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Department of Molecular Genetics, The Ohio State University, Columbus, Ohio,1 Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania2
SUMMARY INTRODUCTION ESTABLISHMENT AND MAINTENANCE OF Cdc42 POLARIZATION The Rho Family GTPase Cdc42 and Its Regulators Cdc42 GTPase. Cdc24, a GEF for Cdc42. Cdc42 GAPs. Rho GDI. Cycling of Cdc42 between the GDP- and GTP-bound states. Regulation of Cdc42 Clustering at Sites of Polarized Growth Establishment of Cdc42 polarization. Maintenance of Cdc42 polarization. (i) The central role of Bem1 in maintaining Cdc42 polarization. (ii) Role of F-actin in maintaining Cdc42 polarization. (iii) Role of exocytosis in maintaining Cdc42 polarization. (iv) A possible mechanism of concentrating Cdc42-GDP at sites of polarized growth. DETERMINATION OF THE AXIS FOR CELL POLARIZATION DURING BUDDING The Rsr1 GTPase Module—Rsr1, Bud2, and Bud5 Coupling of the Rsr1 GTPase Module to the Cdc42 GTPase Module Interaction between Rsr1 and the Cdc42 module. Model for coupling bud site selection to polarity establishment. Spatial Landmarks That Specify the Site for Polarized Growth Axial landmarks. (i) Bud3, Bud4, and septins. (ii) Axl1 and Axl2. Bipolar landmarks. (i) Bud8 and Bud9. (ii) Rax1 and Rax2. (iii) Other proteins necessary for the bipolar budding pattern. Regulation of Cell Type-Specific Budding Patterns Coupling of spatial landmarks to the Rsr1 GTPase module. Model for cell type-specific budding patterns. TEMPORAL CONTROL OF POLARITY ESTABLISHMENT DURING YEAST BUDDING Cdc42 EFFECTORS AND THEIR ROLES IN POLARITY DEVELOPMENT Actin Organization Actin patches and endocytosis. Cdc42 effectors and actin patches. Actin cables and exocytosis. Cdc42 effectors and actin cables. Septin Organization General properties and functions of the septins. Role of Cdc42 in septin organization. Is the role of Cdc42 in septin organization universal? Model for the Roles of Cdc42 in Actin and Septin Organization The Mitotic Exit Network and Cytokinesis OTHER TYPES OF CELL POLARIZATION Cell Polarization during Mating Filamentous Growth OTHER SMALL GTPases AND THEIR ROLES IN POLARIZED GROWTH Rho1 Regulators Rho1 GEFs. Rho1 GAPs. Rho1 Effectors and Biological Responses Rho1 and cell wall assembly. Rho1 and actin organization. Rho1 and exocytosis. Rho3, Rho4, and Polarized Cell Growth Rho3, Rho4, and actin organization. Rho3, Rho4, and exocytosis. What Is the Role of Ras in Polarized Growth? COORDINATION OF Cdc42, Rho, AND Rab DURING POLARIZED GROWTH Polarisome-Based Signaling Hub Exocyst-Based Signaling Hub UNIFYING CONCEPTS IN CELL POLARIZATIONS IN DIFFERENT ORGANISMS Global and Local Cell Polarity Local Activation and Global Inhibition Self-Organization CONCLUDING REMARKS ACKNOWLEDGMENTS REFERENCES
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The budding yeast Saccharomyces cerevisiae is a particularly attractive model organism because it displays pronounced cell polarity in response to intracellular and extracellular cues. Cells of budding yeast undergo polarized growth during various phases of their life cycle, such as budding during vegetative growth, mating between haploid cells of opposite mating types, and filamentous growth (FG) upon deprivation of nutrients such as nitrogen (Fig. 1). There are three cell types in yeast (here we sometimes refer to S. cerevisiae as "yeast," although we realize that other yeasts such as Schizosaccharomyces pombe are somewhat different from S. cerevisiae): a and 
cells (such as normal haploids) and a/ cells (such as normal diploids), which are determined by their mating-type loci (220). Both haploid and diploid cells initiate budding when they reach the critical cell size in the late G1 phase of the cell cycle. Bud growth is initially targeted to the bud tip (apical growth) and then throughout the bud (isotropic growth) until nuclear division and cytokinesis take place (Fig. 1A). The second form of polarized growth occurs when haploid a and cells encounter each other and undergo mating to form diploid a/ cells (Fig. 1B). During both budding and mating, the overall cellular organization is similar, although budding cells have a constriction between mother and bud called the bud neck (Fig. 1A). A specific site for cell polarization during budding is determined by the cell type, whereas cell polarization during mating is chemotropic; i.e., a cell of one mating type responds to a gradient of a peptide mating pheromone secreted by a cell of the opposite mating type (221). The most prominent feature in both processes is the organization of the polarized actin cytoskeleton, which guides secretion towards the bud site or the tip of a mating projection, resulting in polarized cell growth (126, 457).
