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1.
Cdc14 protein phosphatase is critical for late mitosis progression in budding yeast, although its orthologs in other organisms, including mammalian cells, function as stress-responsive phosphatases. We found herein unexpected roles of Cdc14 in autophagy induction after nutrient starvation and target of rapamycin complex 1 (TORC1) kinase inactivation. TORC1 kinase phosphorylates Atg13 to repress autophagy under nutrient-rich conditions, but if TORC1 becomes inactive upon nutrient starvation or rapamycin treatment, Atg13 is rapidly dephosphorylated and autophagy is induced. Cdc14 phosphatase was required for optimal Atg13 dephosphorylation, pre-autophagosomal structure formation, and autophagy induction after TORC1 inactivation. In addition, Cdc14 was required for sufficient induction of ATG8 and ATG13 expression. Moreover, Cdc14 activation provoked autophagy even under normal conditions. This study identified a novel role of Cdc14 as the stress-responsive phosphatase for autophagy induction in budding yeast.  相似文献   

2.
Chromosome morphology in Saccharomyces cerevisiae is only visible at the microscopic level in the ribosomal DNA array (rDNA). The rDNA has been thus used as a model to characterize condensation and segregation of sister chromatids in mitosis. It has been established that the metaphase structure (“loop”) depends, among others, on the condensin complex; whereas its segregation also depends on that complex, the Polo-like kinase Cdc5 and the cell cycle master phosphatase Cdc14. In addition, Cdc14 also drives rDNA hypercondensation in telophase. Remarkably, since all these components are essential for cell survival, their role on rDNA condensation and segregation was established by temperature-sensitive (ts) alleles. Here, we show that the heat stress (HS) used to inactivate ts alleles (25 ºC to 37 ºC shift) causes rDNA loop condensation in metaphase-arrested wild type cells, a result that can also be mimicked by other stresses that inhibit the TORC1 pathway. Because this condensation might challenge previous findings with ts alleles, we have repeated classical experiments of rDNA condensation and segregation, yet using instead auxin-driven degradation alleles (aid alleles). We have undertaken the protein degradation at lower temperatures (25 ºC) and concluded that the classical roles for condensin, Cdc5, Cdc14 and Cdc15 still prevailed. Thus, condensin degradation disrupts rDNA higher organization, Cdc14 and Cdc5 degradation precludes rDNA segregation and Cdc15 degradation still allows rDNA hypercompaction in telophase. Finally, we provide direct genetic evidence that this HS-mediated rDNA condensation is dependent on TORC1 but, unlike the one observed in anaphase, is independent of Cdc14.  相似文献   

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Autophagic degradation of ribosomes is promoted by nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1). Here we show that selective autophagic degradation of ribosomes (called ribophagy) after TORC1 inactivation requires the specific autophagy receptor Atg11. Rim15 protein kinase upregulated ribophagy, while it downregulated non-selective degradation of ribosomes.  相似文献   

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The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is induced when Saccharomyces cerevisiae are starved of glucose. However, when glucose is added to cells that have been starved for 3 days, FBPase is degraded in the vacuole. FBPase is first imported to Vid (vacuole import and degradation) vesicles, and these vesicles then merge with the endocytic pathway. In this report we show that two additional gluconeogenic enzymes, isocitrate lyase and phosphoenolpyruvate carboxykinase, were also degraded in the vacuole via the Vid pathway. These new cargo proteins and FBPase interacted with the TORC1 complex during glucose starvation. However, Tor1p was dissociated from FBPase after the addition of glucose. FBPase degradation was inhibited in cells overexpressing TOR1, suggesting that excessive Tor1p is inhibitory. Both Tco89p and Tor1p were found in endosomes coming from the plasma membrane as well as in retrograde vesicles forming from the vacuole membrane. When TORC1 was inactivated by rapamycin, FBPase degradation was inhibited. We suggest that TORC1 interacts with multiple cargo proteins destined for the Vid pathway and plays an important role in the degradation of FBPase in the vacuole.  相似文献   

10.
Target of rapamycin complexes (TORCs), which are vital for nutrient utilization, contain a catalytic subunit with the phosphatidyl inositol kinase-related kinase (PIKK) motif. TORC1 is required for cell growth, while the functions of TORC2 are less well understood. We show here that the fission yeast Schizosaccharomyces pombe TORC2 has a cell cycle role through determining the proper timing of Cdc2 Tyr15 dephosphorylation and the cell size under limited glucose, whereas TORC1 restrains mitosis and opposes securin-separase, which are essential for chromosome segregation. These results were obtained using the previously isolated TORC1 mutant tor2-L2048S in the phosphatidyl inositol kinase (PIK) domain and a new TORC2 mutant tor1-L2045D, which harbours a mutation in the same site. While mutated TORC1 and TORC2 displayed diminished kinase activity and FKBP12/Fkh1-dependent rapamycin sensitivity, their phenotypes were nearly opposite in mitosis. Premature mitosis and the G2-M delay occurred in TORC1 and TORC2 mutants, respectively. Surprisingly, separase/cut1-securin/cut2 mutants were rescued by TORC1/tor2-L2048S mutation or rapamycin addition or even Fkh1 deletion, whereas these mutants showed synthetic defect with TORC2/tor1-L2045D. TORC1 and TORC2 coordinate growth, mitosis and cell size control, such as Wee1 and Cdc25 do for the entry into mitosis.  相似文献   

