Amyloid-like properties of Saccharomyces cerevisiae cell wall glucantransferase Bgl2p: Prediction and experimental evidences

Glucantransferase Bgl2p is a major conserved cell wall constituent described for a wide range of yeast species. In the baker''s yeast Saccharomyces cerevisiae it is the only non-covalently bound cell wall protein that cannot be released from cell walls by sequential SDS and trypsin treatment. It contains seven amyloidogenic determinants. Circular dichroism analysis and fluorescence spectroscopy with thioflavin T indicate the presence of β-sheet structures in Bgl2p isolates. Bgl2p forms fibrils, a process that is enforced in the presence of other cell wall components. Thus the data obtained is the first evidence for amyloid-like properties of yeast cell wall protein—glucantransferase Bgl2p.Key words: glucantransferase Bgl2p, cell wall amyloid-like protein     

Revealing of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> yeast cell wall proteins capable of binding thioflavin T,a fluorescent dye specifically interacting with amyloid fibrils

Proteins binding thioflavin T leading to its specific fluorescence were discovered in a fraction of noncovalently bound Saccharomyces cerevisiae yeast cell wall mannoproteins. Thioflavin-binding proteins display high resistance to trypsin digestion in solution. These data are the first experimental evidence for the presence of proteins whose properties are characteristic of amyloids in yeast cell wall, except for data on glucanotransferase Bgl2p that has amyloid properties. Our data suggest the anchoring of these proteins in the cell wall by a trypsin-sensitive part of the protein molecule. Experiments with a mutant strain devoid of the BGL2 gene suggest the compensation of absent amyloid-like protein Bgl2p by increase in contents of thioflavin-binding proteins in the cell wall.     

Exo70p mediates the secretion of specific exocytic vesicles at early stages of the cell cycle for polarized cell growth

In budding yeast, two classes of post-Golgi secretory vesicles carrying different sets of cargoes typified by Bgl2p and invertase are delivered to the plasma membrane for secretion. The exocyst is implicated in tethering these vesicles to the daughter cell membrane for exocytosis. In this study, we report that mutations in the exocyst component Exo70p predominantly block secretion of the Bgl2p vesicles. Furthermore, a defect in invertase vesicle trafficking caused by vps1Delta or pep12Delta in the exo70 mutant background is detrimental to the cell. The secretion defect in exo70 mutants was most pronounced during the early budding stage, which affected daughter cell growth. The selective secretion block does not occur at the vesicle formation or sorting stage because the exocytic vesicles are properly generated and protein processing is normal in the exo70 mutants. Our study suggests that Exo70p functions primarily at early stages of the cell cycle in Bgl2p vesicle secretion, which is critical for polarized cell growth.     

Cofilin-mediated sorting and export of specific cargo from the Golgi apparatus in yeast

The mechanism of cargo sorting at the trans-Golgi network (TGN) for secretion is poorly understood. We previously reported the involvement of the actin-severing protein cofilin and the Ca(2+) ATPase secretory pathway calcium ATPase 1 (SPCA1) in the sorting of soluble secretory cargo at the TGN in mammalian cells. Now we report that cofilin in yeast is required for export of selective secretory cargo at the late Golgi membranes. In cofilin mutant (cof1-8) cells, the cell wall protein Bgl2 was secreted at a reduced rate and retained in a late Golgi compartment, whereas the plasma membrane H(+) ATPase Pma1, which is transported in the same class of carriers, reached the cell surface. In addition, sorting of carboxypeptidase Y (CPY) to the vacuole was delayed, and CPY was secreted from cof1-8 cells. Loss of the yeast orthologue of SPCA1 (Pmr1) exhibited similar sorting defects and displayed synthetic sickness with cof1-8. In addition, overexpression of PMR1 restored Bgl2 secretion in cof1-8 cells. These findings highlight the conserved role of cofilin and SPCA1/Pmr1 in sorting of the soluble secretory proteins at the TGN/late Golgi membranes in eukaryotes.     

Structural and functional analysis of hybrid enzymes generated by domain shuffling between Saccharomyces cerevisiae (var. diastaticus) Sta1 glucoamylase and Saccharomycopsis fibuligera Bgl1 β-glucosidase

Saccharomyces cerevisiae Sta1 glucoamylase and Saccharomycopsis fibuligera Bgl1 β-glucosidase, two relevant enzymes from a biotechnological point of view, are proteins with multidomain structure. Starting with homology-based structural models of Sta1 and Bgl1, we have constructed a series of hybrid enzymes by interchanging domains of the two proteins. The first purpose of these constructs was to check available hypotheses about the uncertain biological functions of two domains: the serine/threonine-rich domain (STRD) of Sta1 and a β-sandwich domain present in Bgl1 that we have designated fibronectin-like domain (FLD). While, according to the initial hypothesis, proteins carrying the FLD tend to adhere to the cell wall, our results argued against the idea of an involvement of the STRD in protein secretion that stemmed from the presence of similar domains in different proteins secreted by yeast. The second objective of this work was to increase the enzymatic repertoire by generating enzymes with new structural and functional properties.     

