INTRODUCTION

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The establishment of cell polarity is an important component of the overall process of cellular morphogenesis, the complex process by which the three-dimensional organization of subcellular constituents, which ultimately determines an organism's characteristic growth patterns and shape, is generated and maintained. The generation of cell polarity is critical for the control of many cellular and developmental processes such as shape development in early plant and animal embryogenesis, axon migration and neurite outgrowth in early development, the intracellular movement of organelles and proteins in polarized epithelial cells, the stimulated secretion of neurotransmitters, the directed movement of migratory cells, polarized growth within yeast and fungal cells, and the asymmetric partitioning of new cellular constituents during cell division. Establishment of cell polarity involves the generation of cellular asymmetry through the localized temporal and spatial activation of cellular processes and can be divided into several hierarchical and interdependent events. These events include the initial response to endogenous and/or exogenous signals, the determination of an axis of polarization relative to these signals, and the subsequent asymmetric distribution of cellular components along that axis. At the molecular level, cell polarity is best understood in the budding yeast Saccharomyces cerevisiae, but results from studies in fission yeast Schizosaccharomyces pombe, Drosophila, Caenorhabditis elegans, and cultured mammalian cells strongly suggest that the molecular mechanisms controlling cell polarity in S. cerevisiae are highly conserved in other eukaryotes. Due to the abundance of recent reviews on cell polarity and signaling (26, 69, 77, 100, 126, 170, 177, 202, 216, 218, 246, 281, 346, 347, 350, 382, 464, 465, 467, 511, 520, 587) and on the roles of Rho-type GTPases in these processes (46, 61, 120, 141, 171, 186, 192, 225, 241, 288, 290, 325, 326, 337, 344, 419, 441, 473, 477, 478, 544, 550, 552, 572, 578, 638, 644). I will limit this review to a discussion of the Cdc42p GTPase, its identification, its structure and subcellular localization, its function(s) in controlling cell polarity, and its regulators and effectors.

It is becoming increasingly apparent that the Cdc42p GTPase and other Rho-type GTPases play a vital role in regulating the signal transduction pathways that control the generation and maintenance of cell polarity in many, if not all, eukaryotic cell types. The Cdc42p GTPase signaling module consists of regulators of the guanine nucleotide-bound state of Cdc42p, i.e., guanine nucleotide exchange factors (GEFs), guanine nucleotide dissociation inhibitors (GDIs), and GTPase-activating proteins (GAPs), as well as downstream effectors of Cdc42p function (Table 1). The regulators of the guanine nucleotide-bound state of Cdc42p must respond to a variety of exogenous and/or endogenous signals, thereby activating Cdc42p to a GTP-bound state or inactivating it to a GDP-bound state. A myriad of Cdc42p downstream effectors interact with the activated (GTP-bound) form of Cdc42p, thereby inducing a number of downstream events, including rearrangements of the actin cytoskeletal network and protein kinase-dependent induction of transcription, which are increasingly coming into view. Interactions between the 21-kDa Cdc42p GTPase and this host of regulators and effectors must be controlled in a temporal and spatial manner so that Cdc42p can function at different times within the cell cycle and at different places within the cell. Cdc42p function is also regulated by its subcellular localization, which depends on its prenylation state and interactions with its GDI.

CDC42P STRUCTURE AND FUNCTIONAL DOMAINS

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Identification of Cdc42

The Cdc42p GTPase was first identified from an S. cerevisiae mutant strain carrying a temperature-sensitive (ts) mutation, cdc42-1^ts, that blocked bud formation at 37°C but allowed the cell mass and volume to increase, resulting in greatly enlarged, unbudded cells (2). Although cell division was arrested at 37°C, DNA replication and nuclear division continued into the next cycle, resulting in multinucleate cells as determined by DNA staining with the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI) and mitotic spindle staining with anti-tubulin antibodies. Fluorescence microscopy with rhodamine-conjugated phalloidin showed that the polarized organization of the actin cytoskeleton (i.e., cortical actin distribution to the regions of new cell growth in the bud and actin cables directed into the enlarging bud) was disrupted, indicating that Cdc42p functioned in the organization of the actin cytoskeleton, which is necessary for polarized cell growth. Chitin and other cell surface materials were deposited uniformly throughout the enlarging cell walls, in contrast to their normal polarized patterns of deposition. Growth of the cdc42-1^ts strain at semipermissive temperatures led to a small percentage of cells with elongated buds. Taken together, these observations suggested that Cdc42p controls polarized cell growth during the cell cycle but that isotropic incorporation of new cell wall material was not impaired through the loss of Cdc42p function. Examination of the cdc42 null phenotype in S. cerevisiae and S. pombe indicated that Cdc42p was essential for viability (242, 390).

DNA and predicted amino acid sequence analysis (242) indicated that Cdc42p belongs to the Rho subfamily of the Ras superfamily of GTPases that act as molecular switches in the control of a variety of eukaryotic processes (191, 239, 241, 242, 642) (see below). At about the same time, a ~25-kDa guanine nucleotide binding protein was purified from bovine brain and human placental membranes (140, 461, 582), and peptide sequences from this protein, termed G_p or G25K, showed a high degree of similarity to S. cerevisiae Cdc42p (242, 461). This protein was shown to be a good in vitro substrate for epidermal growth factor (EGF)-stimulated phosphorylation (209), although the in vivo phosphorylation of Cdc42p has not been reported to date. Subsequent analysis of the predicted amino acid sequence from two independent human cDNA isolates indicated the existence of two highly conserved (95% identical) proteins, the ubiquitously expressed Cdc42Hs (525) and the brain isoform G25K (407). The Cdc42Hs and G25K proteins are identical in both nucleotide and predicted amino acid sequences up to amino acid 163 but diverge from residues 163 to 191, suggesting that these isoforms are differential splicing products of a single gene. Structural and/or functional Cdc42p homologs have subsequently been characterized in the pathogenic yeast Candida albicans (394), S. pombe (390), C. elegans (88, 500), Drosophila (336), chicken (Gallus gallus) cochlea (172), mouse (Mus musculus) liver (172) and brain (367), and dog (Canus familiaris) (GenBank accession no. Z49944), and these homologs are 80 to 95% identical in predicted amino acid sequence (241) (see below) (Fig. 1). S. pombe, Drosophila, and C. elegans Cdc42p, as well as Cdc42Hs and G25K, can complement the cdc42-1^ts mutant (88, 390, 407, 507, 525), suggesting that Cdc42p may have conserved functions in these other eukaryotes.

