A Cdc24p-Far1p-Gbetagamma protein complex required for yeast orientation during mating

Oriented cell growth requires the specification of a site for polarized growth and subsequent orientation of the cytoskeleton towards this site. During mating, haploid Saccharomyces cerevisiae cells orient their growth in response to a pheromone gradient overriding an internal landmark for polarized growth, the bud site. This response requires Cdc24p, Far1p, and a heterotrimeric G-protein. Here we show that a two- hybrid interaction between Cdc24p and Gbeta requires Far1p but not pheromone-dependent MAP-kinase signaling, indicating Far1p has a role in regulating the association of Cdc24p and Gbeta. Binding experiments demonstrate that Cdc24p, Far1p, and Gbeta form a complex in which pairwise interactions can occur in the absence of the third protein. Cdc24p localizes to sites of polarized growth suggesting that this complex is localized. In the absence of CDC24-FAR1-mediated chemotropism, a bud site selection protein, Bud1p/Rsr1p, is essential for morphological changes in response to pheromone. These results suggest that formation of a Cdc24p-Far1p-Gbetagamma complex functions as a landmark for orientation of the cytoskeleton during growth towards an external signal.     

Nuclear sequestration of the exchange factor Cdc24 by Far1 regulates cell polarity during yeast mating

Cytoskeletal rearrangements during the cell cycle and in response to signals are regulated by small Rho-type GTPases, but it is not known how these GTPases are activated in a spatial and temporal manner. Here we show that Cdc24, the guanine-nucleotide exchange factor for the yeast GTPase Cdc42, is sequestered in the cell nucleus by Far1. Export of Cdc24 to a site of cell polarization is mediated by two mechanisms. At bud emergence, activation of the G1 cyclin-dependent kinase Cdc28-Cln triggers degradation of Far1 and, as a result, relocation of Cdc24 to the cytoplasm. Cells overexpressing a non-degradable Far1 were unable to polarize their actin cytoskeleton because they failed to relocate Cdc24 to the incipient bud site. In contrast, in response to mating pheromones, the Far1-Cdc24 complex is exported from the nucleus by Msn5. This mechanism ensures that Cdc24 is targeted to the site of receptor-associated heterotrimeric G-protein activation at the plasma membrane, thereby allowing polarization of the actin cytoskeleton along the morphogenetic gradient of pheromone. Either degradation of Far1 or its nuclear export by Msn5 was sufficient for cell growth, suggesting that the two mechanisms are redundant for cell viability. Taken together, our results indicate that Far1 functions as a nuclear anchor for Cdc24. This sequestration regulates cell polarity in response to pheromones by restricting activation of Cdc42 to the site of pheromone receptor activation.     

Nuclear-specific degradation of Far1 is controlled by the localization of the F-box protein Cdc4

Far1 is a bifunctional protein that is required to arrest the cell cycle and establish cell polarity during yeast mating. Here we show that SCF(Cdc4) ubiquitylates Far1 in the nucleus, which in turn targets the multi-ubiquitylated protein to 26S proteasomes most likely located at the nuclear envelope. In response to mating pheromones, a fraction of Far1 was stabilized after its export into the cytoplasm by Ste21/Msn5. Preventing nuclear export destabilized Far1, while conversely cytoplasmic Far1 was stabilized, although the protein was efficiently phosphorylated in a Cdc28-Cln-dependent manner. The core SCF subunits Cdc53, Hrt1 and Skp1 were distributed in the nucleus and the cytoplasm, whereas the F-box protein Cdc4 was exclusively nuclear. A cytoplasmic form of Cdc4 was unable to complement the growth defect of cdc4-1 cells, but it was sufficient to degrade Far1 in the cytoplasm. Our results illustrate the importance of subcellular localization of F-box proteins, and provide an example of how an extracellular signal regulates protein stability at the level of substrate localization.     

The guanine-nucleotide-exchange factor Cdc24p is targeted to the nucleus and polarized growth sites.