Cell surface expression of bacterial esterase A by Saccharomyces cerevisiae and its enhancement by constitutive activation of the cellular unfolded protein response

Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg(-1) protein for Kre1/EstA/Cwp2p and 72 mU mg(-1) protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg(-1) protein for Kre1/EstA/Cwp2p and 1.27 U mg(-1) protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway.     

Amyloidogenic peptides of yeast cell wall glucantransferase Bgl2p as a model for the investigation of its pH-dependent fibril formation

The pH-dependence of the ability of Bgl2p to form fibrils was studied using synthetic peptides with potential amyloidogenic determinants (PADs) predicted in the Bgl2p sequence. Three PADs, FTIFVGV, SWNVLVA and NAFS, were selected on the basis of combination of computational algorithms. Peptides AEGFTIFVGV, VDSWNVLVAG and VMANAFSYWQ, containing these PADs, were synthesized. It was demonstrated that these peptides had an ability to fibrillate at pH values from 3.2 to 5.0. The PAD-containing peptides, except for VDSWNVLVAG, could fibrillate also at pH values from pH 5.0 to 7.6. We supposed that the ability of Bgl2p to form fibrils most likely depended on the coordination of fibrillation activity of the PAD-containing areas and Bgl2p could fibrillate at mild acid and neutral pH values and lose the ability to fibrillate with the increasing of pH values. It was demonstrated that Bgl2p was able to fibrillate at pH value 5.0, to form fibrils of various morphology at neutral pH values and lost the fibrillation ability at pH value 7.6. The results obtained allowed us to suggest a new simple approach for the isolation of Bgl2p from Saccharomyces cerevisiae cell wall.     

Yeast arming by the Aga2p system: effect of growth conditions in galactose on the efficiency of the display and influence of expressing leucine-containing peptides

The amino or carboxy-terminal regions of certain cell wall proteins are capable of anchoring foreign proteins or peptides on the cell wall of the yeast Saccharomyces cerevisiae. This possibility has resulted in the development of a methodology known as yeast display which has powerful applications in biotechnology, pharmacy, and medicine. This work describes the results of experiments in which the agglutinin Aga2p protein is used as an anchor and several leucine-based peptides have been introduced into its N-terminal or C-terminal position. We found that the sequence of these peptides can affect plasmid stability, growth kinetics, and levels of the fusion protein displayed, and we analyzed how the incubation conditions influence these parameters. Besides, we show that the introduction of these small peptides can modify the properties of cell cover; in particular, fusing five or ten leucine residues to the Aga2p protein results in greater hydrophobicity of the cell wall and also in increased resistance to the presence of the organic solvents acetonitrile and ethanol and to high salt concentrations. The introduction of the RLRLL sequence also results in higher resistance to the exposure of yeast cells to NaCl stress.     

A new phenotypic manifestation of deletion of the BGL2 gene encoding the cell-wall glucanotransferase Bgl2p in the yeast Saccharomyces cerevisiae

Deletion of the gene encoding the cell-wall glucanotransferase Bgl2p in Saccharomyces cerevisiae decreases the number of dead cells in the yeast culture incubated in a liquid nutrient medium for more than two days. After storage for three months, only 32% of the wild-type cells were found to be able to produce colonies, whereas all cells with the inactivated BGL2 gene retained this ability. It is suggested that the glucanotransferase Bgl2p plays an important role in the limitation of the reproductive life span of aging yeast cells.     

Schizosaccharomyces pombe ehs1p is involved in maintaining cell wall integrity and in calcium uptake

