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ISSN: 2161-1459
Journal of Clinical & Experimental Pharmacology
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The Non-classical Pathway is the Major Pathway to Secrete Proteins in Saccharomyces cerevisiae

Kathryn R Stein, Bennett J Giardina and Hui-Ling Chiang*

Department of Cellular and Molecular Physiology, Penn State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA

*Corresponding Author:
Hui-Ling Chiang
Department of Cellular and Molecular Physiology
Penn State University College of Medicine
500 University Drive, Hershey, PA 17033, USA
Tel: 717-531-0860
Fax: 717-531-7667
E-mail: [email protected]

Received date: January 28, 2014; Accepted date: April 25, 2014; Published date: April 30, 2014

Citation: Stein KR, Giardina BJ, Chiang H (2014) The Non-classical Pathway is the Major Pathway to Secrete Proteins in Saccharomyces cerevisiae. Clin Exp Pharmacol 4:155. doi:10.4172/2161-1459.1000155

Copyright: © 2014 Stein KR, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Protein secretion is a fundamental process in all living cells. Proteins that contain the ER signal are secreted by the classical pathway, whereas proteins without the ER sequence are secreted via the non-classical pathway. Recent evidence indicates that a large number of signal-less proteins including metabolic enzymes, transcriptional factors, translation factors, heat shock proteins, and anti-oxidant proteins are secreted by a variety of cells that range from bacteria to human. Furthermore, the secretion of gluconeogenic enzymes has been observed in bacteria, fungi, parasites, and mammalian cells. Gluconeogenic enzymes are secreted into the periplasm when Saccharomyces cerevisiae are grown in low glucose. The distribution of gluconeogenic enzymes in the periplasm/ extracellular fraction of Saccharomyces cerevisiae was observed by immuno-TEM, confirmed with an extraction protocol, and identified in a large-scale proteomic study. It is expected that there are other proteins that lack the ER signal sequence and utilize the non-classical pathway to be secreted. Indeed, 92 proteins were identified that were present in the extracellular fraction using proteomics and more than 95% of the proteins do not have the ER sequence. Thus, the non-classical pathway is the major pathway to secrete proteins in Saccharomyces cerevisiae. This review article focuses on the use of multiple techniques including immuno-TEM, an extraction protocol, and proteomics to identify proteins that are present in the extracellular fraction in Saccharomyces cerevisiae, and to compare the dataset produced in yeast with other secretomic studies performed on a range of organisms from bacteria to human cells. Common proteins include metabolic enzymes, heat shock proteins, anti-oxidant proteins, and translation factors. Therefore, the secretion of these proteins is widely observed across species.

Keywords

Non-Classical Secretory Pathway; Proteomics/ Secretomics; Gluconeogenesis; Vacuole Import and Degradation; Fructose-1,6-Bisphosphatase; Malate Dehydrogenase; Isocitrate Lyase; Phosphoenolpyruvate Carboxykinase; Glyceraldehyde-3-Phosphate Dehydrogenase; Cyclophilin A

Abbreviations

FBPase: Fructose-1,6-Bisphosphatase; MDH2: Malate Dehydrogenase; Icl1p: Isocitrate Lyase; Pck1p: Phosphoenolpyruvate Carboxykinase; GAPDH: Glyceraldehyde-3- Phosphate Dehydrogenase; Cpr1p: Cyclophilin A; Vid: Vacuole Import and Degradation

Signal-less Proteins are Secreted via the Non-classical Pathway from Bacteria to Humans

Protein secretion plays an important role in the physiological processes of many organisms, spanning from bacteria to humans. Secretory proteins are required for cell growth, cell differentiation, proliferation, blood coagulation, and immune defense [1-8]. Secretory proteins are also crucial in cancer angiogenesis, invasion, and metastasis [6,9-13]. As such, proteins that are secreted by cancer cells are promising sources for biomarker discovery [6,9-13]. The secreted carcinoembryonic antigen, α-fetoprotein, and a prostate-specific antigen have each been used as cancer-specific markers [6]. Proteins released from viruses, bacteria, fungi, and parasites are critical for pathogen-host interactions and for their survival within hosts [14-21]. Additionally, fungi have thick cell walls that rely on secretory proteins for nutrient scavenging, cell separation, and the formation and maintenance of cell walls [22-29].

Cells can secrete proteins via multiple pathways [1-3,7,19,30,31]. In the classical pathway, proteins are secreted after they are synthesized and translocated into the ER. They are then transported to the Golgi and subsequently secreted after secretory vesicles fuse with the plasma membrane [30,32,33]. In general, these proteins contain the N-terminal signal sequence. Recent evidence indicates that proteins without the ER sequence are also secreted from a variety of cells. The signalless proteins include translation factors, transcriptional regulators, metabolic enzymes, chaperones, and mitochondrial proteins [2,5,7,16-20,23,31,34-37]. Viral proteins such as HIV-Tat and the Herpes simplex tegument protein VP22 do not have the conventional signal sequences and are secreted from cells [2,7]. Mitochondrial matrix protein rhodanese is also released into the culture media when over-expressed in HEK-293 cells [38]. Signal-less nuclear proteins such as Engrailed homeoprotein isoform 2 and HMG proteins are exported from cells [2,3,7]. Heat shock proteins Hsp70, Hsp90, and Hsp97 do not have the ER sequence and constitute the largest protein family in the secretome from adult worms of Schistosoma japonicum [39]. Glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and enolase have been shown by immuno-TEM to be present on the surface of Candida albicans, Saccharomyces cerevisiae [26], and Listeria monocytogenes [20]. Importantly, signal-less proteins can be secreted from cells in a controlled manner [1,3,4,8,40,41]. The protein galectin-1, from a family of proteins involved in cell proliferation, differentiation and apoptosis [8,40,41], is secreted when stimulated by the addition of erythropoietin in the leukemia cell line K-562 [40]. Molecules such as FGF-2, IL-1b, and HMGB1 are released from cells upon stimulation [1,4].

