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Keywords:

  • Enolase;
  • Cell wall;
  • Saccharomyces cerevisiae;
  • Protein export;
  • Yeast

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Non-covalently attached or soluble cell wall proteins of Saccharomyces cerevisiae were extracted using a high pH/2-mercaptoethanol procedure and were separated for peptide sequencing using 2D-PAGE. A partial N- terminal sequence of a major protein spot was obtained and showed high identity with enolase gene products. Western blotting with an anti-enolase antibody confirmed that enolase was present in the cell wall extract. Biotinylation of cells prior to protein extraction with a membrane impermeable biotinylating agent confirmed that the detection was not owing to cell lysis during extraction. Transmission immunoelectron microscopy showed enolase to be present in the cell wall. Enolase contains no known secretion signal that would localize it to the cell wall. Thus S. cerevisiae must have further mechanisms for targeting proteins to the cell wall.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

The cell wall of Saccharomyces cerevisiae, which functions to provide the cell protection against external stresses, consists of two major components, glucan and mannoproteins, and one minor component, chitin [1]. The mannoproteins consist of reducing agent extractable mannoproteins, glucanase extractable mannoproteins and sodium dodecyl sulfate (SDS) extractable mannoproteins [2]. The glucanase extractable mannoproteins have an N-terminal secretion signal sequence, a serine/threonine sequence for glycosylation and a C-terminal glycosylphosphatidylinositol (GPI) attachment site [3]. The completion of the S. cerevisiae genome sequencing project [4] allows in silico analysis of the genome. This analysis suggests that 53 proteins have the GPI, glycosylation and secretion sites and, experimentally, 14 have been found to be cell wall attached [5]. These proteins become covalently attached to glucan in the cell wall by their GPI anchor residues, arriving at the cell wall by the classical secretory pathway. This pathway consists of protein translocation from the cytosol into the endoplasmic reticulum lumen, where the signal peptide sequence is removed and then through vesicles to the Golgi and on to the cell surface [6]. Additionally, treatment with reducing agents releases a set of cell wall proteins: these are largely unidentified species and it is not known if they are classically secreted proteins or not.

In this study, we investigated proteins solubilized under reducing conditions from the yeast cell wall. Using the Saccharomyces Proteome Database [7], we were able to compare our isolated protein against the Saccharomyces proteome and determine any novel localization.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

2.1Strain and growth conditions

S. cerevisiae strain INVSC1 (MATa his3Δ1 leu2 trp1-289 ura3-52) was grown in medium containing 2% (w/v) glucose, 2% (w/v) peptone and 1% (w/v) yeast extract. Cells were grown at 25°C, with shaking at 180 rpm to exponential phase growth.

2.2Preparation of cell wall extracts

Proteins were extracted as described previously [8]. Briefly, the cells were harvested by centrifugation, washed twice in water and then the cell wall components were isolated by resuspending cells in ammonium carbonate (1.6 g l−1) and adding 2% 2-mercaptoethanol (2-ME). Cells were incubated for 30 min at 37°C. The suspensions were centrifuged and the residual cells in the supernatant removed by filtration through a 0.45-μm filter. The supernatant was dialyzed against water (Cellulose tubing 6–8K MW cut-off) for 48 h. The dialysate was then lyophilized and resuspended in Tris buffered saline, pH 7.4. Protein concentrations were determined by the Bradford method [9] using commercial reagents (Bio-Rad, Hercules, CA, USA). Samples were stored at −20°C.

2.3Biotinylation of cell wall proteins

Intact cells were biotinylated with SulfoNHS-LC biotin (Pierce, Rockford, IL, USA), which does not permeate the cell membrane [10], and then washed repeatedly to remove the SulfoNHS-LC biotin. Cell wall proteins were extracted and prepared as described above. Biotinylated proteins (10 μg) from a portion of the extract were further purified by affinity chromatography on immobilized streptavidin (Pierce) following the manufacturer's instructions as described previously [11].

2.4Two-dimensional gel electrophoresis

2D gels were run based upon the procedure of O'Farrell [12]. First dimension tubes for isoelectric focusing were prepared using broad (pI 3–10) and narrow pH (pI 5–7) range ampholytes. 100 μg of protein was loaded in the first dimension. For the second dimension, 12.5% PAGE gels were used [13]. Proteins were transferred from the gel to polyvinyl difluoride (PVDF, Bio-Rad) membrane for sequencing using a semi-dry blotter and the transfer was visualized by staining with Coomassie blue, or silver stain (Bio-Rad Silver Stain kit) [14].