The Schizosaccharomyces pombe mutant ehs1-1 mutant was isolated on the basis of its hypersensitivity to Echinocandin and Calcofluor White, which inhibit cell wall synthesis. The mutant shows a thermosensitive growth phenotype that is suppressed in the presence of an osmotic stabiliser. The mutant also showed other cell wall-associated phenotypes, such as enhanced sensitivity to enzymatic cell wall degradation and an imbalance in polysaccharide synthesis. The ehs1 + gene encodes a predicted integral membrane protein that is 30% identical to Saccharomyces cerevisiae Mid1p, a protein that has been proposed to form part of a calcium channel. As expected for such a function, we found that ehs1+ is involved in intracellular Ca2+ accumulation. High external Ca2+ concentrations suppressed all phenotypes associated with the ehs1 null mutation, suggesting that the cell integrity defects of ehs1 mutants result from inadequate levels of calcium in the cell. We observed a genetic relationship between ehs1+ and the protein kinase C homologue pck2+. pck2+ suppressed all phenotypes of ehs1-1 mutant cells. Overproduction of pck2p is deleterious to wild-type cells, increasing 1,3-beta-D-glucan synthase activity and promoting accumulation of extremely high levels of Ca2+. The lethality associated with pck2p, the increase in 1,3-beta-D-glucan synthase production and the strong Ca2+ accumulation are all dependent on the presence of ehs1p. Our results suggest that in fission yeast ehs1p forms part of a calcium channel that is involved in the cell wall integrity pathway that includes the kinase pck2p.     

Sbe2p and sbe22p, two homologous Golgi proteins involved in yeast cell wall formation

The cell wall of fungal cells is important for cell integrity and cell morphogenesis and protects against harmful environmental conditions. The yeast cell wall is a complex structure consisting mainly of mannoproteins, glucan, and chitin. The molecular mechanisms by which the cell wall components are synthesized and transported to the cell surface are poorly understood. We have identified and characterized two homologous yeast proteins, Sbe2p and Sbe22p, through their suppression of a chs5 spa2 mutant strain defective in chitin synthesis and cell morphogenesis. Although sbe2 and sbe22 null mutants are viable, sbe2 sbe22 cells display several phenotypes indicative of defects in cell integrity and cell wall structure. First, sbe2 sbe22 cells display a sorbitol-remediable lysis defect at 37 degrees C and are hypersensitive to SDS and calcofluor. Second, electron microscopic analysis reveals that sbe2 sbe22 cells have an aberrant cell wall structure with a reduced mannoprotein layer. Finally, immunofluorescence experiments reveal that in small-budded cells, sbe2 sbe22 mutants mislocalize Chs3p, a protein involved in chitin synthesis. In addition, sbe2 sbe22 diploids have a bud-site selection defect, displaying a random budding pattern. A Sbe2p-GFP fusion protein localizes to cytoplasmic patches, and Sbe2p cofractionates with Golgi proteins. Deletion of CHS5, which encodes a Golgi protein involved in the transport of Chs3p to the cell periphery, is lethal in combination with disruption of SBE2 and SBE22. Thus, we suggest a model in which Sbe2p and Sbe22p are involved in the transport of cell wall components from the Golgi apparatus to the cell surface periphery in a pathway independent of Chs5p.     

Cell Surface Expression of Bacterial Esterase A by Saccharomyces cerevisiae and Its Enhancement by Constitutive Activation of the Cellular Unfolded Protein Response

Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg⁻¹ protein for Kre1/EstA/Cwp2p and 72 mU mg⁻¹ protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg⁻¹ protein for Kre1/EstA/Cwp2p and 1.27 U mg⁻¹ protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway.     

Novel strategy for anchorage position control of GPI-attached proteins in the yeast cell wall using different GPI-anchoring domains

The yeast cell surface provides space to display functional proteins. Heterologous proteins can be covalently anchored to the yeast cell wall by fusing them with the anchoring domain of glycosylphosphatidylinositol (GPI)-anchored cell wall proteins (GPI-CWPs). In the yeast cell-surface display system, the anchorage position of the target protein in the cell wall is an important factor that maximizes the capabilities of engineered yeast cells because the yeast cell wall consists of a 100- to 200-nm-thick microfibrillar array of glucan chains. However, knowledge is limited regarding the anchorage position of GPI-attached proteins in the yeast cell wall. Here, we report a comparative study on the effect of GPI-anchoring domain–heterologous protein fusions on yeast cell wall localization. GPI-anchoring domains derived from well-characterized GPI-CWPs, namely Sed1p and Sag1p, were used for the cell-surface display of heterologous proteins in the yeast Saccharomyces cerevisiae. Immunoelectron-microscopic analysis of enhanced green fluorescent protein (eGFP)-displaying cells revealed that the anchorage position of the GPI-attached protein in the cell wall could be controlled by changing the fused anchoring domain. eGFP fused with the Sed1-anchoring domain predominantly localized to the external surface of the cell wall, whereas the anchorage position of eGFP fused with the Sag1-anchoring domain was mainly inside the cell wall. We also demonstrate the application of the anchorage position control technique to improve the cellulolytic ability of cellulase-displaying yeast. The ethanol titer during the simultaneous saccharification and fermentation of hydrothermally-processed rice straw was improved by 30% after repositioning the exo- and endo-cellulases using Sed1- and Sag1-anchor domains. This novel anchorage position control strategy will enable the efficient utilization of the cell wall space in various fields of yeast cell-surface display technology.     

