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Review
. 2014 Sep 3;4(3):608-29.
doi: 10.3390/membranes4030608.

Exocytosis and Endocytosis of Small Vesicles across the Plasma Membrane in Saccharomyces cerevisiae

Kathryn Stein  1 , Hui-Ling Chiang  2
Affiliations
Review

Exocytosis and Endocytosis of Small Vesicles across the Plasma Membrane in Saccharomyces cerevisiae

Kathryn Stein et al. Membranes (Basel). .

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.

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Figures

Figure 1
Figure 1
FBPase is re-distributed from the periplasm to the cytoplasm following glucose addition. Wild-type cells were grown in low glucose media for 3 days and harvested (t = 0 min) (A); or transferred to high glucose media for 30 min and harvested (t = 30 min) (B). Cells were processed and FBPase was visualized by immuno-TEM. Bars: 200 nm, PM: plasma membrane, CW: cell wall. This figure is in a manuscript published in Proteome Science [73].
Figure 2
Figure 2
Glucose induces a rapid decline of small vesicles in the extracellular fraction. Wild-type and ∆end3 cells were glucose starved for 3 days and re-fed with glucose for 30 min. Total extracts were obtained and centrifuged at 3000× g and then at 200,000× g. The 200,000× g pellet fraction was fixed, stained with uranyl acetate, and visualized by TEM (A). Bars: 200 nm. The number of 30–50 nm small vesicles per µm2 at t = 0 and t = 30 min in wild-type (B) and ∆end3 (C) cells was quantified. The number of 100–300 nm large structures per µm2 at t = 0 and t = 30 min in wild-type (D) and ∆end3 (E) cells was quantified. This figure is in a manuscript published in the Journal of Extracellular Vesicles [93].
Figure 3
Figure 3
FBPase, MDH2, Icl1p, Pck1p, GAPDH, and Cpr1p are associated with vesicles in the extracellular fraction. Total extracts were obtained from wild-type cells starved of glucose for 3 days and subjected to ultracentrifugation. The final 200,000× g pellet fraction was aliquoted, and incubated with or without 2% SDS for 30 min. Samples were re-centrifuged at 200,000× g for 2 h following incubation. Western blotting was used to examine the distribution of FBPase, MDH2, Icl1p, Pck1p, GAPDH, and Cpr1p in the 200,000× g supernatant (S) and pellet (P) fractions. This figure is in a manuscript published in the Journal of Extracellular Vesicles [93].
Figure 4
Figure 4
The decline of extracellular FBPase, MDH2, Icl1p, Pck1p, GAPDH, and Cpr1p in response to glucose re-feeding is dependent on END3. Wild-type cells (A) and the ∆end3 cells (B) were starved of glucose for 3 days and re-fed with glucose for 0, 15, and 30 min. Western blotting was used to examine levels of FBPase, MDH2, Icl1p, Pck1p, GAPDH, and Cpr1p in the intracellular (I) and extracellular (E) fractions. This figure is in a manuscript published in the Journal of Extracellular Vesicles [93].
Figure 5
Figure 5
FBPase fails to be internalized in response to glucose in cells lacking the VPS34 gene. The ∆vps34 mutant was starved of glucose for 3 days (A) and then re-fed with glucose for 2 h (B). Cells were processed and examined for the distribution of FBPase using immuno-TEM. This figure is in a manuscript published in the Journal of Biological Chemistry [72].
Figure 6
Figure 6
Vid vesicles are secreted into the periplasm during glucose starvation and are internalized following glucose addition. When yeast cells are grown in low glucose, Vid vesicles that carry gluconeogenic enzymes and non-gluconeogenic enzymes are secreted into the periplasm via the non-classical secretory pathway (A). In the periplasm, 100–300 nm structures are also present (A). In the cytoplasm, FBPase is associated with Vid vesicles. The association is dependent on Ubc1p, Ssa1p/Ssa2p, Vid22p, and Cpr1p. Vid vesicles also aggregate to form large clusters and associate with actin patches. This step requires the VID28 and VID30 genes. Following glucose addition to glucose-starved cells for 30 min, FBPase is internalized (B). The internalization requires the endocytosis gene END3 and the VPS34 gene encoding PI3K (B). The 100–300 nm structures decrease in number but are still observed at the t = 30 min time point. Following internalization, FBPase is delivered to the vacuole to be degraded, while proteins that are not degraded may be recycled. Vid24p, Sec28p, Reg1p/Glc7p, V-ATPase, Ypt7p, HOPS, v-SNARE and t-SNARE are critical for the post-internalization step in the Vid pathway [44,48,98,99,100].
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