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| ESTABLISHMENT AND MAINTENANCE OF Cdc42 POLARIZATION |
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Cdc42 also contains the "Rho insert domain" of 
13 amino acid residues, which is unique to Rho-type GTPases (Fig. 3B). This domain has been implicated in interactions with some of its downstream effectors such as IQGAPs and guanine nucleotide dissociation inhibitors (GDIs) for human Cdc42 (375, 621). However, the crystallographic structure of the Cdc42/Rho GDI complex reveals that the switch I and switch II domains and the geranylgeranyl moiety of Cdc42 interact with Rho GDI (229). Cdc42 also contains the C-terminal CAAX (A is aliphatic; X is any amino acid) box, CTIL, for prenylation with the geranylgeranyl isoprene group at the Cys residue (651). Geranylgeranylation has been found in Cdc42s from all other organisms examined. This posttranslational modification is important for the membrane attachment and function of Cdc42. Cell fractionation experiments indicate that Cdc42 is found in both soluble and particulate pools, with most in the latter fraction, which includes the plasma membrane, secretory vesicles, and other dense materials (651). The particulate pool of Cdc42 is eliminated by mutagenizing the Cys188 in the C-terminal 188CTIL and is decreased in the geranylgeranyltransferase mutant cdc43 (651). Lipid modification of Cdc42 is thus required for its targeting to the particulate pool. Indeed, Cdc42C188S fused to green fluorescent protein (GFP) fails to localize to any internal and plasma membranes (466). A GFP fused to the CAAX motif alone (GFP-188CTIL) localizes to the internal membranes but not to the plasma membranes, while GFP-KKSKKCTIL (the polybasic motif plus the CAAX box) is sufficient for targeting to both membranes. However, GFP-KKSKKCTIL still fails to cluster at sites of polarized growth (466), supporting the idea that Cdc42 clustering may require guanine nucleotide binding (468). Thus, it appears that the CAAX box and its immediate upstream polybasic domain are sufficient for the targeting of Cdc42 to the plasma membrane, while other regions of Cdc42 are likely to be involved in its clustering at sites of polarized growth.
Cdc42 localizes to the plasma membrane and to sites of polarized growth, first to the incipient bud site, the tips of growing buds, and then the mother-bud neck in large-budded cells (466, 651). It is noteworthy that Cdc42 localization to the incipient bud site is not disrupted by incubation with latrunculin A, which sequesters G actin and thus causes a rapid depolymerization of the actin filaments (30). Thus, Cdc42 is likely to arrive to the presumptive bud site independently of the localization or integrity of the actin cytoskeleton during budding (see below for a further discussion of Cdc42 polarization).
The phenotype of a temperature-sensitive (Ts) cdc42 mutant, cdc42-1, has provided the first clue for the function of Cdc42 (8, 217). This mutant fails to form a bud at 37°C but continues DNA replication, nuclear division, and the increase in cell mass and volume, resulting in an enlarged unbudded cell. The actin cytoskeleton is haphazardly distributed in the mutant cells, indicating that Cdc42 is required for the polarized organization of the actin cytoskeleton (8). Mutations of CDC42 or CDC24, which encodes a GDP-GTP exchange factor (GEF) for Cdc42, also prevent targeted secretion so that new cell surface growth is not limited to the bud or mating projection (8, 535). Null mutants of CDC42 or CDC24 are not viable, as expected from their essential role in budding (260). Analyses of many cdc42 mutants have provided much information about the functional domains of Cdc42 and have uncovered a variety of processes in which Cdc42 is involved (84, 183, 259, 293, 394, 465) (see below).
Cdc24, a GEF for Cdc42. As discussed above, CDC24 is also required for bud emergence or the establishment of cell polarity (215, 216). Temperature-sensitive mutations in CDC24 result in the formation of large, round, unbudded cells at the restrictive temperature (215, 216, 534, 535), as is the case with some of the cdc42-Ts mutants, suggesting that CDC42 and CDC24 are likely to function in the same process, i.e., bud emergence (8, 260). These cdc24 mutants exhibit an overall defect in cell polarity resulting in delocalized chitin and mannan deposition throughout the cell surface (473, 534, 535).
Cdc24 contains a Dbl homology (DH) domain (388), which is generally associated with its GEF activity (228). Indeed, Cdc24 displays GEF activity towards Cdc42 (563, 645). Because a hyperactive mutation in CDC42 (cdc42G60D) can bypass the requirement for Cdc24, it is likely that the GEF activity towards Cdc42 is the sole essential function for Cdc24 in polarized growth (84). Cdc24 also contains other highly conserved domains including a calponin homology (CH) domain, which has been implicated in binding to actin in some proteins (551), and the pleckstrin homology (PH) domain, which is thought to bind phosphoinositides (310). A recent genome-wide characterization of all potential PH domains in yeast indicates that the PH domain of Cdc24, like most of the other yeast PH domains, binds phosphoinositides but with no headgroup specificity and with low binding affinity (635). In addition, the PH domain of Cdc24 alone fails to localize to the membrane (635). Thus, the functional significance of the PH domain of Cdc24 is not clear. Cdc24 also has two calcium binding domains (residues 649 to 658 and 820 to 831). It has been reported previously that Bem1, another protein important for polarity establishment, binds directly to Cdc24 and that this interaction is inhibited by Ca2+ in vitro (644), which may explain why CDC24 was also identified from a screen for mutants sensitive to a high level of exogenous calcium (421, 422).
Despite no obvious transmembrane domain or hydrophobic stretches, Cdc24 fractionates to a particulate pool (383, 388, 454). It is possible that Cdc24 associates with membrane through its PH domain and/or through interactions with other proteins on the plasma membrane. Analyses of deletion and point mutations of CDC24 have identified a 56-amino-acid domain (amino acids 647 to 703) as being necessary and sufficient for its localization to sites of polarized growth (572). This domain alone, however, is unable to anchor Cdc24 at these sites or is unable to support a tight association of Cdc24 with the plasma membrane. Anchoring of Cdc24 requires the Cdc24 carboxyl-terminal PC (phox and Cdc) domain that interacts with Bem1 and also requires Bem1, Rsr1, or the potential transmembrane protein Tos2/Ygr221C (572). Not much is known about Tos2 except that it exhibits a two-hybrid interaction with Cdc24 and also localizes to sites of polarized growth (123). Together, these data suggest that Cdc24 localization requires both membrane-specific targeting and subsequent anchoring within a multiprotein complex.
