Small GTPases and the major events in the early stage of budding in S. cerevisiae. Two major structures of actin filaments, actin cables and actin patches, are indicated with red lines and red dots, respectively, to highlight the polarized organization of the actin cyto-skeleton.

Model for the establishment and maintenance of Cdc42 polarization in the absence of a spatial cue. (Top panel) It is thought that activated Cdc42 clusters spontaneously and transiently and becomes stabilized by interactions among its effectors and/or regulators. To enhance and/or maintain the initial Cdc42 polarization at a growth site, at least two positive feedback mechanisms are operating: an actomyosin-based delivery of Cdc42 and a Bem1-based activation of Cdc42. These positive actions are counteracted by endocytosis-mediated dispersal and GDI-mediated extraction of Cdc42 away from the plasma membrane. (Bottom panel) A molecular model for Cdc42 polarization at a growth site. Cdc42 in its GDP-bound state is carried on secretory vesicles, which are transported along actin cables to the growth site by the type V myosin Myo2. The “Cdc42-GDP” cargo is captured by Msb3 and Msb4 or other factors capable of binding to Cdc42-GDP, resulting in the local accumulation of Cdc42-GDP, poised for activation by the GEF Cdc24. The Bem1-based complex functions to bring Cdc42, Cdc24, and the PAK Cla4 together, resulting in the phosphorylation of Cdc24 by Cla4 and in the further accumulation of Cdc42-GTP at the growth site. How endocytosis mediates the dispersal of Cdc42 from the polarization site remains unknown.

Model for the molecular pathway governing axial and bipolar budding in haploid a or α cells and in diploid a/α cells. Although physical interactions have been demonstrated for some proteins, many of them are postulated based on genetic and localization data. The figure also does not imply that all the proteins shown necessarily interact at the same time (see the text for details and Fig. 11 for Cdc42 effectors and their downstream components). (Modified from the supporting online material from reference 273 with permission of the publisher.)

Models for cell type control of budding patterns. (A) Axl1 may act positively on the axial landmark proteins, and bipolar budding may occur as a default in the absence of Axl1 in a/α cells. (B) Axl1 may act indirectly by blocking the function of Rax1 and Rax2, which play a positive role in promoting bipolar budding in the absence of Axl1 in a/α cells. For simplicity, only Axl2, which interacts with Bud5, is shown as an axial landmark (see the text for details).

Role of Cdc42 in polarization of actin cables and exocytosis. Cdc42 regulates the polarization of actin cables by directly interacting with the formin Bni1 and regulating its localization and/or activation. Cdc42 controls polarized secretion mainly via its role in actin cable organization, but it also interacts directly with the exocyst subunit Sec3, endowing the exocyst with one polarization signal. Rho1 and Rho3 also interact with Sec3 and Exo70, respectively, providing the exocyst additional polarization signals. Sec4 (brown) and its GEF, Sec2 (light blue), associate with the secretory vesicles to guarantee Sec4 in its GTP-bound form, which is required for vesicle transport from the mother cell to the bud and also for the interaction with the vesicle-associated partial exocyst through a Sec4-Sec15 interaction (all exocyst subunits are shown in light gray). Upon vesicle arrival at the bud tip, the exocyst components on the vesicle interact with Sec3 and the fraction of Exo70 already localized at the bud tip, resulting in the assembly of a complete exocyst, which is required for vesicle tethering. Thus, an exocyst is formed dynamically for each round of vesicle transport. After fulfilling its function at the plasma membrane, Sec4-GTP is hydrolyzed with the help of its GAPs Msb3 and Msb4, and Sec4-GDP returns to the cytosol. By segregating its GEF and GAPs at distinct locations, the Sec4 GTPase cycle is employed to carry out its exocytic functions efficiently. Msb3, Msb4, and Bni1 are brought to close proximity by interacting with distinct domains of Spa2. SHD, Spa2 homology domain.

