Analysis of the Mechanisms of Action of the Saccharomyces cerevisiae Dominant Lethal cdc42 G12V and Dominant Negative cdc42 D118A Mutations

Abstract

The Saccharomyces cerevisiae Cdc42p GTPase is localized to the plasma membrane and involved in signal transduction mechanisms controlling cell polarity. The mechanisms of action of the dominant negative cdc42 D118A mutant and the lethal, gain of function cdc42 G12V mutant were examined. Cdc42 D118A,C188S p and its guanine-nucleotide exchange factor Cdc24p displayed a temperature-dependent interaction in the twohybrid system, which correlated with the temperature dependence of the cdc42 D118A phenotype and supported a Cdc24p sequestration model for the mechanism of cdc42 D118A action. Five cdc42 mutations were isolated that led to decreased interactions with Cdc24p. The isolation of one mutation (V44A) correlated with the observations that the T35A effector domain mutation could interfere with Cdc42 D118A,C188S p-Cdc24p interactions and could suppress the cdc42 D118A mutation, suggesting that Cdc24p may interact with Cdc42p through its effector domain. The cdc42 G12V mutant phenotypes were suppressed by the intragenic T35A and K183-187Q mutations and in skm1⌬ and cla4⌬ cells but not ste20⌬ cells, suggesting that the mechanism of cdc42 G12V action is through the Skm1p and Cla4p protein kinases at the plasma membrane. Two intragenic suppressors of cdc42 G12V were also identified that displayed a dominant negative phenotype at 16°C, which was not suppressed by overexpression of Cdc24p, suggesting an alternate mechanism of action for these dominant negative mutations.The establishment of cell polarity is crucial for the control of many cellular and developmental processes, such as the generation of cell shape, the intracellular movement of organelles, and the secretion and deposition of new cell surface constituents (1). Polarized growth in the yeast Saccharomyces cerevisiae occurs in response to both internal and external signals, resulting in different morphological structures (2-5). The mechanics of cell polarity initiation during the mitotic cell cycle can be divided into three sequential phases: (i) nonrandom bud site selection; (ii) organization of proteins at the bud site; and (iii) bud emergence and polarized growth. Genetic and biochemical studies have identified over 25 proteins, including several GTPases and components of the actin cytoskeleton, that are involved in the regulation of the cell polarity pathway in S. cerevisiae (1, 6, 7).At least six members of the Ras superfamily of GTPases (Rsr1p/Bud1p, Cdc42p, Rho1p, Rho2p, Rho3p, and Rho4p) are involved in controlling cell polarity in S. cerevisiae. These proteins are active when in the GTP-bound state and inactive in the GDP-bound state (8, 9). The activity of these GTPases is controlled by regulatory proteins, such as guanine-nucleotide exchange factors, GTPase-activating proteins, and guanine-nucleotide dissociation inhibitors, as well as by the intracellular localization of the GTPase. Rsr1p/Bud1p is a member of the Ras subfamily and is responsible for bud site selection at one of the two cell poles, but it is not required for bud emergence or polari...

The genetics of Pak

“…These data suggest that the kinase domain of Skm1 can potentially phosphorylate at least some of the same substrates as Ste20, but that, in the context of the full-length protein, is unable or unavailable to do so (Martin et al, 1997). Interestingly, the lethal effects of expressing activated Cdc42 in budding yeast can be suppressed by deletion of CLA4 or SKM1, but not by deletion of STE20 (Davis et al, 1998). These results suggest that, like the other two yeast Paks, Skm1 is a bona-fide effector of Cdc42, but its precise role remains for the moment obscure.…”

Control of morphogenesis and actin localization by thePenicillium marneffei RAChomolog

“…The D123A mutation is located in a conserved position that, when mutated in other small GTPases, results in a dominant-negative phenotype, whereas the G18V mutation in small GTPases (including RAC homologs) gives rise to a dominant-activated phenotype because the mutant protein is unable to hydrolyse GTP and constitutively interacts with downstream effectors (Davis et al, 1998;Fanto et al, 2000;Kawaski et al, 1999;Kost et al, 1999). Both mutant alleles, as well as a wild-type allele were placed behind the ethanolinducible alcA promoter from A. nidulans (May, 1987).…”

Saccharomyces cerevisiae Cdc42p GTPase Is Involved in Preventing the Recurrence of Bud Emergence during the Cell Cycle

“…Mutational and biochemical characterization of Cdc42p revealed that regulation of the guanine nucleotide state of Cdc42p is essential for its proper function in these processes (9,40). Moreover, biochemical and two-hybrid studies showed that GTP-bound Cdc42 G12V p displayed enhanced interactions with effectors and regulators (4, 5, 8-10, 15, 20, 29, 30, 33, 37).…”

“…Mutational analysis of the Cdc42p effector domain, which consists of amino acids 26 to 50 ( Fig. 1), indicated that this region is required for function and lends specificity to interactions with a number of Cdc42p regulators and effectors (9,23,33). However, how these various specific interactions lead to downstream events such as actin reorganization and bud emergence still needs to be explored.…”

“…Cdc42 V44A p showed altered interactions with Cla4p, Gic1p, Gic2p, and Cdc24p, which led to defects in the apical-isotropic bud growth switch, localization and structural integrity of the septin ring, and cytokinesis and a delay in nuclear division (9,33). To build on the cdc42 V44A study, four additional effector domain mutations, Y32K, F37A, D38E, and Y40C, were introduced into CDC42 and characterized for interactions with regulators and effectors and for functionality.…”