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1.
ACdc25 family protein Lte1 (low temperature essential) is essential for mitotic exit at a lowered temperature and has been presumed to be a guanine nucleotide exchange factor (GEF) for a small GTPase Tem1, which is a key regulator of mitotic exit. We found that Lte1 physically associates with Ras2-GTP both in vivo and in vitro and that the Cdc25 homology domain (CHD) of Lte1 is essential for the interaction with Ras2. Furthermore, we found that the proper localization of Lte1 to the bud cortex is dependent on active Ras and that the overexpression of a derivative of Lte1 without the CHD suppresses defects in mitotic exit of a Deltalte1 mutant and a Deltaras1 Deltaras2 mutant. These results suggest that Lte1 is a downstream effector protein of Ras in mitotic exit and that the Ras GEF domain of Lte1 is not essential for mitotic exit but required for its localization. 相似文献
2.
《Cell cycle (Georgetown, Tex.)》2013,12(7):940-946
Lte1, a protein important for exit from mitosis, localizes to the bud cortex as soon as the bud forms and remains there until cells exit from mitosis. Ras, the Rho GTPase Cdc42 and its effector the protein kinase Cla4 are required for Lte1’s association with the bud cortex. Here we investigate how Ras, and the Cdc42 effector Cla4 regulate the localization of Lte1. We find that Ras2 and Lte1 associate in stages of the cell cycle when Lte1 is phosphorylated and associated with the bud cortex and that this association requires CLA4. Additionally, RAS1 and RAS2 are required for CLA4-dependent Lte1 phosphorylation. Our findings suggest that Cla4-dependent phosphorylation promotes the initial association of Lte1 with Ras at the bud cortex and that Ras is required to stabilize phosphorylated forms of Lte1 at the bud cortex. Our results also raise the interesting possibility that the localization of Lte1 affects the protein’s ability to promote mitotic exit. 相似文献
3.
BACKGROUND: The putative guanine nucleotide exchange factor Lte1 plays an essential role in promoting exit from mitosis at low temperatures. Lte1 is thought to activate a Ras-like signaling cascade, the mitotic exit network (MEN). MEN promotes the release of the protein phosphatase Cdc14 from the nucleolus during anaphase, and this release is a prerequisite for exit from mitosis. Lte1 is present throughout the cell during G1 but is sequestered in the bud during S phase and mitosis by an unknown mechanism. RESULTS: We show that anchorage of Lte1 in the bud requires septins, the cell polarity determinants Cdc42 and Cla4, and Kel1. Lte1 physically associates with Kel1 and requires Kel1 for its localization in the bud, suggesting a role for Kel1 in anchoring Lte1 at the bud cortex. Our data further implicate the PAK-like protein kinase Cla4 in controlling Lte1 phosphorylation and localization. CLA4 is required for Lte1 phosphorylation and bud localization. Furthermore, when overexpressed, CLA4 induces Lte1 phosphorylation and localization to regions of polarized growth. Finally, we show that Cdc14, directly or indirectly, controls Lte1 dephosphorylation and delocalization from the bud during exit from mitosis. CONCLUSION: Restriction of Lte1 to the bud cortex depends on the cortical proteins Cdc42 and Kel1 and the septin ring. Cla4 and Cdc14 promote and demote Lte1 localization at and from the bud cortex, respectively, suggesting not only that the phosphorylation status of Lte1 controls its localization but also indicating that Cla4 and Cdc14 are key regulators of the spatial asymmetry of Lte1. 相似文献
4.
Marco Geymonat Adonis Spanos Geoffroy de Bettignies Steven G. Sedgwick 《The Journal of cell biology》2009,187(4):497-511
Lte1 is a mitotic regulator long envisaged as a guanosine nucleotide exchange factor (GEF) for Tem1, the small guanosine triphosphatase governing activity of the Saccharomyces cerevisiae mitotic exit network. We demonstrate that this model requires reevaluation. No GEF activity was detectable in vitro, and mutational analysis of Lte1’s putative GEF domain indicated that Lte1 activity relies on interaction with Ras for localization at the bud cortex rather than providing nucleotide exchange. Instead, we found that Lte1 can determine the subcellular localization of Bfa1 at spindle pole bodies (SPBs). Under conditions in which Lte1 is essential, Lte1 promoted the loss of Bfa1 from the maternal SPB. Moreover, in cells with a misaligned spindle, mislocalization of Lte1 in the mother cell promoted loss of Bfa1 from one SPB and allowed bypass of the spindle position checkpoint. We observed that lte1 mutants display aberrant localization of the polarity cap, which is the organizer of the actin cytoskeleton. We propose that Lte1’s role in cell polarization underlies its contribution to mitotic regulation. 相似文献
5.