Generation of cellular asymmetry or cell polarity plays a critical role in cell-cycle-regulated morphogenetic processes involving the actin cytoskeleton. The GTPase Cdc42 regulates actin rearrangements and signal transduction pathways in all eukaryotic cells [1], and the temporal and spatial regulation of Cdc42p depends on the activity and targeting of its guanine-nucleotide exchange factor (GEF). Cdc24p, the Saccharomyces cerevisiae GEF for Cdc42p, is found in a particulate fraction and localizes to the plasma membrane [2] [3] at sites of polarized growth [4]. We show that Cdc24p labeled with green fluorescent protein (GFP-Cdc24p) was targeted to pre-bud sites, the tips and sides of enlarging buds, and mating projections in pheromone-treated cells. Unexpectedly, GFP-Cdc24p also localized to the nucleus and GFP-Cdc24p levels diminished before nuclear division followed by its reappearance in divided nuclei and mother-bud necks during cytokinesis. The Cdc24p amino-terminal 283 amino acids were necessary and sufficient for nuclear localization, which depended on the cyclin-dependent-kinase inhibitor Far1p. The Cdc24p carboxy-terminal 289 amino acids were necessary and sufficient for targeting to the pre-bud site, bud, mother-bud neck, and mating projection. Targeting was independent of the Cdc24p-binding proteins Far1p, the GTPase Rsr1p/Bud1p, the scaffold protein Bem1p, and the G(beta) subunit Ste4p. These data are consistent with a temporal and spatial regulation of Cdc24p-dependent activation of Cdc42p during the cell cycle.     

Site-specific regulation of the GEF Cdc24p by the scaffold protein Far1p during yeast mating

Receptor-mediated cell polarization via heterotrimeric G-proteins induces cytoskeletal rearrangements in a variety of organisms. In yeast, Far1p is required for orienting cell growth towards the mating partner by linking activated Gbetagamma to the guanine-nucleotide exchange factor (GEF) Cdc24p, which activates the Rho-type GTPase Cdc42p. Here we investigated the role of Far1p in the regulation of Cdc24p in vivo. Using time-lapse microscopy of mating cells and artificial membrane targeting of Far1p, we show that Far1p is necessary and sufficient to recruit Cdc24p to the plasma membrane. Wild-type Far1p contains a PH-like domain, which is required for its membrane localization in vivo. Interestingly, expression of membrane-targeted Far1p causes toxicity, most likely by activating Cdc42p uniformly at the cell cortex. The ability of full-length Far1p to function as an activator of Cdc24p in vivo requires its interaction with Cdc24p and Gbetagamma. Our results imply that Gbetagamma not only targets Far1p to the correct site but may also trigger a conformational change in Far1p that is required for its ability to activate Cdc24p in vivo.     

Efficient initiation of S-phase in yeast requires Cdc40p, a protein involved in pre-mRNA splicing

The S. cerevisiae CDC40 gene was originally identified as a cell-division-specific gene that is essential only at elevated temperatures. Cells carrying mutations in this gene arrest with a large bud and a single nucleus with duplicated DNA content. Cdc40p is also required for spindle establishment or maintenance. Sequence analysis reveals that CDC40 is identical to PRP17, a gene involved in pre-mRNA splicing. In this paper, we show that Cdc40p is required at all temperatures for efficient entry into S-phase and that cell cycle arrest associated with cdc40 mutations is independent of all the known checkpoint mechanisms. Using immunofluorescence, we show that Cdc40p is localized to the nuclear membrane, weakly associated with the nuclear pore. Our results point to a link between cell cycle progression, pre-mRNA splicing, and mRNA export. Received: 9 April 1998 / Accepted: 10 August 1998     

Assembly of scaffold-mediated complexes containing Cdc42p, the exchange factor Cdc24p, and the effector Cla4p required for cell cycle-regulated phosphorylation of Cdc24p