View larger version (59K): [in this window] [in a new window]   FIG. 1.   Cdc42p mutations and X-ray crystal structure. (A) Comparison of Cdc42p sequences from S. cerevisiae (Sc [242]), C. albicans (Ca [394]), S. pombe (Sp [390]), C. elegans (Ce [88]), D. melanogaster (Dm [336]), chicken (Gd [172]), dog (Cf [GenBank no. Z49944]), mouse (Mm [172]), human (Hs [525]), mouse brain (Mmb [367]), and human brain (G25K [407]). Included are the known Cdc42 mutations mapped onto the primary amino acid sequences and the known functional domains: GTP binding/hydrolysis domains (blue boxes), effector domain (red box), Rho insert domain (purple box), and membrane localization domain (green box). Secondary-structure elements indicated below the sequence are taken from reference 145. (B) Two views of the X-ray crystal structure of Cdc42Hs complexed with GDP (kindly provided by N. Nassar and R. Cerione, Cornell University). Color coding for functional domains is the same as in panel A.

The Cdc42 family of proteins currently has 11 members ranging in size from 190 to 192 amino acids (Fig. 1A). Within this family, there is a very high degree of sequence conservation, ranging from ~75% amino acid identity between C. albicans Cdc42p and the human brain isoform G25K to 100% identity between the dog, mouse, and human Cdc42p and 100% identity between the mouse brain and human brain (G25K) isoforms. Cdc42 proteins display ~40% similarity to other Ras-like GTPases, but this similarity is clustered in the four domains implicated in GTP binding and hydrolysis (Fig. 1, blue boxed domains). The most obvious difference between Cdc42p and Ras protein sequences in these domains is in amino acids 115 to 118. Ras proteins contain the diagnostic sequence Asn-Lys-Xaa-Asp (NKXD, where X is any amino acid), while all Cdc42p proteins contain the signature sequence Thr-Gln-Xaa-Asp (TQXD, with X being predominantly an Ile residue). It has been postulated that these differences may account for the ~10-fold-higher rate of GTP hydrolysis observed with Cdc42p proteins than with Ras proteins (210), but this has not been experimentally tested to date (see "GTPase-activating proteins" below). All Cdc42 proteins contain the C-terminal sequence Cys-Xaa-Xaa-Leu except for the two brain isoforms (Cdc42Mmb and G25K [Fig. 1]), which end in a Phe residue. This conserved domain is necessary for proper membrane anchorage of Cdc42 proteins (see "Prenylation and subcellular localization" below). Much that is known about the functional domains of Cdc42p has been determined by analyzing gain-of-function, loss-of-function, and dominant negative mutations (shown in Fig. 1). A compendium of these mutations, along with their mutant phenotypes, is listed in Table 2.

The numerous cdc42 mutations analyzed to date (see below) have greatly aided in defining functional domains within Cdc42p. However, without the information derived from the crystal structure of purified Cdc42, these mutations do little to clarify the global structure of Cdc42 and hence the multiple interactions between Cdc42 and its regulators and effectors. This problem has recently been resolved with the determination of the solution structure of Cdc42Hs by nuclear magnetic resonance (NMR) spectroscopy techniques (145), along with the determination of the X-ray crystal structure of Cdc42Hs bound to GDP (Fig. 1B) (413a). Several of the more interesting and informative mutations have been mapped onto the crystal structure (Fig. 1), and they highlight the potential functional domains of Cdc42p. The four domains implicated in the binding and hydrolysis of GTP are highlighted in blue in Fig. 1. The structure of these domains is similar to those found in the Ras and Rac crystal structures, highlighting the conservation of structure and function between different guanine nucleotide binding proteins.

Clearly, one of the more interesting and functionally important domains is the effector or switch I domain between residues 26 and 50 (highlighted in red in Fig. 1) (see "Effector domain" below). This domain forms an extended 2-strand/loop structure covering a large proportion of one face of the molecule. Based on its extended structure, it is easy to see how different effectors or regulators of Cdc42p could bind to different subdomains of the effector domain, possibly at the same time, as suggested by the analysis of different effector domain mutations. One effector/regulator could be bound to the N-terminal proximal domain around residue 35, which is in close proximity to the bound nucleotide, while another could be bound to the N-terminal distal region around residue 44. In addition, binding of an effector protein to this domain could interfere with the binding of other effector/regulator proteins to this domain, thereby providing a basis for the regulation of the myriad of Cdc42-dependent cellular processes.

The domain that makes Rho-type GTPases unique within the Ras superfamily is the so-called Rho insert domain (highlighted in purple in Fig. 1). This extra ~13 amino acids is -helical and has been implicated in Cdc42 interactions with one of its downstream effectors, the IQGAPs (379, 605), as well as its GDIs (605). In studies with a chimeric Cdc42Hs in which the insert domain between residues 120 to 139 was replaced with residues 121 to 127 of Ha-Ras, the resulting Cdc42Hs-L8 protein showed a two- to threefold-reduced affinity for the carboxyl-terminal (97-kDa) half of IQGAP1, which contains the GRD Cdc42 binding domain (379, 605) (see "Cdc42p downstream effectors" below). While this Cdc42Hs-L8 protein did not exhibit altered interactions with Rho-GDI from the perspective of the ability of Rho-GDI to extract Cdc42Hs-L8 from membranes, it had a greatly reduced sensitivity to the Rho-GDI-dependent inhibition of GDP dissociation or GTP hydrolysis (605). These results suggest that the insert domain is mediating some of the effects of the Rho-GDI. Rho-GDI binding to Cdc42Hs requires C-terminal prenylation (307) (see "Mammalian GDIs" below), suggesting that Rho-GDI binds to the C-terminal prenylation domain. The fact that the insert domain is on the other side of the Cdc42 molecule from the C-terminal prenylation domain (Fig. 1B) makes it likely that Rho-GDI binding induces a conformational change in the structure of the insert domain, possibly leading to the insert domain shifting its location to block the guanine nucleotide binding pocket, thereby "locking" the Cdc42 protein in either a GDP- or GTP-bound state and inhibiting GDP dissociation or GTP hydrolysis (see "Mammalian GDIs" below). It is interesting that Ras proteins do not have an insert domain and also do not seem to have physiological interactions with GDI proteins. Recently, the insert domain was shown to play a role in the ability of Cdc42 to transform NIH 3T3 fibroblasts (606). The L8 deletion mutation (see above) could intragenically suppress the transforming ability of the Cdc42^F28L mutant protein without affecting its ability to bind GTP, induce Jun N-terminal kinase (JNK) and p21-activated kinase (PAK) activities, or induce filopodium formation (see "Mammals" under "Functional studies" below). These results further highlight the ability of Cdc42p to differentially function in multiple cellular processes through interactions between its different structural domains and downstream effectors (see "Cdc42p downstream effectors" below).