Specific Cell Wall Proteins Confer Resistance to Nisin upon Yeast Cells

The cell wall of a yeast cell forms a barrier for various proteinaceous and nonproteinaceous molecules. Nisin, a small polypeptide and a well-known preservative active against gram-positive bacteria, was tested with wild-type Saccharomyces cerevisiae. This peptide had no effect on intact cells. However, removal of the cell wall facilitated access of nisin to the membrane and led to cell rupture. The roles of individual components of the cell wall in protection against nisin were studied by using synchronized cultures. Variation in nisin sensitivity was observed during the cell cycle. In the S phase, which is the phase in the cell cycle in which the permeability of the yeast wall to fluorescein isothiocyanate dextrans is highest, the cells were most sensitive to nisin. In contrast, the cells were most resistant to nisin after a peak in expression of the mRNA of cell wall protein 2 (Cwp2p), which coincided with the G2 phase of the cell cycle. A mutant lacking Cwp2p has been shown to be more sensitive to cell wall-interfering compounds and Zymolyase (J. M. Van der Vaart, L. H. Caro, J. W. Chapman, F. M. Klis, and C. T. Verrips, J. Bacteriol. 177:3104–3110, 1995). Here we show that of the single cell wall protein knockouts, a Cwp2p-deficient mutant is most sensitive to nisin. A mutant with a double knockout of Cwp1p and Cwp2p is hypersensitive to the peptide. Finally, in yeast mutants with impaired cell wall structure, expression of both CWP1 and CWP2 was modified. We concluded that Cwp2p plays a prominent role in protection of cells against antimicrobial peptides, such as nisin, and that Cwp1p and Cwp2p play a key role in the formation of a normal cell wall.     

The stress response protein Hsp12p increases the flexibility of the yeast Saccharomyces cerevisiae cell wall

The yeast S. cerevisiae cell wall comprising a 10 nm thick layer of polysaccharides, predominantly beta(1,3)-glucan and proteins, is the interface between the cell and the neighbouring environment. As such it is not a static entity but rather one that is dynamically remodelled in response to changes in the environmental conditions. We have recently proposed from studies using yeast cells lacking the gene encoding Hsp12p (Deltahsp12 yeast) and from incorporation of Hsp12p into agarose, used as a model system for the beta-glucan layer of the cell wall, that the hydrophilic stress response cell wall protein Hsp12p acts as a cell wall plasticizer. In this report we have used force spectroscopy to confirm that Deltahsp12 yeast are indeed less flexible than the wild type strain. The spring constant of the cell wall of Deltahsp12 yeast, kcw was determined to be 72+/-3 mN m-1 as compared to 17+/-5 mN m-1 obtained for the wild type strain. A similar result was found on the basis of a quantitative analysis of the electrophoretic mobilities measured for the two yeast strains. Those indicated that the hydrodynamic permeability quantified through the softness parameter of the external layer of Deltahsp12 cells was smaller than the one of wild type cells. We proposed from surface infrared spectroscopy measurements that yeast compensate for the lack of Hsp12p by reducing the carbohydrate/proteins ratio of the cell wall or increasing the cell wall chitin content.     

NPFXD-mediated endocytosis is required for polarity and function of a yeast cell wall stress sensor

The actin-associated protein Sla1p, through its SHD1 domain, acts as an adaptor for the NPFX(1,2)D endocytic targeting signal in yeast. Here we report that Wsc1p, a cell wall stress sensor, depends on this signal-adaptor pair for endocytosis. Mutation of NPFDD in Wsc1p or expression of Sla1p lacking SHD1 blocked Wsc1p internalization. By live cell imaging, endocytically defective Wsc1p was not concentrated at sites of endocytosis. Polarized distribution of Wsc1p to regions of cell growth was lost in the absence of endocytosis. Mutations in genes necessary for endosome to Golgi traffic caused redistribution of Wsc1p from the cell surface to internal compartments, indicative of recycling. Inhibition of Wsc1p endocytosis caused defects in polarized deposition of the cell wall and increased sensitivity to perturbation of cell wall synthesis. Our results reveal that the NPFX(1,2)D-Sla1p system is responsible for directing Wsc1p into an endocytosis and recycling pathway necessary to maintain yeast cell wall polarity. The dynamic localization of Wsc1p, a sensor of the extracellular wall in yeast, resembles polarized distribution of certain extracellular matrix-sensing integrins through endocytic recycling.