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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

Experimental conditions to study glucose effects in regulating protein levels. A, wild-type cells expressing Lia1p-GFP were grown either in YNB-based media containing 2% glucose (I and II) or in YPKG-based media containing 0.5% glucose (III and IV) for 3d. In experiment I, 2% glucose was added directly to the existing YNB culture for 0, 2, and 4 hours. In experiment II, cells were grown in YNB-based media, pelleted and resuspended in fresh YNB with 2% glucose for 0, 2, and 4 hours. In experiment III, cells were grown in YPKG for 3d and 2% glucose was added directly to the existing YPKG media for 0, 2, and 4 hours. In experiment IV, cells were grown in YPKG for 3d, pelleted, and resuspended in YPD media for 0, 2, and 4 hours. Levels of Lia1p-GFP, Fbp1p, and Tpi1p were examined using anti-GFP, anti-Fbp1p, and anti-Tpi1 antibodies. B, wild-type cells were grown in YPKG for 3d and transferred to YPD (left panels) or YP (right panels) for 0, 2, and 4 hours. Levels of Lia1p, Fbp1p, and Tpi1p were examined by Western blotting using anti-GFP, Fbp1p, and Tpi1p antibodies. C, wild-type cells were grown in YPKG for 3d and diluted to OD600 = 0.2/ml in YPD (2% glucose), YPKG (0.5% glucose), or YP (0% glucose). Cell densities were measured at OD600 for 0, 2, 4, 6, and 8 hours using a Beckman spectrophotometer.
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Figure 1: Experimental conditions to study glucose effects in regulating protein levels. A, wild-type cells expressing Lia1p-GFP were grown either in YNB-based media containing 2% glucose (I and II) or in YPKG-based media containing 0.5% glucose (III and IV) for 3d. In experiment I, 2% glucose was added directly to the existing YNB culture for 0, 2, and 4 hours. In experiment II, cells were grown in YNB-based media, pelleted and resuspended in fresh YNB with 2% glucose for 0, 2, and 4 hours. In experiment III, cells were grown in YPKG for 3d and 2% glucose was added directly to the existing YPKG media for 0, 2, and 4 hours. In experiment IV, cells were grown in YPKG for 3d, pelleted, and resuspended in YPD media for 0, 2, and 4 hours. Levels of Lia1p-GFP, Fbp1p, and Tpi1p were examined using anti-GFP, anti-Fbp1p, and anti-Tpi1 antibodies. B, wild-type cells were grown in YPKG for 3d and transferred to YPD (left panels) or YP (right panels) for 0, 2, and 4 hours. Levels of Lia1p, Fbp1p, and Tpi1p were examined by Western blotting using anti-GFP, Fbp1p, and Tpi1p antibodies. C, wild-type cells were grown in YPKG for 3d and diluted to OD600 = 0.2/ml in YPD (2% glucose), YPKG (0.5% glucose), or YP (0% glucose). Cell densities were measured at OD600 for 0, 2, 4, 6, and 8 hours using a Beckman spectrophotometer.

Mentions: Glucose has profound effects in regulating proteins levels. For instance, glucose up-regulates Lia1p involved protein synthesis, while down-regulating enzymes involved in gluconeogenesis. There are different ways to examine glucose effects in regulating protein levels. We used wild-type cells grown either in YNB-based media (yeast nitrogen base with amino acids) or YP based media to study glucose effects in up-regulation of Lia1p and down-regulation of Fbp1p (Figure 1A). In Experiment I, wild-type cells expressing Lia1p-GFP were grown in YNB-based media containing 2% glucose for 3d followed by the addition of 2% glucose directly to the existing culture media for 0, 2, and 4 hours. In experiment II, cells were grown in YNB media containing 2% glucose for 3d. Cells were pelleted and resuspended in fresh YNB with 2% glucose for 0, 2, and 4 hours. In experiment III, cells were grown in YP-based media containing 0.5% glucose (YPKG) for 3d followed by the addition of 2% glucose directly to the existing YPKG media for 0, 2, and 4 hours. In experiment IV, cells were grown in YPKG for 3d. Cells were pelleted and resuspended in YPD media containing 2% glucose for 0, 2, and 4 hours. Equal amounts of cells were harvested at each time point, processed and examined for changes in Lia1p and Fbp1p levels (Figure 1A). In experiments I and II where cells grown in YNB-based media, the level of Liap1 was low with an increase at t = 4 hours. Lia1p levels were higher in cells grown in YPKG. However, Lia1p was not up-regulated in experiment III but was up-regulated rapidly in experiment IV. Fbp1p levels were low in cells grown in YNB at t = 0 and did not show significant changes at the 2 and 4 hour time points (experiments I and II). Fbp1p levels were higher in cells grown in YPKG (experiments III and IV). Additionally, Fbp1p levels decreased slowly in experiment III but decreased rapidly in experiment IV. Tpi1p (triose phosphate isomerase) is a glycolytic enzyme and levels of this protein were similar under all four experimental conditions. Because experiment IV produced a faster increase in Lia1p levels and a faster decrease in Fbp1p levels, this condition was used to study glucose effects for most experiments described in this study unless otherwise specified.


Glucose induces rapid changes in the secretome of Saccharomyces cerevisiae.