In addition to the effector domain, Cdc24p and other GEFs interact with Cdc42 through other domains, including residues 82 to 100, which encompass the 4-strand-3-helix region, and residues 140 to 150, which encompass the 4-helix. The 3- and 4-helices lie on the same face of Cdc42p highlighted by the S86, W97, and G142 residues (Fig. 1B, right). The dominant negative S86P mutation lies in the loop region between the 4-strand and the 3-helix and it interferes with interactions between S. cerevisiae Cdc42p and Cdc24p (116). Interestingly, this loop region makes close contacts with residues in the Rho insert domain (see above), and this region undergoes chemical shift changes in NMR spectroscopy studies upon binding of the nonhydrolyzable GTP analog GMPPCP (145), suggesting that it may be an additional switch region (i.e., switch III). The nature of the S86P dominant negative phenotype is unknown, but it is not due to sequestration of Cdc24p as is the mechanism of action of the D118A mutant allele (116). The W97R (3-helix) and G142S (4-helix) mutations are ts loss-of-function alleles in S. cerevisiae (391) and although their mechanisms of action are unknown, the W97R mutation leads to a bud site selection defect, implicating Cdc42p in the initial selection of a nonrandom bud emergence site. Taken together, these observations show that the face of Cdc42p defined by the 3- and 4-helices plays a critical role in Cdc42p function. The structure and function of the C-terminal membrane localization domain (highlighted in green in Fig. 1) are discussed below (see "Prenylation and subcellular localization").

The Cdc42 domains involved in guanine nucleotide binding and GTP hydrolysis (blue boxes in Fig. 1) have been inferred through structural similarities to domains in other GTPases and through the analysis of activated and dominant negative cdc42 mutations. The initial cdc42 mutations were analyzed in S. cerevisiae (642) and were based on the paradigmatic Ras mutations that led to oncogenic transformation. The cdc42^G12V and cdc42^Q61L mutations are analogous to H-ras mutations that cause a decreased level of intrinsic GTPase activity, thereby shifting the mutant proteins to an "activated" GTP-bound state. In S. cerevisiae, these cdc42 mutations were lethal, resulting in large, multibudded cells with aberrant cortical actin structures localized in multiple buds (642). These phenotypes suggested that the mutant proteins were activated and constitutively interacting with downstream components of the pathway, leading to polarization, albeit incorrectly, of the actin cytoskeleton. The H-ras^D119A mutation also leads to an activated phenotype; however, this phenotype was mechanistically due to an increased GDP dissociation rate, which is thought to result in a higher probability of the protein being in a GTP-bound state due to the higher concentration of GTP than of GDP in the cell. The phenotype of the S. cerevisiae cdc42^D118A mutant was unexpected and different from the cdc42^G12V and cdc42^Q61L activated phenotypes. The cdc42^D118A mutant phenotype was temperature-dependent dominant lethal and resulted in large, round, unbudded cells that were phenotypically similar to cdc42^ts mutants grown at restrictive temperatures. This dominant negative phenotype suggested that the Cdc42^D118A mutant protein could bind and sequester the cellular factor(s) necessary for the budding process (see "S. cerevisiae Cdc24p" below).

Expression of equivalent mutant proteins in S. pombe gave different results (390). First, unlike the activated and dominant negative phenotypes seen in S. cerevisiae, the morphological phenotypes of cells overproducing the cdc42^G12V, cdc42^Q61L, and cdc42^D118A mutant gene products were similar to one another. Second, the mutant constructs did not exert a dominant lethal effect in S. pombe cells. Instead, S. pombe cells overproducing these mutant proteins exhibited an abnormal morphology of enlarged, round or misshapen cells with delocalized cortical actin structures, as opposed to the small, round cellular morphology of cdc42 loss-of-function and dominant negative mutants (390, 447). The cdc42^D118A mutant phenotype also was temperature dependent in S. pombe, suggesting that the mutant protein loses either a critical interaction or its three-dimensional structure upon shift to higher temperature, leading to its mutant morphology. Interestingly, septum formation was still evident in these mutant cells, even though the presence of an organized actin ring was not, suggesting that septation can occur in the absence of an actin ring. However, this point must be clarified by experiments with either actin mutants or actin polymerization inhibitors such as latrunculin A.

While activated or dominant negative cdc42 mutations have not been analyzed in C. elegans to date, expression of the cdc42^G12V allele in Drosophila ovaries led to defects in actin distribution whereas expression of dominant negative cdc42 alleles led to defects in actin organization in imaginal discs and wing hairs (see "Drosophila" under "Functional studies" below), reinforcing a role for Cdc42p in regulating actin function. For a discussion of mutations in mammalian Cdc42p, see "Mammals" under "Functional studies" below.

The so-called effector domain between residues 26 and 48 of the Ras GTPase is required for downstream effector function (370, 460). The effector or switch I domain between residues 26 and 50 is highly conserved among Cdc42 proteins (Fig. 1A) but diverges among closely related but not functionally homologous Rac GTPases (239). The current paradigm is that GTPases bind to GEFs when in the nucleotide-free or GDP-bound state and bind to GAPs and downstream effectors when in the GTP-bound state. Since the switch I and II (residues 60 to 76) domains are the predominant regions of GTPases that change conformation upon binding different guanine nucleotides, it is likely that multiple factors interact with these regions. Given that Cdc42p interacts with multiple downstream effectors along with regulatory factors such as GEFs and GAPs (see "Cdc42p regulators" and "Cdc42p downstream effectors" below), it is likely that the specificity of interaction will be through either different residues within the Cdc42p effector domain, competition between effectors and regulators, and/or interactions at different times in the cell cycle.

