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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

Comparison of proteins identified in our study and the studies by Braconi et al., Insenser et al., Rowe et al., and Oliveira et al. Proteins that were identified by Braconi et al. and Insenser et al. were compared with those identified in iTRAQ1 (A) and iTRAQ2 (B). Proteins identified by Rowe et al. and Oliveira et al. were compared with those identified in iTRAQ1 (C) and iTRAQ2 (D).
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Figure 10: Comparison of proteins identified in our study and the studies by Braconi et al., Insenser et al., Rowe et al., and Oliveira et al. Proteins that were identified by Braconi et al. and Insenser et al. were compared with those identified in iTRAQ1 (A) and iTRAQ2 (B). Proteins identified by Rowe et al. and Oliveira et al. were compared with those identified in iTRAQ1 (C) and iTRAQ2 (D).

Mentions: We performed two iTRAQ experiments and identified a total of 347 proteins that were present in the extracellular fraction in wild-type cells that were grown in YPKG. The vast majority of the proteins identified in this study lack typical ER signal sequence, suggesting that they are secreted via the non-classical pathway. A small number of proteins identified in the current study, including Ape2p, Bgl2p, Exg1p, Scw4p, Suc2p, Pep4p, Prb1p, Prc1p, Pho3p, Pho5p, Pho12p, Ygp1p, and Uth1p, did however contain ER-signal peptides and were thus presumably secreted by the classical pathway. Our observations of the relatively low number of classic pathway secreted proteins are consistent with another secretomic/surfomic study in Saccharomyces cerevisiae in which only 17 proteins of a total of 99 proteins contained a signal sequence[72]. When we compared our iTRAQ1 results with two previous Saccharomyces cerevisiae secretomic/surfomic studies[17,72], 16 proteins overlapped in all 3 studies (Figure 10A). Overlapping proteins included Eno1p, Eno2p, Hxk2p, Pgk1p, Tdh3p, Adh1p, Pdc1p, Hsp82p, Ssa1p, Ssa2p, Ssb1p, Sse1p, Sod1p, Act1p, Bmh1p, and Exg1p. As indicated in the Venn diagram (Figure 10A), 192 proteins were unique to iTRAQ1, whereas 36 proteins were unique to the study by Insenser et al.[72] and 15 proteins by Braconi et al.[17]. We obtained very similar distribution patterns when we compared iTRAQ2 with the studies by Braconi et al. and Insenser et al. (Figure 10B), where the same 16 proteins overlapped. The observed low numbers of overlapping proteins may result from variations in the number of proteins identified in each study. When we compared the proteins we identified with those identified by Rowe et al.[89], 82 proteins overlapped with iTRAQ1 (Figure 10C) and 87 proteins overlapped with iTRAQ2 (Figure 10D). Common proteins included metabolic enzymes, heat shock proteins and proteins involved in oxidative stress, protein folding, and protein translation. Since we used cells that were grown in low glucose, whereas other studies used cells grown in high glucose, the identification of these common proteins in the secretome appears to be independent of the growth conditions. Furthermore, because different methods such as trypsin shaving, biotinylation or extraction with reducing agents were utilized in different studies, these proteins are unlikely to be released into the extracellular fraction due to cell lysis. In our study, both extracted and non-extracted cells were not stained with trypan blue, suggesting that cell lysis is minimal. In contrast, when Triton-X100 was added to the extracted cells and then incubated with trypan blue, high percentages of cells were stained. It was also observed that non-extracted and extracted cells were able to internalize exogenously added FM dye and actively transport it to the vacuole, suggesting that the endocytic pathway is functional in these cells.


Glucose induces rapid changes in the secretome of Saccharomyces cerevisiae.

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

Comparison of proteins identified in our study and the studies by Braconi et al., Insenser et al., Rowe et al., and Oliveira et al. Proteins that were identified by Braconi et al. and Insenser et al. were compared with those identified in iTRAQ1 (A) and iTRAQ2 (B). Proteins identified by Rowe et al. and Oliveira et al. were compared with those identified in iTRAQ1 (C) and iTRAQ2 (D).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 10: Comparison of proteins identified in our study and the studies by Braconi et al., Insenser et al., Rowe et al., and Oliveira et al. Proteins that were identified by Braconi et al. and Insenser et al. were compared with those identified in iTRAQ1 (A) and iTRAQ2 (B). Proteins identified by Rowe et al. and Oliveira et al. were compared with those identified in iTRAQ1 (C) and iTRAQ2 (D).
Mentions: We performed two iTRAQ experiments and identified a total of 347 proteins that were present in the extracellular fraction in wild-type cells that were grown in YPKG. The vast majority of the proteins identified in this study lack typical ER signal sequence, suggesting that they are secreted via the non-classical pathway. A small number of proteins identified in the current study, including Ape2p, Bgl2p, Exg1p, Scw4p, Suc2p, Pep4p, Prb1p, Prc1p, Pho3p, Pho5p, Pho12p, Ygp1p, and Uth1p, did however contain ER-signal peptides and were thus presumably secreted by the classical pathway. Our observations of the relatively low number of classic pathway secreted proteins are consistent with another secretomic/surfomic study in Saccharomyces cerevisiae in which only 17 proteins of a total of 99 proteins contained a signal sequence[72]. When we compared our iTRAQ1 results with two previous Saccharomyces cerevisiae secretomic/surfomic studies[17,72], 16 proteins overlapped in all 3 studies (Figure 10A). Overlapping proteins included Eno1p, Eno2p, Hxk2p, Pgk1p, Tdh3p, Adh1p, Pdc1p, Hsp82p, Ssa1p, Ssa2p, Ssb1p, Sse1p, Sod1p, Act1p, Bmh1p, and Exg1p. As indicated in the Venn diagram (Figure 10A), 192 proteins were unique to iTRAQ1, whereas 36 proteins were unique to the study by Insenser et al.[72] and 15 proteins by Braconi et al.[17]. We obtained very similar distribution patterns when we compared iTRAQ2 with the studies by Braconi et al. and Insenser et al. (Figure 10B), where the same 16 proteins overlapped. The observed low numbers of overlapping proteins may result from variations in the number of proteins identified in each study. When we compared the proteins we identified with those identified by Rowe et al.[89], 82 proteins overlapped with iTRAQ1 (Figure 10C) and 87 proteins overlapped with iTRAQ2 (Figure 10D). Common proteins included metabolic enzymes, heat shock proteins and proteins involved in oxidative stress, protein folding, and protein translation. Since we used cells that were grown in low glucose, whereas other studies used cells grown in high glucose, the identification of these common proteins in the secretome appears to be independent of the growth conditions. Furthermore, because different methods such as trypsin shaving, biotinylation or extraction with reducing agents were utilized in different studies, these proteins are unlikely to be released into the extracellular fraction due to cell lysis. In our study, both extracted and non-extracted cells were not stained with trypan blue, suggesting that cell lysis is minimal. In contrast, when Triton-X100 was added to the extracted cells and then incubated with trypan blue, high percentages of cells were stained. It was also observed that non-extracted and extracted cells were able to internalize exogenously added FM dye and actively transport it to the vacuole, suggesting that the endocytic pathway is functional in these cells.

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