Abstract
When Saccharomyces cerevisiae is starved of glucose, the gluconeogenic enzymes fructose-1,6-bisphosphatase (FBPase), phosphoenolpyruvate carboxykinase, isocitrate lyase, and malate dehydrogenase, as well as the non-gluconeogenic enzymes glyceraldehyde-3-phosphate dehydrogenase and cyclophilin A, are secreted into the periplasm. In the extracellular fraction, these secreted proteins are associated with small vesicles that account for more than 90% of the total number of extracellular structures observed. When glucose is added to glucose-starved cells, FBPase is internalized and associated with clusters of small vesicles in the cytoplasm. Specifically, the internalization of FBPase results in the decline of FBPase and vesicles in the extracellular fraction and their appearance in the cytoplasm. The clearance of extracellular vesicles and vesicle-associated proteins from the extracellular fraction is dependent on the endocytosis gene END3. This internalization is regulated when cells are transferred from low to high glucose. It is rapidly occurring and is a high capacity process, as clusters of vesicles occupy 10%–20% of the total volume in the cytoplasm in glucose re-fed cells. FBPase internalization also requires the VPS34 gene encoding PI3K. Following internalization, FBPase is delivered to the vacuole for degradation, whereas proteins that are not degraded may be recycled.
Highlights
Saccharomyces cerevisiae is an excellent model system to study cellular responses to environmental changes such as temperature, oxidative stress, and the availability of carbon sources [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15]
Glucose represses genes required for mitochondrial functions and genes encoding for gluconeogenic enzymes that include FBP1, ICL1, PCK1, and MLS1 [4,6,17,26,28,29,30]
At the t = 30 min time point, most of the FBPase was found in intracellular structures that contained clusters of vesicles (Figure 1B, arrow). These results suggest that FBPase and vesicles are internalized in response to glucose addition resulting in the appearance of FBPase and vesicles in the cytoplasm in glucose re-fed cells
Summary
Saccharomyces cerevisiae is an excellent model system to study cellular responses to environmental changes such as temperature, oxidative stress, and the availability of carbon sources [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15]. The vacuole import and degradation (Vid) pathway utilizes small vesicles to carry the gluconeogenic enzymes FBPase, MDH2, Pck1p, and Icl1p to the vacuole for degradation following glucose replenishment to glucose-starved cells. As shown by immuno-TEM, FBPase, MDH2, Icl1p, Pck1p, GAPDH, and Cpr1p are secreted into the periplasm in glucose-starved cells. The extraction procedure detects the presence of FBPase, MDH2, Icl1p, Pck1p, GAPDH, and Cpr1p in the extracellular fraction in glucose-starved cells [93]. In glucose-starved cells, FBPase, MDH2, Icl1p, Pck1p, GAPDH, and Cpr1p were present in the extracellular fraction [93]. Having established that the extraction protocol was useful in detecting the presence of FBPase, MDH2, Icl1p, Pck1p, GAPDH, and Cpr1p in the extracellular fraction, this protocol was used to examine whether or not secretion of these proteins into the extracellular fraction was dependent on the duration of starvation. Many of these extracellular proteins found in yeast have been identified in multiple large-scale secretomic/proteomic studies from organisms that ranged from bacteria to humans [75,76,77,78,83,84,85,86], suggesting that the secretion of these proteins via the non-classical pathways is conserved across species
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