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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

Fbp1p is re-distributed from the periplasm to the cytoplasm upon a transfer of cells from YPKG to YPD. Wild-type cells were grown in YPKG for 3d (A, t = 0 min) or transferred to YPD for 30 min (B, t = 30 min). Cells were processed and Fbp1p was visualized by immuno-TEM. The number of gold particles in the cytoplasm and the periplasm was 26 and 162 in t = 0 min wild-type cells (A), and 146 and 3 in t = 30 min wild-type cells (B), respectively. Bars: 200 nm, PM: plasma membrane, CW: cell wall.
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Figure 2: Fbp1p is re-distributed from the periplasm to the cytoplasm upon a transfer of cells from YPKG to YPD. Wild-type cells were grown in YPKG for 3d (A, t = 0 min) or transferred to YPD for 30 min (B, t = 30 min). Cells were processed and Fbp1p was visualized by immuno-TEM. The number of gold particles in the cytoplasm and the periplasm was 26 and 162 in t = 0 min wild-type cells (A), and 146 and 3 in t = 30 min wild-type cells (B), respectively. Bars: 200 nm, PM: plasma membrane, CW: cell wall.

Mentions: We next determined the effects of glucose on the distribution of Fbp1p at the ultra-structural level using immuno-TEM. Wild-type cells were grown in YPKG for 3d and transferred to YPD for 30 min. Cells were processed and thin sections of cells were incubated with affinity purified anti-Fbp1p antibodies followed by goat anti-rabbit secondary antibodies conjugated with 10 nm gold particles. The distribution of Fbp1p was then observed by TEM. When cells were grown in YPKG for 3d, substantial amounts of Fbp1p were in the periplasm (Figure 2A, arrow). Because Fbp1p is degraded in the vacuole following glucose addition, extracellular Fbp1 should be internalized upon a transfer or cells from YPKG to YPD. Indeed, Fbp1p was found in intracellular structures that contained clusters of small vesicles at the t = 30 min time point (Figure 2B, arrow). Thus, transferring cells from low to high glucose causes a rapid redistribution of Fbp1p from the periplasm to the cytoplasm, resulting in a rapid decline in Fbp1p levels in the extracellular fraction.


Glucose induces rapid changes in the secretome of Saccharomyces cerevisiae.

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

Fbp1p is re-distributed from the periplasm to the cytoplasm upon a transfer of cells from YPKG to YPD. Wild-type cells were grown in YPKG for 3d (A, t = 0 min) or transferred to YPD for 30 min (B, t = 30 min). Cells were processed and Fbp1p was visualized by immuno-TEM. The number of gold particles in the cytoplasm and the periplasm was 26 and 162 in t = 0 min wild-type cells (A), and 146 and 3 in t = 30 min wild-type cells (B), respectively. Bars: 200 nm, PM: plasma membrane, CW: cell wall.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3927832&req=5

Figure 2: Fbp1p is re-distributed from the periplasm to the cytoplasm upon a transfer of cells from YPKG to YPD. Wild-type cells were grown in YPKG for 3d (A, t = 0 min) or transferred to YPD for 30 min (B, t = 30 min). Cells were processed and Fbp1p was visualized by immuno-TEM. The number of gold particles in the cytoplasm and the periplasm was 26 and 162 in t = 0 min wild-type cells (A), and 146 and 3 in t = 30 min wild-type cells (B), respectively. Bars: 200 nm, PM: plasma membrane, CW: cell wall.
Mentions: We next determined the effects of glucose on the distribution of Fbp1p at the ultra-structural level using immuno-TEM. Wild-type cells were grown in YPKG for 3d and transferred to YPD for 30 min. Cells were processed and thin sections of cells were incubated with affinity purified anti-Fbp1p antibodies followed by goat anti-rabbit secondary antibodies conjugated with 10 nm gold particles. The distribution of Fbp1p was then observed by TEM. When cells were grown in YPKG for 3d, substantial amounts of Fbp1p were in the periplasm (Figure 2A, arrow). Because Fbp1p is degraded in the vacuole following glucose addition, extracellular Fbp1 should be internalized upon a transfer or cells from YPKG to YPD. Indeed, Fbp1p was found in intracellular structures that contained clusters of small vesicles at the t = 30 min time point (Figure 2B, arrow). Thus, transferring cells from low to high glucose causes a rapid redistribution of Fbp1p from the periplasm to the cytoplasm, resulting in a rapid decline in Fbp1p levels in the extracellular fraction.

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