A fast method to distinguish between fermentative and respiratory metabolisms in single yeast cells
Saccharomyces cerevisiae adapts its metabolism according to nutrient availability. Typically, it rapidly ferments glucose to ethanol, and then shifts to respiration when glucose becomes limited. However, our understanding of the regulation of metabolism is largely based on population averages, whereas nutrient transitions may cause heterogeneous responses at the individual cell level. Although protein expression can be followed at the single-cell level as a proxy for metabolic modes, direct assessment of the contribution of respiration or (respiro)fermentation to energy metabolism is lacking. Here we describe a method to quickly differentiate between fermentative and respiratory metabolisms in individual cells of budding yeast. The method explores the use of the fluorescent FRET-based biosensor yAT1.03 to measure cytosolic ATP, coupled with the respiratory inhibitor Antimycin A. For the method validation, we used cells under fermentative and respiratory regimes from batch and chemostat cultures. Upon Antimycin A addition, we observed a sharp decrease of the normalized FRET ratio for respiratory cells; respirofermentative cells showed no response. Next, we tracked the changes in metabolism during the diauxic shift of a glucose pre-grown batch culture. Following glucose exhaustion, the entire cell population experienced a progressive rise in intracellular ATP produced via respiration, suggesting a uniform and gradual increase in respiratory capacity as cells pick up growth in a medium with ethanol as the sole carbon source. Overall, the combination of yAT1.03 with Antimycin A is a robust tool to distinguish fermentative from respiratory yeast cells, offering a new single-cell opportunity to study yeast metabolism. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/546324v1_figs1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1db7087org.highwire.dtl.DTLVardef@1b8b839org.highwire.dtl.DTLVardef@46d641org.highwire.dtl.DTLVardef@9c4aac_HPS_FORMAT_FIGEXP M_FIG Identification of fermentative and respiratory metabolisms in yeast cells using an ATP sensor coupled with a respiration inhibitor. (a) yAT1.03 consists of a donor (tdTomato) and an acceptor (ymTq2{Delta}11) domain linked by a binding domain with affinity to ATP. When ATP binds to the binding domain, donor and acceptor come together and the Forster energy is transferred from the first to the second domain. When expressed in in vivo cells the sensor allows real time measurements of ATP changes. (b) Depending on the growth conditions, yeast cells expressing yAT1.03 show a distinct response after being pulsed with the respiratory inhibitor Antimycin A (AA). The drop in ATP levels in respiratory cells caused by AA results from the inhibition of the mitochondrial electron transport chain. (c) Distinct metabolic responses to an AA pulse pre-, during and post-diauxic shift reveal distinct metabolic phenotypes. C_FIG