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The evolutionarily conserved Orm1 and Orm2 proteins mediate sphingolipid homeostasis. However, the homologous Orm proteins and the signaling pathways modulating their phosphorylation and function are incompletely characterized. Here we demonstrate that inhibition of nutrient-sensitive target of rapamycin complex 1 (TORC1) stimulates Orm phosphorylation and synthesis of complex sphingolipids in Saccharomyces cerevisiae. TORC1 inhibition activates the kinase Npr1 that directly phosphorylates and activates the Orm proteins. Npr1-phosphorylated Orm1 and Orm2 stimulate de novo synthesis of complex sphingolipids downstream of serine palmitoyltransferase. Complex sphingolipids in turn stimulate plasma membrane localization and activity of the nutrient scavenging general amino acid permease 1. Thus activation of Orm and complex sphingolipid synthesis upon TORC1 inhibition is a physiological response to starvation.  相似文献   

13.
The completion of chromosome segregation during anaphase requires the hypercondensation of the ~1-Mb rDNA array, a reaction dependent on condensin and Cdc14 phosphatase. Using systematic genetic screens, we identified 29 novel genetic interactions with budding yeast condensin. Of these, FOB1, CSM1, LRS4, and TOF2 were required for the mitotic condensation of the tandem rDNA array localized on chromosome XII. Interestingly, whereas Fob1 and the monopolin subunits Csm1 and Lrs4 function in rDNA condensation throughout M phase, Tof2 was only required during anaphase. We show that Tof2, which shares homology with the Cdc14 inhibitor Net1/Cfi1, interacts with Cdc14 phosphatase and its deletion suppresses defects in mitotic exit network (MEN) components. Consistent with these genetic data, the onset of Cdc14 release from the nucleolus was similar in TOF2 and tof2Δ cells; however, the magnitude of the release was dramatically increased in the absence of Tof2, even when the MEN pathway was compromised. These data support a model whereby Tof2 coordinates the biphasic release of Cdc14 during anaphase by restraining a population of Cdc14 in the nucleolus after activation of the Cdc14 early anaphase release (FEAR) network, for subsequent release by the MEN.  相似文献   

14.
Target of rapamycin (TOR) kinase controls cell growth and metabolism in response to nutrient availability. In the fission yeast Schizosaccharomyces pombe, TOR complex 1 (TORC1) promotes vegetative growth and inhibits sexual differentiation in the presence of ample nutrients. Here, we report the isolation and characterization of mutants with similar phenotypes as TORC1 mutants, in that they initiate sexual differentiation even in nutrient‐rich conditions. In most mutants identified, TORC1 activity is downregulated and the mutated genes are involved in tRNA expression or modification. Expression of tRNA precursors decreases when cells undergo sexual differentiation. Furthermore, overexpression of tRNA precursors prevents TORC1 downregulation upon nitrogen starvation and represses the initiation of sexual differentiation. Based on these observations, we propose that tRNA precursors operate in the S. pombe TORC1 pathway to switch growth mode from vegetative to reproductive.  相似文献   

15.
《Autophagy》2013,9(8):854-862
It has been reported in various model organisms that autophagy and the target of rapamycin complex 1 (TORC1) signaling are strongly involved in eukaryotic cell aging and decreasing TORC1 activity extends longevity by an autophagy-dependent mechanism. Thus, to expand our knowledge of the regulation of eukaryotic cell aging, it is important to understand the relationship between TORC1 signaling and autophagy. Many researchers have shown that TORC1 represses autophagy under normal growth conditions, and TORC1 inactivation contributes to the upregulation of autophagy. However, it is poorly understood how autophagy is regulated or terminated when starvation is prolonged. Here, we report that bidirectional regulation between autophagy and TORC1 exists in the yeast Saccharomyces cerevisiae. We show that mutant cells with weak TORC1 activity maintain autophagy longer than wild-type cells, and TORC1 is partially reactivated under ongoing nitrogen starvation by an autophagy-dependent mechanism. In addition, we found that Atg13 is gradually rephosphorylated during prolonged nitrogen starvation, and the kinase activity of Atg1 is required for Atg13 rephosphorylation. Our data suggest that TORC1 can be substantially, if not fully, reactivated in an autophagy-dependent manner under ongoing starvation, and that partially reactivated TORC1 eventually plays a role in the attenuation of autophagy.  相似文献   

