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.     

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.     

PIG-S and PIG-T, essential for GPI anchor attachment to proteins, form a complex with GAA1 and GPI8

Many eukaryotic cell surface proteins are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). The GPI transamidase mediates GPI anchoring in the endoplasmic reticulum, by replacing a protein's C-terminal GPI attachment signal peptide with a pre-assembled GPI. During this transamidation reaction, the GPI transamidase forms a carbonyl intermediate with a substrate protein. It was known that the GPI transamidase is a complex containing GAA1 and GPI8. Here, we report two new components of this enzyme: PIG-S and PIG-T. To determine roles for PIG-S and PIG-T, we disrupted these genes in mouse F9 cells by homologous recombination. PIG-S and PIG-T knockout cells were defective in transfer of GPI to proteins, particularly in formation of the carbonyl intermediates. We also demonstrate that PIG-S and PIG-T form a protein complex with GAA1 and GPI8, and that PIG-T maintains the complex by stabilizing the expression of GAA1 and GPI8. Saccharomyces cerevisiae Gpi16p (YHR188C) and Gpi17p (YDR434W) are orthologues of PIG-T and PIG-S, respectively.     

高效絮凝素毕赤酵母表面展示系统的构建

为了获得高效的脂肪酶毕赤酵母表面展示系统,利用来自酿酒酵母絮凝素蛋白Flo1的N端874个氨基酸残基(FS)和C端的1101个氨基酸残基(FL)作为锚定蛋白分别构建了2套载体系统.带有前肽的米黑根毛霉脂肪酶(ProRML)克隆到构建的2套展示载体中,使米黑根毛霉脂肪酶(RML)分别以N端锚定或C端锚定的方式实现在毕赤酵母细胞表面的展示.利用RMLC端的Flag标签,通过流式细胞术和激光扫描共聚焦显微镜检测2套系统中RML在酵母表面的展示情况.研究发现,N端锚定于酵母表面的展示酶FSR以pNPC为底物时,水解活力达到了105.3U/g,大约为C端锚定的展示酶FLR活力的2倍.同时FSR比FLR具有更宽的温度、pH作用范围和更好的热稳定性.与游离酶和固定化酶相比,展示酶FSR也表现出更为优良的热稳定性.结果提示,基于Flo1N端锚定的展示系统更适合展示活性中心近C端的脂肪酶,推动了展示酶的进一步研究和开发.     

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.     

Determination and physiological roles of the glycosylphosphatidylinositol lipid remodelling pathway in yeast

In the yeast Saccharomyces cerevisiae, glycosylphosphatidylinositol (GPI)‐anchored proteins play important roles in cell wall biogenesis/assembly and the formation of lipid microdomains. The lipid moieties of mature GPI‐anchored proteins in yeast typically contain either ceramide moieties or diacylglycerol. Recent studies have identified that the GPI phospholipase A₂ Per1p and O‐acyltransferase Gup1p play essential roles in diacylglycerol‐type lipid remodelling of GPI‐anchored proteins, while Cwh43p is involved in the remodelling of lipid moieties to ceramide. It has been generally proposed that phosphatidylinositol with diacylglycerol containing a C26 saturated fatty acid, which is generated by the sequential activity of Per1p and Gup1p, is converted to inositolphosphorylceramide by Cwh43p. In this report, we constructed double‐mutant strains defective in lipid remodelling and investigated their growth phenotypes and the lipid moieties of GPI‐anchored proteins. Based on our analyses of single‐ and double‐mutants of proteins involved in lipid remodelling, we demonstrate that an alternative pathway, in which lyso‐phosphatidylinositol generated by Per1p is used as a substrate for Cwh43p, is involved in the remodelling of GPI lipid moieties to ceramide when the normal sequential pathway is inhibited. In addition, mass spectrometric analysis of lipid species of Flag‐tagged Gas1p revealed that Gas1p contains ceramide moieties in its GPI anchor.     