Exit from mitosis must not occur prior to partitioning of chromosomes between daughter cells. We find that the GTP binding protein Tem1, a regulator of mitotic exit, is present on the spindle pole body that migrates into the bud during S phase and mitosis. Tem1's exchange factor, Lte1, localizes to the bud. Thus, Tem1 and Lte1 are present in the same cellular compartment (the bud) only after the nucleus enters the bud during nuclear division. We also find that the presence of Tem1 and Lte1 in the bud is required for mitotic exit. Our results suggest that the spatial segregation of Tem1 and Lte1 ensures that exit from mitosis only occurs after the genetic material is partitioned between mother and daughter cell. 相似文献
6.
Castillon GA Adames NR Rosello CH Seidel HS Longtine MS Cooper JA Heil-Chapdelaine RA 《Current biology : CB》2003,13(8):654-658
In Saccharomyces cerevisiae, the spindle position checkpoint ensures that cells do not exit mitosis until the mitotic spindle moves into the mother/bud neck and thus guarantees that each cell receives one nucleus [1-6]. Mitotic exit is controlled by the small G protein Tem1p. Tem1p and its GTPase activating protein (GAP) Bub2p/Bfa1p are located on the daughter-bound spindle pole body. The GEF Lte1p is located in the bud. This segregation helps keep Tem1p in its inactive GDP state until the spindle enters the neck. However, the checkpoint functions without Lte1p and apparently senses cytoplasmic microtubules in the mother/bud neck [7-9]. To investigate this mechanism, we examined mutants defective for septins, which compose a ring at the neck [10]. We found that the septin mutants sep7Delta and cdc10Delta are defective in the checkpoint. When movement of the spindle into the neck was delayed, mitotic exit occurred, inappropriately leaving both nuclei in the mother. In sep7Delta and cdc10Delta mutants, Lte1p is mislocalized to the mother. In sep7Delta, but not cdc10Delta, mutants, inappropriate mitotic exit depends on Lte1p. These results suggest that septins serve as a diffusion barrier for Lte1p, and that Cdc10p is needed for the septin ring to serve as a scaffold for a putative microtubule sensor. 相似文献
7.
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
8.
In budding yeast, the spindle position checkpoint (SPC) delays mitotic exit until the mitotic spindle moves into the neck between the mother and bud. This checkpoint works by inhibiting the mitotic exit network (MEN), a signaling cascade initiated and controlled by Tem1, a small GTPase. Tem1 is regulated by a putative guanine exchange factor, Lte1, but the function and regulation of Lte1 remains poorly understood. Here, we identify novel components of the checkpoint that operate upstream of Lte1. We present genetic evidence in agreement with existing biochemical evidence for the molecular mechanism of a pathway that links microtubule-cortex interactions with Lte1 and mitotic exit. Each component of this pathway is required for the spindle position checkpoint to delay mitotic exit until the spindle is positioned correctly. 相似文献
9.
The spindle position checkpoint in Saccharomyces cerevisiae delays mitotic exit until the spindle has moved into the mother-bud neck, ensuring that each daughter cell inherits a nucleus. The small G protein Tem1p is critical in promoting mitotic exit and is concentrated at the spindle pole destined for the bud. The presumed nucleotide exchange factor for Tem1p, Lte1p, is concentrated in the bud. These findings suggested the hypothesis that movement of the spindle pole through the neck allows Tem1p to interact with Lte1p, promoting GTP loading of Tem1p and mitotic exit. However, we report that deletion of LTE1 had little effect on the timing of mitotic exit. We also examined several mutants in which some cells inappropriately exit mitosis even though the spindle is within the mother. In some of these cells, the spindle pole body did not interact with the bud or the neck before mitotic exit. Thus, some alternative mechanism must exist to coordinate mitotic exit with spindle position. In both wild-type and mutant cells, mitotic exit was preceded by loss of cytoplasmic microtubules from the neck. Thus, the spindle position checkpoint may monitor such interactions. 相似文献
10.