Gluconeogenic Enzymes are Secreted from Bacteria, Fungi, Parasites, and Animal Cells

Gluconeogenic enzymes are secreted from various organisms including bacteria, fungi, parasites, and animal cells [14,18-20,36,42]. Fructose-1,6-bisphosphatase (FBPase) is a key gluconeogenic enzyme and is found in secretomes from Bacillus anthracis [14] and Clonorchis sinensis [36]. Infection with Clonorchis sinensis is an emergent public health problem in many countries in Asia [36]. Adult Clonorchis sinensis worms dwell in bile ducts and cause various hepatic and biliary diseases. Secreted FBPase is found in sera from mice infected with Trichinella spiralis [36]. Moreover, secreted FBPase is detected in sera in humans infected with Ascaris lumbricoides, Taenia asiatica, Taenia solium, and Taeniarhynchus saginatus [36]. Malate dehydrogenase is another gluconeogenic enzyme and is detected in the secretomes from Bacillus anthracis, Clonorchis sinensis [36], and Schistosoma mansoni [18]. Similarly, the enzyme, phosphoenolpyruvate carboxykinase, is identified in the secretomes from Clonorchis sinensis, Echinostoma caproni, and Schistosoma mansoni [18,19,36]. Additionally, secreted isocitrate lyase is necessary for full virulence of Mycobacterium tuberculosis and of the plant-pathogenic fungus Leptosphaeria maculans [43,44]. These enzymes were also found in extracellular vesicles released from Histoplasma capsultatum [45]. Furthermore, they were present in exosomes isolated from a mouse insulinoma cell line [42]. Thus, the secretion of gluconeogenic enzymes is widely observed from bacteria to animal cells. Because gluconeogenic enzymes do not contain signals for the classical pathway, they are secreted via the non-classical pathway.

In Saccharomyces cerevisiae, gluconeogenic enzymes are secreted into the periplasm when cells are grown in media low in glucose [46-55]. These gluconeogenic enzymes include FBPase that converts fructose- 1,6-bisphosphate to fructose-6-phosphate, malate dehydrogenase (MDH2) that catalyzes the interconversion of malate and oxaloacetate, isocitrate lyase (Icl1p) that produces succinate and glyoxylate from isocitrate, and phosphoenolpyruvate carboxykinase (Pck1p) which coverts oxaloacetate to phosphoenolpyruvate. The secretion of gluconeogenic enzymes is dependent on the duration of starvation [56,57]. FBPase is not secreted in cells that are starved of glucose for 1 day. However, FBPase secretion increases as cells are starved of glucose for 3 days [56,57]. These results suggest that the secretion of FBPase is related to the depletion of glucose or FBPase substrates in the cells.

Prolonged Glucose-starved Cells Re-fed with Glucose Utilize Vacuole Import and Degradation Pathway to Degrade Gluconeogenic Enzymes

FBPase has been used extensively to study glucose-induced inactivation and degradation in Saccharomyces cerevisiae [49,58-61]. FBPase expression is induced when yeast cells are grown in media containing low glucose. Following glucose addition to glucose-starved cells, this protein is inactivated and subsequently degraded [61-70]. FBPase is either ubiquitinated and degraded in the proteasome or phosphorylated and degraded in the vacuole. The site of degradation is dependent on the duration of starvation. When glucose is added to cells that are starved for one day, this protein is degraded in the proteasome [71]. However, when glucose is added to cells that are starved for three days, FBPase is degraded in the vacuole via the Vacuole-import and degradation (Vid) pathway [61-67,71-73]. The other gluconeogenic enzymes MDH2, Icl1p, and Pck1p are also targeted to the vacuole for degradation via the same Vid pathway when glucose-starved cells are re-fed with glucose [64,71].

For the vacuole pathway, glucose induces a transient increase in cAMP levels which activates the RAS2-PKA signaling pathway. This leads to the inactivation and subsequent degradation of FBPase in the vacuole [49,58-61]. Prior to targeting to the vacuole, FBPase is associated with multiple FBPase-containing structures that have been purified to near homogeneity. The first structures to be purified were called Vid vesicles [74]. These round vesicles are 30-50 nm in diameter and exist as free and aggregated forms in the cytoplasm [74]. The second and third FBPase-containing structures were called small and large Vid/ endosomes, respectively, because they differed in size [63]. Immuno- TEM studies indicated that Vid/endosomes contained clusters of Vid vesicles [63]. Furthermore, FBPase was associated with numerous Vid vesicles inside these Vid/endosomes [63]. These results suggest that Vid vesicles are used to transport FBPase to different locations for the vacuole import and degradation pathway.