MCD4 encodes a conserved endoplasmic reticulum membrane protein essential for glycosylphosphatidylinositol anchor synthesis in yeast

Glycosylphosphatidylinositol (GPI)-anchored proteins are cell surface-localized proteins that serve many important cellular functions. The pathway mediating synthesis and attachment of the GPI anchor to these proteins in eukaryotic cells is complex, highly conserved, and plays a critical role in the proper targeting, transport, and function of all GPI-anchored protein family members. In this article, we demonstrate that MCD4, an essential gene that was initially identified in a genetic screen to isolate Saccharomyces cerevisiae mutants defective for bud emergence, encodes a previously unidentified component of the GPI anchor synthesis pathway. Mcd4p is a multimembrane-spanning protein that localizes to the endoplasmic reticulum (ER) and contains a large NH2-terminal ER lumenal domain. We have also cloned the human MCD4 gene and found that Mcd4p is both highly conserved throughout eukaryotes and has two yeast homologues. Mcd4p's lumenal domain contains three conserved motifs found in mammalian phosphodiesterases and nucleotide pyrophosphases; notably, the temperature-conditional MCD4 allele used for our studies (mcd4-174) harbors a single amino acid change in motif 2. The mcd4-174 mutant (1) is defective in ER-to-Golgi transport of GPI-anchored proteins (i.e., Gas1p) while other proteins (i.e., CPY) are unaffected; (2) secretes and releases (potentially up-regulated cell wall) proteins into the medium, suggesting a defect in cell wall integrity; and (3) exhibits marked morphological defects, most notably the accumulation of distorted, ER- and vesicle-like membranes. mcd4-174 cells synthesize all classes of inositolphosphoceramides, indicating that the GPI protein transport block is not due to deficient ceramide synthesis. However, mcd4-174 cells have a severe defect in incorporation of [3H]inositol into proteins and accumulate several previously uncharacterized [3H]inositol-labeled lipids whose properties are consistent with their being GPI precursors. Together, these studies demonstrate that MCD4 encodes a new, conserved component of the GPI anchor synthesis pathway and highlight the intimate connections between GPI anchoring, bud emergence, cell wall function, and feedback mechanisms likely to be involved in regulating each of these essential processes. A putative role for Mcd4p as participating in the modification of GPI anchors with side chain phosphoethanolamine is also discussed.     

Comparison of cell wall proteins of Saccharomyces cerevisiae as anchors for cell surface expression of heterologous proteins.

The carboxyl-terminal regions of five cell wall proteins (Cwp1p, Cwp2p, Ag alpha 1p, Tip1p, and Flo1p) and three potential cell wall proteins (Sed1p, YCR89w, and Tir1p) all proved capable of immobilizing alpha-galactosidase in the cell wall of Saccharomyces cerevisiae. The fraction of the total amount of fusion protein that was localized to the cell wall varied depending on the anchor domain used. The highest proportion of cell wall incorporation was achieved with Cwp2p, Ag alpha 1p, or Sed1p as an anchor. Although 80% of these fusion proteins were incorporated in the cell wall, the total production of alpha-galactosidase-Ag alpha 1p was sixfold lower than that of alpha-galactosidase-Cwp2p and eightfold lower than that of alpha-galactosidase-Sed1p. Differences in mRNA levels were not responsible for this discrepancy, nor was an intracellular accumulation of alpha-galactosidase-Ag alpha 1p detectable. A lower translation efficiency of the alpha-galactosidase-AG alpha 1 fusion construct is most likely to be responsible for the low level of protein production. alpha-Galactosidase immobilized by the carboxyl-terminal 67 amino acids of Cwp2p was most effective in the hydrolysis of the high-molecular-weight substrate guar gum from Cyamopsis tetragonoloba. This indicates that the use of a large anchoring domain does not necessarily result in a better exposure of the immobilized enzyme to the exterior of the yeast cell.     

Identification of six complementation classes involved in the biosynthesis of glycosylphosphatidylinositol anchors in Saccharomyces cerevisiae