Cdc24 interacts with a number of proteins including Rsr1, Cdc42, Bem1, and Far1 (47, 70, 434, 644). Domains of Cdc24 that interact with some of its binding partners have been mapped. The conserved PB1 (phox and Bem) domain of Bem1 interacts with the PC motif-containing region at the C terminus of Cdc24 (47, 69, 249, 565). The mating-specific alleles of cdc24, which fail to interact with Far1 but are not defective in budding, have been mapped in the CH domain, suggesting that Far1 interacts with the CH domain of Cdc24 (70, 408, 409). Which domain of Cdc24 interacts with Rsr1 is less clear. Rsr1 in its GTP-bound state can interact with the C-terminal half of Cdc24 in vitro (434), and this interaction is necessary for the localization of Cdc24 to the presumptive bud site (436). Consistent with these data, the C-terminal half of Cdc24 is important for its localization to the site of polarized growth, whereas the N-terminal region is required for its localization to the nucleus (571, 572). On the other hand, it has been shown that Rsr1 exhibits a two-hybrid interaction with the CH domain of Cdc24 (523), which overlaps with the Far1 binding domain located near the N terminus. It is possible that more than one domain of Cdc24 interacts with Rsr1 and that each study may have overlooked the other binding site. Several questions remain. For example, does the CH domain interact directly with Rsr1 in a GTP-dependent manner? Does Rsr1 interact with more than one domain of Cdc24 at the same time in vivo? Is the Rsr1-Cdc24 interaction important only for guiding Cdc24 to the proper bud site, or does it also affect the GEF activity of Cdc24? It has been reported that Cdc24 inhibits both the intrinsic and the GTPase-activating protein-stimulated GTPase activity of Rsr1, suggesting that Cdc24 acts as a GTPase inhibitor protein for Rsr1 (644). However, this notion is somewhat surprising given that Rsr1 does not exhibit a high intrinsic GTPase activity in vitro (even in the absence of Cdc24) (435), and thus, the physiological significance of this observation is unclear.
Cdc24 localizes to the presumptive bud site in the late G1 phase and to sites of polarized growth during the cell cycle (408, 522, 571). In wild-type cells, the localization of Cdc24 to the presumptive bud site is likely to occur through the interaction with Rsr1-GTP (434, 436, 644). However, Cdc24 still localizes to a single site, although at a random location, in the absence of Rsr1 in the late G1 phase, indicating that other mechanisms operate in the Cdc24 clustering in cells lacking Rsr1. In the late M and early G1 phases, Cdc24 localizes to the nucleus through the interaction with Far1 (411, 522). The export of Cdc24 from the nucleus is triggered either by entry into the cell cycle or by mating pheromones. Activation of the cyclin-dependent kinase (CDK) Cdc28 by G1 cyclin Cln2 triggers the degradation of Far1, and as a result, Cdc24 is relocated from the nucleus to the presumptive bud site (196). This Cdc28/Cln2-triggered relocation of Cdc24 defines one step for the temporal regulation of polarity establishment (see Temporal Control of Polarity Establishment during Yeast Budding for further discussion).
Isolation of the mating-specific alleles of CDC24 uncovers important roles of Cdc24 in polarity establishment as well as cell fusion during mating (see below ["Cell Polarization during Mating"]). Certain alleles of CDC24 also exhibit the bud site selection defect (534, 535), while others display sensitivity to high-calcium growth media (421, 422). In addition, certain cdc24 alleles are also sensitive to high-Na+ growth media and exhibit synthetic lethality with a vacuolar ATPase subunit mutant, vma5 (611), suggesting that Cdc24 might be involved in Na+ tolerance and in vacuole function. However, the role of Cdc24 in calcium-mediated regulation or in vacuole function is not well established. The genetic interaction between CDC24 and the genes involved in vacuole morphology or function is likely to be related to its role as a GEF for Cdc42 since Cdc42 is implicated in vacuole membrane fusion (402). Cdc42 promotes the assembly of the actin cytoskeleton (see below), which is also involved in vacuole inheritance or movement (224).
Cdc42 GAPs. There are four potential GTPase-activating proteins (GAPs) for Cdc42: Bem2 (46, 282), Bem3 (645), Rga1/Dbm1 (91, 550), and Rga2 (181, 536). In vitro GAP assays indicate that Bem2 acts on Cdc42 (357) and Rho1 (446); Bem3 acts mainly on Cdc42 and, to a lesser extent, on Rho1 (645, 646); and both Rga1 and Rga2 act on Cdc42 (181, 536). These in vitro assays, together with two-hybrid interaction data (536, 550), suggest that Bem3, Rga1, and Rga2 are more specific to Cdc42, whereas Bem2 is a GAP for both Cdc42 and Rho1 (Fig. 3A) (see the section on Rho1 GAPs below for more discussion on Bem2).