In budding yeast cells, the cytoskeletal polarization and depolarization events that shape the bud are triggered at specific times during the cell cycle by the cyclin-dependent kinase Cdc28p. Polarity establishment also requires the small GTPase Cdc42p and its exchange factor, Cdc24p, but the mechanism whereby Cdc28p induces Cdc42p-dependent polarization is unknown. Here we show that Cdc24p becomes phosphorylated in a cell cycle-dependent manner, triggered by Cdc28p. However, the role of Cdc28p is indirect, and the phosphorylation appears to be catalyzed by the p21-activated kinase family member Cla4p and also depends on Cdc42p and the scaffold protein Bem1p. Expression of GTP-Cdc42p, the product of Cdc24p-mediated GDP/GTP exchange, stimulated Cdc24p phosphorylation independent of cell cycle cues, raising the possibility that the phosphorylation is part of a feedback regulatory pathway. Bem1p binds directly to Cdc24p, to Cla4p, and to GTP-bound Cdc42p and can mediate complex formation between these proteins in vitro. We suggest that Bem1p acts to concentrate polarity establishment proteins at a discrete site, facilitating polarization and promoting Cdc24p phosphorylation at specific times during the cell cycle.     

Characterization of Cdc47p-minichromosome maintenance complexes in Saccharomyces cerevisiae: identification of Cdc45p as a subunit.

Cdc47p is a member of the minichromosome maintenance (MCM) family of polypeptides, which have a role in the early stages of chromosomal DNA replication. Here, we show that Cdc47p assembles into stable complexes with two other members of the MCM family, Cdc46p and Mcm3p. The assembly of Cdc47p into complexes with Cdc46p does not appear to be cell cycle regulated, making it unlikely that these interactions per se are a rate-limiting step in the control of S phase. Cdc45p is also shown to interact with Cdc47p in vivo and to be a component of high-molecular-weight MCM complexes in cell lysates. Like MCM polypeptides, Cdc45p is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae; however, Cdc45p remains in the nucleus throughout the cell cycle, whereas MCMs are nuclear only during G1. We characterize two mutations in CDC47 and CDC46 which arrest cells with unduplicated DNA as a result of single base substitutions. The corresponding amino acid substitutions in Cdc46p and Cdc47p severely reduce the ability of these polypeptides to assemble in a complex with each other in vivo and in vitro. This argues that assembly of Cdc47p into complexes with other MCM polypeptides is important for its role in the initiation of chromosomal DNA replication.     

Caspase-3-mediated cleavage of Cdc6 induces nuclear localization of p49-truncated Cdc6 and apoptosis

We show that Cdc6, an essential initiation factor for DNA replication, undergoes caspase-3-mediated cleavage in the early stages of apoptosis in HeLa cells and SK-HEP-1 cells induced by etoposide, paclitaxel, ginsenoside Rh2, or tumor necrosis factor-related apoptosis-inducing ligand. The cleavage occurs at the SEVD442/G motif and generates an N-terminal truncated Cdc6 fragment (p49-tCdc6) that lacks the carboxy-terminal nuclear export sequence. Cdc6 is known to be phosphorylated by cyclin A-cyclin dependent kinase 2 (Cdk2), an event that promotes its exit from the nucleus and probably blocks it from initiating inappropriate DNA replication. In contrast, p49-tCdc6 translocation to the cytoplasm is markedly reduced under the up-regulated conditions of Cdk2 activity, which is possibly due to the loss of nuclear export sequence. Thus, truncation of Cdc6 results in an increased nuclear retention of p49-tCdc6 that could act as a dominant negative inhibitor of DNA replication and its accumulation in the nucleus could promote apoptosis. Supporting this is that the ectopic expression of p49-tCdc6 not only promotes apoptosis of etoposide-induced HeLa cells but also induces apoptosis in untreated cells. Thus, the caspase-mediated cleavage of Cdc6 creates a truncated Cdc6 fragment that is retained in the nucleus and induces apoptosis.     

