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1.
Interaction between a Ras and a Rho GTPase couples selection of a growth site to the development of cell polarity in yeast
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Kozminski KG Beven L Angerman E Tong AH Boone C Park HO 《Molecular biology of the cell》2003,14(12):4958-4970
Polarized cell growth requires the coupling of a defined spatial site on the cell cortex to the apparatus that directs the establishment of cell polarity. In the budding yeast Saccharomyces cerevisiae, the Ras-family GTPase Rsr1p/Bud1p and its regulators select the proper site for bud emergence on the cell cortex. The Rho-family GTPase Cdc42p and its associated proteins then establish an axis of polarized growth by triggering an asymmetric organization of the actin cytoskeleton and secretory apparatus at the selected bud site. We explored whether a direct linkage exists between the Rsr1p/Bud1p and Cdc42p GTPases. Here we show specific genetic interactions between RSR1/BUD1 and particular cdc42 mutants defective in polarity establishment. We also show that Cdc42p coimmunoprecipitated with Rsr1p/Bud1p from yeast extracts. In vitro studies indicated a direct interaction between Rsr1p/Bud1p and Cdc42p, which was enhanced by Cdc24p, a guanine nucleotide exchange factor for Cdc42p. Our findings suggest that Cdc42p interacts directly with Rsr1p/Bud1p in vivo, providing a novel mechanism by which direct contact between a Ras-family GTPase and a Rho-family GTPase links the selection of a growth site to polarity establishment. 相似文献
2.
Site-specific activation of the Rho-type GTPase Cdc42p by its guanine-nucleotide exchange factor (GEF) Cdc24p is critical for the establishment of cell polarity. Here we show that binding of Cdc24p to the small GTPase Rsr1p/Bud1p is required for its recruitment to the incipient bud site. Rsr1p/Bud1p binds to the CH-domain of Cdc24p, which is essential for its function in vivo. We have identified a cdc24-mutant allele, which is specifically defective for bud-site selection. Our results suggest that Cdc24p is auto-inhibited by an intramolecular interaction with its carboxy-terminal PB1-domain. Rsr1p/Bud1p appears to activate the GEF activity of Cdc24p in vivo, possibly by triggering a conformational change that dissociates the PB1-domain from its intramolecular binding site. Genetic experiments suggest that Bem1p functions as a positive regulator of Cdc24p by binding to the PB1-domain of Cdc24p, thereby preventing its re-binding to the intramolecular inhibitory site. Taken together, our results support a two-step molecular mechanism for the site-specific activation of Cdc24p, which involves Rsr1p/Bud1p and the adaptor protein Bem1p. 相似文献
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Sung-Hee Ahn Brian T. Tobe Jonathan N. Fitz Gerald Shannon L. Anderson Adriana Acurio Stephen J. Kron 《Molecular biology of the cell》2001,12(11):3589-3600
The yeast cyclin-dependent kinase Cdc28p regulates bud morphogenesis and cell cycle progression via the antagonistic activities of Cln and Clb cyclins. Cln G1 cyclins direct polarized growth and bud emergence, whereas Clb G2 cyclins promote isotropic growth of the bud and chromosome segregation. Using colony morphology as a screen to dissect regulation of polarity by Cdc28p, we identified nine point mutations that block the apical-isotropic switch while maintaining other functions. Like a clb2 Delta mutation, each confers tubular bud shape, apically polarized actin distribution, unipolar budding, and delayed anaphase. The mutations are all suppressed by CLB2 overexpression and are synthetically lethal with a CLB2 deletion. However, defects in multiple independent pathways may underlie their common phenotype, because the mutations are scattered throughout the CDC28 sequence, complement each other, and confer diverse biochemical properties. Glu12Gly, a mutation that alters a residue involved in Swe1p inhibition of Cdc28p, was unique in being suppressed by deficiency of SWE1 or CLN1. With wild-type CDC28, filament formation induced by CLN1 overexpression was markedly decreased in a SWE1 deletion. These results suggest that Swe1p, via inhibition of Clb2p/Cdc28p, may mediate much of the effect of Cln1p on filamentous morphogenesis. 相似文献
5.