Why does Cdc42 need multiple GAPs? Are the GAPs involved in distinct functions, or do they share a redundant role? Can they also carry out a specific biological function as a part of the Cdc42 effector? Answers to these questions are not yet clear. In contrast to CDC42, none of the genes encoding the putative Cdc42 GAPs is essential. BEM3 was originally isolated as a multicopy suppressor of a bem2-Ts mutant, which is defective in bud emergence (645). RGA1/DBM1 was identified in a genetic screen designed to isolate mutants that activate the pheromone response pathway in the absence of the Ste4 Gß subunit (550) and also as a dominant suppressor of a bem2-Ts mutant (91). Deletion of RGA1 results in increased expression of a FUS1::lacZ reporter gene. Another potential Cdc42 GAP, Rga2, was identified through its homology to Rga1 (536, 550). The rga1
bem3 mutant in some strain backgrounds produces a high percentage of elongated cells, while the single mutant of either rga1 or rga2 does not produce such abnormally shaped cells (83, 181, 536). A bem3 mutant also produces a low percentage of abnormally shaped cells, such as cells that are peanut or finger shaped, depending on strain backgrounds. The rga1 bem3 double mutant and the rga1 rga2 bem3 triple mutant show more misshapen cells than the bem3 single mutant. Deletion of RGA1 also leads to an increase in the bipolar budding pattern in haploids instead of the axial budding pattern (91, 536), suggesting that Rga1 has a distinct function in bud site selection that is not shared by Rga2 or Bem3. It is not clear why a mutation of a Cdc42 GAP leads to a defect specifically in the axial budding pattern. Distinct phenotypes of strains lacking RGA1, RGA2, or BEM3 led to a suggestion that each GAP may regulate different functions of Cdc42, although quantitative differences in the GAP activities of the mutants may contribute to the overall phenotype (536). Importantly, the strain lacking all three GAPs, rga1 rga2 bem3, displays a much more elongated bud morphology and increased induction of FUS1-LacZ than any single or double GAP mutants (although the extent of these defects seems to vary depending on strain backgrounds) (83, 181, 536), both of which are consistent with more activation of Cdc42. Rga1, Rga2, and Bem3 may thus play some overlapping roles in regulating morphogenesis and mitogen-activated protein (MAP) kinase (MAPK) activation during the mating response, presumably via their GAP activity towards Cdc42.
The localization of Bem3 and Rga2 is similar to that of Cdc42: both Bem3 and Rga2 were observed at the presumptive bud site, the tips of small buds, and the mother-bud necks of cells late in the cell cycle, although no distinct signal was detected at intermediate stages (83). Rga1 exhibits a distinct localization pattern. It localizes to the presumptive bud site and to the cortex of a tiny bud rather than only at the bud tip. Unlike Bem3 and Rga2, Rga1 localizes as a ring to the bud site of the neck in cells with a medium or large bud. Later in the cell cycle, Rga1 localizes to the neck as a double ring, with approximately equal intensities for both rings. Localization of these Cdc42 GAPs to the neck, but not to the presumptive bud site or bud tip, depends on septins (83). A large-scale localization study indicated that Bem2 is found at the bud neck and the cytoplasm (237). It remains to be determined whether the localization pattern of each GAP contributes to the functional differences in Cdc42 GAPs. In summary, Cdc42 GAPs are likely to play differential and overlapping roles in regulating polarized cell growth via their GAP activity. An important question is how Cdc42 GAPs are temporally and spatially regulated in the cell cycle so that they can coordinately regulate Cdc42 activity.
Rho GDI. Rho GDI (GDP dissociation inhibitor) is known to display three biochemical activities: inhibiting the dissociation of GDP from Cdc42, Rho, and Rac (95, 165, 312); inhibiting the intrinsic and GAP-stimulated GTPase activity of Cdc42, Rho, and Rac (95, 208, 214); and extracting Cdc42, Rac, and Rho from cellular membranes into cytosol (232, 416). The first and third activities inhibit GDP/GTP exchange and decrease the membrane pool of small GTPases, respectively, leading to the perception that Rho GDIs act as negative regulators of these GTPases. In contrast, the second activity maintains Cdc42, Rho, and Rac in their GTP-bound form, suggesting that Rho GDI may also play a positive role in the functions of these GTPases. The C-terminal lipid modification of Rho GTPases is essential for their binding to the GDIs (208, 229, 232) (Fig. 3B). In addition, the switch I and switch II regions of Rho GTPases, which bind to their GEFs, GAPs, and effectors, and the polybasic region, which is required for their targeting to the plasma membrane, are also involved in their interactions with the GDIs (110, 229, 259) (Fig. 3B). Unlike Rab GDIs, which bind preferentially to the GDP-bound form of Rab GTPases (17, 448), Rho GDIs bind equally well to both the GTP- and the GDP-bound forms of Cdc42, Rho, and Rac (208, 416). Rab GDIs also play an important role in delivering and/or loading Rab GTPases to target membranes (177, 447, 448), whereas such a role may not exist for Rho GDIs (120), as Cdc42 and Rac mutants deficient in their interactions with Rho GDIs appear to target to plasma membranes and elicit normal biological responses (167, 174, 175).
Rdi1, the only known Rho GDI in S. cerevisiae (365), can efficiently extract Cdc42 and Rho1 from the vacuolar membrane (133) and can also extract Cdc42 from other membranes including the plasma membrane (468, 563). Deletion of RDI1 does not produce any detectable phenotypes (365) and does not affect the clustering of Cdc42 at the sites of polarized growth (468). Rho1 and Cdc42 were found in the cytosol of the rdi1 cells to a similar extent as in wild-type cells, suggesting that other mechanisms or other GDI-like activities are responsible for the cycling of these GTPases between membranes and the cytosol (288). Overexpression of Rdi1 causes a slightly rounder cell morphology in some strain backgrounds (563) but causes lethality in other strain backgrounds (365) for reasons that are not known. In a cdc24-Ts mutant where the activation of Cdc42 is compromised, overexpression of Rdi1 causes lethality with cells arrested as large, round, unbudded cells, which is indicative of a loss of cell polarity (563). These overexpression phenotypes are consistent with Rdi1 being a negative regulator of Cdc42.
Rdi1 localizes to the sites of polarized growth in the cell cycle, the tip of a small bud and the mother-bud neck during cytokinesis (468). Unlike Cdc42 (466), Rdi1 does not appear to localize to the presumptive bud site and internal membranes (468). The Rdi1 localization pattern is consistent with the possibility that there might be a pool of Cdc42-GDP at sites of polarized growth. This notion is supported by the observation that the GDP-locked form of Cdc42, Cdc42D57Y, clusters at the presumptive bud site as efficiently as the wild type and the active form of Cdc42 (605).