Analysis of the role of phosphorylation in fission yeast Cdc13p/cyclinB function

The Cdk1p-cyclin B complex drives entry into mitosis in all eukaryotes. Cdc13p is the single essential cyclin in Schizosaccharomyces pombe and a member of the cyclin B family. Cdc13p abundance rises during G(2)-phase and falls as cells progress through mitosis and G(1). Cdc13p degradation, mediated by the anaphase-promoting complex, is an important mechanism of Cdk1p inhibition and mitotic exit. Cdk1p-cyclin B1 complexes shuttle between the nucleus and cytoplasm, and preventing nuclear accumulation of Cdk1p-cyclin B1 in mammalian cells appears to be one mechanism of preventing entry into mitosis during a DNA damage-induced checkpoint delay. In vertebrates, phosphorylation plays a key role in regulating the intracellular distribution of cyclins. Previous mass spectrometric analysis identified sites of Cdc13p phosphorylation. Here, we have confirmed that these sites are the sole in vivo Cdc13p phosphorylation sites and have studied the role that phosphorylation plays in Cdc13p localization and function. Our data indicate that Cdc13p accumulates in the nucleolus in response to G(2) checkpoint delays, rather than in the cytoplasm, and that phosphorylation plays no role in Cdc13p localization or function.     

Distinct nuclear and cytoplasmic functions of the S. pombe Cdc14-like phosphatase Clp1p/Flp1p and a role for nuclear shuttling in its regulation

Cdc14-like phosphatases regulate a variety of cell cycle events by dephosphorylating CDK sites. Their cell cycle-dependent changes in localization may be important to carry out distinct functions. Work in budding and fission yeast suggested that Cdc14-like phosphatases are inhibited by nucleolar sequestration. In S. cerevisiae, Cdc14p is released from the nucleolus by the FEAR network and Cdk1, whereas the S. pombe CDC14-like phosphatase Clp1p (also known as Flp1p) is released at mitotic entry by an unknown mechanism. The mitotic exit network (MEN) in S. cerevisiae and its homologous network, the septation initiation network (SIN), in S. pombe act through an unknown mechanism to keep the phosphatase out of the nucleolus in late mitosis. SIN-dependent cytoplasmic maintenance of Clp1p is thought to be essential for the cytokinesis checkpoint, which blocks further rounds of nuclear division until cytokinesis is completed. By targeting Clp1p to the nucleus or the cytoplasm, we demonstrate distinct functions for these pools of Clp1p in chromosome segregation and cytokinesis, respectively. Our results further suggest that the SIN does not keep Clp1p out of the nucleolus by regulating nucleolar affinity, as proposed for S. cerevisiae Cdc14p, but instead, Clp1p may be regulated by nuclear import/export.     

Cdc24 regulates nuclear shuttling and recruitment of the Ste5 scaffold to a heterotrimeric G protein in Saccharomyces cerevisiae

The Saccharomyces cerevisiae guanine nucleotide exchange factor Cdc24 regulates polarized growth by binding to Cdc42, a Rho-type GTPase that has many effectors, including Ste20 kinase, which activates multiple MAPK cascades. Here, we show that Cdc24 promotes MAPK signaling during mating through interactions with Ste5, a scaffold that must shuttle through the nucleus and bind to the beta subunit (Ste4) of a G protein for Ste20 to activate the tethered MAPK cascade. Ste5 was basally recruited to growth sites of G1 phase cells independently of Ste4. Loss of Cdc24 inhibited nuclear import and blocked basal and pheromone-induced recruitment of Ste5. Ste5 was not basally recruited and the MAPK Fus3 was not basally activated in the presence of a Cdc24 mutant (G168D) that still activates Cdc42, suggesting that Cdc24 regulates Ste5 and the associated MAPK cascade through a function that is not dependent on its guanine nucleotide exchange factor activity. Consistent with this, Cdc24 bound Ste5 and coprecipitated with Ste4 independently of Far1 and Ste5. Loss of Cdc24 decreased Ste5-Ste4 complex formation, and loss of Ste4 stimulated Cdc24-Ste5 complex formation. Collectively, these findings suggest that Cdc24 mediates site-specific localization of Ste5 to a heterotrimeric G protein and may therefore ensure localized activation of the associated MAPK cascade.     

