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Glucose induces rapid changes in the secretome of Saccharomyces cerevisiae.

Giardina BJ, Stanley BA, Chiang HL - Proteome Sci (2014)

Bottom Line: Most of these proteins did not contain typical ER-Golgi signal sequences.Therefore, we conclude that the secretome undergoes dynamic changes during transition from glucose-deficient to glucose-rich media.Most of these extracellular proteins do not contain typical ER signal sequences, suggesting that they are secreted via the non-classical pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Penn State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA. hxc32@psu.edu.

ABSTRACT

Background: Protein secretion is a fundamental process in all living cells. Proteins can either be secreted via the classical or non-classical pathways. In Saccharomyces cerevisiae, gluconeogenic enzymes are in the extracellular fraction/periplasm when cells are grown in media containing low glucose. Following a transfer of cells to high glucose media, their levels in the extracellular fraction are reduced rapidly. We hypothesized that changes in the secretome were not restricted to gluconeogenic enzymes. The goal of the current study was to use a proteomic approach to identify extracellular proteins whose levels changed when cells were transferred from low to high glucose media.

Results: We performed two iTRAQ experiments and identified 347 proteins that were present in the extracellular fraction including metabolic enzymes, proteins involved in oxidative stress, protein folding, and proteins with unknown functions. Most of these proteins did not contain typical ER-Golgi signal sequences. Moreover, levels of many of these proteins decreased upon a transfer of cells from media containing low to high glucose media. Using an extraction procedure and Western blotting, we confirmed that the metabolic enzymes (glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, glucose-6-phosphate dehydrogenase, pyruvate decarboxylase), proteins involved in oxidative stress (superoxide dismutase and thioredoxin), and heat shock proteins (Ssa1p, Hsc82p, and Hsp104p) were in the extracellular fraction during growth in low glucose and that the levels of these extracellular proteins were reduced when cells were transferred to media containing high glucose. These proteins were associated with membranes in vesicle-enriched fraction. We also showed that small vesicles were present in the extracellular fraction in cells grown in low glucose. Following a transfer from low to high glucose media for 30 minutes, 98% of these vesicles disappeared from the extracellular fraction.

Conclusions: Our data indicate that transferring cells from low to high glucose media induces a rapid decline in levels of a large number of extracellular proteins and the disappearance of small vesicles from the extracellular fraction. Therefore, we conclude that the secretome undergoes dynamic changes during transition from glucose-deficient to glucose-rich media. Most of these extracellular proteins do not contain typical ER signal sequences, suggesting that they are secreted via the non-classical pathway.

No MeSH data available.


Related in: MedlinePlus

Extracted cells transport the vital dye FM to the vacuole membrane. (A-D), wild-type cells were grown in YPKG for 3d (t = 0) or transferred to YPD for 30 min (t = 30). Cells were divided and half of the cells were extracted. Non-extracted and extracted cells at the t = 0 and t = 30 min time points were incubated with or without 2% Triton X-100 (TX) for 30 min followed by incubation with trypan blue for 30 min. Cells were examined using a light microscope. (E-L), wild-type cells were grown in YPKG for 3d (t = 0) or transferred to glucose for 30 min (t = 30) and harvested. Cells were incubated in the absence or presence of Triton X-100 (TX) and incubated with FM for 3 hours. The distribution of FM was observed by fluorescence microscopy in non-extracted t = 0 cells (E), t = 30 cells (G), TX treated t = 0 cells (F), and TX treated t = 30 cells (H). The same amounts of t = 0 and t = 30 min cells were subjected to the extraction procedure and treated in the absence (I and K) or presence of Triton X-100 (J and L). Cells were incubated with FM and the distribution of FM was examined by fluorescence microscopy.
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Figure 3: Extracted cells transport the vital dye FM to the vacuole membrane. (A-D), wild-type cells were grown in YPKG for 3d (t = 0) or transferred to YPD for 30 min (t = 30). Cells were divided and half of the cells were extracted. Non-extracted and extracted cells at the t = 0 and t = 30 min time points were incubated with or without 2% Triton X-100 (TX) for 30 min followed by incubation with trypan blue for 30 min. Cells were examined using a light microscope. (E-L), wild-type cells were grown in YPKG for 3d (t = 0) or transferred to glucose for 30 min (t = 30) and harvested. Cells were incubated in the absence or presence of Triton X-100 (TX) and incubated with FM for 3 hours. The distribution of FM was observed by fluorescence microscopy in non-extracted t = 0 cells (E), t = 30 cells (G), TX treated t = 0 cells (F), and TX treated t = 30 cells (H). The same amounts of t = 0 and t = 30 min cells were subjected to the extraction procedure and treated in the absence (I and K) or presence of Triton X-100 (J and L). Cells were incubated with FM and the distribution of FM was examined by fluorescence microscopy.