A predominant binding partner for the effector domain is the CRIB (for "Cdc42/Rac interactive binding") domain (also known as the PBD, GBD, or PAK domain [see Table 3]) found in many Cdc42p downstream effectors, including the PAK family of protein kinases (59). The highest-efficiency binding domain in the CRIB-containing PAK protein was residues 70 to 118, thereby defining the optimal CRIB domain as these 48 amino acid residues (558). In these studies, it was also shown that this domain interacted with Cdc42p at a ~3- to 10-fold-higher affinity than it interacted with Rac and that it interacted with activated (Q61L) alleles at a 5- to 10-fold-higher affinity than it interacted with the wild type, reinforcing the notion that CRIB-containing interacting proteins function as downstream effectors. A recent study in which NMR spectroscopy was used to probe the interactions between Cdc42Hs and 46 amino acids of the PAK CRIB domain showed that the CRIB binding domain surface on Cdc42Hs was the 2 switch I domain and part of the loop between the 1-helix and 2-strand (185). In addition, nuclear Overhauser effect contacts suggested that the formation of an intermolecular -sheet was the basis for the Cdc42Hs-CRIB domain interactions. The CRIB domain of the Wiskott-Aldrich syndrome protein (WASP) downstream effector (see "Cdc42p downstream effectors" below) was dissected by a variety of biophysical techniques including fluorescence spectroscopy, surface plasmon resonance, circular dichroism, and NMR spectroscopy (498). The results indicated that a core 26-amino-acid fragment (residues 221 to 257) was necessary for binding to GST-Cdc42, but higher affinity binding was observed with a larger (120-amino-acid) fragment (residues 201 to 321), suggesting that the CRIB domain was necessary but not sufficient for high-level binding. In addition, these studies suggested that the isolated CRIB domain does not exhibit any apparent secondary structure; it is unknown if the CRIB domain would form a secondary structure, possibly -strands (see above), within the context of the entire protein.

Mutations that disrupt the interaction between Cdc42p and downstream effectors should define the effector domain and should suppress dominant activated cdc42^G12V mutant phenotypes. The T35A allele was thought to be a paradigmatic effector domain mutation in that it could interfere with the ability of S. cerevisiae Cdc42p to interact with the PAK family of protein kinases and could suppress the dominant-activated cdc42^G12V mutant but could not complement the loss-of-function cdc42-1^ts allele. However, the T35A mutation also suppressed the dominant negative S. cerevisiae cdc42^D118A allele and interfered with two-hybrid protein interactions between Cdc42^D118Ap and the Cdc24p GEF (116), suggesting that the effector domain may also interact with the Cdc24p GEF. Corroborating this hypothesis are the results obtained with the Y32K and F37E mutations in the Cdc42Hs effector domain, which caused a loss of Cdc24-stimulated GDP dissociation (323), and the Cdc42Hs F28L mutation, which led to rapid nucleotide exchange and transformation of NIH 3T3 cells similar to that seen with the Cdc24p homolog Dbl (327). Interestingly, the T25K, N26D, and Y40K mutations within the Cdc42Hs effector domain did not show a loss of Cdc24-stimulated GDP dissociation (323), which could be a function of the individual mutational changes or could indicate a level of specificity at the individual amino acid residue for interactions with GEFs.

The S. cerevisiae cdc42^V44A mutation represents a new class of effector domain mutations in that it could complement the cdc42-1^ts allele (475a), suggesting that it did not lead to a nonfunctional protein; it also interfered with interactions with the upstream effector Cdc24p (116). In addition, the cdc42^V44A mutant displayed a morphological phenotype of elongated buds with multiple nuclei, which is suggestive of either a delay at the apical/isotropic switch and morphogenesis checkpoint (see "S. cerevisiae" under "Functional studies" below) and/or a defect in cytokinesis (475a). The V44A mutation interfered with two-hybrid protein interactions between Cdc42p and the S. cerevisiae Cla4p PAK-like kinase but not the Ste20p or Skm1p PAK-like kinases and also between Cdc42p and the Gic1p and Gic2p downstream effectors but not the Bnip or Iqg1p scaffold proteins (see "Cdc42p downstream effectors" below). All of these proteins contain CRIB domains, suggesting that the effector domain may differentially interact with multiple CRIB domain-containing effectors. This hypothesis is substantiated by mutations in the Cdc42Hs effector domain that differentially affected interactions with mammalian downstream effectors (291, 379) (see "Cdc42p downstream effectors" below). The Y40C mutation interfered with interactions between Cdc42p and downstream PAKs and other proteins containing CRIB domains, leading to a loss of p65^PAK kinase activation in transfected COS fibroblasts, but it did not affect Cdc42p-dependent actin reorganization, as evidenced by normal filopodium and integrin complex formation in Swiss 3T3 cells (291). The D38E mutation interfered with in vitro binding to an mPAK-3 CRIB domain peptide (310) but had no effect on the binding of two other downstream effectors, IQGAP1 and IQGAP2. The Cdc42Hs F37A mutation did not affect interactions with CRIB-containing proteins or actin reorganization (291). Taken together, these results suggest that there are different classes of effector domain mutations that can be distinguished by their morphological phenotypes and protein-protein interactions. These different mutations may define interactions with different effectors or regulators, thereby allowing us to dissect the multiple pathways leading from Cdc42p (see "Cdc42p downstream effectors" below). Given the possibility that the Cdc42p effector domain interacts with CRIB-containing proteins through formation of an intermolecular -sheet (see above), it is likely that the nature and orientation of amino acid side chains emanating from the 2 strand (Fig. 1) have an influence on this differential binding. It should be noted, however, that not all Cdc42p-interacting proteins contain recognizable CRIB domains, suggesting that there may be multiple mechanisms by which proteins interact with Cdc42p.

In addition to the effector/switch I domain mutations that affect interactions between Cdc42 and its GEFs, there are mutations in other domains that affect either binding to GEFs or GEF-induced GDP dissociation. Mutations within the switch II domain, including D63L, D65K, R66D, R68A, P69A, and F78L, did not affect Cdc24-stimulated GDP dissociation, but the Q116K mutation in the Cdc42 signature GTP binding domain did (323). Analysis of chimeric Cdc42Hs-RhoA proteins indicated that residues 82 to 120 and 121 to 155 are necessary for Cdc24 responsiveness (323). Within the first domain, the S86P and S89P mutations in S. cerevisiae Cdc42p led to a cold-sensitive, dominant negative phenotype and, in the case of S86P, led to a loss of interaction with Cdc24p (116). Similar mutations in Drosophila Cdc42p also led to a dominant negative phenotype (see "Drosophila" under "Functional studies" below). Other mutations that led to a loss of interaction with Cdc24p in S. cerevisiae included the I117S mutation within the Cdc42 signature GTP binding domain and the T138A and L165S mutations (116). In addition, the C157R mutation within one of the highly conserved GTP binding domains led to a partially cold-sensitive dominant negative phenotype. The C-terminal membrane localization domain (see "Prenylation and subcellular localization" below) does not seem to be necessary for GEF interactions (323). Interestingly, the S86, S89, and T138 residues all lie on the same face of the Cdc42 protein (Fig. 1B), suggesting that this may be a conserved interactional interface between Cdc42p and its GEFs.

