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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

Transferring cells from YPKG to YPD causes a rapid disappearance of small vesicles from the extracellular fraction. (A and B) Wild-type cells were grown in YPKG or transferred to YPD for 30 minutes and extracted. Total extracts from t = 0 (A) and t = 30 (B) cells were stained with uranyl acetate and visualized by transmission electron microscope as described in Methods. Bars: 200 nm. (C) Quantification of the number of 30–50 nm small vesicles per μm2 in total extracts in t = 0 and t = 30 cells. Mean and SD were derived from counting the number of small vesicles using 3 images taken from extracts isolated from t = 0 and t = 30 cells. (D) Quantification of the number of 100–300 nm structures per μm2 in total extracts in t = 0 and t = 30 cells. Mean and SD were obtained from counting the number of 100–300 nm structures from 3 images taken from total extracts isolated from t = 0 and t = 30 cells.
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Figure 8: Transferring cells from YPKG to YPD causes a rapid disappearance of small vesicles from the extracellular fraction. (A and B) Wild-type cells were grown in YPKG or transferred to YPD for 30 minutes and extracted. Total extracts from t = 0 (A) and t = 30 (B) cells were stained with uranyl acetate and visualized by transmission electron microscope as described in Methods. Bars: 200 nm. (C) Quantification of the number of 30–50 nm small vesicles per μm2 in total extracts in t = 0 and t = 30 cells. Mean and SD were derived from counting the number of small vesicles using 3 images taken from extracts isolated from t = 0 and t = 30 cells. (D) Quantification of the number of 100–300 nm structures per μm2 in total extracts in t = 0 and t = 30 cells. Mean and SD were obtained from counting the number of 100–300 nm structures from 3 images taken from total extracts isolated from t = 0 and t = 30 cells.

Mentions: The extraction procedure employed in the current work has been used to study aspects of protein composition in the extracellular fraction[2,64,77]. However, the contents of the extracted materials had not previously been visualized at the ultra-structural level. Small vesicles called exosomes are released from a variety of mammalian cells. In yeast, Fbp1p is associated with small vesicles that are 30–50 nm in diameter[88]. Because of the size of these vesicles, they can only be visualized by EM techniques. We thus performed TEM studies to examine whether or not small vesicles were present in the extracellular fraction in cells grown in YPKG. Wild-type cells were grown in YPKG for 3d and harvested (t = 0 min), or transferred to YPD for 30 min (t = 30 min) and subjected to the same extraction procedure. Extracted materials were centrifuged at 3,000 × g and then at 200,000 × g. The 200,000 × g pellets from the t = 0 and t = 30 cells were stained with uranyl acetate followed by visualization using TEM (Figure 8A and8B). When total extracts were prepared from t = 0 cells, at least two types of structures were observed. Small vesicles of 30–50 nm in diameter and large structures of 100–300 nm in diameter were identified (Figure 8A, arrows). Quantification of these structures indicated that total extracts from cells grown in YPKG consisted of approximately 93.9% small vesicles and 6.1% large structures. However, when total extracts were isolated from t = 30 cells, very few small vesicles were observed, whereas the 100–300 nm large structures still remained (Figure 8B, arrows). The number of small vesicles in total extracts was 218.3 ± 13.5 per μm2 at t = 0 min and was 4.5 ±1.3 per μm2 at t = 30 min (Figure 8C). The number of 100–300 nm large structures was 14.3 ± 1.7 per μm2 at t = 0 min and was 10.3 ±1.1 per μm2 at t = 30 min (Figure 8D). Therefore, approximately 98% of the small vesicles disappeared within 30 min following glucose replenishment in YPD. In contrast, only about 28% of the large structures had disappeared at the 30 min time point. This rapid disappearance of small vesicles from the extracellular fraction at t = 30 min demonstrates another example of rapid changes in the secretome/extracellular fraction during transition from glucose-deficient to glucose-rich media.


Glucose induces rapid changes in the secretome of Saccharomyces cerevisiae.

