The nutrient-responsive CDK Pho85 primes the Sch9 kinase for its activation by TORC1.

Yeast cells maintain an intricate network of nutrient signaling pathways enabling them to integrate information on the availability of different nutrients and adjust their metabolism and growth accordingly. Cells that are no longer capable of integrating this information, or that are unable to make the necessary adaptations, will cease growth and eventually die. Here, we studied the molecular basis underlying the synthetic lethality caused by loss of the protein kinase Sch9, a key player in amino acid signaling and proximal effector of the conserved growth-regulatory TORC1 complex, when combined with either loss of the cyclin-dependent kinase (CDK) Pho85 or loss of its inhibitor Pho81, which both have pivotal roles in phosphate sensing and cell cycle regulation. We demonstrate that it is specifically the CDK-cyclin pair Pho85-Pho80 or the partially redundant CDK-cyclin pairs Pho85-Pcl6/Pcl7 that become essential for growth when Sch9 is absent. Interestingly, the respective three CDK-cyclin pairs regulate the activity and distribution of the phosphatidylinositol-3 phosphate 5-kinase Fab1 on endosomes and vacuoles, where it generates phosphatidylinositol-3,5 bisphosphate that serves to recruit both TORC1 and its substrate Sch9. In addition, Pho85-Pho80 directly phosphorylates Sch9 at Ser726, and to a lesser extent at Thr723, thereby priming Sch9 for its subsequent phosphorylation and activation by TORC1. The TORC1-Sch9 signaling branch therefore integrates Pho85-mediated information at different levels. In this context, we also discovered that loss of the transcription factor Pho4 rescued the synthetic lethality caused by loss of Pho85 and Sch9, indicating that both signaling pathways also converge on Pho4, which appears to be wired to a feedback loop involving the high-affinity phosphate transporter Pho84 that fine-tunes Sch9-mediated responses.

The Hsp70 homolog Ssb affects ribosome biogenesis via the TORC1-Sch9 signaling pathway.

The Hsp70 Ssb serves a dual role in de novo protein folding and ribosome biogenesis; however, the mechanism by which Ssb affects ribosome production is unclear. Here we establish that Ssb is causally linked to the regulation of ribosome biogenesis via the TORC1-Sch9 signaling pathway. Ssb is bound to Sch9 posttranslationally and required for the TORC1-dependent phosphorylation of Sch9 at T737. Also, Sch9 lacking phosphorylation at T737 displays significantly reduced kinase activity with respect to targets involved in the regulation of ribosome biogenesis. The absence of either Ssb or Sch9 causes enhanced ribosome aggregation. Particularly with respect to proper assembly of the small ribosomal subunit, SSB and SCH9 display strong positive genetic interaction. In combination, the data indicate that Ssb promotes ribosome biogenesis not only via cotranslational protein folding, but also posttranslationally via interaction with natively folded Sch9, facilitating access of the upstream kinase TORC1 to Sch9-T737.The yeast Hsp70 homolog Ssb is a chaperone that binds translating ribosomes where it is thought to function primarily by promoting nascent peptide folding. Here the authors find that the ribosome biogenesis defect associated with the loss of Ssb is attributable to a specific disruption in TORC1 signaling rather than defects in ribosomal protein folding.

The yeast Hsp70 homolog Ssb: a chaperone for general de novo protein folding and a nanny for specific intrinsically disordered protein domains.

Activation of the heterotrimeric kinase SNF1 via phosphorylation of a specific residue within the α subunit is essential for the release from glucose repression in the yeast Saccharomyces cerevisiae. When glucose is available, SNF1 is maintained in the dephosphorylated, inactive state by the phosphatase Glc7-Reg1. Recent findings suggest that Bmh and Ssb combine their unique client-binding properties to interact with the regulatory region of the SNF1 α subunit and by that stabilize a conformation of SNF1, which is accessible for Glc7-Reg1-dependent dephosphorylation. Together, the 14-3-3 protein Bmh and the Hsp70 homolog Ssb comprise a novel chaperone module, which is required to maintain proper glucose repression in the yeast S. cerevisiae.

The Hsp70 homolog Ssb and the 14-3-3 protein Bmh1 jointly regulate transcription of glucose repressed genes in Saccharomyces cerevisiae.

Chaperones of the Hsp70 family interact with a multitude of newly synthesized polypeptides and prevent their aggregation. Saccharomyces cerevisiae cells lacking the Hsp70 homolog Ssb suffer from pleiotropic defects, among others a defect in glucose-repression. The highly conserved heterotrimeric kinase SNF1/AMPK (AMP-activated protein kinase) is required for the release from glucose-repression in yeast and is a key regulator of energy balance also in mammalian cells. When glucose is available the phosphatase Glc7 keeps SNF1 in its inactive, dephosphorylated state. Dephosphorylation depends on Reg1, which mediates targeting of Glc7 to its substrate SNF1. Here we show that the defect in glucose-repression in the absence of Ssb is due to the ability of the chaperone to bridge between the SNF1 and Glc7 complexes. Ssb performs this post-translational function in concert with the 14-3-3 protein Bmh, to which Ssb binds via its very C-terminus. Raising the intracellular concentration of Ssb or Bmh enabled Glc7 to dephosphorylate SNF1 even in the absence of Reg1. By that Ssb and Bmh efficiently suppressed transcriptional deregulation of Δreg1 cells. The findings reveal that Ssb and Bmh comprise a new chaperone module, which is involved in the fine tuning of a phosphorylation-dependent switch between respiration and fermentation.

Mangiferin, a dietary xanthone protects against mercury-induced toxicity in HepG2 cells.

Mercury is one of the noxious heavy metal environmental toxicants and is a cause of concern for human exposure. Mangiferin (MGN), a glucosylxanthone found in Mangifera indica, reported to have a wide range of pharmacological properties. The objective of this study was to evaluate the cytoprotective potential of MGN, against mercury chloride (HgCl(2) ) induced toxicity in HepG2 cell line. The cytoprotective effect of MGN on HgCl(2) induced toxicity was assessed by colony formation assay, while antiapoptotic effect by fluorescence microscopy, flow cytometric DNA analysis, and DNA fragmentation pattern assays. Further, the cytoprotective effect of MGN against HgCl(2) toxicity was assessed by using biochemical parameters like reduced glutathione (GSH), glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT) by spectrophotometrically, mitochondrial membrane potential by flowcytometry and the changes in reactive oxygen species levels by DCFH-DA spectrofluoremetric analysis. A significant increase in the surviving fraction was observed with 50 µM of MGN administered two hours prior to various concentrations of HgCl(2) . Further, pretreatment of MGN significantly decreased the percentage of HgCl(2) induced apoptotic cells. Similarly, the levels of ROS generated by the HgCl(2) treatment were inhibited significantly (P < 0.01) by MGN. MGN also significantly (P < 0.01) inhibited the HgCl(2) induced decrease in GSH, GST, SOD, and CAT levels at all the post incubation intervals. Our study demonstrated the cytoprotective potential of MGN, which may be attributed to quenching of the ROS generated in the cells due to oxidative stress induced by HgCl(2) , restoration of mitochondrial membrane potential and normalization of cellular antioxidant levels.