A number of VID (vacuole import and degradation) genes are required for the degradation of FBPase in the vacuole. As mentioned above, Vid vesicles exist as free forms and aggregated forms in the cytoplasm. In wild-type cells, Vid vesicle proteins such as Vid24p, Sec28p, and Vid30p are present in these aggregates and associated with actin patches. The VID28 gene is involved in the formation of Vid vesicle aggregates in the cytoplasm [75]. In cells lacking the VID28 gene, Vid vesicles failed to aggregate and these Vid proteins were secreted [75]. FBPase is secreted in both wild-type and the Δvid28 mutant cells.

Immuno-TEM Reveals the Presence of Gluconeogenic Enzymes in the Periplasm in Cells Grown in Low Glucose

Immuno-TEM was used to demonstrate that gluconeogenic enzymes FBPase, MDH2, Icl1p, and Pck1p are secreted into the periplasm in cells grown in low glucose. Wild-type cells co-expressing Icl1p tagged with HA and Pck1p tagged with Myc were grown in media containing low glucose for 3 days and processed for Western blot or immuno-TEM [76]. Sec28p is a COPI coatomer subunit and is distributed mainly in the intracellular fraction. The distribution of Sec28p, FBPase, MDH2, Icl1p-HA, and Pck1p-Myc was examined using polyclonal antibodies directed against Sec28p, FBPase, MDH2, HA, and Myc antibodies. When total lysates from wild-type cells were blotted with polyclonal antibodies against these proteins, they all reacted with a single protein band at the predicted molecular weight (Figure 1A).

clinical-experimental-pharmacology-antibodies

Figure 1: FBPase, MDH2, Icl1p, Pck1p, GAPDH, and Cpr1p are in the periplasm. (A), Total lysates from wild-type cells expressing Icl1p-HA and Pck1p- Myc were examined for the specificities of antibodies by Western blotting. These antibodies reacted with a single band at the predicted molecular weights for Sec28p, FBPase, MDH2, Icl1p-HA, Pck1p-Myc, GAPDH, and Cpr1p. (B), Wild-type cells expressing Icl1p-HA and Pck1p-Myc were grown in low glucose for 3 days and fixed. Cells were processed and embedded in LR White resin. Thin sections of cells (10 nm) were incubated with or without primary antibodies against Sec28p, FBPase, MDH2, Icl1p-HA, Pck1p-Myc, GAPDH, and Cpr1p in whole cells. Thin sections were then incubated with secondary goat anti-rabbit antibodies conjugated with 10 nm gold particles. Enlargements of the periplasm of the whole cells are shown. PM: Plasma membrane; CW: Cell wall; Bars: 200 nm. (This figure is in a manuscript published in Communicative and Integrative Biology, 2013 [76]).

The distribution of gluconeogenic enzymes was determined by incubating thin sections of wild-type cells with or without affinity purified primary antibodies followed by goat anti-rabbit secondary antibodies conjugated with 10 nm gold particles (Figure 1B). In the absence of primary antibodies, there were very few gold particles observed between the cell wall and the plasma membrane. Sec28p was not detected in the periplasm. In contrast, substantial amounts of FBPase, MDH2, Icl1p-HA, and Pck1p-Myc were observed in the periplasm (Figure 1B). Quantification of gold particles indicated that 76.7% of FBPase was in the periplasm and 33.3% was in the cytoplasm. Furthermore, 36.9% of MDH2, 46.9% of Icl1p-HA, and 57.6% of Pck1pmyc were also distributed in the periplasm.

Non-gluconeogenic enzymes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cyclophilin A (Cpr1p) are also secreted into the periplasm in glucose-starved cells (Figure 1B). GAPDH is a cell surface protein [77,78], whereas Cpr1p has been identified in culture media in Saccharomyces cerevisiae in large-scale secretomic/ extraction studies [79,80]. Polyclonal antibodies against GAPDH and Cpr1p recognized single protein bands at the predicted molecular weight (Figure 1A). Moreover, these proteins were found in the periplasm in cells grown in low glucose (Figure 1B). Quantification of gold particles indicated that 33.7% of the GAPDH and 42.7% of Cpr1p were distributed in the periplasm. Taken together, 33.7-76.7% of FBPase, MDH2, Icl1p, Pck1p, GAPDH, and Cpr1p are secreted into the periplasm in cells grown in low glucose.

The Use of an Extraction Procedure to Extract Periplasmic Proteins from Whole Cells

Because of the distribution of FBPase, MDH2, Icl1p, Pck1p, GAPDH, and Cpr1p in the periplasm, it is necessary to extract these proteins from this location. Wild-type cells were grown in low glucose for 3 days and pelleted by a low speed centrifugation. Following centrifugation, culture media was removed and cells were incubated with the extraction buffer containing β-mercaptoethanol and Tris with pH 9.4 at 37°C [25,81]. This extraction method was selected, because it is easy to perform and it effectively extracts known periplasmic proteins from whole cells. Furthermore, extracted cells internalize exogenously added molecules and maintain the integrity of the plasma membrane. It has been utilized to study the secretion of a mammalian galectin-1 expressed in Saccharomyces cerevisiae [82]. A similar method has also been used to identify proteins associated with the cell wall in Candida albicans [28,83]. The following control experiments indicated that proteins that are known to be in the extracellular fraction are extracted effectively from whole cells without causing non-specific leakage of proteins into the extracellular fraction.