16.
In the budding yeast Saccharomyces cerevisiae, Cdc14 is sequestered within the nucleolus before anaphase entry through its association with Net1/Cfi1, a nucleolar protein. Protein phosphatase PP2ACdc55 dephosphorylates Net1 and keeps it as a hypophosphorylated form before anaphase. Activation of the Cdc fourteen early anaphase release (FEAR) pathway after anaphase entry induces a brief Cdc14 release from the nucleolus. Some of the components in the FEAR pathway, including Esp1, Slk19, and Spo12, inactivate PP2ACdc55, allowing the phosphorylation of Net1 by mitotic cyclin-dependent kinase (Cdk) (Clb2-Cdk1). However, the function of another FEAR component, the Polo-like kinase Cdc5, remains elusive. Here, we show evidence indicating that Cdc5 promotes Cdc14 release primarily by stimulating the degradation of Swe1, the inhibitory kinase for mitotic Cdk. First, we found that deletion of SWE1 partially suppresses the FEAR defects in cdc5 mutants. In contrast, high levels of Swe1 impair FEAR activation. We also demonstrated that the accumulation of Swe1 in cdc5 mutants is responsible for the decreased Net1 phosphorylation. Therefore, we conclude that the down-regulation of Swe1 protein levels by Cdc5 promotes FEAR activation by relieving the inhibition on Clb2-Cdk1, the kinase for Net1 protein.  相似文献   

17.
Shin CS  Huh WK 《Autophagy》2011,7(8):854-862
It has been reported in various model organisms that autophagy and the target of rapamycin complex 1 (TORC1) signaling are strongly involved in eukaryotic cell aging and decreasing TORC1 activity extends longevity by an autophagy-dependent mechanism. Thus, to expand our knowledge of the regulation of eukaryotic cell aging, it is important to understand the relationship between TORC1 signaling and autophagy. Many researchers have shown that TORC1 represses autophagy under normal growth conditions, and TORC1 inactivation contributes to the upregulation of autophagy. However, it is poorly understood how autophagy is regulated or terminated when starvation is prolonged. Here, we report that bidirectional regulation between autophagy and TORC1 exists in the yeast Saccharomyces cerevisiae. We show that mutant cells with weak TORC1 activity maintain autophagy longer than wild-type cells, and TORC1 is partially reactivated under ongoing nitrogen starvation by an autophagy-dependent mechanism. In addition, we found that Atg13 is gradually rephosphorylated during prolonged nitrogen starvation, and the kinase activity of Atg1 is required for Atg13 rephosphorylation. Our data suggest that TORC1 can be substantially, if not fully, reactivated in an autophagy-dependent manner under ongoing starvation, and that partially reactivated TORC1 eventually plays a role in the attenuation of autophagy.  相似文献   