Glycosylphosphatidylinositol (GPI)-anchored membrane proteins are synthesized by the posttranslational attachment of a preformed glycolipid to newly made glycoproteins. alpha-Agglutinin is a GPI- anchored glycoprotein that gets expressed at the cell surface of MAT alpha cells after induction with type a mating factor. Mutants affecting the biosynthesis of GPI anchors were obtained by selecting for the absence of alpha-agglutinin from the cell wall after induction with a-factor at 37 degrees C. 10 recessive mutants were grouped into 6 complementation classes, gpi4 to gpi9. Mutants are considered to be deficient in the biosynthesis of GPI anchors, since each mutant accumulates an abnormal, incomplete GPI glycolipid containing either zero, two, or four mannoses. One mutant accumulates a complete precursor glycolipid, suggesting that it might be deficient in the transfer of complete precursor lipids to proteins. When labeled with [2- 3H]inositol, mutants accumulate reduced amounts of radiolabeled GPI- anchored proteins, and the export of the GPI-anchored Gas1p out of the ER is severely delayed in several mutant strains. On the other hand, invertase and acid phosphatase are secreted by all but one mutant. All mutants show an increased sensitivity to calcofluor white and hygromycin B. This suggests that GPI-anchored proteins are required for the integrity of the yeast cell wall.     

The β-1,6-glucan containing side-chain of cell wall proteins of Saccharomyces cerevisiae is bound to the glycan core of the GPI moiety

Abstract Cell wall proteins of Saccharomyces cerevisiae are anchored by means of a β-1,6-glucan-containing side-chain. It is not known whether this chain is linked to the protein part (e.g. through carbohydrate side-chains) or to the glycosylphosphatidylinositol (GPI) moiety of cell wall proteins. An IgA protease recognition site was introduced in Cwp2p, a β-1,6-glucosylated cell wall protein, immediately N-terminal from the omega amino acid (the attachment site of the GPI moiety). Proteolytic cleavage of this site revealed that the β-1,6-glucan epitope was not linked to the protein part. We conclude that neither N - or O -glycosylation is involved in β-glucosylation of cell wall proteins. This confirms that the glycan core of the GPI moiety is the probable β-1,6-glucan attachment site.     

Glycosyl phosphatidylinositol-dependent cross-linking of alpha- agglutinin and beta 1,6-glucan in the Saccharomyces cerevisiae cell wall

The cell adhesion protein alpha-agglutinin is bound to the outer surface of the Saccharomyces cerevisiae cell wall and mediates cell- cell contact in mating. alpha-Agglutinin is modified by addition of a glycosyl phosphatidylinositol (GPI) anchor as it traverses the secretory pathway. The presence of a GPI anchor is essential for cross- linking into the wall, but the fatty acid and inositol components of the anchor are lost before cell wall association (Lu, C.-F., J. Kurjan, and P. N. Lipke, 1994. A pathway for cell wall anchorage of Saccharomyces cerevisiae alpha-agglutinin. Mol. Cell. Biol. 14:4825- 4833). Cell wall association of alpha-agglutinin was accompanied by an increase in size and a gain in reactivity to antibodies directed against beta 1,6-glucan. Several kre mutants, which have defects in synthesis of cell wall beta 1,6-glucan, had reduced molecular size of cell wall alpha-agglutinin. These findings demonstrate that the cell wall form of alpha-agglutinin is covalently associated with beta 1,6- glucan. The alpha-agglutinin biosynthetic precursors did not react with antibody to beta 1,6-glucan, and the sizes of these forms were unaffected in kre mutants. A COOH-terminal truncated form of alpha- agglutinin, which is not GPI anchored and is secreted into the medium, did not react with the anti-beta 1,6-glucan. We propose that extracellular cross-linkage to beta 1,6-glucan mediates covalent association of alpha-agglutinin with the cell wall in a manner that is dependent on prior addition of a GPI anchor to alpha-agglutinin.     

Two endoplasmic reticulum (ER) membrane proteins that facilitate ER-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins

Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes in Saccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for "delayed GPI-anchored protein transport"), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Delta dgt1Delta cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting that LAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non-GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Delta dgt1Delta cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Delta dgt1Delta cells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins.     

Brittle Culm1, a COBRA-Like Protein,Functions in Cellulose Assembly through Binding Cellulose Microfibrils

Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1), a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI) anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM) at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD) assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs) function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity.     