酵母交配过程的极性生长及其调控机制

酿酒酵母单倍体细胞能够与相反交配型的单倍体细胞发生交配。交配时酿酒酵母放弃原有出芽位点,根据信息素的浓度梯度,重新选择生长位点,向相反交配型细胞伸出突起进行极性生长。交配因子受体指导选择交配突起的位点,通过G蛋白激活Ste20p,将信号经由Ste11p、Ste7p和Fus3p组成的MAPK模块传递到Far1p和Ste12p等因子,调控相关基因的转录,抑制原有的出芽位点,选择新的生长位点,并使细胞周期停止在G1期,G蛋白与Cdc24p、Cdc42p和Bem1p等蛋白作用,聚集在细胞,使得肌协蛋白细胞骨架在交配突起处聚集,呈极性化分布,使细胞发生极性生长。     

Crm1-Mediated Nuclear Export of Cdc14 is Required for the Completion of Cytokinesis in Budding Yeast

The mitotic exit network (MEN) controls the exit from mitosis in budding yeast. The proline-directed phosphatase, Cdc14p, is a key component of MEN and promotes mitotic exit by activating the degradation of Clb2p and by reversing Cdk-mediated mitotic phosphorylation. Cdc14p is sequestered in the nucleolus during much of the cell cycle and is released in anaphase from the nucleolus to the nucleoplasm and cytoplasm to perform its functions. Release of Cdc14p from the nucleolus during anaphase is well understood. In contrast, less is known about the mechanism by which Cdc14p is released from the nucleus to the cytoplasm. Here we show that Cdc14p contains a leucine-rich nuclear export signal (NES) that interacts with Crm1p physically. Mutations in the NES of Cdc14p allow Clb2p degradation and mitotic exit, but cause abnormal morphology and cytokinesis defects at non-permissive temperatures. Cdc14p localizes to the bud neck, among other cytoplasmic structures, following its release from the nucleolus in late anaphase. This bud neck localization of Cdc14p is disrupted by mutations in its NES and by the leptomycin B-mediated inhibition of Crm1p. Our results suggest a requirement for Crm1p-dependent nuclear export of Cdc14p in coordinating mitotic exit and cytokinesis in budding yeast.     

Use of bimolecular fluorescence complementation to study in vivo interactions between Cdc42p and Rdi1p of Saccharomyces cerevisiae

Saccharomyces cerevisiae Cdc42p functions as a GTPase molecular switch, activating multiple signaling pathways required to regulate cell cycle progression and the actin cytoskeleton. Regulatory proteins control its GTP binding and hydrolysis and its subcellular localization, ensuring that Cdc42p is appropriately activated and localized at sites of polarized growth during the cell cycle. One of these, the Rdi1p guanine nucleotide dissociation inhibitor, negatively regulates Cdc42p by extracting it from cellular membranes. In this study, the technique of bimolecular fluorescence complementation (BiFC) was used to study the dynamic in vivo interactions between Cdc42p and Rdi1p. The BiFC data indicated that Cdc42p and Rdi1p interacted in the cytoplasm and around the periphery of the cell at the plasma membrane and that this interaction was enhanced at sites of polarized cell growth during the cell cycle, i.e., incipient bud sites, tips and sides of small- and medium-sized buds, and the mother-bud neck region. In addition, a ring-like structure containing the Cdc42p-Rdi1p complex transiently appeared following release from G1-phase cell cycle arrest. A homology model of the Cdc42p-Rdi1p complex was used to introduce mutations that were predicted to affect complex formation. These mutations resulted in altered BiFC interactions, restricting the complex exclusively to either the plasma membrane or the cytoplasm. Data from these studies have facilitated the temporal and spatial modeling of Rdi1p-dependent extraction of Cdc42p from the plasma membrane during the cell cycle.     