Role of Cdc42 in mitotic exit. (A) The MEN in S. cerevisiae. The activation of Tem1 leads to the activation of a kinase cascade, resulting in the release of the phosphatase Cdc14 from the nucleolus to the nucleus, which regulates mitotic exit and cytokinesis independently. The regulators for Tem1 GTPase cycling and the localization of the key components of the MEN are indicated. (B) In normal anaphase cells, Tem1 and its GAP localize to the daughter-bound SPB (old SPB) (SPB is indicated by a solid black circle). This localization pattern ensures that Tem1 is activated only after the spindle penetrates into the bud, where Lte1 (+), the putative GEF for Tem1, is localized at the cortex. The septins and the mother cell cortex-localized inhibitor Kin4 (−) provide additional layers of regulation to further restrict the activation of Tem1 in the bud. (C) Cdc42 regulates the MEN by multiple means through its role in septin ring assembly (see the section on Cdc42 and septin organization) and its effectors Cla4, Gic1, and Gic2 to affect the localization of Lte1, through Ste20 to activate the MEN in parallel to Lte1 by an undefined mechanism, and through blocking the binding of the GAP Bub2 to Tem1 (see the text for further details).

Signaling pathways leading to pseudohyphal growth. Nitrogen starvation is sensed by Ras2, which controls the expression of the cell surface flocculin Flo11 via two distinct pathways, the cAMP-dependent PKA pathway (in blue) (AC, adenylyl cyclase) and the MAPK pathway (in green). PKA activates the transcriptional activator Flo8 and inactivates the transcriptional repressor Sfl1, both of which contribute to the increased expression of Flo11. Ras2 somehow activates Cdc42, which activates the MAPK cascade via its effector, Ste20. The MAP kinase Kss1 inactivates the repressors Dig1 and Dig2, thereby allowing Ste12 and Tec1 to activate the expression of Flo11 as well as of other proteins involved in cell elongation. As in budding and mating, Cdc42 regulates polarized actin organization via its effectors (in red), thereby contributing to the elongated cell morphology. In response to the nitrogen starvation signal, Sok2 negatively regulates the expression of the transcription factors Phd1 and Swi5, which activate Flo11 expression directly or via the daughter cell-specific transcription factor Ash1. The two transcription factors Swi5 and Ace2 activate the expression of the endoglucanase Egt2 and the endochitinase Cts1. Decreased expression of these key enzymes that are required for cell separation after cytokinesis causes cell attachment during filamentous growth.

Model for roles of Rho3 in exocytosis and actin organization. Activated Rho3 interacts with Myo2 and Exo70 to effect vesicle transport and vesicle tethering during exocytosis. Activated Rho3 and Rho4 (not depicted here) may activate the formins directly via their binding to the RBD of the formins (dashed line with a question mark). No physical interaction between these Rho proteins and the formins, except for the Rho4-Bnr1 interaction, has been reported. Alternatively, Rho3 and Rho4 could activate the formins indirectly and also affect actin patch polarization via their role in exocytosis (see the section on Cdc42 polarization). The specific GEFs and the GAPs, except Rgd1, of Rho3 and Rho4 are not known.

Polarized growth in the life cycle of the budding yeast S. cerevisiae. Polarized growth occurs during budding, mating, and nutritional starvation. Nutritional starvation triggers filamentous growth, which can be divided into invasive growth (exhibited by haploid cells) and pseudohyphal growth (exhibited by diploid cells). Numbers in panel C indicate the order of each budding event. The direction of growth is indicated by red arrows.

