Screening for glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in Saccharomyces cerevisiae

Open reading frames in the genome of Saccharomyces cerevisiae were screened for potential glycosylphosphatidylinositol (GPI)-attached proteins. The identification of putative GPI-attached proteins was based on three criteria: the presence of a GPI-attachment signal sequence, a signal sequence for secretion and a serine- or threonine-rich sequence. In all, 53 ORFs met these three criteria and 38 were further analyzed as follows. The sequence encoding the 40 C-terminal amino acids of each was fused with the structural gene for a reporter protein consisting of a secretion signal, α-galactosidase and a hemagglutinin (HA) epitope, and examined for the ability to become incorporated into the cell wall. On this basis, 14 of fusion proteins were classified as GPI-dependent cell wall proteins because cells expressing these fusion proteins: (i) had high levels of α-galactosidase activity on their surface; (ii) released significant amounts of the fusion proteins from the membrane on treatment with phosphatidylinositol-specific phospholipase C (PI-PLC); and (iii) released fusion proteins from the cell wall following treatment with laminarinase. Of the 14 identified putative GPI-dependent cell wall proteins, 12 had novel ORFs adjacent to their GPI-attachment signal sequence. Amino acid sequence alignment of the C-terminal sequences of the 12 ORFs, together with those of known cell wall proteins, reveals some sequence similarities among them.     

An α-galactosidase with an essential function during leaf development

The putative α-galactosidase gene HvSF11 of barley, previously shown to be expressed during dark induced senescence, is expressed in the growing/elongating zone of primary foliage leaves of barley. The amino acid sequence deduced from the full length HvSF11 cDNA contains a hydrophobic signal sequence at the N-terminus. Phylogenetic relationship of the HvSF11 encoded barley α-galactosidase to other α-galactosidases revealed high homology with the α-galactosidase encoded by the gene At5g08370 from Arabidopsis thaliana. We have isolated two independent heterozygous At5g08370 T-DNA insertion mutants from Arabidopsis thaliana, both of which have a higher number of rosette leaves with a curly surface leaf morphology and delayed flowering time in comparison to wildtype plants. Localization of the Arabidopsis α-galactosidase protein via GUS-tag revealed that the protein is associated with the cell wall. This result was confirmed by immunological detection of the orthologous barley protein in a protein fraction derived from cell walls of barley leaves. It is concluded that the α-galactosidase proteins from barley and Arabidopsis might fulfill an important role in leaf development by functioning in cell wall loosening and cell wall expansion.

---

     

Efficient glycosylphosphatidylinositol (GPI) modification of membrane proteins requires a C-terminal anchoring signal of marginal hydrophobicity

Many plasma membrane proteins are anchored to the membrane via a C-terminal glycosylphosphatidylinositol (GPI) moiety. The GPI anchor is attached to the protein in the endoplasmic reticulum by transamidation, a reaction in which a C-terminal GPI-attachment signal is cleaved off concomitantly with addition of the GPI moiety. GPI-attachment signals are poorly conserved on the sequence level but are all composed of a polar segment that includes the GPI-attachment site followed by a hydrophobic segment located at the very C terminus of the protein. Here, we show that efficient GPI modification requires that the hydrophobicity of the C-terminal segment is "marginal": less hydrophobic than type II transmembrane anchors and more hydrophobic than the most hydrophobic segments found in secreted proteins. We further show that the GPI-attachment signal can be modified by the transamidase irrespective of whether it is first released into the lumen of the endoplasmic reticulum or is retained in the endoplasmic reticulum membrane.     