2.5Western blotting

Standard Western blotting techniques were employed [15]. To detect for the biotinylation of protein, nitrocellulose membranes were treated with a 1/3000 dilution of peroxidase conjugated Extravidin (Sigma, St. Louis, MO, USA). To detect enolase, nitrocellulose membranes were treated with a 1/100 dilution of IgA monoclonal anti-enolase antibody (a gift from H. Buckley). A 1/100 dilution of peroxidase conjugated goat anti-mouse IgA antibody was used as the secondary antibody. All blots were washed in Tris buffered saline at pH 7.4 with 0.05% Tween 20 and developed with 4-chloro-1-naphthol and hydrogen peroxide [16].

2.6Protein sequencing

Proteins from the PVDF membranes were sequenced partially by Edman degradation at the Texas Tech University Biotechnology Core Facility. Proteins were identified from the partial peptide sequence using Saccharomyces Genome Database [7].

2.7Electron microscopy (EM)

EM was performed to further localize enolase. Samples were freeze substituted as described previously [17]. The sections were treated with the primary antibody (monoclonal anti-enolase) and a goat anti-mouse IgA colloidal gold conjugated antibody (EY Laboratories, San Mateo, CA, USA, 10 nm gold particle size). As negative controls, a primary irrelevant antibody and no primary antibody were used. Grids were stained with uranyl acetate and lead citrate. Samples were observed with a Hitachi H-600 microscope at 75 kV at a magnification of ×30 000. Gold particles were counted for five cells in each sample. Statistical analysis was performed by analysis of the variance (ANOVA) with Bonferroni's multiple comparison test.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

3.1Extraction of cell wall proteins using bicarbonate buffer/2-ME

The extracted cell wall proteins were separated using 2D-PAGE. A silver stained 2D gel is shown in Fig. 1. A duplicate, unstained gel was transferred to PVDF and one major band (MW 47K) was partially sequenced. The peptide sequence n-AVSKVYARxVYDxRGNPTVE-c was obtained, where ‘x’ represents ambiguous residues. This peptide showed identity (with the 17 unambiguous residues) to the enolases (2-phosphoglycerate dehydratases), Eno1 and Eno2.

image

Figure 1. A silver stained 2D gel of the cell wall proteins extracted under high pH/reducing conditions. The first dimension was focused using ampholytes (pI range 3–10). The second dimension is a 12.5% SDS-poly acrylamide gel. The acid (H+) and alkali (OH) ends of the gel are marked. The spot marked with the arrow (MW 47 000) was sequenced.

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3.2Confirmation that enolase is a cell wall protein

The above results suggest that enolase is found in the cell wall. Protein blotting was employed to further confirm the presence of enolase in the cell wall. Fig. 2 (lane 1) shows, in a Western blot, that enolase was detected in a cell wall extract of the yeast cells using an anti-enolase antibody. To address the concern that the enolase could be released owing to cell lysis during the extraction procedure, and thus be detected as a cell wall artifact, cells were biotinylated with a cell membrane impermeable agent (SulfoNHS-LC Biotin) to label cell wall proteins prior to extraction.

image

Figure 2. Analysis of the biotinylated proteins. Unfractionated biotinylated cell wall extract (lane 1 and 4), and biotinylated proteins purified by streptavidin affinity chromatography (lanes 2 and 3) were separated by SDS-PAGE and transferred to nitrocellulose. Lanes 1 and 2 were developed as a Western blot, using anti-enolase antibody and show extracted biotinylated proteins (lane 1) and streptavidin purified biotinylated proteins (lane 2). Lanes 3 and 4 were developed using Extravidin and show the profile of extracted biotinylated proteins (lane 4) and streptavidin purified biotinylated proteins (lane 3). Lane M represents prestained molecular mass standards (Bio-Rad, MW of 203, 116, 83, 48.7, 33.4, 28.2, 20.7 and 7.6 kDa). The 48.7-kDa MW standard is indicated. The arrow indicates the detection of the enolase protein.

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A Western blot of biotinylated cell wall proteins detected with an anti-enolase antibody is shown in Fig. 2. Lane 1 shows non-purified, biotinylated, cell wall proteins. Biotinylated proteins were streptavidin bead purified and similarly detected with anti-enolase (lane 2). Enolase was detected in both lanes 1 and 2. The profile of streptavidin purified biotinylated proteins, detected with Extravidin is shown in lane 3. Lane 4 shows the profile of non-purified biotinylated proteins detected with Extravidin. Therefore, it can be concluded that enolase was present on or in the cell wall prior to extraction and its detection was not artifactual owing to cell lysis.