Mentions: We first determined whether or not cells that were subjected to the extraction procedure retained the ability to exclude trypan blue from cells (Figure 3A-D). Trypan blue dye permeates dead cells and is therefore an indicator of cell viability. Wild-type cells were grown in YPKG for 3d (t = 0) or transferred to YPD for 30 min (t = 30). Cells were then treated with or without Triton X-100 and incubated with trypan blue for 30 min. Cells were then observed by light microscopy. In t = 0 and t = 30 cells, the dye did not stain cells (Figure 3A and C, left panels). However, when Triton X-100 was added to t = 0 and t = 30 cells followed by incubation with trypan blue, about 60-90% of the cells were stained (Figure 3A and3C, right panels). We also used t = 0 and t = 30 cells that were extracted and then incubated with trypan blue. The majority of extracted cells were not stained (Figure 3B and3D, left panels). In contrast, when Triton X-100 was added to the extracted cells followed by incubation with trypan blue, about 60-90% of the cells were stained (Figure 3B and3D, right panels).


Glucose induces rapid changes in the secretome of Saccharomyces cerevisiae.

Giardina BJ, Stanley BA, Chiang HL - Proteome Sci (2014)

Extracted cells transport the vital dye FM to the vacuole membrane. (A-D), wild-type cells were grown in YPKG for 3d (t = 0) or transferred to YPD for 30 min (t = 30). Cells were divided and half of the cells were extracted. Non-extracted and extracted cells at the t = 0 and t = 30 min time points were incubated with or without 2% Triton X-100 (TX) for 30 min followed by incubation with trypan blue for 30 min. Cells were examined using a light microscope. (E-L), wild-type cells were grown in YPKG for 3d (t = 0) or transferred to glucose for 30 min (t = 30) and harvested. Cells were incubated in the absence or presence of Triton X-100 (TX) and incubated with FM for 3 hours. The distribution of FM was observed by fluorescence microscopy in non-extracted t = 0 cells (E), t = 30 cells (G), TX treated t = 0 cells (F), and TX treated t = 30 cells (H). The same amounts of t = 0 and t = 30 min cells were subjected to the extraction procedure and treated in the absence (I and K) or presence of Triton X-100 (J and L). Cells were incubated with FM and the distribution of FM was examined by fluorescence microscopy.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3927832&req=5