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

Transferring cells from YPKG to YPD causes a rapid disappearance of small vesicles from the extracellular fraction. (A and B) Wild-type cells were grown in YPKG or transferred to YPD for 30 minutes and extracted. Total extracts from t = 0 (A) and t = 30 (B) cells were stained with uranyl acetate and visualized by transmission electron microscope as described in Methods. Bars: 200 nm. (C) Quantification of the number of 30–50 nm small vesicles per μm2 in total extracts in t = 0 and t = 30 cells. Mean and SD were derived from counting the number of small vesicles using 3 images taken from extracts isolated from t = 0 and t = 30 cells. (D) Quantification of the number of 100–300 nm structures per μm2 in total extracts in t = 0 and t = 30 cells. Mean and SD were obtained from counting the number of 100–300 nm structures from 3 images taken from total extracts isolated from t = 0 and t = 30 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3927832&req=5

Figure 8: Transferring cells from YPKG to YPD causes a rapid disappearance of small vesicles from the extracellular fraction. (A and B) Wild-type cells were grown in YPKG or transferred to YPD for 30 minutes and extracted. Total extracts from t = 0 (A) and t = 30 (B) cells were stained with uranyl acetate and visualized by transmission electron microscope as described in Methods. Bars: 200 nm. (C) Quantification of the number of 30–50 nm small vesicles per μm2 in total extracts in t = 0 and t = 30 cells. Mean and SD were derived from counting the number of small vesicles using 3 images taken from extracts isolated from t = 0 and t = 30 cells. (D) Quantification of the number of 100–300 nm structures per μm2 in total extracts in t = 0 and t = 30 cells. Mean and SD were obtained from counting the number of 100–300 nm structures from 3 images taken from total extracts isolated from t = 0 and t = 30 cells.
Mentions: The extraction procedure employed in the current work has been used to study aspects of protein composition in the extracellular fraction[2,64,77]. However, the contents of the extracted materials had not previously been visualized at the ultra-structural level. Small vesicles called exosomes are released from a variety of mammalian cells. In yeast, Fbp1p is associated with small vesicles that are 30–50 nm in diameter[88]. Because of the size of these vesicles, they can only be visualized by EM techniques. We thus performed TEM studies to examine whether or not small vesicles were present in the extracellular fraction in cells grown in YPKG. Wild-type cells were grown in YPKG for 3d and harvested (t = 0 min), or transferred to YPD for 30 min (t = 30 min) and subjected to the same extraction procedure. Extracted materials were centrifuged at 3,000 × g and then at 200,000 × g. The 200,000 × g pellets from the t = 0 and t = 30 cells were stained with uranyl acetate followed by visualization using TEM (Figure 8A and8B). When total extracts were prepared from t = 0 cells, at least two types of structures were observed. Small vesicles of 30–50 nm in diameter and large structures of 100–300 nm in diameter were identified (Figure 8A, arrows). Quantification of these structures indicated that total extracts from cells grown in YPKG consisted of approximately 93.9% small vesicles and 6.1% large structures. However, when total extracts were isolated from t = 30 cells, very few small vesicles were observed, whereas the 100–300 nm large structures still remained (Figure 8B, arrows). The number of small vesicles in total extracts was 218.3 ± 13.5 per μm2 at t = 0 min and was 4.5 ±1.3 per μm2 at t = 30 min (Figure 8C). The number of 100–300 nm large structures was 14.3 ± 1.7 per μm2 at t = 0 min and was 10.3 ±1.1 per μm2 at t = 30 min (Figure 8D). Therefore, approximately 98% of the small vesicles disappeared within 30 min following glucose replenishment in YPD. In contrast, only about 28% of the large structures had disappeared at the 30 min time point. This rapid disappearance of small vesicles from the extracellular fraction at t = 30 min demonstrates another example of rapid changes in the secretome/extracellular fraction during transition from glucose-deficient to glucose-rich media.

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