In all organisms examined, Cdc42p is prenylated with a C₂₀ geranylgeranyl isoprene group at a C-terminal Cys residue, and this prenylation is necessary for the membrane attachment of Cdc42p. S. cerevisiae and S. pombe Cdc42p fractionated into both soluble and particulate fractions, suggesting that Cdc42p can exist in two cellular pools (390, 643). S. cerevisiae Cdc42p was found predominantly in the particulate fraction, but a significant soluble pool, up to ~20% in some instances, could be observed. Given the existence of GDI proteins in S. cerevisiae and mammalian cells that can interact with Cdc42p and extract Cdc42p from membranes (see "Guanine nucleotide dissociation inhibitors" below), the soluble pool of S. cerevisiae Cdc42p is probably either nonprenylated or complexed with the rho-GDI protein Rdi1p (267, 373). The particulate form of Cdc42p could be solubilized by added detergent but not by added NaCl or urea, suggesting that Cdc42p was tightly associated with either a cellular membrane or a cytoskeletal complex. When synchronous cultures were used, the fractionation pattern of S. cerevisiae Cdc42p did not appear to vary through the cell cycle (643). However, recent studies indicating that Cdc42p functions at different places in the cell at different times of the cell cycle (see below) suggest that fractionation patterns may not be a very sensitive measure of Cdc42p localization.

By using Cdc42p-specific antibodies in immunofluorescence and immunoelectron microscopy, S. cerevisiae Cdc42p was localized to the plasma membrane at sites of polarized growth (642, 643). These sites coincided with the sites of cortical actin localization and included invaginations of the plasma membrane at the site of bud emergence, the tips of growing buds, and the tips of mating projections in pheromone-arrested cells (3, 259). This localization pattern was consistent with Cdc42p functioning in controlling polarized cell growth during the mitotic cell cycle and mating. Recently, functional green fluorescent protein (GFP)-Cdc42p fusion proteins have been localized to the mother-bud neck region in S. cerevisiae and the septum area in S. pombe, suggesting that Cdc42p also plays a role in cytokinesis and/or septation in both yeasts (508a). This localization of Cdc42p at different sites of polarized growth during the S. cerevisiae cell cycle, which is mirrored by the localization of the actin cytoskeletal network, suggests that the subcellular localization of Cdc42p is under temporal and spatial control during the cell cycle. It should be noted that Cdc42p localization to sites of polarized growth was not disrupted by incubation with the actin-depolymerizing drug latrunculin-A (21), suggesting that Cdc42p localization occurs independently of actin localization and of the structural integrity of the actin cytoskeleton.

S. cerevisiae Cdc42p contains the C-terminal ¹⁸³Lys-Lys-Ser-Lys-Lys-Cys-Thr-Ile-Leu sequence which is essential for the membrane localization of Cdc42p. In the Cdc42Hs NMR and crystal structures, this region forms a flexible tether that is separated from the body of Cdc42Hs by two Pro residues at positions 179 and 180 (145) (Fig. 1B), thereby allowing the bulk of Cdc42p to be sterically unhindered by membrane attachment and accessible for binding to other proteins. This region is modified by geranylgeranylation at the Cys¹⁸⁸ residue (indicated by an underbar in the above sequence), which is necessary for its anchoring within the plasma membrane (147, 643). This is thought to be followed by proteolytic cleavage of the last three amino acids and carboxyl methylation of the now C-terminal Cys residue, although this has not been experimentally shown with S. cerevisiae Cdc42p (see below). The geranylgeranylation is deemed necessary because the C188S mutation, which renders the protein incapable of being prenylated, can intragenically suppress the dominant lethality associated with the cdc42^G12V, cdc42^Q61L, and cdc42^D118A mutations (642) and because the S. cerevisiae and S. pombe Cdc42^C188S mutant proteins, either by themselves or as GFP-Cdc42^C188Sp fusion proteins, are nonfunctional, delocalized proteins that fractionate almost exclusively into soluble pools (508a, 642, 643). Whether this prenylation is sufficient for Cdc42p targeting to the sites of polarized growth is not known.

An increase in soluble S. cerevisiae Cdc42p was observed in cdc43-2^ts (643) and cdc43-5^ts (434) mutant cell extracts, suggesting that membrane localization of Cdc42p depended on Cdc43p-dependent geranylgeranylation. Cdc43p was originally identified by ts mutations that led to cell cycle arrest of large, unbudded cells, a phenotype similar to cdc42^ts mutants (2). In addition, cdc43^ts cdc42^ts double mutants displayed a synthetic lethal phenotype at 23°C, suggesting that these gene products may interact. Another mutation in CDC43, designated cal1-1, was identified by its calcium-dependent phenotype (437); the cal1-1 mutant required 100 mM CaCl₂ for growth. The predicted amino acid sequence of the CDC43/CAL1 gene (240, 435) showed significant similarity to the S. cerevisiae DPR1/RAM1 gene (173), which encoded the  subunit of the protein farnesyltransferase (FTase), which modified the C termini of Ras GTPases. Type I protein geranylgeranyltransferase (GGTase I) activity was reconstituted from Escherichia coli cells that overproduced both Cdc43p and Ram2p (377, 539, 540), and S. cerevisiae GGTase I activity was decreased in cdc43^ts and ram2 mutants but not ram1 mutants (147, 377, 434), indicating that Cdc43p and Ram2p encoded the  and  subunits, respectively, of the S. cerevisiae GGTase I. The Ram2p  subunit also acts as the  subunit for the S. cerevisiae FTase (214), which may account for the in vitro and in vivo cross-specificity that is observed between FTase and GGTase I activities in S. cerevisiae (66, 565). S. cerevisiae GGTase I is an Mg²⁺-requiring Zn²⁺ metalloenzyme (377, 539), but it can also function with Ca²⁺ as the only divalent cation (377). Added Ca²⁺ could not rescue the reduced in vitro GGTase I activity from cal1-1 mutant cell extracts (434), but only 20 mM CaCl₂ was added, as opposed to the 100 mM CaCl₂ needed to rescue the in vivo cal1-1 growth defect (435, 437). The essential targets for S. cerevisiae GGTase I seem to be Cdc42p and Rho1p, because certain cdc43/cal1 alleles can be suppressed by overexpression of one or both of these GTPases (434, 439).

