Evolutionarily conserved target of rapamycin (TOR) complex 1 (TORC1) responds to nutrients, especially amino acids, to promote cell growth. In the yeast Saccharomyces cerevisiae, various nitrogen sources activate TORC1 with different efficiencies, although the mechanism remains elusive. Leucine, and perhaps other amino acids, was reported to activate TORC1 via the heterodimeric small GTPases Gtr1-Gtr2, the orthologues of the mammalian Rag GTPases. More recently, an alternative Gtr-independent TORC1 activation mechanism that may respond to glutamine was reported, although its molecular mechanism is not clear. In studying the nutrient-responsive TORC1 activation mechanism, the lack of an in vitro assay hinders associating particular nutrient compounds with the TORC1 activation status, whereas no in vitro assay that shows nutrient responsiveness has been reported. In this study, we have developed a new in vitro TORC1 kinase assay that reproduces, for the first time, the nutrient-responsive TORC1 activation. This in vitro TORC1 assay recapitulates the previously predicted Gtr-independent glutamine-responsive TORC1 activation mechanism. Using this system, we found that this mechanism specifically responds to L-glutamine, resides on the vacuolar membranes, and involves a previously uncharacterized Vps34-Vps15 phosphatidylinositol (PI) 3-kinase complex and the PI-3-phosphate [PI(3)P]-binding FYVE domain-containing vacuolar protein Pib2. Thus, this system was proved to be useful for dissecting the glutamine-responsive TORC1 activation mechanism.KEYWORDS Saccharomyces cerevisiae, TOR kinase, TORC1, Vps34, glutamine, in vitro kinase assay T he target of rapamycin (TOR) is an evolutionarily conserved protein kinase that regulates cell growth as the catalytic component of rapamycin-sensitive TOR complex 1 (TORC1) (1, 2). TORC1 is activated by nutrients, particularly amino acids, in all tested eukaryotes, and active TORC1 promotes cell growth by activating anabolic processes, including synthesis of protein, lipids, and nucleotides and by inhibiting catabolic processes, such as autophagy. In mammals, TORC1 consists of mammalian/ mechanistic TOR (mTOR), raptor, mammalian Lst8 (mLst8), DEP domain-containing mTOR-interacting protein (DEPTOR), and PRAS40, whereas in the yeast Saccharomyces cerevisiae, TORC1 consists of Tor1 or Tor2, the raptor orthologue Kog1, the mLst8 orthologue Lst8, and Tco89. Recent studies have revealed an evolutionarily conserved amino acid-responsive TORC1 activation mechanism, in which heterodimeric small GTPases, RagA or RagB (RagA/B)-RagC/D in mammals and Gtr1-Gtr2 in yeast, play a central role (3)(4)(5). The mammalian Rag heterodimer is anchored to the lysosomal membrane through the Ragulator complex. An amino acid stimulus induces conversion of RagA/B from the GDP-bound state to the GTP-bound state, and the GTP-bound form directly binds to TORC1 (3, 4), thereby recruiting TORC1 to the lysosomal membrane Citation Tanigawa M, Maeda T. 2017. An in vitro TORC1 kinase assay that recapitulates the...

bThe yeast high-osmolarity glycerol response (HOG) mitogen-activated protein (MAP) kinase pathway is activated in response to hyperosmotic stress via two independent osmosensing branches, the Sln1 branch and the Sho1 branch. While the mechanism by which the osmosensing machinery activates the downstream MAP kinase cascade has been well studied, the mechanism by which the machinery senses and responds to hyperosmotic stress remains to be clarified. Here we report that inhibition of the de novo sphingolipid synthesis pathway results in activation of the HOG pathway via both branches. Inhibition of ergosterol biosynthesis also induces activation of the HOG pathway. Sphingolipids and sterols are known to be tightly packed together in cell membranes to form partitioned domains called rafts. Raft-enriched detergent-resistant membranes (DRMs) contain both Sln1 and Sho1, and sphingolipid depletion and hyperosmotic stress have similar effects on the osmosensing machinery of the HOG pathway: dissociation of an Sln1-containing protein complex and elevated association of Sho1 with DRMs. These observations reveal the sphingolipid-mediated regulation of the osmosensing machinery of the HOG pathway.

TOR complex 1 (TORC1) is an evolutionarily-conserved protein kinase that controls cell growth and metabolism in response to nutrients, particularly amino acids. In mammals, several amino acid sensors have been identified that converge on the multi-layered machinery regulating Rag GTPases to trigger TORC1 activation; however, these sensors are not conserved in many other organisms including yeast. Previously, we reported that glutamine activates yeast TORC1 via a Gtr (Rag ortholog)-independent mechanism involving the vacuolar protein Pib2, although the identity of the supposed glutamine sensor and the exact TORC1 activation mechanism remain unclear. In this study, we successfully reconstituted glutamine-responsive TORC1 activation in vitro using only purified Pib2 and TORC1. In addition, we found that glutamine specifically induced a change in the folding state of Pib2. These findings indicate that Pib2 is a glutamine sensor that directly activates TORC1, providing a new model for the metabolic control of cells.

In poplar, we identified proteins homologous to yeast proteins involved in osmosensing multistep phosphorelay Sln1p-Ypd1p-Ssk1p. This finding led us to speculate that Populus cells could sense osmotic stress by a similar mechanism. This study focuses on first and second protagonists of this possible pathway: a histidine-aspartate kinase (HK1), putative osmosensor and histidine phosphotransfer proteins (HPt1 to 10), potential partners of this HK. Characterization of HK1 showed its ability to homodimerize in two-hybrid tests and to act as an osmosensor with a kinase activity in yeast, by functional complementation of sln1Δ sho1Δ strain. Moreover, in plant cells, plasma membrane localization of HK1 is shown. Further analysis on HPts allowed us to isolate seven new cDNAs, leading to a total of 10 different HPts identified in poplar. Interaction tests showed that almost all HPts can interact with HK1, but two of them exhibit stronger interactions, suggesting a preferential partnership in poplar. The importance of the phosphorylation status in these interactions has been investigated with two-hybrid tests carried out with mutated HK1 forms. Finally, in planta co-expression analysis of genes encoding these potential partners revealed that only three HPts are co-expressed with HK1 in different poplar organs. This result reinforces the hypothesis of a partnership between HK1 and these three preferential HPts in planta. Taken together, these results shed some light on proteins partnerships that could be involved in the osmosensing pathway in Populus.

The budding yeast Saccharomyces cerevisiae alters its gene expression profile in response to changing environmental conditions. The Pho85 kinase, one of the yeast cyclin-dependent kinases (CDK), is known to play an important role in the cellular response to alterations in parameters such as nutrient levels and salinity. Several genes whose expression is regulated, either directly or indirectly, by the Rim101 transcription factor become constitutively activated when Pho85 function is absent,. Because Rim101 is responsible for adaptation to alkaline conditions, this observation suggests an interaction between Pho85 and Rim101 in the response to alkaline stress. We have found that Pho85 affects neither RIM101 transcription, the proteolytic processing that is required for Rim101 activation, nor Rim101 stability. Rather, Pho85 regulates the nuclear accumulation of active Rim101, possibly via phosphorylation. Additionally, we report that Pho85 and the transcription factor Pho4 are necessary for adaptation to alkaline conditions and that PTK2 activation by Pho4 is involved in this process. These findings illustrate novel roles for the regulators of the PHO system when yeast cells cope with various environmental stresses potentially threatening their survival.