Control experiments for these extraction experiments included a soluble cell wall protein Scw11p [84], a cytosolic protein Tps2p [85], and a plasma membrane protein Sur7p [86,87]. If the extraction causes non-specific leakage of proteins, Tps2p or Sur7p may be released into the extracellular fraction. However, if the extraction procedure works, the soluble cell wall protein Scw11p should be extracted, whereas Tps2p and Sur7p should not be extracted. In addition, the distribution of Tps2p and Sur7p should not be altered following the extraction protocol.

Wild-type cells expressing GFP protein that was fused to Scw11p, Tps2p, or Sur7p were grown in low-glucose media for 3 days and then transferred to medium containing high glucose. The expression of these proteins was examined using Western blotting and their distribution before and after extraction was determined using fluorescence microscopy. Scw11p-GFP was expressed in cells grown in low glucose and in cells that were re-fed with glucose for 2 hours (Figure 2A). To extract Scw11p, cells expressing Scw11p-GFP were subjected to the extraction procedure. Following extraction, proteins that were released into the supernatant were precipitated with TCA, washed and solubilized in SDS buffer to form the fraction referred to as the Extracellular (E) fraction. The remaining cell-associated fraction were pelleted and lysed. Proteins were solubilized in SDS-PAGE buffer. This fraction was called the Intracellular (I) fraction. Before extraction, Scw11p-GFP exhibited a diffuse signal throughout the entire cell similar to that reported in the Saccharomyces cerevisiae GFP database (Figure 2B). Following the extraction procedure, most of the GFP signal decreased, suggesting that this protein had been extracted from whole cells. Consistent with this idea, the majority of the Scw11p-GFP was detected in the extracellular fraction following the extraction procedure (Figure 2C).

clinical-experimental-pharmacology-extracellular

Figure 2: The extraction protocol does not cause non-specific leakage of proteins into the extracellular fraction. (A), Wild-type cells expressing Scw11p- GFP were grown in YPKG for 3 days and examined for expression of proteins following glucose addition for 0 and 2 hours using Western blotting with anti-GFP antibodies. (B), Cells expressing Scw11p-GFP were glucose starved and taken for fluorescence imaging before extraction. Extracellular proteins were extracted and cells were visualized by fluorescence microscopy after extraction. (C), Cells expressing Scw11p-GFP were glucose starved and subjected to the extraction procedure. Following extraction, the distribution of Scw11p-GFP in the Intracellular (I) fraction and the Extracellular fraction (E) was examined by Western blotting. Wild-type cells that expressed Tps2p-GFP were examined for protein expression (D), GFP distribution before and after extraction by fluorescence microscopy (E), and the distribution in I and E fractions following extraction by Western blotting. (F), Wild-type cells expressing Sur7p-GFP were examined for expression (G), GFP distribution before and after extraction (H), and the distribution in the I and E fractions after extraction (I).

Tps2p is the phosphatase subunit of the trehalose-6-phosphate synthase/phosphatase complex [85] and is distributed mostly in the cytosol. If the extraction protocol causes non-specific leakage of Tps2p, significant amounts of Tps2p should be detected in the extracellular fraction. Tps2p-GFP was expressed in glucose-starved cells and in cells that were shifted to glucose for 2 hours (Figure 2D). Tps2p-GFP showed a strong cytosolic signal before extraction (Figure 2E). A similar cytosolic distribution was also observed after the extraction. Moreover, the majority of Tps2p-GFP was in the intracellular fraction following the extraction protocol (Figure 2F). Thus, the extraction procedure does not cause non-specific leakage of Tps2p into the extracellular fraction.

Sur7p is a component of eisosomes on the plasma membrane [86,87]. Sur7p-GFP was expressed in cells grown in low glucose, and its levels did not increase following the addition of glucose (Figure 2G). Fluorescence studies indicated that Sur7p-GFP was distributed in punctate structures at the cell periphery before and after extraction (Figure 2H). Finally, following the extraction, Sur7p was detected in the intracellular fraction only (Figure 2I). Thus, this extraction protocol effectively extracted Scw11p into the extracellular fraction. Additionally, this procedure does not alter the distribution of Sur7p and Tps2p in the cell and does not cause the release of these proteins into the extracellular fraction.