Endoplasmic reticulum localized PerA is required for cell wall integrity,azole drug resistance,and virulence in Aspergillus fumigatus

GPI‐anchoring is a universal and critical post‐translational protein modification in eukaryotes. In fungi, many cell wall proteins are GPI‐anchored, and disruption of GPI‐anchored proteins impairs cell wall integrity. After being synthesized and attached to target proteins, GPI anchors undergo modification on lipid moieties. In spite of its importance for GPI‐anchored protein functions, our current knowledge of GPI lipid remodelling in pathogenic fungi is limited. In this study, we characterized the role of a putative GPI lipid remodelling protein, designated PerA, in the human pathogenic fungus Aspergillus fumigatus. PerA localizes to the endoplasmic reticulum and loss of PerA leads to striking defects in cell wall integrity. A perA null mutant has decreased conidia production, increased susceptibility to triazole antifungal drugs, and is avirulent in a murine model of invasive pulmonary aspergillosis. Interestingly, loss of PerA increases exposure of β‐glucan and chitin content on the hyphal cell surface, but diminished TNF production by bone marrow‐derived macrophages relative to wild type. Given the structural specificity of fungal GPI‐anchors, which is different from humans, understanding GPI lipid remodelling and PerA function in A. fumigatus is a promising research direction to uncover a new fungal specific antifungal drug target.     

Identification of a missing link in glycosylphosphatidylinositol anchor biosynthesis in mammalian cells.

A large number of mammalian proteins are anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Biosynthetic intermediates of the GPI anchor have been identified in mammalian cells. The early GPI precursors are sensitive to phosphatidylinositol (PI)-specific phospholipase C (PLC). However, all of the later GPI precursors, which contain 1 or more mannose residues, are PI-PLC-resistant, suggesting that there is another unidentified precursor. Here, we report the identification of this missing link. This GPI precursor can only be labeled with glucosamine and inositol, and is resistant to PI-PLC but sensitive to GPI-phospholipase D. It accumulates in large quantity only in mutants which are defective in the addition of the first mannose residue to the elongating GPI core. Thus, fatty acylation of glucosaminylphosphatidylinositol, to render it PI-PLC-resistant, is an obligatory step in the biosynthesis of mammalian GPI anchor precursors.     

Modular domain structure in the Candida glabrata adhesin Epa1p,a beta1,6 glucan-cross-linked cell wall protein

The yeast pathogen Candida glabrata adheres avidly to cultured human epithelial cells. This interaction depends on the expression of EPA1, which encodes a lectin belonging to a large family of GPI-anchored glucan-cross-linked cell wall proteins (GPI-CWPs) found in diverse fungal species. To understand the relationship between different domains of EPA1 and its function, we have mapped functional domains of Epa1p and analysed their contribution to Epa1p function. We found that the N-terminal third of the protein contains the ligand-binding domain, and that the GPI anchor is essential both for cross-linking in the cell wall and for Epa1p-mediated adherence. We also found that the C-terminal Ser/Thr-rich domain, characteristic of many GPI-CWPs, was absolutely essential for function. Although Epa1p derivatives lacking the Ser/Thr domain were expressed abundantly in the cell wall, they were localized to internal layers of the cell wall; such constructs were unable to mediate adherence. The outer layer of the yeast cell wall is known to act as a permeability barrier; we found that the C-terminal Ser/Thr-rich region was absolutely required to project the N-terminal domain of Epa1p through this permeability barrier and into the external environment. Thus, the Ser/Thr-rich domain of Epa1p and, presumably, of other related GPI-CWPs serves an essential structural role in localization of the protein at the external surface of the yeast cell where it can interact with its ligand. In conclusion, Epa1p has a modular structure, with each domain serving a distinct and essential role in the function of the adhesin.     

GPI‐anchored human placental alkaline phosphatase has a nonpolarized distribution on the cell surface of mouse cerebellar granule neurons in vitro