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

Experimental conditions to study glucose effects in regulating protein levels. A, wild-type cells expressing Lia1p-GFP were grown either in YNB-based media containing 2% glucose (I and II) or in YPKG-based media containing 0.5% glucose (III and IV) for 3d. In experiment I, 2% glucose was added directly to the existing YNB culture for 0, 2, and 4 hours. In experiment II, cells were grown in YNB-based media, pelleted and resuspended in fresh YNB with 2% glucose for 0, 2, and 4 hours. In experiment III, cells were grown in YPKG for 3d and 2% glucose was added directly to the existing YPKG media for 0, 2, and 4 hours. In experiment IV, cells were grown in YPKG for 3d, pelleted, and resuspended in YPD media for 0, 2, and 4 hours. Levels of Lia1p-GFP, Fbp1p, and Tpi1p were examined using anti-GFP, anti-Fbp1p, and anti-Tpi1 antibodies. B, wild-type cells were grown in YPKG for 3d and transferred to YPD (left panels) or YP (right panels) for 0, 2, and 4 hours. Levels of Lia1p, Fbp1p, and Tpi1p were examined by Western blotting using anti-GFP, Fbp1p, and Tpi1p antibodies. C, wild-type cells were grown in YPKG for 3d and diluted to OD600 = 0.2/ml in YPD (2% glucose), YPKG (0.5% glucose), or YP (0% glucose). Cell densities were measured at OD600 for 0, 2, 4, 6, and 8 hours using a Beckman spectrophotometer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3927832&req=5

Figure 1: Experimental conditions to study glucose effects in regulating protein levels. A, wild-type cells expressing Lia1p-GFP were grown either in YNB-based media containing 2% glucose (I and II) or in YPKG-based media containing 0.5% glucose (III and IV) for 3d. In experiment I, 2% glucose was added directly to the existing YNB culture for 0, 2, and 4 hours. In experiment II, cells were grown in YNB-based media, pelleted and resuspended in fresh YNB with 2% glucose for 0, 2, and 4 hours. In experiment III, cells were grown in YPKG for 3d and 2% glucose was added directly to the existing YPKG media for 0, 2, and 4 hours. In experiment IV, cells were grown in YPKG for 3d, pelleted, and resuspended in YPD media for 0, 2, and 4 hours. Levels of Lia1p-GFP, Fbp1p, and Tpi1p were examined using anti-GFP, anti-Fbp1p, and anti-Tpi1 antibodies. B, wild-type cells were grown in YPKG for 3d and transferred to YPD (left panels) or YP (right panels) for 0, 2, and 4 hours. Levels of Lia1p, Fbp1p, and Tpi1p were examined by Western blotting using anti-GFP, Fbp1p, and Tpi1p antibodies. C, wild-type cells were grown in YPKG for 3d and diluted to OD600 = 0.2/ml in YPD (2% glucose), YPKG (0.5% glucose), or YP (0% glucose). Cell densities were measured at OD600 for 0, 2, 4, 6, and 8 hours using a Beckman spectrophotometer.
Mentions: Glucose has profound effects in regulating proteins levels. For instance, glucose up-regulates Lia1p involved protein synthesis, while down-regulating enzymes involved in gluconeogenesis. There are different ways to examine glucose effects in regulating protein levels. We used wild-type cells grown either in YNB-based media (yeast nitrogen base with amino acids) or YP based media to study glucose effects in up-regulation of Lia1p and down-regulation of Fbp1p (Figure 1A). In Experiment I, wild-type cells expressing Lia1p-GFP were grown in YNB-based media containing 2% glucose for 3d followed by the addition of 2% glucose directly to the existing culture media for 0, 2, and 4 hours. In experiment II, cells were grown in YNB media containing 2% glucose for 3d. Cells were pelleted and resuspended in fresh YNB with 2% glucose for 0, 2, and 4 hours. In experiment III, cells were grown in YP-based media containing 0.5% glucose (YPKG) for 3d followed by the addition of 2% glucose directly to the existing YPKG media for 0, 2, and 4 hours. In experiment IV, cells were grown in YPKG for 3d. Cells were pelleted and resuspended in YPD media containing 2% glucose for 0, 2, and 4 hours. Equal amounts of cells were harvested at each time point, processed and examined for changes in Lia1p and Fbp1p levels (Figure 1A). In experiments I and II where cells grown in YNB-based media, the level of Liap1 was low with an increase at t = 4 hours. Lia1p levels were higher in cells grown in YPKG. However, Lia1p was not up-regulated in experiment III but was up-regulated rapidly in experiment IV. Fbp1p levels were low in cells grown in YNB at t = 0 and did not show significant changes at the 2 and 4 hour time points (experiments I and II). Fbp1p levels were higher in cells grown in YPKG (experiments III and IV). Additionally, Fbp1p levels decreased slowly in experiment III but decreased rapidly in experiment IV. Tpi1p (triose phosphate isomerase) is a glycolytic enzyme and levels of this protein were similar under all four experimental conditions. Because experiment IV produced a faster increase in Lia1p levels and a faster decrease in Fbp1p levels, this condition was used to study glucose effects for most experiments described in this study unless otherwise specified.

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