Extracted Cells Re-grow in Rich Media and Actively Transport the Vital Dye FM 4-64 to the Vacuole Membrane

Cells grown in low glucose for 3 days are in the G0 stage of the cell cycle. When diluted in YPD media containing high glucose, these cells are able to re-enter the cell cycle and divide. Cells that were extracted also resumed growth and divided in YPD media. To measure cell growth, wild-type cells that were grown in low glucose for 3 days were diluted to OD600=0.2/ml in YPD containing 2% glucose or in YP without glucose. Cell densities were measured at OD600 for 0-24 hours using a spectrophotometer. When cells were diluted in YPD containing 2% glucose, cells increased their densities following a lag (Figure 3A). In contrast, when they were diluted in YP without glucose, cells did not increase densities, indicating that the presence of glucose in the media is required for cells to re-grow. Cells that were extracted and then diluted in YPD also increased cell densities following a lag (Figure 3B). These extracted cells did not increase densities when diluted in YP containing no glucose. Hence, cells that were extracted are able to regrow in rich media containing high glucose.

clinical-experimental-pharmacology-cell-densities

Figure 3: Extracted cells increase cell densities in YPD containing 2% glucose. Cells were grown in YPKG for 3 days until OD600=4-5/ml. Half of the cells were extracted, while the remaining half were not extracted. Both non-extracted (A) and extracted (B) cells were diluted to OD600=0.2/ml in 10 ml YPD containing 2% glucose or 10 ml YP without glucose. Cell densities were measured at OD600 for 0, 2, 4, 6, 8, and 24 hours using a Beckman spectrophotometer.

In addition, cells that were extracted are able to internalize exogenously added vital dye into the cells. FM 4-64 (FM) is a fluorescent dye that is internalized from the media and transported to the vacuole in live cells [88]. When wild-type cells were grown in YPKG (Yeast extracts-peptone-potassium acetate-0.5% glucose) for 3 days and then incubated with FM for 3 hours, this dye was on the vacuole membrane in ring-like circles as observed by fluorescence microscopy (Figure 4A). However, when these cells were treated with Triton X-100 and then incubated with FM, this dye was not observed on the vacuole membrane (Figure 4B), indicating that the integrity of the plasma membrane is required for cells to internalize FM and transport it to the vacuole membrane. When cells were extracted and then incubated with FM, this dye was transported correctly to the vacuole membrane (Figure 4C). However, when these extracted cells were treated with Triton X-100 and then incubated with FM, this dye was not on the vacuole membrane (Figure 4D). Thus, the extraction procedure does not impair the ability of the cells to internalize and transport this dye to the vacuole membrane. Because internalization of FM requires the integrity of the plasma membrane, this indicates that the plasma membrane is intact in cells that were extracted.

clinical-experimental-pharmacology-vacuole-membrane

Figure 4: Extracted cells internalize and transport the vital dye FM to the vacuole membrane. Wild-type cells were grown in YPKG for 3 days and divided. Half of the cells were subjected to the extraction procedure, while the other half was not extracted. Non-extracted and extracted cells were then treated with or without Triton X-100 (TX) for 30 min. Cells were centrifuged and resuspended in YP containing 1 μl FM (16 mg/ml) for 3 hours. The distribution of FM in non-extracted cells (A), non-extracted cells treated with TX (B), extracted cells (C), and extracted cells treated with TX (D) was observed by a Zeiss microscope.

The Extraction Procedure Confirms the Distribution of FBPase, MDH2, Icl1p, Pck1p, GAPDH, and Cpr1p in the Extracellular Fraction

Having established that this extraction procedure does not cause non-specific leakage of proteins into the extracellular fraction, and does not impair the ability of cells to re-grow and to transport FM to the vacuole, this protocol was used to confirm the presence of FBPase, MDH2, Icl1p, Pck1p, GAPDH, and Cpr1p in the extracellular fraction. Wild-type cells were grown in low glucose for 3 days and subjected to the extraction procedure. Following extraction, the distribution of proteins in the intracellular and extracellular fractions was determined. Scw4p is another soluble cell wall protein and was used as a control for proteins that are distributed in the extracellular fraction. Sec28p was used as a control for proteins that reside in the intracellular fraction. As expected, most of the Scw4p was in the extracellular fraction, while Sec28p was distributed mainly in the intracellular fraction (Figure 5). Significant amounts of FBPase, MDH2, Icl1p-HA, Pck1p-Myc, GAPDH, and Cpr1p were in the extracellular fraction. Under this experimental condition, Tco89p and Vps34p signaling molecules involved in glucose regulation of gluconeogenic enzymes are distributed mainly in the intracellular fraction [56,75]. Tco89p is a subunit of the TORC1 complex [89] required for the degradation of FBPase [64]. Vps34p is the major phosphatidylinositol 3-kinase in yeast [90].

clinical-experimental-pharmacology-Western-blotting

Figure 5: The extraction protocol confirms the presence of FBPase, MDH2, Icl1p, Pck1p, GAPDH, and Cpr1p in the extracellular fraction. Wildtype cells expressing Scw4p-GFP were grown in YPKG containing low glucose for three days and extracellular proteins were extracted. The distribution of Scw4p-GFP in the intracellular and extracellular fractions was examined by Western blotting using anti-GFP antibodies. Wild-type cells co-expressing Icl1p-HA and Pck1p-Myc were grown in low glucose media for 3 days and extracted. The distribution of Sec28p, FBPase, MDH2, Icl1p-HA, Pck1p- Myc, GAPDH, and Cpr1p in the intracellular and extracellular fractions was examined by Western blotting using anti-Sec28p, anti-FBPase, anti-HA, anti- Myc, anti-GAPDH and anti-Cpr1p antibodies. (This figure is in a manuscript published in Communicative and Integrative Biology, 2013 [76]).