Yeast cells organize their actin cytoskeleton in a highly polarized manner during vegetative growth. The Ras-like GTPase Rsr1/Bud1 and its regulators are required for selection of a specific site for growth. Here we showed that Rsr1/Bud1 was broadly distributed on the plasma membrane and highly concentrated at the incipient bud site and polarized growth sites. We also showed that localization of Cdc24, a guanine nucleotide exchange factor for the Cdc42 GTPase, to the proper bud site was dependent on Rsr1/Bud1. Surprisingly, Rsr1/Bud1 also localized to intracellular membranes. A mutation in the lysine repeat in the hypervariable region of Rsr1/Bud1 specifically abolished its plasma membrane localization, whereas a mutation at the CAAX motif eliminated both plasma membrane and internal membrane association of Rsr1/Bud1. Thus the lysine repeat and the CAAX motif of Rsr1/Bud1 are important for its localization to the plasma membrane and to the polarized growth sites. This localization of Rsr1/Bud1 is essential for its function in proper bud site selection because both mutations resulted in random bud site selection. 相似文献
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Yeast G1 cyclins CLN1 and CLN2 and a GAP-like protein have a role in bud formation. 总被引:22,自引:2,他引:20
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Cyclin-dependent protein kinases have a central role in cell cycle regulation. In Saccharomyces cerevisiae, Cdc28 kinase and the G1 cyclins Cln1, 2 and 3 are required for DNA replication, duplication of the spindle pole body and bud emergence. These three independent processes occur simultaneously in late G1 when the cells reach a critical size, an event known as Start. At least one of the three Clns is necessary for Start. Cln3 is believed to activate Cln1 and Cln2, which can then stimulate their own accumulation by means of a positive feedback loop. They (or Cln3) also activate another pair of cyclins, Clb5 and 6, involved in initiating S phase. Little is known about the role of Clns in spindle pole body duplication and budding. We report here the isolation of a gene (CLA2/BUD2/ERC25) that codes for a homologue of mammalian Ras-associated GTPase-activating proteins (GAPs) and is necessary for budding only in cln1 cln2 cells. This suggests that Cln1 and Cln2 may have a direct role in bud formation. 相似文献
8.
An essential G1 function for cyclin-like proteins in yeast 总被引:118,自引:0,他引:118
Cyclins were discovered in marine invertebrates based on their dramatic cell cycle periodicity. Recently, the products of three genes associated with cell cycle progression in S. cerevisiae were found to share limited homology with cyclins. Mutational elimination of the CLN1, CLN2, and DAF1/WHI1 products leads to cell cycle arrest independent of cell type, while expression of any one of the genes allows cell proliferation. Using strains where CLN1 was expressed conditionally, the essential function of Cln proteins was found to be limited to the G1 phase. Furthermore, the ability of the Cln proteins to carry out this function was found to decay rapidly upon cessation of Cln biosynthesis. The data are consistent with the hypothesis that Cln proteins activate the Cdc28 protein kinase, shown to be essential for the G1 to S phase transition in S. cerevisiae. Because of the apparent functional redundancy of these genes, DAF1/WHI1 has been renamed CLN3. 相似文献
9.