A role for cell polarity proteins in mitotic exit 总被引:4,自引:0,他引:4
The budding yeast mitotic exit network (MEN) is a signal transduction cascade that controls exit from mitosis by facilitating the release of the cell cycle phosphatase Cdc14 from the nucleolus. The G protein Tem1 regulates MEN activity. The Tem1 guanine nucleotide exchange factor (GEF) Lte1 associates with the cortex of the bud and activates the MEN upon the formation of an anaphase spindle. Thus, the cell cortex has an important but ill-defined role in MEN regulation. Here, we describe a network of conserved cortical cell polarity proteins that have key roles in mitotic exit. The Rho-like GTPase Cdc42, its GEF Cdc24 and its effector Cla4 [a member of the p21-activated kinases (PAKs)] control the initial binding and activation of Lte1 to the bud cortex. Moreover, Cdc24, Cdc42 and Ste20, another PAK, probably function parallel to Lte1 in facilitating mitotic exit. Finally, the cell polarity proteins Kel1 and Kel2 are present in complexes with both Lte1 and Tem1, and negatively regulate mitotic exit. 相似文献
11.
Kar9p-independent microtubule capture at Bud6p cortical sites primes spindle polarity before bud emergence in Saccharomyces cerevisiae
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Spindle orientation is critical for accurate chromosomal segregation in eukaryotic cells. In the yeast Saccharomyces cerevisiae, orientation of the mitotic spindle is achieved by a program of microtubule-cortex interactions coupled to spindle morphogenesis. We previously implicated Bud6p in directing microtubule capture throughout this program. Herein, we have analyzed cells coexpressing GFP:Bud6 and GFP:Tub1 fusions, providing a kinetic view of Bud6p-microtubule interactions in live cells. Surprisingly, even during the G1 phase, microtubule capture at the recent division site and the incipient bud is dictated by Bud6p. These contacts are eliminated in bud6 delta cells but are proficient in kar9 delta cells. Thus, Bud6p cues microtubule capture, as soon as a new cell polarity axis is established independent of Kar9p. Bud6p increases the duration of interactions and promotes distinct modes of cortical association within the bud and neck regions. In particular, microtubule shrinkage and growth at the cortex rarely occur away from Bud6p sites. These are the interactions selectively impaired at the bud cortex in bud6 delta cells. Finally, interactions away from Bud6p sites within the bud differ from those occurring at the mother cell cortex, pointing to the existence of an independent factor controlling cortical contacts in mother cells after bud emergence. 相似文献
12.
Dynamic localization and function of Bni1p at the sites of directed growth in Saccharomyces cerevisiae
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Ozaki-Kuroda K Yamamoto Y Nohara H Kinoshita M Fujiwara T Irie K Takai Y 《Molecular and cellular biology》2001,21(3):827-839
Formin homology (FH) proteins are implicated in cell polarization and cytokinesis through actin organization. There are two FH proteins in the yeast Saccharomyces cerevisiae, Bni1p and Bnr1p. Bni1p physically interacts with Rho family small G proteins (Rho1p and Cdc42p), actin, two actin-binding proteins (profilin and Bud6p), and a polarity protein (Spa2p). Here we analyzed the in vivo localization of Bni1p by using a time-lapse imaging system and investigated the regulatory mechanisms of Bni1p localization and function in relation to these interacting proteins. Bni1p fused with green fluorescent protein localized to the sites of cell growth throughout the cell cycle. In a small-budded cell, Bni1p moved along the bud cortex. This dynamic localization of Bni1p coincided with the apparent site of bud growth. A bni1-disrupted cell showed a defect in directed growth to the pre-bud site and to the bud tip (apical growth), causing its abnormally spherical cell shape and thick bud neck. Bni1p localization at the bud tips was absolutely dependent on Cdc42p, largely dependent on Spa2p and actin filaments, and partly dependent on Bud6p, but scarcely dependent on polarized cortical actin patches or Rho1p. These results indicate that Bni1p regulates polarized growth within the bud through its unique and dynamic pattern of localization, dependent on multiple factors, including Cdc42p, Spa2p, Bud6p, and the actin cytoskeleton. 相似文献
13.