Cycling of Cdc42 between the GDP- and GTP-bound states.
CDC24, which encodes the only known GEF for Cdc42 (563, 645), is an essential gene (215, 388, 534), suggesting that the exchange of GTP for GDP is essential for Cdc42 function. Three cdc42 alleles, cdc42G12V, cdc42Q61L, and cdc42D118A, which contain mutations in the putative GTP-binding and hydrolysis domains, cause dominant dosage-dependent lethality, suggesting that GTP hydrolysis by Cdc42 is essential for its normal function (650). Consistent with this interpretation, the triple mutant of Cdc42 GAPs, rga1 rga2 bem3, is defective in bud morphogenesis and septin organization (see below for details) (83, 181, 536). Cdc42 still possesses GTPase activity to some extent in the rga1 rga2 bem3 mutant (645), consistent with the fact that the phenotype of the GAP triple mutant is less severe than that of the mutants expressing the GTP-locked form of Cdc42. It is possible that there is an additional GAP (such as Bem2) for Cdc42 and/or that the intrinsic GTPase activity of Cdc42 is sufficient for cell survival.
Cdc42 GTPase appears to function in two different modes: one, like Ras, to turn on signaling pathways in its GTP-bound state and another, like the translation elongation factor EF-Tu (EF-1 in eukaryotes), to assemble a macromolecular structure such as the septin ring (181). The cycle of nucleotide binding and GTP hydrolysis is critical for the latter mode of action, as was also proposed for Sec4 (601) and Rsr1 (434, 435, 484). The cycling feature of Cdc42 can best explain the paradoxical phenotypes of the cdc42G60D mutation, which makes Cdc42 hyperactive and causes multiple buddings per cell cycle yet is completely recessive in terms of the budding phenotype (84). Perhaps the Cdc42G60D mutant protein may cycle more slowly than wild-type Cdc42. If rapid cycling of Cdc42 is required for establishing and maintaining a single site for budding in each cell, wild-type Cdc42 should outcompete the mutant Cdc42 for recruiting effectors and other downstream components to a single site, thus suppressing the mutant phenotype (84). This hypothesis may explain, at least in part, the recessiveness of the cdc42G60D allele. This hypothesis is also supported by a recent observation: fluorescence recovery after photobleaching (FRAP) analysis indicates that wild-type Cdc42 at the presumptive bud site recovers much more quickly after photobleaching than Cdc42Q61L and Cdc42D57Y, which are expected to be the GTP- or GDP-locked Cdc42, respectively (605). However, it is likely that there are some intrinsic differences between the cdc42G60D and cdc42Q61L mutants, since the former is recessive, whereas the latter is dominant.
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mutants, the budding process does not seem to be compromised, except that the mutant cells bud at a random site (45, 87). This spontaneous cell polarization process in the apparent absence of spatial cues is called "symmetry breaking." Cdc42 polarization also occurs normally once per cell cycle at a random location in rsr1 cells (245, 466, 604, 605, 651). Thus, there is a default pathway leading to Cdc42 polarization in the absence of putative upstream events such as bud site selection. Other mechanisms must operate in rsr1 (and presumably also in wild-type) cells to establish and maintain Cdc42 polarization. Cdc42 polarization can occur in the rsr1 and bem1 single mutants but not in the rsr1 bem1 double mutant (245) or in bem1 cells in which filamentous actin (F-actin) has been disrupted by latrunculin A (LatA) (605). Cdc42 still polarizes to a single random site when bud site selection, F-actin, and microtubules are simultaneously disrupted (245). Thus, the predetermined spatial cue, F-actin, and the microtubule are not essential for Cdc42 polarization per se, although it does not rule out the possibility that these components act cooperatively to enhance Cdc42 polarization. Taken together, these results indicate that bud site selection proteins, the signaling protein Bem1, which binds to Cdc42-GTP (58), and F-actin all share an essential role in achieving a polarization state of Cdc42 (245, 605). One possibility is that the spontaneous clustering of activated Cdc42 in the absence of a spatial cue initiates Cdc42 polarization, but Bem1 or F-actin plays a crucial role in stabilizing, amplifying, and/or maintaining this initial polarization state. Various active forms of Cdc42, Cdc42G12V (196, 466) and Cdc42Q61L (604), are able to polarize in G1-arrested cells in the presence of endogenous Cdc42. Cdc42G60D, as the sole source of Cdc42 in the cell, can also cluster on the plasma membrane randomly at more than one site, resulting in multiple budding events per nuclear cycle (84). Thus, it has been proposed that the activated Cdc42 becomes clustered through stochastic movement on the plasma membrane (84, 604) and that this initial clustering of Cdc42 could be stabilized by the interactions with Cdc42 effectors in the cell cortex (84).
Maintenance of Cdc42 polarization. Cdc42 is highly dynamic at the sites of polarized growth, as indicated by FRAP analysis (605), suggesting that the Cdc42 concentration at these sites has to be dynamically maintained. Because GTP- or GDP-locked Cdc42 recovers more slowly than wild-type Cdc42 after photobleaching, cycling between the two nucleotide-bound states of Cdc42 is likely to be important for its dynamic accumulation at sites of polarized growth (605).
