Endocytosis of the plasma membrane carboxylate transporter Jen1 inSaccharomyces cerevisiaeunder alkali stress: a mechanism for preventing loss of essential metabolites

Abstract

AbstractWhen grown on lactate as a sole carbon source,Saccharomyces cerevisiaecells alkalinize their surroundings. This alkalinization coincides with endocytosis of the Jen1 lactate transporter. Previous studies showed that this process depends on a functional Jen1 transporter and involves Bul1 α-arrestin and an active TORC1 complex. However, it remained unclear whether the internalization of Jen1 triggered by prolonged growth on lactate was exclusively due to the increase in external pH. To further investigate the regulatory mechanisms underlying the alkali stress-induced Jen1 endocytosis,S. cerevisiaecells stably expressing aJEN1-GFPfusion were studied in aerobic carbon-limited continuous cultures subjected to a linear increase of the culture pH. We found that, in these controlled bioreactor cultures, Jen1 internalization is strongly dependent on extracellular pH, showing a sharp transition at pH values of 7.0 to 7.2. This pH threshold coincides with wash-out of the chemostat cultures and with release of pyruvate and acetate. Transcriptome analysis revealed upregulation of genes involved in oxidative phosphorylation around this threshold, which is consistent with higher cellular energy requirements or energy metabolism being compromised. The originality of this study lies in the use of continuous cultures to analyse the endocytosis of a yeast nutrient transporter. This approach allowed the rigorous study of cell responses to a specific alteration in a single environmental condition, such as changes in pH.