Knaus M Cameroni E Pedruzzi I Tatchell K De Virgilio C Peter M 《The EMBO journal》2005,24(17):3000-3011
Regulated interactions between microtubules (MTs) and the cell cortex control MT dynamics and position the mitotic spindle. In eukaryotic cells, the adenomatous polyposis coli/Kar9p and dynein/dynactin pathways are involved in guiding MT plus ends and MT sliding along the cortex, respectively. Here we identify Bud14p as a novel cortical activator of the dynein/dynactin complex in budding yeast. Bud14p accumulates at sites of polarized growth and the mother-bud neck during cytokinesis. The localization to bud and shmoo tips requires an intact actin cytoskeleton and the kelch-domain-containing proteins Kel1p and Kel2p. While cells lacking Bud14p function fail to stabilize the pre-anaphase spindle at the mother-bud neck, overexpression of Bud14p is toxic and leads to elongated astral MTs and increased dynein-dependent sliding along the cell cortex. Bud14p physically interacts with the type-I phosphatase Glc7p, and localizes Glc7p to the bud cortex. Importantly, the formation of Bud14p-Glc7p complexes is necessary to regulate MT dynamics at the cortex. Taken together, our results suggest that Bud14p functions as a regulatory subunit of the Glc7p type-I phosphatase to stabilize MT interactions specifically at sites of polarized growth. 相似文献
14.
Gic2p may link activated Cdc42p to components involved in actin polarization, including Bni1p and Bud6p (Aip3p)
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Gic2p is a Cdc42p effector which functions during cytoskeletal organization at bud emergence and in response to pheromones, but it is not understood how Gic2p interacts with the actin cytoskeleton. Here we show that Gic2p displayed multiple genetic interactions with Bni1p, Bud6p (Aip3p), and Spa2p, suggesting that Gic2p may regulate their function in vivo. In support of this idea, Gic2p cofractionated with Bud6p and Spa2p and interacted with Bud6p by coimmunoprecipitation and two-hybrid analysis. Importantly, localization of Bni1p and Bud6p to the incipient bud site was dependent on active Cdc42p and the Gic proteins but did not require an intact actin cytoskeleton. We identified a conserved domain in Gic2p which was necessary for its polarization function but dispensable for binding to Cdc42p-GTP and its localization to the site of polarization. Expression of a mutant Gic2p harboring a single-amino-acid substitution in this domain (Gic2p(W23A)) interfered with polarized growth in a dominant-negative manner and prevented recruitment of Bni1p and Bud6p to the incipient bud site. We propose that at bud emergence, Gic2p functions as an adaptor which may link activated Cdc42p to components involved in actin organization and polarized growth, including Bni1p, Spa2p, and Bud6p. 相似文献
15.
Bfa1p and Bub2p are spindle checkpoint proteins that likely have GTPase activation activity and are associated with the budding yeast spindle pole body (SPB). Here, we show that Bfa1p and Bub2p bind the Ras-like GTPase Tem1p, a component of the mitotic exit network, to the cytoplasmic face of the SPB that enters the bud, whereas the GDP/GTP exchange factor Lte1p is associated with the cortex of the bud. Migration of the SPB into the bud probably allows activation of Tem1p through Lte1p, thereby linking nuclear migration with mitotic exit. Since components of the Bub2p checkpoint are conserved in other organisms, we propose that the position of the SPB or mammalian centrosome controls the timing of mitotic exit. 相似文献
16.
The spindle position checkpoint (SPC) ensures correct mitotic spindle position before allowing mitotic exit in the budding yeast Saccharomyces cerevisiae. In a candidate screen for checkpoint genes, we identified bud2Δ as deficient for the SPC. Bud2 is a GTPase activating protein (GAP), and the only known substrate of Bud2 was Rsr1/Bud1, a Ras-like GTPase and a central component of the bud-site-selection pathway. Mutants lacking Rsr1/Bud1 had no checkpoint defect, as did strains lacking and overexpressing Bud5, a guanine-nucleotide exchange factor (GEF) for Rsr1/Bud1. Thus, the checkpoint function of Bud2 is distinct from its role in bud site selection. The catalytic activity of the Bud2 GAP domain was required for the checkpoint, based on the failure of the known catalytic point mutant Bud2(R682A) to function in the checkpoint. Based on assays of heterozygous diploids, bud2(R682A), was dominant for loss of checkpoint but recessive for bud-site-selection failure, further indicating a separation of function. Tem1 is a Ras-like protein and is the critical regulator of mitotic exit, sitting atop the mitotic exit network (MEN). Tem1 is a likely target for Bud2, supported by genetic analyses that exclude other Ras-like proteins. 相似文献
17.