Identification of Extracellular Proteins Using Proteomics

Given that the extraction method was able to detect the presence of FB Pase, MDH2, Icl1p, Pck1p, GAPDH, and Cpr1p in the extracellular fraction, this protocol was used to extract extracellular proteins for a large-scale proteomic analysis. Wild-type cells were grown in low glucose for 3 days, extracellular proteins were obtained, precipitated and digested with trypsin. The resulting tryptic peptides were then subjected to SCX (Strong cation exchange) separation followed by reverse phase C18 nano flow-LC separation. Eluant was spotted onto MALDI target plates and analyzed in a data-dependent manner on an ABI 5800 MALDI TOF-TOF. Protein identification was accomplished using the Paragon Algorithm [91] in Protein Pilot TM 4.0 software (AB Sciex). A total of 92 proteins with an estimated local False Discovery Rate (FDR) of less than 5% were identified in the extracellular fraction. These proteins were further classified into different functional groups as defined by the Saccharomyces genome database (Table 1). A high percentage of proteins (18%) were involved in carbohydrate metabolism and in amino acid metabolism (14%). Proteins that have roles in the generation of precursor metabolites, in protein folding, in translation and in stress were also identified. Gluconeogenic enzymes MDH2, Icl1p, and Pck1p were identified in the extracellular fraction. Likewise, non-gluconeogenic enzymes GAPDH and Cpr1p were also identified in the extracellular fraction. Heat shock proteins Ssa1p and Ssa2p found in the cell wall of Saccharomyces cerevisiae using immunofluorescence microscopy [27] were also identified as extracellular proteins (Table 1). Proteins involved in the metabolism of alcohol, amino acids, purines/ pyrimidines, fatty acids, glycerol, and acetate were identified as well (Table 1). Additionally, several of the heat shock proteins, anti-oxidant proteins, ribosomal proteins, translation initiation factors, translation elongation factors, and proteins with unknown functions have been identified (Table 1).