The glycosyl phosphatidylinositol (GPI) lipid anchor, which directs GPI‐anchored proteins to the apical cell surface in certain polarized epithelial cell types, has been proposed to act as an axonal protein targeting signal in neurons. However, as several GPI‐anchored proteins have been found on both the axonal and somatodendritic cell‐surface domains of a variety of neuronal cell types, the role of the GPI anchor in protein localization to the axon remains unclear. To begin to address the role of the GPI anchor in neuronal protein localization, we used a replication‐incompetent retroviral vector to express a model GPI‐anchored protein, human placental alkaline phosphatase (hPLAP), in early postnatal mouse cerebellar granule neurons developing in vitro. Purified granule neurons were cultured in large mitotically active cellular reaggregates to allow retroviral infection of undifferentiated, proliferating granule neuron precursors. To more easily visualize hPLAP localization during the sequence of differentiation of single postmitotic granule neurons, reaggregates were dissociated following infection, plated as high‐density monolayers, and maintained for 1–9 days under serum‐free culture conditions. As we previously demonstrated for uninfected granule neurons developing in monolayer culture, hPLAP‐expressing granule neurons likewise developed in vitro through a series of discrete temporal stages highly similar to those observed in situ. hPLAP‐expressing granule neurons first extended either a single neurite or two axonal processes, and subsequently attained a mature, well‐polarized morphology consisting of multiple short dendrites and one or two axons that extended up to 3 mm across the culture substratum. hPLAP was expressed uniformly on the entire cell surface at each stage of granule neuron differentiation. Thus, it appears that the GPI anchor is not sufficient to confer axonal localization to an exogenous GPI‐anchored protein expressed in a well‐polarized primary neuronal cell type in vitro; other signals, such asthose present in the extracellular domain of these proteins, may be necessary for the polarized targeting or retention of axon‐specific GPI‐anchored proteins. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 119–141, 1999     

Deletion of Intragenic Tandem Repeats in Unit C of FLO1 of Saccharomyces cerevisiae Increases the Conformational Stability of Flocculin under Acidic and Alkaline Conditions

Flocculation is an attractive property for Saccaromyces cerevisiae, which plays important roles in fermentation industry and environmental remediation. The process of flocculation is mediated by a family of cell surface flocculins. As one member of flocculins, Flo1 is characterized by four families of repeats (designated as repeat units A, B, C and D) in the central domain. It is generally accepted that variation of repeat unit A in length in Flo1 influences the degree of flocculation or specificity for sugar recognization. However, no reports were observed for other repeat units. Here, we compared the flocculation ability and its sensitivity to environmental factors between yeast strain YSF1 carrying the intact FLO1 gene and yeast strains carrying the derived forms of FLO1 with partial or complete deletion of repeats in unit C. No obvious differences in flocculation ability and specificity of carbohydrate recognition were observed among these yeast strains, which indicates the truncated flocculins can stride across the cell wall and cluster the N-terminal domain on the surface of yeast cells as the intact Flo1 thereby improving intercellular binding. However, yeast strains with the truncated flocculins required more mannose to inhibit completely the flocculation, displayed broad tolerance of flocculation to pH fluctuation, and the fewer the repeats in unit C, the stronger adaptability of flocculation to pH change, which was not relevant to the position of deletion. This suggests that more stable active conformation is obtained for flocculin by deletion the repeat unit C in the central domain of Flo1, which was validated further by the higher hydrophobicity on the surface of cells of YSF1c with complete deletion of unit C under neutral and alkaline conditions and the stabilization of GFP conformation by fusion with flocculin with complete deletion of unit C in the central domain.     

The Fate of PrP GPI‐Anchor Signal Peptide is Modulated by P238S Pathogenic Mutation

Glycosylphosphatidylinositol (GPI)‐anchored proteins are localized to the plasma membrane via a C‐terminally linked GPI anchor. The GPI anchor is added concomitantly to the cleavage of the carboxy‐terminal GPI‐anchor signal sequence, thereby causing the release of a C‐terminal hydrophobic peptide, whose fate has not yet been investigated. Here we followed the fate of the GPI‐attachment signal of the prion protein (PrP), a protein implicated in various types of transmissible neurodegenerative spongiform encephalopathies (TSE). The PrP GPI‐anchor signal sequence shows a remarkable and unusual degree of conservation across the species and contains two point mutations (M232R/T and P238S) that are responsible for genetic forms of prion disorders. We show that the PrP GPI‐anchor signal peptide (SP), but not the one from an unrelated GPI‐anchored protein (folate receptor), undergoes degradation via the proteasome. Moreover, the P238S point mutation partially protects the PrP GPI‐anchor SP from degradation. Our data provide the first attempt to address the fate of a GPI‐anchor SP and identify a role for the P238S mutation, suggesting the possibility that the PrP GPI‐anchor SP could play a role in neurodegenerative prion diseases.