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
18.
Formins are conserved proteins that nucleate actin assembly and tightly associate with the fast growing barbed ends of actin filaments to allow insertional growth. Most organisms express multiple formins, but it has been unclear whether they have similar or distinct activities and how they may be regulated differentially. We isolated and compared the activities of carboxyl-terminal fragments of the only two formins expressed in Saccharomyces cerevisiae, Bni1 and Bnr1. Bnr1 was an order of magnitude more potent than Bni1 in actin nucleation and processive capping, and unlike Bni1, Bnr1 bundled actin filaments. Profilin bound directly to Bni1 and Bnr1 and regulated their activities similarly. However, the cell polarity factor Bud6/Aip3 specifically bound to and stimulated Bni1, but not Bnr1. This was unexpected, since previous two-hybrid studies suggested Bud6 interacts with both formins. We mapped Bud6 binding activity to specific residues in the carboxyl terminus of Bni1 that are adjacent to its diaphanous autoregulatory domain (DAD). Fusion of the carboxyl terminus of Bni1 to Bnr1 conferred Bud6 stimulation to a Bnr1-Bni1 chimera. Thus, Bud6 differentially stimulates Bni1 and not Bnr1. We found that Bud6 is up-regulated during bud growth, when it is delivered to the bud tip on Bni1-nucleated actin cables. We propose that Bud6 stimulation of Bni1 promotes robust cable formation, which in turn delivers more Bud6 to the bud tip, reinforcing polarized cell growth through a positive feedback loop. 相似文献
19.
The differential roles of budding yeast Tem1p, Cdc15p, and Bub2p protein dynamics in mitotic exit
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Molk JN Schuyler SC Liu JY Evans JG Salmon ED Pellman D Bloom K 《Molecular biology of the cell》2004,15(4):1519-1532
In the budding yeast Saccharomyces cerevisiae the mitotic spindle must be positioned along the mother-bud axis to activate the mitotic exit network (MEN) in anaphase. To examine MEN proteins during mitotic exit, we imaged the MEN activators Tem1p and Cdc15p and the MEN regulator Bub2p in vivo. Quantitative live cell fluorescence microscopy demonstrated the spindle pole body that segregated into the daughter cell (dSPB) signaled mitotic exit upon penetration into the bud. Activation of mitotic exit was associated with an increased abundance of Tem1p-GFP and the localization of Cdc15p-GFP on the dSPB. In contrast, Bub2p-GFP fluorescence intensity decreased in mid-to-late anaphase on the dSPB. Therefore, MEN protein localization fluctuates to switch from Bub2p inhibition of mitotic exit to Cdc15p activation of mitotic exit. The mechanism that elevates Tem1p-GFP abundance in anaphase is specific to dSPB penetration into the bud and Dhc1p and Lte1p promote Tem1p-GFP localization. Finally, fluorescence recovery after photobleaching (FRAP) measurements revealed Tem1p-GFP is dynamic at the dSPB in late anaphase. These data suggest spindle pole penetration into the bud activates mitotic exit, resulting in Tem1p and Cdc15p persistence at the dSPB to initiate the MEN signal cascade. 相似文献
20.
Anastasia SD Nguyen DL Thai V Meloy M MacDonough T Kellogg DR 《The Journal of cell biology》2012,197(1):89-104
Addition of new membrane to the cell surface by membrane trafficking is necessary for cell growth. In this paper, we report that blocking membrane traffic causes a mitotic checkpoint arrest via Wee1-dependent inhibitory phosphorylation of Cdk1. Checkpoint signals are relayed by the Rho1 GTPase, protein kinase C (Pkc1), and a specific form of protein phosphatase 2A (PP2A(Cdc55)). Signaling via this pathway is dependent on membrane traffic and appears to increase gradually during polar bud growth. We hypothesize that delivery of vesicles to the site of bud growth generates a signal that is proportional to the extent of polarized membrane growth and that the strength of the signal is read by downstream components to determine when sufficient growth has occurred for initiation of mitosis. Growth-dependent signaling could explain how membrane growth is integrated with cell cycle progression. It could also control both cell size and morphogenesis, thereby reconciling divergent models for mitotic checkpoint function. 相似文献