Regulation of Cdc42 in S. cerevisiae. (A) Cdc42 activity is regulated by at least three types of regulators: the GEF Cdc24; the GAPs Bem3, Rga1, Rga2, and Bem2; and the GDI Rdi1. The GEF and perhaps the GAPs are likely to mediate the regulation of Cdc42 by most internal and external signals. Once activated, Cdc42 regulates the organization of the actin cytoskeleton and the septins and interacts with components of the exocytic machinery. The polarized actin cytoskeleton guides exocytosis, leading to polarized cell growth. (B) Structure motifs of S. cerevisiae Cdc42 and their potential binding partners based on studies of Cdc42 from yeast to mammals. The switch I region (which is also known as the Ras-like effector region), the switch II region, the Rho insert domain (which is present only in Rho-type GTPases), the polybasic (PB) region (¹⁸³KKSKK¹⁸⁷), the CAAX box (¹⁸⁸CAIL¹⁹¹), and their binding partners are indicated. The cysteine residue in the CAAX box is modified by prenylation (wavy line), and the last three amino acids are cleaved off, followed by carboxymethylation (see the text for details).

Cell type-specific budding patterns in S. cerevisiae. (A) Axial and bipolar patterns of cell division as observed in cells growing on a solid surface. The axes of cell polarity are indicated with red arrows. (B) Patterns of bud scars on the yeast cell surface resulting from the two modes of budding. On each cell, a single birth scar marks the pole at which the cell was attached to its mother. A bud scar shown as a blue ring marks a division site on the mother cell surface. Bud scars can be visualized by staining with calcofluor dye or by scanning electron microscopy. In the axial pattern, scars form a continuous chain as shown in the two cells on the left. In the bipolar pattern, scars cluster around the poles: the birth pole (proximal pole) and the pole opposite the birth end (distal pole). (Modified from reference 85 with permission. © 1999 by Annual Reviews.)

Model of how the Rsr1/Bud1 GTPase cycle directs polarity establishment to a specific site. In step 1, Bud5 exchanges GDP for GTP on Rsr1. In step 2, Rsr1-GTP associates with Cdc24 and Cdc42 and guides them to the bud site. In step 3, Bud2 activates Rsr1 to hydrolyze the GTP bound to Rsr1. In step 4, Dissociation of GDP-bound Rsr1 from Cdc24 may activate Cdc24, which catalyzes the exchange of GDP for GTP on Cdc42. Cdc42-GTP then triggers actin assembly and exocyst localization to establish an axis of polarity. Bud5 at the bud site may convert Rsr1 to a GTP-bound state (dashed line), allowing for another cycle of signal transduction (see the text for details). (Adapted from reference 292 with permission of the publisher.)

Localization of Cdc42 and Cdc42-controlled actin and septin organization. (A) Cdc42 localization (green) and the directionality of cell growth (blue arrows) in the cell cycle are indicated. Components of the exocytic machinery such as the exocyst display a similar localization pattern as Cdc42. A cell cycle-triggered switch from apical growth to isotropic growth occurs around the G₂/M transition. (B) Actin organization in the cell cycle. Actin patches (red spots), actin cables (red lines), and the actin ring (red circle) are indicated. The short, branched actin filaments in the patches are nucleated by the Arp2/3 complex and its activators, whereas the linear actin filaments in the cables and the rings are nucleated by the formins Bni1 (brown) and Bnr1 (yellow), whose localization patterns are also indicated. Presumably, Bni1 exists in the bud cortex during the isotropic growth phase at G₂/M phase, but its concentration is too low to be detected by fluorescence microscopy. Thus, Bni1 is drawn as a dashed line around the bud cortex at the G₂/M phase. It is not clear whether Bnr1 still localizes at the bud neck after the actomyosin ring contraction and is thus indicated as a dashed line in telophase cells. (C) Septin organization in the cell cycle. Septin collar or hourglass (purple) formation involves at least three steps: septin recruitment, which requires Cdc42-GTP, its effectors Gic1 and Gic2, and possibly the polarisome; septin ring assembly, which requires Cdc42 cycling between the GDP- and GTP-bound states, its GAPs, and possibly its effector Cla4; and maturation of the septin ring into a collar, which requires Cla4 and GTP binding of the septins.