The exchange factor Cdc24 is required for cell fusion during yeast mating

During Saccharomyces cerevisiae mating, chemotropic growth and cell fusion are critical for zygote formation. Cdc24p, the guanine nucleotide exchange factor for the Cdc42 G protein, is necessary for oriented growth along a pheromone gradient during mating. To understand the functions of this critical Cdc42p activator, we identified additional cdc24 mating mutants. Two mating-specific mutants, the cdc24-m5 and cdc24-m6 mutants, each were isolated with a mutated residue in the conserved catalytic domain. The cdc24-m6 mutant responds normally to pheromone and orients its growth towards a mating partner yet accumulates prezygotes during mating. cdc24-m6 prezygotes have two apposed intact cell walls and do not correctly localize proteins required for cell fusion, despite normal exocytosis. Our results indicate that the exchange factor Cdc24p is necessary for maintaining or restricting specific proteins required for cell fusion to the cell contact region during mating.     

The cyclin-dependent kinase Cdc28p regulates distinct modes of Cdc6p proteolysis during the budding yeast cell cycle

BACKGROUND: Cdc28p, the major cyclin-dependent kinase in budding yeast, prevents re-replication within each cell cycle by preventing the reassembly of Cdc6p-dependent pre-replicative complexes (pre-RCs) once origins have fired. Cdc6p is a rapidly degraded protein that must be synthesised in each cell cycle and is present only during the G1 phase. RESULTS: We found that, at different times in the cell cycle, there are distinct modes of Cdc6p proteolysis. Before Start, Cdc6p proteolysis did not require either the anaphase-promoting complex (APC/C) or the SCF complex, which mediate the major cell cycle regulated ubiquitination pathways, nor did it require Cdc28p activity or any of the potential Cdc28p phosphorylation sites in Cdc6p. In fact, the activation of B cyclin (Clb)-Cdc28p kinase inactivated this pathway of Cdc6p degradation later in the cell cycle. Activation of the G1 cyclins (Clns) caused Cdc6p degradation to become extremely rapid. This degradation required the SCF(CDC4) and Cdc28p consensus sites in Cdc6p, but did not require Clb5 and Clb6. Later in the cell cycle, SCF(CDC4)-dependent Cdc6p proteolysis remained active but became less rapid. CONCLUSIONS: Levels of Cdc6p are regulated in several ways by the Cdc28p cyclin-dependent kinase. The Cln-dependent elimination of Cdc6p, which does not require the S-phase-promoting cyclins Clb5 and Clb6, suggests that the ability to assemble pre-RCs is lost before, not concomitant with, origin firing.     

Cdc42p GDP/GTP cycling is necessary for efficient cell fusion during yeast mating

The highly conserved small Rho G-protein, Cdc42p plays a critical role in cell polarity and cytoskeleton organization in all eukaryotes. In the yeast Saccharomyces cerevisiae, Cdc42p is important for cell polarity establishment, septin ring assembly, and pheromone-dependent MAP-kinase signaling during the yeast mating process. In this study, we further investigated the role of Cdc42p in the mating process by screening for specific mating defective cdc42 alleles. We have identified and characterized novel mating defective cdc42 alleles that are unaffected in vegetative cell polarity. Replacement of the Cdc42p Val36 residue with Met resulted in a specific cell fusion defect. This cdc42[V36M] mutant responded to mating pheromone but was defective in cell fusion and in localization of the cell fusion protein Fus1p, similar to a previously isolated cdc24 (cdc24-m6) mutant. Overexpression of a fast cycling Cdc42p mutant suppressed the cdc24-m6 fusion defect and conversely, overexpression of Cdc24p suppressed the cdc42[V36M] fusion defect. Taken together, our results indicate that Cdc42p GDP-GTP cycling is critical for efficient cell fusion.