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Yeast Gtr1p and its human homolog RRAG A belong to the Ras-like small G-protein superfamily and genetically interact with RCC1, a guanine nucleotide exchange factor for Ran GTPase. Little is known regarding the function of Gtr1p. We performed yeast two-hybrid screening using Gtr1p as the bait to find interacting proteins. Rpc19p, a shared subunit of RNA polymerases I and III, associated with Gtr1p. The association of Gtr1p with Rpc19p occurred in a GTP-form-specific manner. RRAG A associated with RPA16 (human Rpc19p homolog) in a GTP-form-specific manner, suggesting that the association is conserved during evolution. Ribosomal RNA and tRNA synthesis were reduced in the gtr1Delta strain expressing the GDP form of Gtr1p, but not the GTP form of Gtr1p. Gel-filtration studies revealed an accumulation of the smaller Rpc19p-containing complex, but not of A135, in the gtr1Delta strain. Here, we propose that Gtr1p is involved in RNA polymerase I and III assembly by its association with Rpc19p and could be a mediator that links growth regulatory signals with ribosome biogenesis.  相似文献   

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The target of rapamycin (TOR) kinase integrates nutritional and stress signals to coordinately control cell growth in all eukaryotes. TOR associates with highly conserved proteins to constitute two distinct signaling complexes termed TORC1 and TORC2. Inactivation of TORC1 by rapamycin negatively regulates protein synthesis in most eukaryotes. Here, we report that down-regulation of TOR signaling by rapamycin in the model green alga Chlamydomonas reinhardtii resulted in pronounced phosphorylation of the endoplasmic reticulum chaperone BiP. Our results indicated that Chlamydomonas TOR regulates BiP phosphorylation through the control of protein synthesis, since rapamycin and cycloheximide have similar effects on BiP modification and protein synthesis inhibition. Modification of BiP by phosphorylation was suppressed under conditions that require the chaperone activity of BiP, such as heat shock stress or tunicamycin treatment, which inhibits N-linked glycosylation of nascent proteins in the endoplasmic reticulum. A phosphopeptide localized in the substrate-binding domain of BiP was identified in Chlamydomonas cells treated with rapamycin. This peptide contains a highly conserved threonine residue that might regulate BiP function, as demonstrated by yeast functional assays. Thus, our study has revealed a regulatory mechanism of BiP in Chlamydomonas by phosphorylation/dephosphorylation events and assigns a role to the TOR pathway in the control of BiP modification.  相似文献   

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Background

Ribosomal protein S6 kinase 1(S6K1) is an evolutionary conserved kinase that is activated in response to growth factors and viral stimuli to influence cellular growth and proliferation. This downstream effector of target of rapamycin (TOR) signaling cascade is known to be directly activated by TOR- kinase mediated hydrophobic motif (HM) phosphorylation at Threonine 412 (T412). Selective loss of this phosphorylation by inactivation of TOR kinase or activation/recruitment of a phosphatase has accordingly been implicated in mediating inhibition by rapamycin.

Findings

We present evidence that baculovirus driven expression of S6K1 in insect cells (Sf9) fails to activate the enzyme and instead renders it modestly active representing 4-6 folds less activity than its fully active mammalian counterpart. Contrary to the contention that viral infection activates TOR signaling pathway, we report that BVr enzyme fails to exhibit putative TOR dependent phosphorylation at the HM and the resultant phosphorylation at the activation loop (AL) of the enzyme, correlating with the level of activity observed. Surprisingly, the BVr enzyme continued to exhibit sensitivity to rapamycin that remained unaffected by mutations compromised for TOR phosphorylation (T412A) or deletions compromised for TOR binding (ΔNH 2-46/ΔCT104).

Conclusions

These data together with the ability of the BVr enzyme to resist inactivation by phosphatases indicate that inhibition by rapamycin is not mediated by any phosphorylation event in general and TOR dependent phosphorylation in particular.  相似文献   

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Target of rapamycin (TOR) is a central regulator of cellular and organismal growth in response to nutrient conditions. In a genetic screen for novel TOR interactors in Drosophila melanogaster, we have identified the clathrin-uncoating ATPase Hsc70-4, which is a key regulator of endocytosis. We present genetic evidence that TOR signaling stimulates bulk endocytic uptake and inhibits the targeted endocytic degradation of the amino acid importer Slimfast. Thus, TOR simultaneously down-regulates aspects of endocytosis that inhibit growth and up-regulates potential growth-promoting functions of endocytosis. In addition, we find that disruption of endocytosis leads to changes in TOR and phosphatidylinositol-3 kinase activity, affecting cell growth, autophagy, and rapamycin sensitivity. Our data indicate that endocytosis acts both as an effector function downstream of TOR and as a physiologically relevant regulator of TOR signaling.  相似文献   

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Mitogenic and nutritional signals must be integrated for a cell to grow. The target of rapamycin (TOR) is emerging as an effector for signals which indicate to the cell whether the external environment is conducive for growth. Use of the immunosuppressant rapamycin, a bacterial macrolide, has been instructive in identifying potential signaling components downstream of TOR, leading to the observation that both protein synthesis and turnover are under TOR control. The central issues concerning TOR are the identification of the proliferative and anti-proliferative signals which mediate its function and the mechanisms by which these signals are transduced to downstream molecules.  相似文献   

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Eukaryotic RNA polymerases are multisubunit assemblies, whose enzymatic function in the nucleus is intensively studied. However, little is known about the biogenesis of the three RNA polymerases and coupling to nucleo-cytoplasmic transport. Here, we show that Rpc128, the second largest subunit of RNA polymerase III, was mislocalized to the cytoplasm, when a short sequence in the N-terminal domain was deleted. Importantly, nuclear import of other, but not all, RNA polymerase III subunits was impaired in this RPC128DeltaN mutant. These data suggest that RNA polymerase III subunits are not imported independently into the nucleus but may require preassembly into cytoplasmic subcomplexes for coordinated nuclear uptake. We expect these studies to be a starting point to dissect the complex biogenesis pathway of eukaryotic RNA polymerases.  相似文献   

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