(i) The central role of Bem1 in maintaining Cdc42 polarization. Bem1 binds to Cdc24, Cdc42-GTP, and Cla4, a PAK known to be an effector of Cdc42 (58, 196, 446, 644). It has been suggested that this protein complex enhances Cdc42 polarization and is thus involved in both the establishment and the maintenance of Cdc42 polarization. Cdc28/G1 cyclin complexes trigger Cdc42 activation indirectly through Cdc24, a GEF for Cdc42. Activated Cdc42 binds to Bem1, which, in turn, binds to Cdc24 and Cla4, and Cla4 phosphorylates Cdc24 (58, 69, 196, 245). This cascade of events may result in the accumulation of more Cdc24 and Cdc42 at sites of polarized growth. Thus, it has been proposed that the components of the Bem1-mediated protein complex constitute a positive feedback loop to establish and maintain Cdc42 polarization (69, 245) (see below [Temporal Control of Polarity Establishment during Yeast Budding] for further discussion).
(ii) Role of F-actin in maintaining Cdc42 polarization. Two major filamentous actin structures found in yeast are actin cables and actin patches, which are required for polarized exocytosis and endocytosis, respectively (457). Cdc42 polarizes normally in cells treated with latrunculin A (30, 246), which disrupts all F-actin structures, suggesting that Cdc42 polarization can be established and maintained in the absence of F-actin. However, when cells are treated with a less potent drug, latrunculin B (which disrupts all actin cables but not all actin patches), Cdc42 fails to cluster at the sites of polarized growth, suggesting that endocytosis may be involved in Cdc42 dispersal from its polarization site. In addition, in mutants conditionally defective in actin cable formation or in post-Golgi vesicle transport, Cdc42 polarization can occur initially but cannot be maintained (246). Thus, it appears that once Cdc42 polarization is established in an F-actin-independent manner, actin cable-mediated delivery of Cdc42 is required to counteract the actin patch-mediated dispersal of Cdc42 such that a dynamic pool of Cdc42 can be maintained at the sites of polarized growth.
(iii) Role of exocytosis in maintaining Cdc42 polarization.
Cdc42 polarization is not maintained in a number of mutants that are defective at various stages of exocytosis. For example, Cdc42 fails to maintain polarization in the tropomyosin mutant tpm2 tpm1-2 (246, 460, 604, 637), the type V myosin mutants myo2-16 (246) and myo2-66 (604), and the late sec mutants sec4-8 and sec5-24 (637). These data indicate that Cdc42 is delivered to sites of polarized growth through exocytosis and that actomyosin-based vesicle transport and the subsequent vesicle tethering and/or fusion are required for maintaining Cdc42 polarization. It is noteworthy that the establishment of Cdc42 polarization is independent of polarized exocytosis, as Cdc42 polarization can occur in cells treated with LatA (30, 246) as well as in cells carrying tropomyosin and myo2 mutations (246, 460, 604, 637), all of which block polarized secretion but not secretion per se. In summary, current data support the view that Cdc42 polarization initiates polarized growth, while polarized exocytosis reinforces or maintains polarized growth by delivering more polarity factors, including Cdc42, to sites of polarized growth.
(iv) A possible mechanism of concentrating Cdc42-GDP at sites of polarized growth.
The Rsr1 GTPase module is likely to be involved in the recruitment of Cdc42 to the proper bud site (292). In rsr1 cells, however, a mechanism involving Msb3 and Msb4 is likely to operate. Msb3 and Msb4 may also carry out a similar function in wild-type cells as well, but it becomes more apparent in rsr1 cells. Msb3 and Msb4, a pair of homologous proteins each containing a Rab GAP domain (12, 13, 168), function as dosage-dependent suppressors of cdc24 and cdc42 mutants (51, 168, 563). Although a deletion of either MSB3 or MSB4 does not produce any obvious defect, the deletion of both genes results in a large, round mother with small buds, and a significant fraction of the double mutant cells has a disorganized actin cytoskeleton (32, 51). Thus, Msb3 and Msb4 may function in the same pathway as Cdc42. Msb3 and Msb4 interact with Spa2, a scaffold protein of the "polarisome" (563) (see below). Spa2 localizes to the presumptive bud site prior to Start in the late G1 phase, and this localization is dependent upon Cdc42 but not its GEF Cdc24, suggesting that Cdc42-GDP may play some role in bud site assembly (470). Interestingly, Msb3 and Msb4 bind specifically to Cdc42-GDP and Rho1-GDP but not Rho3 and Rho4 (563). Like Cdc42, Msb3 and Msb4 localize to sites of polarized growth (51, 168). Together, these results have led to a hypothesis that Msb3 and Msb4 are involved in recruiting Cdc42 from the cytosol and/or capturing Cdc42 from the secretory pathway, increasing a local pool of Cdc42-GDP at the sites of polarized growth, which is poised for activation by the GEF Cdc24 (563). Other functions of Msb3 and Msb4, including their GAP activity towards Rab GTPases and their functions in the Rho1 pathway, will be discussed below.
| DETERMINATION OF THE AXIS FOR CELL POLARIZATION DURING BUDDING |
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cells bud in the axial pattern in which both mother and daughter cells select a bud site immediately adjacent to their previous division site. In contrast, diploid a/ cells bud in the bipolar pattern: mother cells select a bud site adjacent to their daughter or on the opposite end of the cell, whereas daughter cells almost exclusively choose a bud site directed away from their mother (89, 158, 222) (Fig. 5A). These two distinct patterns of budding reflect genetic programming of cell polarization. The choice of a bud site determines the axis of cell polarity and ultimately the cell division plane, which is perpendicular to the axis of cell polarity. These patterns of cell division result in characteristic shapes of microcolonies on a solid surface (Fig. 5A) and distinct patterns of bud scars, which mark the sites of cell division on the mother cell surface (Fig. 5B). In cells undergoing axial budding, the division site is likely marked by a spatial signal(s) that specifies the location of the new bud site (89). Since the starvation and refeeding of axially budding cells result in the formation of a new bud at a nonaxial site, the spatial signal for the axial budding pattern appears to be transient in that it lasts only from one budding event to the next (89). Despite the transient nature of the axial spatial cues, haploid a or cells exhibit the axial pattern with remarkably high fidelity during continuous logarithmic growth. Unlike the transient nature of the axial spatial cue, the bipolar spatial cue(s) appears to consist of persistent cortical markers that are present at both poles of diploid a/ cells (89).
