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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

Levels of extracellular proteins involved in different functions decrease upon a transfer from YPKG to YPD. Wild-type cells expressing Tdh1p-GFP, Pgk1p-GFP, Zwf1p-GFP, Pdc1p-GFP, Sod1p-GFP, Trx1p-GFP, Ssa1p-GFP, Hsc82p-GFP, and Hsp104p-GFP were transferred from YPKG to YPD for 0, 15, and 30 min. The distribution of GFP tagged proteins in the intracellular and extracellular fractions was examined by Western blotting.
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Figure 7: Levels of extracellular proteins involved in different functions decrease upon a transfer from YPKG to YPD. Wild-type cells expressing Tdh1p-GFP, Pgk1p-GFP, Zwf1p-GFP, Pdc1p-GFP, Sod1p-GFP, Trx1p-GFP, Ssa1p-GFP, Hsc82p-GFP, and Hsp104p-GFP were transferred from YPKG to YPD for 0, 15, and 30 min. The distribution of GFP tagged proteins in the intracellular and extracellular fractions was examined by Western blotting.

Mentions: According to our iTRAQ data, t30/t0 ratios were reduced for Tdh1p, Pgk1p, Zwf1p, and Pdc1p (Additional file2: Table S2). Our iTRAQ results also indicated that Sod1p, Trx1p, Ssa1p, Hsc82p, and Hsp104p decreased their t30/t0 ratios following a transfer of cells to YPD. To confirm that these extracellular proteins reduced their levels following glucose addition in YPD, wild-type cells expressing GFP tagged proteins were grown in YPKG media and transferred to YPD for 0, 15, and 30 min. Cells were subjected to the extraction procedure and the distribution of GFP-tagged proteins in the intracellular and extracellular fractions was determined by Western blotting using anti-GFP antibodies (Figure 7). At t = 0 min, Tdh1p, Pgk1p, Zwf1p, and Pdc1p were detectable in the extracellular fraction. Following a transfer of cells from YPKG to YPD for up to 30 min, their levels in the extracellular fraction decreased rapidly. In a similar manner, Sod1p, Trx1p, Ssa1p, Hsc82p, and Hsp104p were in the extracellular fraction when cells were grown in YPKG. Levels of these proteins in the extracellular fraction decreased after a shift of cells to YPD for 30 min. Taken together, these proteins that are involved in different biological functions show very similar distribution characteristics. They are all present in the extracellular fraction during growth in YPKG and their levels in the extracellular fraction are all reduced following a transfer of cells to YPD for 30 min.


Glucose induces rapid changes in the secretome of Saccharomyces cerevisiae.

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

Levels of extracellular proteins involved in different functions decrease upon a transfer from YPKG to YPD. Wild-type cells expressing Tdh1p-GFP, Pgk1p-GFP, Zwf1p-GFP, Pdc1p-GFP, Sod1p-GFP, Trx1p-GFP, Ssa1p-GFP, Hsc82p-GFP, and Hsp104p-GFP were transferred from YPKG to YPD for 0, 15, and 30 min. The distribution of GFP tagged proteins in the intracellular and extracellular fractions was examined by Western blotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Levels of extracellular proteins involved in different functions decrease upon a transfer from YPKG to YPD. Wild-type cells expressing Tdh1p-GFP, Pgk1p-GFP, Zwf1p-GFP, Pdc1p-GFP, Sod1p-GFP, Trx1p-GFP, Ssa1p-GFP, Hsc82p-GFP, and Hsp104p-GFP were transferred from YPKG to YPD for 0, 15, and 30 min. The distribution of GFP tagged proteins in the intracellular and extracellular fractions was examined by Western blotting.
Mentions: According to our iTRAQ data, t30/t0 ratios were reduced for Tdh1p, Pgk1p, Zwf1p, and Pdc1p (Additional file2: Table S2). Our iTRAQ results also indicated that Sod1p, Trx1p, Ssa1p, Hsc82p, and Hsp104p decreased their t30/t0 ratios following a transfer of cells to YPD. To confirm that these extracellular proteins reduced their levels following glucose addition in YPD, wild-type cells expressing GFP tagged proteins were grown in YPKG media and transferred to YPD for 0, 15, and 30 min. Cells were subjected to the extraction procedure and the distribution of GFP-tagged proteins in the intracellular and extracellular fractions was determined by Western blotting using anti-GFP antibodies (Figure 7). At t = 0 min, Tdh1p, Pgk1p, Zwf1p, and Pdc1p were detectable in the extracellular fraction. Following a transfer of cells from YPKG to YPD for up to 30 min, their levels in the extracellular fraction decreased rapidly. In a similar manner, Sod1p, Trx1p, Ssa1p, Hsc82p, and Hsp104p were in the extracellular fraction when cells were grown in YPKG. Levels of these proteins in the extracellular fraction decreased after a shift of cells to YPD for 30 min. Taken together, these proteins that are involved in different biological functions show very similar distribution characteristics. They are all present in the extracellular fraction during growth in YPKG and their levels in the extracellular fraction are all reduced following a transfer of cells to YPD for 30 min.

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