The Cln3 cyclin homolog of Saccharomyces cerevisiae functions to promote cell cycle START for only a short time following its synthesis. Cln3 protein is highly unstable and is stabilized by C-terminal truncation. Cln3 binds to Cdc28, a protein kinase catalytic subunit essential for cell cycle START, and Cln3 instability requires Cdc28 activity. The long functional lifetime and the hyperactivity of C-terminally truncated Cln3 (Cln3-2) relative to those of full-length Cln3 are affected by mutations in CDC28: the functional lifetime of Cln3-2 is drastically reduced by the cdc28-13 mutation at the permissive temperature, and the cdc28-4 mutation at the permissive temperature completely blocks the function of Cln3-2 while only partially reducing the function of full-length Cln3. Thus, sequences in the C-terminal third of Cln3 might help stabilize functional Cdc28-Cln3 association, as well as decreasing the lifetime of the Cln3 protein. These and other results strongly support the idea that Cln proteins function to activate Cdc28 at START. 相似文献
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Asymmetrically localized Bud8p and Bud9p proteins control yeast cell polarity and development
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Diploid strains of the budding yeast Saccharomyces cerevisiae change the pattern of cell division from bipolar to unipolar when switching growth from the unicellular yeast form (YF) to filamentous, pseudohyphal (PH) cells in response to nitrogen starvation. The functions of two transmembrane proteins, Bud8p and Bud9p, in regulating YF and PH cell polarity were investigated. Bud8p is highly concentrated at the distal pole of both YF and PH cells, where it directs initiation of cell division. Asymmetric localization of Bud8p is independent of the Rsr1p/Bud1p GTPase. rsr1/bud1 mutations are epistatic to bud8 mutations, placing Rsr1p/Bud1p downstream of Bud8p. In YF cells, Bud9p is also localized at the distal pole, yet deletion of BUD9 favours distal bud initiation. In PH cells, nutritional starvation for nitrogen efficiently prevents distal localization of Bud9p. Because Bud8p and Bud9p proteins associate in vivo, we propose Bud8p as a landmark for bud initiation at the distal cell pole, where Bud9p acts as inhibitor. In response to nitrogen starvation, asymmetric localization of Bud9p is averted, favouring Bud8p-mediated cell division at the distal pole. 相似文献
12.
To identify cyclin-dependent kinase mutants with relaxed cyclin requirements, CDC28 alleles were selected that could rescue a yeast strain expressing as its only CLN G1 cyclin a mutant Cln2p (K129A,E183A) that is defective for Cdc28p binding. Rescue of this strain by mutant CDC28 was dependent upon the mutant cln2-KAEA, but additional mutagenesis and DNA shuffling yielded multiply mutant CDC28-BYC alleles (bypass of CLNs) that could support highly efficient cell cycle initiation in the complete absence of CLN genes. By gel filtration chromatography, one of the mutant Cdc28 proteins exhibited kinase activity associated with cyclin-free monomer. Thus, the mutants' CLN bypass activity might result from constitutive, cyclin-independent activity, suggesting that Cdk targeting by cyclins is not required for cell cycle initiation. 相似文献
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《The Journal of cell biology》1993,120(6):1305-1320
Analysis of cell cycle regulation in the budding yeast Saccharomyces cerevisiae has shown that a central regulatory protein kinase, Cdc28, undergoes changes in activity through the cell cycle by associating with distinct groups of cyclins that accumulate at different times. The various cyclin/Cdc28 complexes control different aspects of cell cycle progression, including the commitment step known as START and mitosis. We found that altering the activity of Cdc28 had profound effects on morphogenesis during the yeast cell cycle. Our results suggest that activation of Cdc28 by G1 cyclins (Cln1, Cln2, or Cln3) in unbudded G1 cells triggers polarization of the cortical actin cytoskeleton to a specialized pre-bud site at one end of the cell, while activation of Cdc28 by mitotic cyclins (Clb1 or Clb2) in budded G2 cells causes depolarization of the cortical actin cytoskeleton and secretory apparatus. Inactivation of Cdc28 following cyclin destruction in mitosis triggers redistribution of cortical actin structures to the neck region for cytokinesis. In the case of pre-bud site assembly following START, we found that the actin rearrangement could be triggered by Cln/Cdc28 activation in the absence of de novo protein synthesis, suggesting that the kinase may directly phosphorylate substrates (such as actin-binding proteins) that regulate actin distribution in cells. 相似文献
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We screened for mutations that resulted in lethality when the G1 cyclin Cln2p was overexpressed throughout the cell cycle
in Saccharomyces cerevisiae. Mutations in five complementation groups were found to give this phenotype, and three of the mutated genes were identified
as MEC1, NUP170, and CDC14. Mutations in CDC14 may have been recovered in the screen because Cdc14p may reduce the cyclin B (Clb)-associated Cdc28 kinase activity in late
mitosis, and Cln2p may normally activate Clb-Cdc28 kinase activity by related mechanisms. In agreement with the idea that
cdc14 mutations elevate Clb-Cdc28 kinase activity, deletion of the gene for the Clb-Cdc28 inhibitor Sic1 caused synthetic lethality
with cdc14-1, as did the deletion of HCT1, which is required for proteolysis of Clb2p. Surprisingly, deletion of the gene for the major B-type cyclin, CLB2, also caused synthetic lethality with the cdc14-1 mutation. The clb2 cdc14 strains arrested with replicated but unseparated DNA and unseparated spindle pole bodies; this phenotype is distinct from
the late mitotic arrest of the sic1::TRP1 cdc14-1 and the cdc14-1 hct1::LEU2 double mutants and of the cdc14 CLN2 overexpressor. We found genetic interactions between CDC14 and the replication initiator gene CDC6, extending previous observations of interactions between the late mitotic function of Cdc14p and control of DNA replication.