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The localization of Rsr1, Bud2, and Bud5 is consistent with their functions at the presumptive bud site in each cell type. Rsr1 fused to GFP localizes to the plasma membrane and then becomes concentrated at sites of polarized growth, first to the presumptive bud site, the bud tip, and then the mother-bud neck at a later stage of the cell cycle (436). GFP-Rsr1 is also present at the internal organelle membranes, particularly the vacuolar membrane (436). Subcellular fractionation of Rsr1 is also consistent with its localization pattern (434). Localization of Rsr1 to the plasma membrane and to the sites of polarized growth requires both the CAAX box and the polylysine residues near the C terminus (436). The replacement of Cys269 with Ser in the CAAX box of Rsr1 abolishes all membrane association of Rsr1, whereas the replacement of all Lys residues at positions 260 to 264 with Ser disrupts the localization of Rsr1 to the plasma membrane and to the sites of polarized growth but not to the internal organelle membrane (436). Since both mutations cause random budding (436), localization of Rsr1 to the plasma membrane and to the sites of polarized growth is necessary for its function in bud site selection, whereas its localization to the internal organelle membrane is not sufficient for its function. It is not known whether the localization of Rsr1 to the internal membrane indicates other unknown functions of Rsr1 or reflects intermediate locations during its delivery to the plasma membrane.
Bud2 localizes in a patch at the incipient bud site in the late G1 phase: an axial bud site in haploid a or cells or either the proximal or distal pole of diploid a/ cells (437). Bud2 localizes to the mother-bud neck after bud emergence and then delocalizes around the G2/M phase in all cell types (358, 437). In haploid a or cells, Bud5 localizes to the presumptive bud site in G1, to the tip of growing buds after bud emergence, and then to the mother-bud neck around the G2/M phase. In the late M phase, Bud5-GFP appears as a double ring at the neck, which splits into two single rings upon cell separation, and both mother and daughter cells inherit a single ring. Thus, most of the newly born G1 cells have the Bud5 ring at the division site (273, 358). This localization pattern of Bud5 is similar to that of Axl2, which is required for the axial budding pattern (see below), throughout the cell cycle. Bud5-GFP exhibits distinct localization patterns in diploid a/ cells, particularly during G1 and M phases. Before bud emergence, Bud5-GFP is present at both poles: as a ring at one pole, which is the previous division site, and in a patch at the opposite pole, which becomes a new bud site. After bud emergence, Bud5-GFP localizes throughout the periphery of the bud, as seen in haploid cells. At a later stage of the cell cycle, Bud5-GFP localizes to the neck and one pole of the mother cell (and/or bud tip), while a small percentage of cells shows a Bud5-GFP signal only at the neck (273, 358). Taken together, these specific patterns of localization of Bud2 and Bud5 are probably important for proper bud site selection, since the overexpression of Bud2 or Bud5 results in the mislocalization of each protein and causes random budding (273). Consistent with this notion, some alleles of BUD5 that disrupt the proper localization of Bud5 only in a/ cells are specifically defective in the bipolar budding pattern (273). Mislocalization of Bud5 in other bud mutants suggests that Bud5 localizes to specific sites in each cell type through the interaction with the cell type-specific landmark (272, 273) (see below).
The interaction between Rsr1 GTPase and Cdc42 GTPase is particularly interesting. RSR1 was identified as an allele-specific dosage suppressor of a cdc42 allele (cdc42-118) that is defective in polarity establishment (292). This suppression requires the "Ras-like effector domain" of Rsr1 and the cycling of Rsr1 between its GDP- and GTP-bound states (292). In addition, an rsr1 deletion mutant was found to be synthetic lethal at 30°C with cdc42-118 (292). Rsr1 also physically interacts with Cdc42, and this interaction appears to be enhanced in the presence of Cdc24 (292). Interestingly, the rsr1-7 mutant, which carries mutations in the polylysine repeat near the C terminus of Rsr1, suppresses a cdc24 mutant but fails to suppress a cdc42 mutant. The mutation may thus disrupt the interaction between Rsr1 and Cdc42 but not the interaction between Rsr1 and Cdc24. These data are consistent with the idea that the interaction between Rsr1 and Cdc42 is likely to be direct, rather than being bridged by Cdc24, a GEF for Cdc42 (292). These findings provide a novel mechanism of action of GTPases controlling polarity establishment. Interestingly, GTPase heterodimerization as a mechanism for activating GTPase signaling in bacteria (518) and plants (607) has recently been reported.
What would the physiological significance of the interaction between Rsr1 and Cdc42 be? The interaction between Rsr1 and Cdc42 may contribute to the localization of Cdc42 to the proper bud site, although it has not been directly addressed. The genetic interaction between RSR1 and CDC42 suggests that Rsr1 functions not only in selection of a growth site but also in polarity establishment. The latter role of Rsr1 becomes phenotypically apparent when polarity establishment is compromised as in a cdc42-Ts mutant (292). In addition, RSR1 is essential in the absence of GIC1 and GIC2, which encode two related targets of Cdc42 that are involved in polarity establishment (278). Cells lacking all three genes, RSR1, GIC1, and GIC2, fail to undergo bud emergence. A detailed analysis of live cells by high-resolution microscopy also indicates that Rsr1 is required for selecting and stabilizing the polarity axis in the G1 phase of the cell cycle (426). Thus, it is likely that Rsr1 has a role in polarity establishment, although the exact role of Rsr1 in polarity establishment is yet to be determined.