Name B. anthracis (14) L. monocytogenes (20) S. aureus (17) C. albicans (95) C. albicans (28) S. cerevisiae (28) S. cerevisiae (29) S. cerevisiae (94) K. lactis (22) C. sinensis (36) B. malayi (16) S. japonicum (39) E. caproni (19) S. mansoni (18) ASMC (34) HepG2 (12) HNOSCC (9) Glioblastoma (11)
A. Glycolysis/Gluconeogenesis                                    
Pgi1p, Phosphoglucose isomerase               +       +            
Fba1p, Fructose 1,6-bisphosphate aldolase   + + + + +   + +     + + + +   + +
Tpi1p, Triose phosphate isomerase +   +     +   + + + + + +     + +  
Tdh1p, Glyceraldehyde-3-phosphate dehydrogenase + +   + + + + +       + + + +   +  
Tdh3p, Glyceraldehyde-3-phosphate dehydrogenase + +   + + + + +       + + + +   +  
Pgk1p, 3-Phosphoglycerate kinase   + + + + + + + +     + + + + +   +
Gpm1p, Tetrameric phosphoglycerate mutase + +   + + +   + +     +   + + +   +
Eno1p, Enolase + + + + + + + + + + + + + + + + + +
Eno2p, Enolase + + + + + + + + + + + + + + + + + +
Cdc19p, Pyruvate kinase       + + + + +       +     +   + +
Pck1p, Phosphoenolpyruvate carboxykinase                   +     + +        
Mdh2p, Malate dehydrogenase cytosolic isozyme +     +                   +       +
Icl1p, Isocitrate lyase +                                  
B. TCA Cycle                                    
Lpd1p, Dihydrolipoamide dehydrogenase   +               +                
Aco1p, Aconitase                                   +
Idh1p,  Isocitrate dehydrogenase                                   +
Idh2p,  Isocitrate dehydrogenase                                   +
Idp1p, NADP-specific isocitrate dehydrogenase                                    
Lsc1p, Alpha subunit of succinyl-CoA ligase                                    
Sdh1p, Subunit of succinate dehydrogenase                                   +
Sdh2p, subunit of succinate dehydrogenase         +                          
Fum1p, Fumarase                                    
Mdh1p, Mitochondrial malate dehydrogenase +     +               +           +
C. Electron Transport                                    
Cor1p, Subunit of the ubiquinol-cytochrome c reductase       +                            
Qcr2p, Ubiquinol-cytochrome C reductase subunit 2       +                            
Atp1p, Alpha subunit of mitochondrial F1F0 ATP synthase         +         +               +
Atp2p, Beta subunit of mitochondrial F1F0 ATP synthase       +           +       + +     +
Atp14p, Subunit of ATP synthase     +                              
D. Pentose Phosphate Pathway                                    
Tkl1p, Transketolase         +     +       +   +        
E. Purine/Pyrimidine Metabolism                                    
Adk1p, Adenylate kinase               +                   +
Rnr4p, Ribonucleotide-diphosphate reductase             +                      
Ura1p, Dihydroorotate dehydrogenase                                    
F. Fermentation                                    
Pdc1p, Pyruvate decarboxylase       + + + + +                    
Adh1p, Alcohol dehydrogenase I       + + + + +                   +
Adh2p, Alcohol dehydrogenase II         + +                       +
Adh3p, Mitochondria alcohol dehydrogenase isozyme III                                   +
Ald4p, Mitochondria aldehyde dehydrogenase                   +   + +         +
G. Metabolism                                    
Ach1p, CoA transferase                                    
Etr1p, 2-Enoyl thioester reductase                                    
Rhr2p, DL-glycerol-3-phosphatase       +                            
Sec14p, PtdIns/PtdCho transfer protein                                    
H. Amino Acid Metabolism                                    
Aro8p, Aromatic aminotransferase I                                    
Met17p, Methionine and cysteine synthase + +                                
Met6p, Methionine Synthase       +       +                    
Mmf1p, Mitochondrial transamination of isoleucine                                    
I. Heat Shock Proteins                                    
Hsp12p, 12KD heat shock protein                 +                  
Hsp26p, Small heat shock protein (sHSP)                                    
Hsp60p, Tetradecameric mitochondrial chaperonin       +                 +   +      
Ssa1p, HSP70 family   +     + + + + +   + +   + +     +
Ssa2p, HSP70 family   +     + + + + +   + +   + +     +
Ssa4p, HSP70 family   +     + + + + +   + +   + +     +
Ssb1p, Member of HSP70 family     + + + + + + +                 +
Ssb2p, Member of HSP70 family     + + + + + + +                 +
Hsc82p, Hsp90 family (nearly identical to Hsp82)         +   + +         +       + +
Sba1p, Co-chaperone that regulates Hsp90 family               +                    
Sse1p, Co-chaperone that regulates Hsp90 family       +     + +                    
Hsp104p, Heat shock protein, HSP100 gene family                                    
Sis1p, Type II HSP40 co-chaperone, DnaJ family               +                   +
Sti1p, Hsp90 co-chaperone                                    
Mge1p, Mitochondrial matrix co-chaperone                                    
J. Protein Folding                                    
Cpr1p, Cytoplasmic cyclophilin                 +     +       + + +
Cpr3p, Mitochondrial cyclophilin                                   +
Fpr1p, FK506-binding protein proline rotamase                               +    
K. Oxidation Response                                    
Ahp1p, Thiol-specific peroxiredoxin     +     +   +           +   +   +
Ctt1p, Cytosolic catalase T                                    
Oye2p, Conserved NADPH oxidoreductase               +                    
Prx1p, Mitochondrial peroxiredoxin       +                     +      
Sod1p,  Superoxide dismutase   + +       + +     + +       +   +
Sod2p,  Superoxide dismutase   + +       + +     + +       +   +
Tsa1p, Thioredoxin peroxidase       + +     +     + +   +   + +  
L. Transcription                                    
Wtm1p, Transcriptional modulator                                    
M. Translation                                    
Rps31p, Ribosomal Protein of the Small subunit 40S                 +                  
Rpl25p, Large (60S) ribosomal subunit                                    
Tif2p, Translation initiation factor             +                      
Efb1p, Elongation factor   +   +                     +     +
Eft2p, Translation elongation factor 2 (EF-2)   +                         +   + +
Tef2p, Translational elongation factor EF-1 alpha   +     +     +       +   + +      
N. Cytoskeleton                                    
Act1p, Actin, structural protein       +     + +   +   + + + + + + +
O. Other                                    
Asc1p, G-protein beta subunit       +       +                   +
Bmh1p, 14-3-3 gene family         + + + +     + + + + +   + +
Bmh2p, 14-3-3 gene family         + + + +     + + + + +   + +
Cdc48p, ATPase         +                         +
Dug1p, Cys-Gly metallo-di-peptidase                                   +
Inh1p, ATPase Inhibitor                                   +
Pet9p, ADP/ATP carrier, mitochondrial membrane                                    
Ptr3p, Plasma membrane amino acid sensor system                                    
Shs1p, Seventh Homolog of Septin                                   +
Tfp1p, Vacuolar Membrane ATPase       +                     +      
Uba1p, Ubiquitin activating enzyme                                    
Ynk1p, Nucleoside diphosphate kinase +     +                            
Gre1p, Hydrophilin of unknown function                                    
Om45p, Mitochondrial outer membrane protein                                    
Ypr127wp, Putative pyridoxal reductase                                    

Table 1: Comparison of proteins identified using the extraction procedure with those identified in secretomic studies from different organisms.