Model for the role of Cdc42 in the polarized organization of the actin cytoskeleton and septins. Cdc42 controls the polarized organization of the actin cables by directly interacting and possibly activating the formin Bni1 and also by indirectly affecting the bud neck localization of the other formin Bnr1 via its role in septin recruitment and/or ring/collar assembly. Cdc42 controls polarized septin organization in late G₁ via its effectors Gic1/Gic2, Bni1, and Cla4. Ste20, another effector of Cdc42, could participate in both the actin and the septin organization by phosphorylating Bni1. Actin cables may mediate the role of Bni1 and perhaps the polarisome in septin organization by guiding the delivery of a putative factor, “X,” which may be directly involved in septin recruitment as indicated by the question mark. Cdc42 effectors could also be involved in enhancing Cdc42 polarization at growth sites by interacting with each other. For simplicity, this type of potential positive feedback mechanism is not depicted here (see the text for a detailed description of the model).

Role of Cdc42 in cell polarization during mating. (A) In response to a gradient of mating pheromones secreted by cells of the opposite mating type, haploid a or α cells polarize to form the mating projection (stage 1). Cdc42 and other polarity proteins localize to the tip of the mating projection. Actin cables and patches are also polarized towards the tip. Septins, which may be involved in defining membrane domains, localize as patches at the base of the mating projection. The signaling events at the shmoo tip, indicated by the black box in the a cell at stage 1, are illustrated in panel B. During cell fusion (stage 2), the membrane protein Fus1 localizes to the cell-cell contact zone. Together with Cdc42, Bni1, and the polarisome component Spa2, Fus1 is involved in the localized delivery of secretory vesicles (black circles) to the cell-cell contact zone, leading to the eventual cell fusion. (B) Upon pheromone binding, the receptor Ste2 clusters and activates the heterotrimeric G protein, which anchors to the plasma membrane via the lipid-modified tails (indicated by the wavy line) of its α (Gpa1) and γ (Ste18) subunits. The GTP-bound α subunit dissociates from the β (Ste4)-γ complex. The βγ complex then triggers the activation of the MAPK cascade, resulting in transcriptional activation and G₁ arrest. The activation of Gβγ also triggers polarized growth towards the pheromone gradient. The blue lines indicate protein-protein interactions (see the text for details). Rho1 has been shown to interact with the β subunit and is thus recruited to the shmoo tip. See Fig. 15 for the Rho1 pathway. For simplicity, extensive cross talks between the MAPK cascade and actin organization are not indicated here.

Signaling pathways mediating the roles of Rho1 in cell wall assembly and actin organization. Cell wall stresses are sensed by the five type I membrane proteins Wsc1, Wsc2, Wsc3, Mid2, and Mtl1, with the major players indicated by the thick lines and boldface type. These sensors then activate the GEFs Rom1 and Rom2, which in turn activate Rho1. Activated Rho1 controls cell wall assembly in two distinct ways: as the regulatory subunit of the glucan synthases Fks1 and Fks2 and by activating the Pkc1-MAPK-mediated CWI pathway, which increases the expression of cell wall synthetic enzymes. Rho1-GTP also controls polarized exocytosis as indicated in Fig. 10. Rho1-GTP also regulates actin ring formation via Bni1 to effect cytokinesis. Mss4, the PI4P-5K, synthesizes the plasma membrane pool of PI4,5P2, which recruits the Rho1 GEFs to the plasma membrane, thus contributing to Rho1 activation. PI4,5P2 and Tor2 are involved in recruiting and/or maintaining the pair of PH domain-containing proteins, Slm1 and Slm2, at the plasma membrane. These Slm proteins are thought to function in actin organization via Pkc1 but not the MAPK pathway. In response to heat stress, Rho1-Pkc1 is thought to function in transient actin depolarization, whereas the MAPK pathway is thought to function in subsequent actin repolarization during the recovery phase (see the text for further details). SBF, Swi4/Swi6 cell cycle box binding factors.

Possible coordination mechanisms among small GTPases during polarized cell growth. (A) Polarisome-based signaling hub. Linkages between Cdc42 and Rho1 that are suggested by preliminary genetic and two-hybrid interactions are not depicted here. (B) Exocyst-based signaling hub (see the text for details).