Acetate and hypertonic stress stimulate vacuole membrane fission using distinct mechanisms

Vacuoles in plants and fungi play critical roles in cell metabolism and osmoregulation. To support these functions, vacuoles change their morphology, e.g. they fragment when these organisms are challenged with draught, high salinity or metabolic stress (e.g. acetate accumulation). In turn, morphology reflects an equilibrium between membrane fusion and fission that determines size, shape and copy number. By studying Saccharomyces cerevisiae and its vacuole as models, conserved molecular mechanisms responsible for fusion have been revealed. However, a detailed understanding of vacuole fission and how these opposing processes respond to metabolism or osmoregulation remain elusive. Herein we describe a new fluorometric assay to measure yeast vacuole fission in vitro. For proof-of-concept, we use this assay to confirm that acetate, a metabolic stressor, triggers vacuole fission and show it blocks homotypic vacuole fusion in vitro. Similarly, hypertonic stress induced by sorbitol or glucose caused robust vacuole fission in vitro whilst inhibiting fusion. Using wortmannin to inhibit phosphatidylinositol (PI)-kinases or rGyp1-46 to inactivate Rab-GTPases, we show that acetate stress likely targets PI signaling, whereas osmotic stress affects Rab signaling on vacuole membranes to stimulate fission. This study sets the stage for further investigation into the mechanisms that change vacuole morphology to support cell metabolism and osmoregulation.

Automated summary

Vacuoles in plants and fungi have critical roles in cell metabolism and osmoregulation. They change their morphology in response to stressors like drought, high salinity, or metabolic stress. This study focuses on understanding vacuole fission and its response to metabolism and osmoregulation. The researchers developed a new assay to measure yeast vacuole fission in vitro and used it to confirm that acetate stress triggers fission and inhibits fusion. They also found that hypertonic stress promotes fission while inhibiting fusion and identified the signaling pathways involved. This study provides a foundation for further research on the mechanisms of vacuole morphology changes in cell metabolism and osmoregulation.