyne & Bretscher, 2000). The authors of Article 16 used two-hybrid analysis to demonstrate this proposed complex and to explore specific interactions among the components of the complex.

1. Diagram the FAR1 'bait' construction, the BEM1 'prey' construction, and the reporter gene used to demonstrate an interaction between Farlp and Bemlp.

2. What experimental data supports the statement, 'Farlp preferentially bound to Cdc42p in its active GTP-bound state, . . .'?

3. What experimental data are presented to support the specificity of the Farlp-Cdc42p interaction?

4. What experimental data indicate that the Farlp interaction with Cdc42p is not direct but is mediated via Bemlp?

5. What pairs of constructs would you test to explore the interactions of Ste20p with the components of this complex? Be sure to examine the possibility that the interactions you detect are not direct and propose how you might test this.

6. Comment on the following. Interactions between two heterologous (nonyeast) proteins detected using the yeast two-hybrid system are most probably direct.

7. Figure 2 shows the two-hybrid results obtained with the farl-s mutant alleles B4 and H7. Discuss how these results are consistent with the phenotype exhibited by strains containing these mutations.

8. The authors cite unpublished results describing STE4 mutant alleles that cause defects in mating but not pheromone signaling. Based on the results described in this article, propose a possible mechanism for this class of ste4 mutation. Outline a series of experiments including two-hybrid analysis and at least one other genetic approach that you might use to investigate this novel class of STE4 alleles to support your hypothesis.

REFERENCES

Chant, J., K. Corrado, J.R. Pringle, & I. Herskowitz (1991) Yeast BUD5, encoding a putative GDP-GTP exhange factor, is necessary for bud site selection and interacts with bud formation gene BEM1. Cell 65: 1213-1224.

Chenevert, J., K. Corrado, A. Bender, J. Pringle, & I. Herskowitz (1992) A yeast gene (BEM1) required for cell polarization whose product contains two SH# domains. Nature 356: 77-79.

Chenevert, J., N. Valtz, & I. Herskowitz (1994) Identification of genes required for normal pheromone-induced cell polarization in Saccharomyces cerevisiae. Genetics 136: 1287-1297.

Elion, E.A. (2000) Pheromone response, mating and cell biology. Curr. Opin. Microbiol. 3: 573-581.

Johnson, D.I. (1999) Cdc42: An essential Rho-type GTPase controlling eukaryotic cell polarity. Microbiol. Mol. Biol. Rev. 63: 54-105.

Leeuw, T., A. Fourest-Lieuvin, C. Wu, J. Chenevert, K. Clark, M. Whiteway, D.Y. Thomas, & E. Leberer (1995) Pheromone response in yeast: association of Bemlp with proteins of the MAP kinase cascade and actin. Science 270: 1210-1203.

Lyons, D.M., S.K. Mahanty, K.Y. Choi, M. Manandhar, & E.A. Elion (1996) The SH3-domain protein Bern 1 coordinates mitogen-activated protein kinase cascade activation with cell cycle control in Saccharomyces cerevisiae. Mol. Cell. Biol. 16: 4095-4106.

Park, H.O., E. Bi, J.R. Pringle, & I. Herskowitz (1997) Two active states of the Ras-related Budl/Rsrl protein bind to different effectors to determine yeast cell polarity. Proc. Natl Acad. Sci. USA 94: 4463-4468.

Peterson, J., Y. Zheng, L. Bender, A. Myers, R. Cerione, & A. Bender (1994) Interactions between the Bud emergence proteins Bemlp and Bem2p and Rho-type GTPase in yeast. J. Cell Biol. 127: 1395-1406.

Pruyne, D. & A. Bretscher (2000) Polarization of cell growth in yeast. I. Establishment and maintenance of polarized states. J. Cell Sci. 113: 365-375.

Takai, Y„ T. Sasaki, & T. Matozaki (2001) Small GTP-binding proteins. Physiol. Rev. 81: 515-524.

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