Figure 3: Extracted cells transport the vital dye FM to the vacuole membrane. (A-D), wild-type cells were grown in YPKG for 3d (t = 0) or transferred to YPD for 30 min (t = 30). Cells were divided and half of the cells were extracted. Non-extracted and extracted cells at the t = 0 and t = 30 min time points were incubated with or without 2% Triton X-100 (TX) for 30 min followed by incubation with trypan blue for 30 min. Cells were examined using a light microscope. (E-L), wild-type cells were grown in YPKG for 3d (t = 0) or transferred to glucose for 30 min (t = 30) and harvested. Cells were incubated in the absence or presence of Triton X-100 (TX) and incubated with FM for 3 hours. The distribution of FM was observed by fluorescence microscopy in non-extracted t = 0 cells (E), t = 30 cells (G), TX treated t = 0 cells (F), and TX treated t = 30 cells (H). The same amounts of t = 0 and t = 30 min cells were subjected to the extraction procedure and treated in the absence (I and K) or presence of Triton X-100 (J and L). Cells were incubated with FM and the distribution of FM was examined by fluorescence microscopy.
Mentions: We first determined whether or not cells that were subjected to the extraction procedure retained the ability to exclude trypan blue from cells (Figure 3A-D). Trypan blue dye permeates dead cells and is therefore an indicator of cell viability. Wild-type cells were grown in YPKG for 3d (t = 0) or transferred to YPD for 30 min (t = 30). Cells were then treated with or without Triton X-100 and incubated with trypan blue for 30 min. Cells were then observed by light microscopy. In t = 0 and t = 30 cells, the dye did not stain cells (Figure 3A and C, left panels). However, when Triton X-100 was added to t = 0 and t = 30 cells followed by incubation with trypan blue, about 60-90% of the cells were stained (Figure 3A and3C, right panels). We also used t = 0 and t = 30 cells that were extracted and then incubated with trypan blue. The majority of extracted cells were not stained (Figure 3B and3D, left panels). In contrast, when Triton X-100 was added to the extracted cells followed by incubation with trypan blue, about 60-90% of the cells were stained (Figure 3B and3D, right panels).

Bottom Line: Most of these proteins did not contain typical ER-Golgi signal sequences.Therefore, we conclude that the secretome undergoes dynamic changes during transition from glucose-deficient to glucose-rich media.Most of these extracellular proteins do not contain typical ER signal sequences, suggesting that they are secreted via the non-classical pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Penn State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA. hxc32@psu.edu.

ABSTRACT

Background: Protein secretion is a fundamental process in all living cells. Proteins can either be secreted via the classical or non-classical pathways. In Saccharomyces cerevisiae, gluconeogenic enzymes are in the extracellular fraction/periplasm when cells are grown in media containing low glucose. Following a transfer of cells to high glucose media, their levels in the extracellular fraction are reduced rapidly. We hypothesized that changes in the secretome were not restricted to gluconeogenic enzymes. The goal of the current study was to use a proteomic approach to identify extracellular proteins whose levels changed when cells were transferred from low to high glucose media.

Results: We performed two iTRAQ experiments and identified 347 proteins that were present in the extracellular fraction including metabolic enzymes, proteins involved in oxidative stress, protein folding, and proteins with unknown functions. Most of these proteins did not contain typical ER-Golgi signal sequences. Moreover, levels of many of these proteins decreased upon a transfer of cells from media containing low to high glucose media. Using an extraction procedure and Western blotting, we confirmed that the metabolic enzymes (glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, glucose-6-phosphate dehydrogenase, pyruvate decarboxylase), proteins involved in oxidative stress (superoxide dismutase and thioredoxin), and heat shock proteins (Ssa1p, Hsc82p, and Hsp104p) were in the extracellular fraction during growth in low glucose and that the levels of these extracellular proteins were reduced when cells were transferred to media containing high glucose. These proteins were associated with membranes in vesicle-enriched fraction. We also showed that small vesicles were present in the extracellular fraction in cells grown in low glucose. Following a transfer from low to high glucose media for 30 minutes, 98% of these vesicles disappeared from the extracellular fraction.

Conclusions: Our data indicate that transferring cells from low to high glucose media induces a rapid decline in levels of a large number of extracellular proteins and the disappearance of small vesicles from the extracellular fraction. Therefore, we conclude that the secretome undergoes dynamic changes during transition from glucose-deficient to glucose-rich media. Most of these extracellular proteins do not contain typical ER signal sequences, suggesting that they are secreted via the non-classical pathway.

No MeSH data available.


Related in: MedlinePlus