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The Target Of Rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol-3-kinase-related kinases (PIKKs). TOR proteins are found at the core of two distinct evolutionarily conserved complexes, TORC1 and TORC2. Disruption of TORC1 or TORC2 results in characteristically dissimilar phenotypes. TORC1 is a major cell growth regulator, while the cellular roles of TORC2 are not well understood. In the fission yeast Schizosaccharomyces pombe, Tor1 is a component of the TORC2 complex, which is particularly required during starvation and various stress conditions. Our genome-wide gene expression analysis of Δtor1 mutants indicates an extensive similarity with chromatin structure mutants. Consistently, TORC2 regulates several chromatin-mediated functions, including gene silencing, telomere length maintenance, and tolerance to DNA damage. These novel cellular roles of TORC2 are rapamycin insensitive. Cells lacking Tor1 are highly sensitive to the DNA-damaging drugs hydroxyurea (HU) and methyl methanesulfonate, similar to mutants of the checkpoint kinase Rad3 (ATR). Unlike Rad3, Tor1 is not required for the cell cycle arrest in the presence of damaged DNA. Instead, Tor1 becomes essential for dephosphorylation and reactivation of the cyclin-dependent kinase Cdc2, thus allowing reentry into mitosis following recovery from DNA replication arrest. Taken together, our data highlight critical roles for TORC2 in chromatin metabolism and in promoting mitotic entry, most notably after recovery from DNA-damaging conditions. These data place TOR proteins in line with other PIKK members, such as ATM and ATR, as guardians of genome stability.The TOR protein kinase is a major cell growth regulator that links cellular growth with cell divisions (18, 42, 64, 65). TOR is an atypical protein kinase conserved from yeast to humans that was isolated as the target of the immunosuppressive and anticancer drug rapamycin (28). TOR proteins can be found in two distinct complexes, known as TORC1 and TORC2 (27, 64). These complexes mediate their distinct cellular functions via phosphorylation and activation of different sets of AGC-like kinases, including mammalian p70S6K, downstream of TORC1, and AKT/protein kinase B (PKB) downstream of TORC2 (18). TORC1 in mammals contains mTOR (Tor1 or Tor2 in Saccharomyces cerevisiae; Tor2 in Schizosaccharomyces pombe) and the Raptor protein (Kog1 in S. cerevisiae; Mip1 in S. pombe). TORC1 in many different eukaryotes plays a central role in the control of growth (mass accumulation) in response to external stimuli, particularly nutrient availability. Disruption of TORC1, either by mutating its components or by rapamycin treatment, can lead to a starvation-like phenotype (64). The cellular roles of TORC2, on the other hand, are less well defined. TORC2 in mammals contains mTOR (Tor2 in S. cerevisiae; Tor1 in S. pombe) together with Rictor (Avo3 in S. cerevisiae; Ste20 in S. pombe) and mSin1 (Avo1 in S. cerevisiae; Sin1 in S. pombe). TORC2 plays a role in regulating the actin cytoskeleton and cell wall integrity pathway in S. cerevisiae (3, 15, 27), a function that is at least partially conserved in human cells (17, 47).Fission yeast contains two TOR homologues, Tor1 and Tor2 (59), which form the TORC2 and TORC1 complexes, respectively (14, 32). Disruption tor2+ (TORC1) mimics nitrogen starvation responses (1, 14, 32, 56, 57, 62), while disruption of tor1+ (TORC2) results in pleiotropic defects, including elongated cells, sensitivity to osmotic and oxidative stress, inability to execute developmental processes in response to nutrient depletion, and a decrease in amino acid uptake (16, 22, 59). Tor1 regulates cell survival under stress conditions and starvation responses via the AGC protein kinase Gad8, a putative homologue of mammalian AKT/PKB (16).In budding yeast and mammalian cells, TORC1 mediates the rapamycin-sensitive signaling branch while TORC2 is far less sensitive to inhibition by this drug (27, 48). Curiously, rapamycin does not inhibit growth of S. pombe cells but partially inhibits sexual development and amino acid uptake (60-62). Inhibition of amino acid uptake is likely a result of inhibiting Tor1 (61, 62). Accordingly, a tor1 rapamycin-defective allele (tor1S1834E) confers rapamycin resistance to strains that are dependent on amino acid uptake for their growth (61). Yet rapamycin also induces a response similar to that for a shift from rich to poor nitrogen conditions, an effect that may involve inhibition of both Tor1 and Tor2 (41).While other members of the phosphatidylinositol-3-kinase-related kinase (PIKK) family of proteins, such as ATM and ATR, have been shown to play central roles in the DNA damage response, little is known about roles that TOR proteins might play in such processes. Recently it was shown that the rapamycin-sensitive TORC1 complex participates in regulating cell survival under DNA-damaging conditions (24, 42, 49). Currently, no such role has been attributed to TORC2.Here we show that Tor1 (TORC2) is critical for cell survival under DNA-damaging conditions, gene silencing at heterochromatic regions, and telomere length maintenance and for regulation of cell cycle progression. Since the TOR complexes are highly conserved in evolution, this novel TORC2 function may also be conserved in other organisms.  相似文献   

20.
The target of rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol 3-kinase-related kinases. TOR proteins are found at the core of two evolutionary conserved complexes, known as TORC1 and TORC2. In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. TORC2 has been implicated in a wide variety of functions; however, the signals that regulate TORC2 activity have so far remained obscure. TORC2 has one known direct substrate, the AGC kinase Gad8, which is related to AKT in human cells. Gad8 is phosphorylated by TORC2 at Ser-546 (equivalent to AKT Ser-473), leading to its activation. Here, we show that glucose is necessary and sufficient to induce Gad8 Ser-546 phosphorylation in vivo and Gad8 kinase activity in vitro. The glucose signal that activates TORC2-Gad8 is mediated via the cAMP/PKA pathway, a major glucose-sensing pathway. By contrast, Pmk1, similar to human extracellular signal-regulated kinases and a major stress-induced mitogen activated protein kinase (MAPK) in fission yeast, inhibits TORC2-dependent Gad8 phosphorylation and activation. Inhibition of TORC2-Gad8 also occurs in response to ionic or osmotic stress, in a manner dependent on the cAMP/PKA and Pmk1-MAPK signaling pathways. Our findings highlight the significance of glucose availability in regulation of TORC2-Gad8 and indicate a novel link between the cAMP/PKA, Pmk1/MAPK, and TORC2-Gad8 signaling.  相似文献   

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