Comparisons with Secretomic Studies from Other Organisms

Proteins that were identified in other secretomic studies from different organisms were compared with those identified in Saccharomyces cerevisiae. Bacillus anthracis is a Gram-positive, rodshaped bacterium. It is the etiologic agent of anthrax and is a disease of wildlife, livestock, and humans [14]. Listeria monocytogenes is a Gram-positive rod-shaped bacterium that causes severe foodborne infections in animals and humans. Listeria monocytogenes is able to spread directly from cell to cell, thus evading the immune system [20]. Staphylococcus aureus is a Gram-positive bacterium and is an opportunistic pathogen that causes a range of life-threatening diseases [17,92]. These pathogens secrete proteins that are required for their adaptation to the environment, for adhesion, and for invasion [17,20,92]. The dimorphic fungus Candida albicans is an opportunistic pathogen of humans and is capable of switching its cell morphology from yeast to hyphal form [28,83,93]. Kluyveromyces lactis grows as a yeast form and is often haploid [22,37]. Saccharomyces cerevisiae is nonpathogenic and can grow as haploid or diploid forms [29,94]. Parasites such as Clonorchis sinensis, Brugia malayi, Schistosoma japonicum, Echinostoma caproni, and Schistosoma mansoni enter a wide range of invertebrate and vertebrate hosts [15,16,18,19,36,39]. They secrete a number of proteins during infection that cause a down-modulation of the host immune response in favor of the maintenance of their biological cycles [15,16,18,19,36].

The yeast results were also compared with those reported for animal cells such as the Arterial Smooth Muscle Cells (ASMC) that secrete a large number of proteins involved in maintaining tone in vessel walls, repairing wounds, and development [34]. Finally, identified proteins from yeast were compared with those reported for cancer cell lines. These included the HepG2 human hepatoma, the Head and Neck/ Oral Squamous Cell Carcinoma (HNOSCC), and glioblastoma (LN18, U98, U118 and U87) cell lines [9-12]. These cancer cells secrete a large number of proteins into their surroundings during growth and invasion [9-12]. As shown in Table 1, common proteins that are secreted from other organisms include glycolytic enzymes, heat shock proteins, elongation factors, oxidative enzymes, cyclophilins, and actin [6,10,12,18-20,36,93]. Among glycolytic enzymes, enolase is the most frequently identified protein followed by 3-phosphoglycerate kinase, fructose-1,6-bisphosphate aldolase, phosphoglucose isomerase, phosphoglucose mutase and GAPDH. Among the heat shock proteins, the Hsp70p family is the most frequently identified. Thus, the secretion of these signal-less proteins is widely observed across species.

Conclusion Remarks

Multiple techniques including immuno-TEM, an extraction procedure, and proteomics have been used to study extracellular proteins that are secreted in Saccharomyces cerevisiae. Using immuno- TEM, significant amounts of gluconeogenic enzymes (FBPase, MDH2, Icl1p, and Pck1p), as well as non-gluconeogenic enzymes (GAPDH and Cpr1p), were observed to be secreted into the periplasm. Because of their distribution in the periplasm, an extraction protocol is needed to release these proteins from this location.

Control experiments indicated that the extraction protocol does not cause non-specific leakage of Tps2p and Sur7p into the extracellular fraction, while successfully extracting most of the Scw11p from whole cells. Fluorescence studies indicated that the distribution of Tps2p and Sur7p was not altered by the extraction procedures. Cells that were extracted were also able to re-grow and increase cell densities following a dilution in YPD with a growth curve similar to non-extracted cells. In addition, cells that were extracted retained the ability to internalize the fluorescent dye FM and actively transport it to the vacuole membrane. Because the integrity of the plasma membrane is needed for FM to be internalized and transported to the vacuole, the plasma membrane should be intact in cells that were extracted. Most importantly, this extraction protocol was able to detect the presence of FBPase, MDH2, Icl1p, Pck1p, GAPDH, and Cpr1p in the extracellular fraction. Under the same conditions, other molecules such as Sec28p, Vid24p, Vid30p, Vps34p, and Tco89p were distributed mainly in the intracellular fraction [56,57,89]. Therefore, this extraction method is effective for extracting periplasmic proteins from whole cells. For proteins that are secreted directly into the culture media, they can be collected from the culture media without the use of this method.

Among the extracellular proteins that were identified by proteomics, fewer than 5% of the identified proteins contain an N-terminal signal sequence. Since more than 95% of the extracellular proteins lack the signal sequence, the non-classical pathway is the major pathway to secrete proteins and the classical pathway is the minor pathway. This conclusion is consistent with a previous Saccharomyces cerevisiae study in which 99 proteins were identified in the secretome and only 17 proteins contained a signal sequence [94].

It is interesting that extracellular proteins that are secreted from cells are involved in so many diverse functions. The secretion of metabolic enzymes may be dependent on the physiological state of the cells. In addition, metabolic enzymes may be secreted as enzymatically active forms [23,77,83]. When GAPDH from Candida albicans is fused with invertase from Saccharomyces cerevisiae, activities of both GAPDH and invertase are detected on the cell surface of Saccharomyces cerevisiae. Secreted proteins may perform different functions outside the cells. Glycolytic enzymes have been identified as immunogens during infection by Candida albicans [28,83,95]. GAPDH and enolase bind to various mammalian proteins such as lysozyme, fibronectin, actin, myosin and plasmin [28,83,93]. These secreted glycolytic enzymes may be involved in invasion by pathogens during infection.

In summary, immuno-TEM, the extraction protocol, and proteomics have been instrumental in producing a large amount of information needed to study extracellular proteins that are secreted in Saccharomyces cerevisiae. Many of the identified extracellular proteins from Saccharomyces cerevisiae are also secreted from other organisms, suggesting that secretion of these proteins is conserved across species. As more research tools become available in the future, more genes and proteins that play important roles in the secretion of extracellular proteins can be identified.

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