3.3Localization of enolase in the cell wall by immunogold electron microscopy

To localize enolase in the cell wall, cells were freeze substituted and prepared for immunogold electron microscopy. Fig. 3 shows a transmission electron micrograph of a sectioned yeast cell. The colloidal gold particles are found primarily in the cytoplasm. Gold particles were found also within and on the surface of the cell wall layer. The detection of enolase within the cell wall is a further confirmation that enolase is a bona fide cell wall protein. Analysis of the particles on the cell wall of the cells revealed a mean of 9 (S.D.=1.58) particles per cell for the sample. No gold particles were counted on the irrelevant antibody control and a mean of 0.2 particles per cell (S.D.=0.45) when only the secondary antibody was used. The distribution of the gold in the experiment compared to the negative controls was statistically significant (p<0.0001).

image

Figure 3. A transmission electron micrograph of yeast cell prepared by freeze substitution, viewed at ×27 600 magnification. The monoclonal antibody anti-enolase was used following procedure described in Section 2. The anti-enolase was detected using colloidal gold (10 nm particle size) conjugated anti-mouse IgA (bar represents 0.2 μm).

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4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

In eukaryotes, proteins for classical secretion are synthesized usually as precursors with a cleavable signal peptide at (or near to) the N-terminus of the polypeptide. This export mechanism is conserved in both prokaryotes and eukaryotes [18]. Using a secretory mutant (Sec18ts) in yeast, which blocks classical secretion, it has been shown the some high molecular mass proteins which are extracted under reducing conditions are classically secreted (they are not present under restrictive conditions), but some proteins remain, which suggests that some of the proteins reach the cell wall via non-classical pathways. Alternatives to the classical secretory pathway have been reported for eukaryotes: in mammalian cells, interleukin 1β has been reported to be secreted while lacking a known amino terminal signal sequence [19] and in yeast, mating factor a is secreted and lacks the classical secretory signal peptides. Its secretion is directed by Ste6, a member of the ABC protein superfamily [20]. Another family of proteins implicated in non-classical secretion is the proteins Nce101, Nce102 and Nce103 [21]. These proteins, which are not ABC family members, bear some structural similarity with ‘miniTEXANS’ (toxin extruders) in bacterial systems, in that Nce102 is a multimembrane spanning protein of similar size to the ‘miniTEXANS’. One possible function of the non-classical pathway of secretion is the removal of potentially toxic proteins from the cytoplasm. The alternative pathway may offer cells a method for protein export when classical secretion is arrested (by heat shock, for example). Some alternative protein secretion pathways have been reviewed [22] and proteins such as hsp70 species and their partners are implicated in protein trafficking and may offer a non-vesicular system for protein transport [23].

Enolase, one of the most abundant enzymes in the cytosol, was found in the cell wall of S. cerevisiae, while lacking the typical signal peptide sequence. The presence of glycolytic enzymes in yeast cell walls is not without precedence [17]. In C. albicans, enolase has been found to be associated with glucan in the inner layers of the cell wall [24] and is a major antigen in systemic infections of candidiasis. In C. albicans, three other glycolytic enzymes have been identified in the cell wall: phosphoglycerate kinase (PGK), glyceraldehyde phosphate dehydrogenase (GAPDH), which shows laminin and fibronectin binding properties, and alcohol dehydrogenase (ADH) (reviewed in [25]). GAPDH has also been reported on the cell wall of Kluyveromyces lactis where it functions as a flocculin. It is possible therefore, that enolase may have an alternative function on the cell wall also. Hsp70 species, also lacking classical secretory signal sequences, have also been found on the cell walls of both S. cerevisiae and C. albicans.

In summary, enolase is, in addition to its cytoplasmic location, a cell wall protein which may arrive at the cell wall via a, currently unknown, pathway. The presence of enolase, which lacks known secretion signals, supports the hypotheses of alternative secretory pathways and may offer new insights into protein transport in yeasts and other eukaryotes.

After the submission of this manuscript, Pardo et al. reported that enolase secreted by protoplasts and its presence in the medium was not owing to cell lysis [26].

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

We are indebted deeply to M. Grimson and M.C. Hastert for their help with the electron microscopy and to H. Buckley for the anti-enolase monoclonal antibody.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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