TORC1 Determines Fab1 Lipid Kinase Function at Signaling Endosomes and Vacuoles

Organelles of the endomembrane system maintain their identity and integrity during growth or stress conditions by homeostatic mechanisms that regulate membrane flux and biogenesis. At lysosomes and endosomes, the Fab1 lipid kinase complex and the nutrient-regulated target of rapamycin complex 1 (TORC1) control the integrity of the endolysosomal homeostasis and cellular metabolism. Both complexes are functionally connected as Fab1-dependent generation of PI(3,5)P2 supports TORC1 activity. Here, we identify Fab1 as a target of TORC1 on signaling endosomes, which are distinct from multivesicular bodies, and provide mechanistic insight into their crosstalk. Accordingly, TORC1 can phosphorylate Fab1 proximal to its PI3P-interacting FYVE domain, which causes Fab1 to shift to signaling endosomes, where it generates PI(3,5)P2. This, in turn, regulates (1) vacuole morphology, (2) recruitment of TORC1 and the TORC1-regulatory Rag GTPase-containing EGO complex to signaling endosomes, and (3) TORC1 activity. Thus, our study unravels a regulatory feedback loop between TORC1 and the Fab1 complex that controls signaling at endolysosomes

Identification of a small molecule yeast TORC1 inhibitor with a flow cytometry-based multiplex screen

TOR (target of rapamycin) is a serine/threonine kinase, evolutionarily conserved from yeast to human, which functions as a fundamental controller of cell growth. The moderate clinical benefit of rapamycin in mTOR-based therapy of many cancers favors the development of new TOR inhibitors. Here we report a high throughput flow cytometry multiplexed screen using five GFPtagged yeast clones that represent the readouts of four branches of the TORC1 signaling pathway in budding yeast. Each GFP-tagged clone was differentially color-coded and the GFP signal of each clone was measured simultaneously by flow cytometry, which allows rapid prioritization of compounds that likely act through direct modulation of TORC1 or proximal signaling components. A total of 255 compounds were confirmed in dose-response analysis to alter GFP expression in one or more clones. To validate the concept of the high throughput screen, we have characterized CID 3528206, a small molecule most likely to act on TORC1 as it alters GFP expression in all five GFP clones in an analogous manner to rapamycin. We have shown that CID 3528206 inhibited yeast cell growth, and that CID 3528206 inhibited TORC1 activity both in vitro and in vivo with EC50s of 150 nM and 3.9 μM, respectively. The results of microarray analysis and yeast GFP collection screen further support the notion that CID 3528206 and rapamycin modulate similar cellular pathways. Together, these results indicate that the HTS has identified a potentially useful small molecule for further development of TOR inhibitors

Effect of elevated CO2 on lycopene content of tomato (Lycopersicon lycopersicum L. Karsten) fruits

Recently several studies have focused on the antioxidant activity of lycopene such as quenching of singlet oxygen and scavenging of peroxyl radicals. These properties may play a role in the prevention of different cancer and heart diseases. Tomato is one of the most important sources of lycopene. The main information on the effect of environmental parameters on quality and health-retaining constituents of tomato fruit is mostly related to temperature (air- and fruit canopy temperature) and light effects that might provide a stress to the fruit. Nowadays little is know about the direct effect of elevated CO2.The aim of the present work was to evaluate the effects of elevated CO2 in Perspex open top chambers (OTC) on the lycopene content of tomato fruit.Experiments on the effects of elevated CO2 concentrations showed mixed results. In this work it was found that concentrations of lycopene in a fruit decreased significantly when elevated CO2 was used. Elevated nitrogen sources generated only slight, but not significant difference in the lycopene concentration of tomato fruit

Long-term response of the nematode community to elevated atmospheric CO2 in a temperate dry grassland soil

Long-term effects of the elevated atmospheric CO2 on biosphere have been in focus of research since the last few decades. In this experiment undisturbed soil monoliths of loess grassland were exposed to an elevated CO2 environment (two-times the ambient CO2 level) for a period of six years with the aid of the open top chamber method. Control without a chamber and CO2 elevation was applied as well. Elevated CO2 level had very little impact on soil food web. It did not influence either root and microbial biomass or microbial and nematode community structure. The only significant response was that density of the bacterial feeder genus Heterocephalobus increased in the chamber with elevated CO2 concentration. Application of the open top chambers initiated more changes on nematodes than the elevated CO2 level. Open top chamber (OTC) method decreased nematode density (total and plant feeder as well) to less than half of the original level. Negative effect was found on the genus level in the case of fungal feeder Aphelenchoides , plant feeder Helicotylenchus and Paratylenchus . It is very likely that the significantly lower belowground root biomass and partly its decreased quality reflected by the increased C/N ratio are the main responsible factors for the lower density of the plant feeder nematodes in the plots of chambers. According to diversity profiles, MI and MI(2–5) parameters, nematode communities in the open top chambers (both on ambient and elevated CO2 level) seem to be more structured than those under normal circumstances six years after start of the experiment

Elevated CO2 affects the content of glomalin related soil protein in xeric temperate loess and temperate semi-desert sand grasslands

Monoliths of temperate loess grassland and temperate semi-desert sand grassland have been exposed to elevated CO2 (700 μmol mol-1 ) and present ambient CO2 concentration in a 6-year open top chamber (OTC) experiment. In loess grassland elevated CO2 increased both biomass and vegetation cover, whereas there was no similar effect found in semi-desert grassland. The content of glomalin related soil protein (GRSP) increased in both loess and sand grasslands under CO2 enrichment (early summer aspect). The increase was higher in the case of easily extractable fraction (EEG), representing 14.7 and 22.2% of the chambered control’s EEG, for loess and sand grassland respectively. In the case of total glomalin the increase was much lower 7.9% (loess) and 2.6% (sand). On the basis of differences between elevated and ambient CO2 treatment we could conclude that elevated CO2 promoted C-deposition in xeric temperate grassland in early summer. Increases of EEG indicate an efficient partitioning of the recently fixed carbon to the soil

Effects of irrigation on community composition and carbon uptake in Pannonian loess grassland monoliths

Grassland ecosystems in the Carpathian Basin may be particularly vulnerable to current and predicted changes in precipitation, and ecosystem responses to potential effects of water are not well understood. To examine how water addition can affect the species composition and structure, and CO2 -flux of a Central European natural steppe plant community, grassland monoliths were irrigated for three consecutive years at Gödöllő, from 2002 through 2004. The loess grassland studied by ex situ is a characteristic plant association of Hungary and similar vegetation can be found in other temperate regions. The treatment consisted of spray irrigation during night-time only in the growing season as well as aboveground biomass removal twice per year. Interannual and intraannual dynamics of species richness, Shannon Diversity, percentage cover, and different functional groups (monocots/dicots; plant life forms; social behaviour types; C4/C3 plants), and Net Ecosystem CO2. Exchange in treated and untreated permanent plots, were studied simultaneously. To measure NEE and water vapour at stand level a self-developed, portable, non-destructive open chamber system (d=60cm) was used. The majority of the examined parameters varied considerably among years at both irrigated and control, but concerning carbon fluxes water addition effects were evident in dry periods only. At the treated plots, in general species richness, Shannon Diversity, the number of plant life forms and social behaviour types, the ratio of dicots and C4 plants declined with addition of water. Our study proved that decline in species richness and Shannon diversity is not necessarily followed by the reduction of stand physiological (synphysiological) processes