Identification of novel membrane-binding domains in multiple yeast Cdc42 effectors

Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Molecular Biology of the Cell (Impact Factor: 4.47). 01/2008; 18(12):4945-56. DOI: 10.1091/mbc.E07-07-0676
Source: PubMed


The Rho-type GTPase Cdc42 is a central regulator of eukaryotic cell polarity and signal transduction. In budding yeast, Cdc42 regulates polarity and mitogen-activated protein (MAP) kinase signaling in part through the PAK-family kinase Ste20. Activation of Ste20 requires a Cdc42/Rac interactive binding (CRIB) domain, which mediates its recruitment to membrane-associated Cdc42. Here, we identify a separate domain in Ste20 that interacts directly with membrane phospholipids and is critical for its function. This short region, termed the basic-rich (BR) domain, can target green fluorescent protein to the plasma membrane in vivo and binds PIP(2)-containing liposomes in vitro. Mutation of basic or hydrophobic residues in the BR domain abolishes polarized localization of Ste20 and its function in both MAP kinase-dependent and independent pathways. Thus, Cdc42 binding is required but is insufficient; instead, direct membrane binding by Ste20 is also required. Nevertheless, phospholipid specificity is not essential in vivo, because the BR domain can be replaced with several heterologous lipid-binding domains of varying lipid preferences. We also identify functionally important BR domains in two other yeast Cdc42 effectors, Gic1 and Gic2, suggesting that cooperation between protein-protein and protein-membrane interactions is a prevalent mechanism during Cdc42-regulated signaling and perhaps for other dynamic localization events at the cell cortex.

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    • "We took the study of this new regulatory region one step further by showing that ND2 interacts with the Cdc42p effector Gic2p (path 4 in Figure 8; Brown et al., 1997; Chen et al., 1997). Gic2p and its homologue, Gic1p, contain a CRIB domain (Burbelo et al., 1995) and a basic-rich domain that binds membranes (Takahashi and Pryciak, 2007); their closest mammalian homologues are the Binder of Rho GTPases (BORG) proteins (Joberty et al., 1999). Gic1p and Gic2p have been implicated in polarization of both the actin and septin cytoskeletons (Brown et al., 1997; Chen et al., 1997; Bi et al., 2000; Jaquenoud and Peter, 2000; Iwase et al., 2006). "
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    ABSTRACT: Actin filaments are dynamically reorganized to accommodate ever-changing cellular needs for intracellular transport, morphogenesis, and migration. Formins, a major family of actin nucleators, are believed to function as direct effectors of Rho GTPases, such as the polarity regulator Cdc42p. However, the presence of extensive redundancy has made it difficult to assess the in vivo significance of the low-affinity Rho GTPase-formin interaction and specifically whether Cdc42p polarizes the actin cytoskeleton via direct formin binding. Here we exploit a synthetically rewired budding yeast strain to eliminate the redundancy, making regulation of the formin Bni1p by Cdc42p essential for viability. Surprisingly, we find that direct Cdc42p-Bni1p interaction is dispensable for Bni1p regulation. Alternative paths linking Cdc42p and Bni1p via "polarisome" components Spa2p and Bud6p are also collectively dispensable. We identify a novel regulatory input to Bni1p acting through the Cdc42p effector, Gic2p. This pathway is sufficient to localize Bni1p to the sites of Cdc42p action and promotes a polarized actin organization in both rewired and wild-type contexts. We suggest that an indirect mechanism linking Rho GTPases and formins via Rho effectors may provide finer spatiotemporal control for the formin-nucleated actin cytoskeleton.
    Molecular biology of the cell 08/2012; 23(19):3814-26. DOI:10.1091/mbc.E12-05-0400 · 4.47 Impact Factor
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    • "Regulates actin and septin organizations, polarity establishment, exocytosis, cell wall remodeling and many other processes Audhya and Emr 2002; Wild et al. 2004; He et al. 2007; Takahashi and Pryciak 2007; Orlando et al. 2008; Zhang et al. 2008; Bertin et al. 2010; Garrenton et al. 2010 Cdc42-GTP at the cortical site, in turn, recruits more Cdc24- Bem1-Cla4 complexes and the positive cycle continues. The second positive feedback loop for Cdc42 polarization involves an actomyosin-based transport system (Figure 3B, bottom) (Wedlich-Soldner et al. 2003). "
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    ABSTRACT: Asymmetric cell division, which includes cell polarization and cytokinesis, is essential for generating cell diversity during development. The budding yeast Saccharomyces cerevisiae reproduces by asymmetric cell division, and has thus served as an attractive model for unraveling the general principles of eukaryotic cell polarization and cytokinesis. Polarity development requires G-protein signaling, cytoskeletal polarization, and exocytosis, whereas cytokinesis requires concerted actions of a contractile actomyosin ring and targeted membrane deposition. In this chapter, we discuss the mechanics and spatial control of polarity development and cytokinesis, emphasizing the key concepts, mechanisms, and emerging questions in the field.
    Genetics 06/2012; 191(2):347-87. DOI:10.1534/genetics.111.132886 · 5.96 Impact Factor
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    • "Putative osmosensors Hkr1 and Msb2 that lie upstream of the Sho1 branch are postulated to directly sense the extracellular osmolarity [12]. Cdc42 interacts with and activates membrane associated Ste20 or Cla4 [13]. In addition, Cdc42 is able to bind the Ste11-Ste50-Opy2 complex (targeted to the membrane by Opy2) to bring activated Ste20 or Cla4 to their substrate Ste11 [14]. "
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    ABSTRACT: Saccharomyces cerevisiae senses hyperosmotic conditions via the HOG signaling network that activates the stress-activated protein kinase, Hog1, and modulates metabolic fluxes and gene expression to generate appropriate adaptive responses. The integral control mechanism by which Hog1 modulates glycerol production remains uncharacterized. An additional Hog1-independent mechanism retains intracellular glycerol for adaptation. Candida albicans also adapts to hyperosmolarity via a HOG signaling network. However, it remains unknown whether Hog1 exerts integral or proportional control over glycerol production in C. albicans. We combined modeling and experimental approaches to study osmotic stress responses in S. cerevisiae and C. albicans. We propose a simple ordinary differential equation (ODE) model that highlights the integral control that Hog1 exerts over glycerol biosynthesis in these species. If integral control arises from a separation of time scales (i.e. rapid HOG activation of glycerol production capacity which decays slowly under hyperosmotic conditions), then the model predicts that glycerol production rates elevate upon adaptation to a first stress and this makes the cell adapts faster to a second hyperosmotic stress. It appears as if the cell is able to remember the stress history that is longer than the timescale of signal transduction. This is termed the long-term stress memory. Our experimental data verify this. Like S. cerevisiae, C. albicans mimimizes glycerol efflux during adaptation to hyperosmolarity. Also, transient activation of intermediate kinases in the HOG pathway results in a short-term memory in the signaling pathway. This determines the amplitude of Hog1 phosphorylation under a periodic sequence of stress and non-stressed intervals. Our model suggests that the long-term memory also affects the way a cell responds to periodic stress conditions. Hence, during osmohomeostasis, short-term memory is dependent upon long-term memory. This is relevant in the context of fungal responses to dynamic and changing environments. Our experiments and modeling have provided an example of identifying integral control that arises from time-scale separation in different processes, which is an important functional module in various contexts.
    BMC Research Notes 05/2012; 5(1):258. DOI:10.1186/1756-0500-5-258
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