Real-time monitoring of the sugar sensing in Saccharomyces cerevisiae indicates endogenous mechanisms for xylose signaling.

BACKGROUND: The sugar sensing and carbon catabolite repression in Baker's yeast Saccharomyces cerevisiae is governed by three major signaling pathways that connect carbon source recognition with transcriptional regulation. Here we present a screening method based on a non-invasive in vivo reporter system for real-time, single-cell screening of the sugar signaling state in S. cerevisiae in response to changing carbon conditions, with a main focus on the response to glucose and xylose. RESULTS: The artificial reporter system was constructed by coupling a green fluorescent protein gene (yEGFP3) downstream of endogenous yeast promoters from the Snf3p/Rgt2p, SNF1/Mig1p and cAMP/PKA signaling pathways: HXT1p/2p/4p; SUC2p, CAT8p; TPS1p/2p and TEF4p respectively. A panel of eight biosensors strains was generated by single copy chromosomal integration of the different constructs in a W303-derived strain. The signaling biosensors were validated for their functionality with flow cytometry by comparing the fluorescence intensity (FI) response in the presence of high or nearly depleted glucose to the known induction/repression conditions of the eight different promoters. The FI signal correlated with the known patterns of the selected promoters while maintaining a non-invasive property on the cellular phenotype, as was demonstrated in terms of growth, metabolites and enzyme activity. CONCLUSIONS: Once verified, the sensors were used to evaluate the signaling response to varying conditions of extracellular glucose, glycerol and xylose by screening in 96-well microtiter plates. We show that these yeast strains, which do not harbor any recombinant pathways for xylose utilization, are lacking a signaling response for extracellular xylose. However, for the HXT2p/4p sensors, a shift in the flow cytometry population dynamics indicated that internalized xylose does affect the signaling. These results suggest that the previously observed effects of this pentose on the S. cerevisiae physiology and gene regulation can be attributed to xylose and not only to a lack of glucose.

Glycolytic Enzymes Localize to Synapses under Energy Stress to Support Synaptic Function.

Changes in neuronal activity create local and transient changes in energy demands at synapses. Here we discover a metabolic compartment that forms in vivo near synapses to meet local energy demands and support synaptic function in Caenorhabditis elegans neurons. Under conditions of energy stress, glycolytic enzymes redistribute from a diffuse localization in the cytoplasm to a punctate localization adjacent to synapses. Glycolytic enzymes colocalize, suggesting the ad hoc formation of a glycolysis compartment, or a "glycolytic metabolon," that can maintain local levels of ATP. Local formation of the glycolytic metabolon is dependent on presynaptic scaffolding proteins, and disruption of the glycolytic metabolon blocks the synaptic vesicle cycle, impairs synaptic recovery, and affects locomotion. Our studies indicate that under energy stress conditions, energy demands in C. elegans synapses are met locally through the assembly of a glycolytic metabolon to sustain synaptic function and behavior. VIDEO ABSTRACT.