Another localization determinant consists of the four Lys residues that are next to the C-terminal prenylated Cys residue. This polylysine region is not found in most Ras-like GTPases, and its positive charges may be interacting with negatively charged components, either protein or phospholipid, at the membrane site to play a role in enhancing membrane association or specific targeting of Cdc42p. A similar polylysine domain is found in the K-Ras protein and is important for membrane localization; altering the Lys residues to Gln results in delocalized K-Ras protein (196, 197). In addition, the analogous polylysine domain in Rac1 was recently shown to be important for interactions with PAK effector kinases (266). Mutating the four Lys residues to Gln in S. cerevisiae Cdc42p, creating the cdc42^K183-187Q mutant protein, led to a partial delocalization of the mutant protein (116), suggesting that this domain played a role in targeting or anchoring Cdc42p to the plasma membrane. The K183-187Q mutation could intragenically suppress the dominant lethal cdc42^G12V mutant, and expression of the cdc42^K183-187Q mutant gene on a plasmid could complement the cdc42-1^ts mutant. The ability of the cdc42^K183-187Q mutant gene to complement the cdc42-1^ts mutant (in contrast to the nonfunctional cdc42^C188S mutant gene, which cannot complement the cdc42-1^ts mutant [642]), together with the partial delocalization of the mutant protein, suggested that the K183-187Q mutation had an intermediate effect on Cdc42p function and that the polylysine domain of Cdc42p was necessary but not sufficient for complete plasma membrane localization. Another interesting mutation in this domain, the cdc42^K186R mutant allele, exhibited a ts loss-of-function phenotype in S. cerevisiae (391) and displayed a morphological phenotype of elongated buds and multiple nuclei suggestive of either a delay at the morphogenesis checkpoint (see "S. cerevisiae" under "Functional studies" below) and/or a cytokinesis defect at permissive temperatures (106a). The nature of this mutation (Lys to Arg) suggested that these phenotypes were not due to a change in charge or conformation of the protein but more probably were due to altered interactions with another protein. However, recent results indicate that this mutant protein has an increased intrinsic GTPase activity (see "GTPase-activating proteins" below), suggesting that improper negative regulation of this mutant protein may be playing a role in its phenotypes. Therefore, the mechanism by which this mutant protein exerts its effects remains to be fully elucidated.

All known Cdc42 proteins contain the C-terminal sequence Cys-Xaa-Xaa-Leu, with the exception of the mouse and human brain G25K isoforms, which end in a Phe residue instead of Leu (Fig. 1). Mammalian Cdc42p posttranslational modifications have been analyzed biochemically with protein purified from bovine brain cells (22, 612, 613), cultured murine erythroleukemia cells (354), rat and human pancreatic islet cells (274), or rat kidney cells (45), not with recombinant protein, and so it is unclear whether these studies were performed on the Cdc42Hs or G25K isoform. Regardless, it is clear that the membrane-bound form of mammalian Cdc42p is geranylgeranylated at the Cys residue, the last three amino acids are proteolytically removed, and the now C-terminal prenylated Cys residue is carboxyl methylated, resulting in a protein with a S-(all-trans-geranylgeranyl) cysteine methyl ester at its C terminus (612). These modifications are necessary for membrane localization, and, as with S. cerevisiae Cdc42p, mammalian Cdc42p fractionates to both particulate and soluble pools (45, 354).

Carboxyl methylation of soluble Cdc42p from bovine brain (22), rat kidney cells (45), or pancreatic islet cells (274) seems to be GTP stimulated, but methylation of the membrane-bound form is not (613), presumably because the membrane-bound form is already GTP bound. The methyltransferase activity from brain extracts (613) and insulin-secreting cells (318) was membrane bound. Recently, a human myeloid prenylcysteine carboxyl methyltransferase with in vitro activity against Cdc42Hs was shown to localize to the endoplasmic reticulum membrane (114); the S. cerevisiae Ste14p prenylcysteine carboxyl methyltransferase is also found in the endoplasmic reticulum membrane (490), but it has not been shown to have in vitro or in vivo activity against Cdc42p. Interestingly, addition of glucose to pancreatic islet cells extracts stimulated the carboxyl methylation of Cdc42p (274), and inhibition of Cdc42p function by Clostridium difficile toxins A or B resulted in reductions in glucose-stimulated insulin secretion (273), suggesting that Cdc42p may play a role in glucose-stimulated insulin secretion.

Using affinity-purified anti-Cdc42 antibodies, Cdc42p from rabbit liver was shown to associate with a membrane fraction highly enriched in Golgi membranes (137). In pancreatic islet cells (274) and rat kidney cells (45), Cdc42 was predominantly cytosolic, but it was translocated to the particulate pool upon addition of guanosine 5'-(3-O-thio)triphosphate (GTPS). In NR-6 fibroblasts and rat kidney cells, Cdc42p localized to a perinuclear region that coincided with markers for the Golgi complex including the 110-kDa subunit of the coatomer complex -COP (137). This localization was rapidly altered to general cytosolic localization upon addition of brefeldin A (BFA), a drug which inhibits vesicle formation at the Golgi membrane by inhibiting the guanine nucleotide exchange activity for the Arf GTPase, suggesting that Cdc42p may play a role in or be subject to intracellular membrane trafficking events. BFA-induced delocalization of Cdc42p was inhibited by AlF₄ and by expression of GTPase-defective Arf, while expression of a dominant negative Arf mutant resulted in BFA-independent delocalization of Cdc42p. These results suggest that association of Cdc42p with Golgi membranes is dependent on the guanine nucleotide-bound state of the Arf GTPase. It should be noted that in these experiments, the NR-6 fibroblasts and rat kidney cells did not exhibit polarized growth patterns to a region of their cell periphery. In human HeLa cells transiently transfected with a epitope-tagged Cdc42^G12V protein, the epitope-tagged protein localized to focal complexes and to regions of polarized growth within the Cdc42^G12V-induced peripheral actin microspikes (PAMs) (see "Mammals" under "Functional studies" below) and colocalized with actin and PAKs within these PAMs (127, 356). In addition, HA-tagged Cdc42^G12V protein co-localized with the IQGAP1 downstream effector to cell-cell contact sites of Madin-Darby canine kidney cells (282).

In Drosophila wing disc epithelial cells, Cdc42p localized in a polarized manner to the basal and apical regions (128). In elongating cells, Cdc42p was restricted to the apical and basal membranes, but in nonelongating cells, it was found on lateral membranes as well. This localization pattern was also seen for the actin cytoskeleton, again providing a mechanistic link between Cdc42 and actin rearrangements. C. elegans Cdc42p was shown to fractionate predominantly to a particulate fraction from mixed-stage populations of C. elegans cells and to localize in a polarized manner to both the circumferential and longitudinal boundaries of hypodermal cells during hypodermal cell fusion in embryo elongation, in a pattern similar to that of the C. elegans PAK homolog (87).