We also describe genetic interactions between CDC28 and CDC14.
Received: 24 May 1999 / Accepted: 19 October 1999 相似文献
17.
Knaus M Pelli-Gulli MP van Drogen F Springer S Jaquenoud M Peter M 《The EMBO journal》2007,26(21):4501-4513
Site-specific activation of the Rho-type GTPase Cdc42p is critical for the establishment of cell polarity. Here we investigated the role and regulation of the GTPase-activating enzymes (GAPs) Bem2p and Bem3p for Cdc42p activation and actin polarization at bud emergence in Saccharomyces cerevisiae. Bem2p and Bem3p are localized throughout the cytoplasm and the cell cortex in unbudded G1 cells, but accumulate at sites of polarization after bud emergence. Inactivation of Bem2p results in hyperactivation of Cdc42p and polarization toward multiple sites. Bem2p and Bem3p are hyperphosphorylated at bud emergence most likely by the Cdc28p-Cln2p kinase. This phosphorylation appears to inhibit their GAP activity in vivo, as non-phosphorylatable Bem3p mutants are hyperactive and interfere with Cdc42p activation. Taken together, our results indicate that Bem2p and Bem3p may function as global inhibitors of Cdc42p activation during G1, and their inactivation by the Cdc28p/Cln kinase contributes to site-specific activation of Cdc42p at bud emergence. 相似文献
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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. 相似文献
20.
Mechanisms controlling subcellular localization of the G(1) cyclins Cln2p and Cln3p in budding yeast 总被引:1,自引:0,他引:1
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Different G1 cyclins confer functional specificity to the cyclin-dependent kinase (Cdk) Cdc28p in budding yeast. The Cln3p G1 cyclin is localized primarily to the nucleus, while Cln2p is localized primarily to the cytoplasm. Both binding to Cdc28p and Cdc28p-dependent phosphorylation in the C-terminal region of Cln2p are independently required for efficient nuclear depletion of Cln2p, suggesting that this process may be physiologically regulated. The accumulation of hypophosphorylated Cln2 in the nucleus is an energy-dependent process, but may not involve the RAN GTPase. Phosphorylation of Cln2p is inefficient in small newborn cells obtained by elutriation, and this lowered phosphorylation correlates with reduced Cln2p nuclear depletion in newborn cells. Thus, Cln2p may have a brief period of nuclear residence early in the cell cycle. In contrast, the nuclear localization pattern of Cln3p is not influenced by Cdk activity. Cln3p localization requires a bipartite nuclear localization signal (NLS) located at the C terminus of the protein. This sequence is required for nuclear localization of Cln3p and is sufficient to confer nuclear localization to green fluorescent protein in a RAN-dependent manner. Mislocalized Cln3p, lacking the NLS, is much less active in genetic assays specific for Cln3p, but more active in assays normally specific for Cln2p, consistent with the idea that Cln3p localization explains a significant part of Clnp functional specificity. 相似文献