Model for coupling bud site selection to polarity establishment. By analogy to Sec4 guiding vesicle targeting to the plasma membrane (61, 601), a scheme in which the Rsr1 GTPase cycle orchestrates bud site assembly to the proper bud site has been proposed (Fig. 7) (292, 434). The model hypothesizes that the cycle between Rsr1-GTP and Rsr1-GDP coupled with the differential affinities of each of these species for binding partners may trigger the ordered assembly of a complex at the bud site. First, Bud5 catalyzes the conversion of Rsr1 from the GDP-bound state to the GTP-bound state at the site where a cortical cue is located (step 1). Rsr1-GTP associates with Cdc24 and Cdc42 and guides these proteins to the presumptive bud site where Bud2 (and Bud5) localizes (step 2). Bud2 activates GTP hydrolysis by Rsr1, resulting in the dissociation of Cdc24 from Rsr1 (step 3). Cdc24 then catalyzes the conversion of GDP-Cdc42 to GTP-Cdc42 (step 4), which then triggers actin cytoskeleton assembly and targeted secretion. Finally, the action of Bud5 may recycle Rsr1, thus allowing further shuttling of the essential components for bud site assembly. Bem1 might join the complex through an interaction with Cdc24, Cdc42, or Rsr1-GDP (58, 69, 434, 446, 644). Several rounds of this cycle may be necessary to assemble critical levels of Cdc42-GTP and its associated proteins at the proper bud site. The model hypothesizes that the ordered assembly of a complex through multiple protein-protein interactions might ensure the establishment of polarity at a correct location. This model provides a simplified view for linking the bud site selection machinery to the Cdc42 module, but the details remain to be tested.
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Axial landmarks.
The axial budding pattern depends on a transient cortical marker that involves a group of proteins such as Bud3, Bud4, Axl1, and Axl2/Bud10 (hereafter called Axl2) (4, 87, 88, 163, 207, 476, 491). Mutations in BUD3, BUD4, AXL1, and AXL2 result in bipolar budding in haploid a or cells but do not affect normal bipolar budding of diploid a/ cells. Genetic and localization data support the view that the cycle of assembly and disassembly of a protein complex at the mother-bud neck provides a spatial memory of the position from one cell cycle to the next, acting as an inherited landmark for axial budding.
(i) Bud3, Bud4, and septins. Bud3 and Bud4 localize to the mother-bud neck at or after the G2 phase (88, 491). Prior to cytokinesis, Bud3 and Bud4 appear as a double ring encircling the mother-bud neck, which splits into two single rings, one on each progeny, after cell division (88, 491). Septins, a family of related proteins including Cdc3, Cdc10, Cdc11, Cdc12, and Shs1/Sep7 (335), also play an important role in axial budding. Some alleles of septin genes, such as cdc10-10 and cdc11-6, are defective in axial budding (88, 152). Extra copies of BUD4 suppress the temperature-sensitive growth of a cdc12 mutant (491). Localization of Bud3 and Bud4 depends on the integrity of septins (88, 491), which localize as a ring to the incipient bud site and as a collar to the neck of budded cells (153, 202, 279, 336) (see below). Thus, Bud3 and Bud4 are likely to assemble at the mother-bud neck through a direct interaction with septins during the G2 and M phases, whereas the assembly of septins and the bud site complex at the axial bud site in the subsequent division cycle is likely to occur through the action of the Rsr1 and Cdc42 GTPase modules during the G1 phase (88, 181, 491). However, molecular details of the assembly of the axial landmark and the mechanisms by which the septins determine the localization of Bud3 and/or Bud4 are not known. Understanding the biochemical properties of Bud3 and Bud4 will shed light on their structural and/or regulatory role in axial budding.
(ii) Axl1 and Axl2.
Axl1 and Axl2 are also required for the axial pattern but not for the bipolar pattern (163, 207, 476). Interestingly, Axl1 is expressed in a and cells but not in a/ cells, and ectopic expression of AXL1 increases axial budding in a/ cells (163). Although Axl1 is likely to be a key component for the cell type-specific budding patterns, how Axl1 functions in axial budding is yet to be established. Axl1 shares homology with the insulin-degrading enzyme family of endoproteases and is also required for processing of the mating pheromone a-factor precursor (4). Amino acid substitutions within the presumptive active site of Axl1 cause defects in the processing of the a-factor precursor but do not perturb bud site selection (4), suggesting that the protease activity of Axl1 is not important for bud site selection.
Axl2 is a transmembrane glycoprotein with an N-terminal signal sequence and a transmembrane domain in the middle. Axl2 is thus predicted to have the type I membrane topology, similar to that of integrin (476). As expected from the predicted structure, Axl2 is heavily glycosylated at the N-terminal half of the protein. Axl2 exhibits no similarity to the ligand-binding or catalytic domains of known transmembrane receptors. It is possible that Axl2 functions in a manner analogous to that of noncatalytic receptors, such as integrins, for which clustering appears to be important for sending a signal to the downstream components (207). However, it remains a possibility that a specific extracellular ligand for Axl2 exists, such as a component of the cell wall. It is also not known whether Axl2 undergoes clustering and the formation of oligomeric complex like the integrin or other extracellular matrix receptors and whether such clustering is required for the axial pattern.
Unlike Bud3 and Bud4, Axl1 and Axl2 are detectable before the