In summary, Cdc42 proteins are membrane bound through their posttranslational modifications and are localized to either internal membranes or the plasma membrane at locations where polarized events are occurring. While prenylation is necessary for membrane anchorage, it is not known if it is sufficient for proper targeting. The mechanism by which Cdc42 proteins are targeted to appropriate membranes in regions of polarized cell growth is unknown, but it is likely to be through protein-protein or protein-lipid interactions at the site.

S. cerevisiae alters its morphology in response to both exogenous and endogenous signals, leading to either bud emergence and enlargement during the mitotic cell cycle, mating-projection ("shmoo") formation through the mating/pheromone response pathway in response to exogenous mating-factor pheromones, pseudohyphal formation and filamentous growth in response to starvation conditions, or spore formation during meiosis. Cdc42p has been implicated in regulating the first three processes but not in sporulation to date. The mechanisms by which Cdc42p regulates the generation of, and switching between, these different morphogenetic patterns is still unclear, but Cdc42p interactions with the actin cytoskeleton play a critical role in this regulation. The functional connection between Cdc42p and the cortical actin cytoskeleton has recently been reinforced by the observation that Cdc42p can stimulate actin polymerization in permeabilized S. cerevisiae cells (324).

Mitotic cell cycle. The morphological changes that occur during the S. cerevisiae mitotic cell cycle can be divided into five sequential phases: (i) selection of a nonrandom bud emergence site and the organization of the protein machinery at that bud site, including rearrangement of the cortical actin cytoskeleton; (ii) bud emergence and polarized growth towards and within the emerging bud; (iii) a switch from apical to isotropic bud growth (the "apical-isotropic switch" [see below]); (iv) cytokinesis, septum formation, and cell separation; and (v) isotropic growth of undersized daughter cells after cell separation prior to the initiation of their next cell cycle (reviewed in references 77, 126, and 465) (Fig. 2). A variety of data suggest that Cdc42p can function at multiple stages of the cell cycle. As mentioned above (see "Cdc42p structure and functional domains"), the initial characterization of S. cerevisiae Cdc42p suggested that it plays a role in the actin-dependent generation of cell polarity during the process of bud emergence. Subsequent analysis of ts, dominant activated, and dominant negative cdc42 alleles substantiated this inference and suggested an additional function in the initial selection of the site of bud emergence. Analysis of the cdc42^V44A and cdc42^K186R mutant alleles, along with the subcellular localization of Cdc42p to the mother-bud neck region in large-budded cells, raises the possibility that Cdc42 functions either within the apical-isotropic switch at a morphogenesis checkpoint (Fig. 2) (see below) and/or in controlling actin-dependent events that occur during cytokinesis and septum formation. In addition, GTP-bound Cdc42p functions with the mitotic cyclin Clb2p-Cdc28p kinase complex to lead to the mitosis-specific phosphorylation of several substrates (see below). A potential model that is consistent with the proposed roles for Cdc42p throughout the cell cycle is presented in Fig. 3; a detailed discussion of individual protein components of the model can be found under the individual protein subsections in "Cdc42p regulators" and "Cdc42p downstream effectors" below.

View larger version (34K): [in this window] [in a new window]   FIG. 2.   Polarized cell growth during the S. cerevisiae and S. pombe cell cycles. See the text for details.

View larger version (59K): [in this window] [in a new window]   FIG. 3.   Molecular and cytological model for polarized cell growth in S. cerevisiae. (A) Molecular model for the Cdc42-dependent processes during the S. cerevisiae cell cycle. Shaded boxes attached to GTPases are isoprenyl groups. The stars by Ste20 in the bud emergence complex and Cla4 in the cytokinesis complex indicate that they are the likely PAK functioning at this stage of the cell cycle. See the text for details. (B) Cytological model for bud emergence and cytokinesis. Cdc42 complexes are the same as in panel A. See the text for details.

Mating pathway. The S. cerevisiae mating pathway is a classic signal transduction pathway in which an extracellular signal (peptide pheromone) binds to a G-protein coupled transmembrane receptor, thereby activating a MAP kinase cascade that ultimately leads to a number of cellular events, including the transcriptional induction of genes necessary for the mating process, a G₁ arrest as a prelude to cell-cell fusion and karyogamy, and the generation of unique morphological structures (mating projections or shmoos) that are the sites of contact for cell-cell fusion and mating (for reviews, see references 26, 297, 350, and 368) (Fig. 4A). The notion that Cdc42p plays a role in the S. cerevisiae mating pathway came from the analysis of cdc42 mutants, the subcellular localization of Cdc42p, and its interactions with the Ste20p protein kinase. The mating efficiencies of loss-of-function cdc42 mutants were reduced, and signaling through the pheromone response pathway was diminished (527, 626), while expression of the dominant activated cdc42^G12V mutant allele led to a modest (two- to threefold) increase in signaling as assayed by FUS1-lacZ expression (9), suggesting that Cdc42p plays a role in the activation of the pheromone response MAP kinase cascade (see below). In addition, Cdc42p localized to the tips of mating projections in pheromone-arrested cells (643), suggesting that it plays a direct role in pheromone-induced morphological changes.

View larger version (50K): [in this window] [in a new window]   FIG. 4.   Comparison of Cdc42 interactions and dependent processes in S. cerevisiae (A), S. pombe (B), and mammals (C). Color and shape coding is given in the box at the bottom of figure. Two-headed arrows indicate physical interactions. Single-headed arrows indicate pathways; dotted arrows indicate potential involvement in pathways. For simplicity, not all components of the actin cytoskeleton or JNK kinase cascade are shown. See the text for details.

Pseudohyphal and invasive growth. S. cerevisiae cells can alter their morphogenetic patterns in response to starvation conditions, leading to filamentous growth and the generation of pseudohyphae (for reviews, see references 26, 167, 297, 349, and 350). Diploid cells respond to nitrogen starvation by altering their cell cycles, budding patterns, cell shape, and cell separation patterns, resulting in polarized elongated budded cells that resemble fungal hyphae (168, 280). Haploid cells can also be induced to filamentous growth, which is manifested as invasive growth into agar plates (43, 487). A detailed mutational analysis of actin mutants indicated that the actin cytoskeleton plays a critical role in various aspects of pseudohyphal growth (63). The primary signaling route leading to pseudohyphal growth involves the Ras2 GTPase signaling through Cdc42p to several components of the pheromone response MAP kinase cascade, including Ste20p, Ste11p, Ste7p, and Kss1p, thereby activating the Ste12p transcription factor which, together with the Tec1 transcription factor, induces the expression of genes necessary for filamentous growth (Fig. 4A) (168, 330, 348, 351, 405, 487, 488).

In the rod-shaped fission yeast S. pombe, there are three switches in polarized cell growth patterns during the cell cycle (Fig. 2). First, selective and polar growth is initiated at the beginning of the cell cycle at the "old end" of the cell, which is the end distal to the previous division site (reviewed in reference 425). This growth occurs at the end of the cylindrical cell and can be monitored by staining with the dye Calcofluor and by the presence of cortical actin dots (366, 376). Second, after ~0.3 of the cell cycle, a switch in polarized growth, referred to as new-end takeoff, occurs from unidirectional at the old end to bidirectional at both ends (Fig. 2). This growth pattern is visualized by the appearance of both Calcofluor staining and cortical actin dots at the new end and depends on the cell attaining a minimal length and completing the S phase. Third, bipolar growth continues until ~0.75 of the cell cycle, at which time cortical actin reorganizes to the site of septum formation and end growth ceases, resulting in a constant-length stage of the cell cycle. Following cytokinesis and cell separation, polarity must be re-established at the old end as a prelude to unipolar growth in the next cell cycle.

The S. pombe Cdc42p homolog (cdc42⁺) was isolated from an S. pombe cDNA library by functional complementation of the S. cerevisiae cdc42-1^ts mutation (390). The predicted amino acid sequence of S. pombe Cdc42p is 85% identical to those of both S. cerevisiae and human Cdc42p (Fig. 1). Disruption of cdc42⁺ showed that the gene was essential for growth. The S. pombe cdc42 loss-of-function phenotype was originally determined by generating a null allele in a haploid strain that was complemented by the wild-type allele on a plasmid and then inducing plasmid loss to uncover the loss-of-function phenotype (390). The morphological phenotype consisted of small, round, dense, uninucleate cells, which is strikingly different from that associated with cdc42 loss-of-function alleles in S. cerevisiae (2, 642) (see above). The S. pombe cdc42 null phenotype suggested that macromolecular synthesis continued but incorporation of new cellular material into an enlarging cell was inhibited, hence the small, dense, dead cells. Similar morphologies, as well as reduced mating efficiencies, were observed with the cdc42^T17N dominant negative allele (447), suggesting that Cdc42p functions within the mating pathway as well (see below). The uninucleate, 1C phenotype, as assayed by DAPI staining and fluorescence-activated cell sorter analysis, indicated that the mitotic cell cycle was blocked in G₁ phase, which is also different from the S. cerevisiae arrest phenotype of multinucleate cells. It is likely that the cell cycle coordination between DNA synthesis and Cdc42 function is more tightly regulated in S. pombe. Taken together, these data are consistent with Cdc42p functioning in the targeting and incorporation of new growth at the old end in G₁ phase, possibly by affecting protein secretion or secretory-vesicle fusion to the plasma membrane.

Recent data indicating that S. pombe Cdc42p localizes to the septum region (381a) raises the possibility that Cdc42p plays a direct role in septum formation. In S. pombe, cytokinesis begins in early M phase with the assembly of the actin-based medial ring followed by septum formation and cell separation (reviewed in reference 176). Given that GFP-Cdc42p in S. pombe localizes to the medial area in a ring-like structure in some cells that do not have a visible septum, Cdc42p may play a role in the early steps of medial ring formation prior to septum formation. Interestingly, S. pombe Cdc12p, a homolog of the S. cerevisiae Cdc42-interacting protein Bni1p (139, 232) (see "Cdc42p downstream effectors" below), has also been implicated in medial ring formation (74).

To date, two potential downstream effectors of Cdc42p has been characterized in S. pombe, the Pak1p/Shk1p (364, 447) and Pak2p/Shk2p (515, 616) protein kinases (see "PAK-like kinases" below). Pak1p/Shk1p is a CRIB domain-containing ~72-kDa serine/threonine protein kinase that belongs to the PAK family of Cdc42-interacting protein kinases. It can autophosphorylate on Ser and Thr residues and binds preferentially to Cdc42p-GTP. The physiological significance of these interactions was supported by the morphological abnormalities associated with the overexpression or absence of Pak1p and the synthetic-overdose phenotypes observed when overexpressing activated or dominant negative cdc42 alleles together with wild-type or kinase-defective pak1 mutants. Deletion of pak1 is lethal, resulting in small, round cells, a morphology reminiscent of cdc42 null mutants. This result indicates that Pak1p provides an essential function in the cell polarity pathway, which is different from its S. cerevisiae homologs Ste20p, Cla4p, and Skm1p. Pak1p and Cdc42p were also required for mating in S. pombe (see "PAK-like kinases" below). Taken together, the data are consistent with Pak1p being a downstream effector of Cdc42p in the cell polarity and mating pathways (Fig. 4B). Pak2p/Shk2p is a nonessential protein with the greatest similarity in predicted amino acid sequence to S. cerevisiae Cla4p and Skm1p (515). Its role as a downstream effector of Cdc42p is unclear (see "PAK-like kinases" below).

The Candida albicans CDC42 gene was identified by degenerate oligonucleotide PCR and isolated from a C. albicans genomic library by DNA-DNA hybridization to the PCR probe (394). C. albicans Cdc42p is 87.8% identical to S. cerevisiae Cdc42p throughout the entire coding region (Fig. 1A), and DNA-DNA hybridizations suggested that CDC42 is single copy. Analysis of mRNA levels indicated that there is a transient increase in Cdc42p expression in the dimorphic switch to bud emergence, suggesting that C. albicans Cdc42p plays a role in this process. C. albicans homologs of the S. cerevisiae cell polarity proteins Rsr1p/Bud1p (608), Rho1p (272), Cla4p (299), Ste20p and Ste7 (268, 296), Fus3p/Kss1p (110, 595), and Ste12p (329, 352) have also been identified, and many of them have been implicated in hyphal formation and candidiasis. It remains to be seen if Cdc42p also functions in pseudohyphal formation in C. albicans, as it does in S. cerevisiae.

The Caenorhabditis elegans Cdc42 gene seems to be more highly expressed in embryonic cells than in larvae or adults, and when expressed in S. cerevisiae, it could complement the cdc42-1^ts allele (88), indicating that it is a functional homolog. The C. elegans Cdc42 protein, expressed as a glutathione S-transferase (GST) fusion, could bind and hydrolyze GTP at rates comparable to the human Cdc42 protein (88). By using anti-Cdc42 antibodies, Cdc42p was shown to fractionate predominantly to a particulate fraction from mixed-stage populations of C. elegans cells and to localize to both the circumferential and longitudinal boundaries of hypodermal cells during hypodermal cell fusion in embryo elongation, in a pattern similar to that of t