Genetic immobilization of cellulase on the cell surface of Saccharomyces cerevisiae

We tried genetically to immobilize cellulase protein on the cell surface of the yeast Saccharomyces cerevisiae in its active form. A cDNA encoding FI-carboxymethylcellulase (CMCase) of the fungus Aspergillus aculeatus, with its secretion signal peptide, was fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast α-agglutinin, a protein involved in mating and covalently anchored to the cell wall. The plasmid constructed containing this fusion gene was introduced into S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase activity was detected in the cell pellet fraction. The CMCase protein was solubilized from the cell wall fraction by glucanase treatment but not by sodium dodecyl sulphate treatment, indicating the covalent binding of the fusion protein to the cell wall. The appearance of the fused protein on the cell surface was further confirmed by immunofluorescence microscopy and immunoelectron microscopy. These results proved that the CMCase was anchored on the cell wall in its active form. Received: 19 March 1997 / Received revision: 19 May 1997 / Accepted: 1 June 1997     

Pmt1 mannosyl transferase is involved in cell wall incorporation of several proteins in Saccharomyces cerevisiae

We constructed hybrid proteins containing a plant α-galactosidase fused to various C-terminal moieties of the hypoxic Srp1p; this allowed us to identify a cell wall-bound form of Srp1p. We showed that the last 30 amino acids of Srp1p, but not the last 16, contain sufficient information to signal glycosyl-phosphatidylinositol anchor attachment and subsequent cell wall anchorage. The cell wall-bound form was shown to be linked by means of a β1,6-glucose-containing side-chain. Pmt1p enzyme is known as a protein-O-mannosyltransferase that initiates the O-glycosidic chains on proteins. We found that a pmt1 deletion mutant was highly sensitive to zymolyase and that in this strain the α-galactosidase–Srp1 fusion proteins, an α-galactosidase–Sed1 hybrid protein and an α-galactosidase–α-agglutinin hybrid protein were absent from both the membrane and the cell wall fractions. However, the plasma membrane protein Gas1p still receives its glycosyl-phosphatidylinositol anchor in pmt1 cells, and in this mutant strain an α-galactosidase–Cwp2 fusion protein was found linked to the cell wall but devoid of β1,6-glucan side-chain, indicating an alternative mechanism of cell wall anchorage.     

Xanthomonas campestris pv. campestris lpsI and lpsJ genes encoding putative proteins with sequence similarity to the α- and β-subunits of 3-oxoacid CoA-transferases are involved in LPS biosynthesis

A 2.1-kb SmaI-EcoRI DNA fragment upstream of the xanA and xanB genes of Xanthomonas campestris pv. campestris carries two ORFs encoding putative proteins with sequence similarities to the α- and β-subunits of 3-oxoacid-CoA transferases. The two ORFs were termed lpsI and lpsJ because strains carrying appropriate mutations showed an autoagglutination phenotype and because lipopolysaccharides of these mutant strains were altered according to silver-stained polyacrylamide gels. The monosaccharide composition of the exopolysaccharide xanthan produced by lpsI and lpsJ mutants remained unchanged. Received: 29 March 1997 / Accepted: 21 July 1997     

A new α-galactosidase from symbiotic Flavobacterium sp. TN17 reveals four residues essential for α-galactosidase activity of gastrointestinal bacteria

The α-galactosidase gene, galA17, was cloned from Flavobacterium sp. TN17, a symbiotic bacterium isolated from the gut of Batocera horsfieldi larvae. The 2,205-bp full-length gene encodes a 734-residue polypeptide (GalA17) containing a putative 28-residue signal peptide and a catalytic domain belonging to glycosyl hydrolase family 36 (GH 36). The deduced amino acid sequence of galA17 was most similar to a putative α-galactosidase from Pedobacter sp. BAL39 (EDM38577; 66.6% identity) and a characterized α-galactosidase from Carnobacterium piscicola BA (AAL27305; 30.1%). Phylogenetic analysis revealed that GalA17 was similar to GH 36 α-galactosidases from symbiotic bacteria sharing two putative catalytic motifs, KWD and SDXXDXXXR, in which D480, S548, D549, and R556 were essential for α-galactosidase activity based on site-directed mutagenesis. Purified recombinant GalA17 showed apparent optimal activity at pH 5.5 and 45°C; exhibited strong resistance to digestion by trypsin, α-chymotrypsin, collagenase, and proteinase K; and efficiently hydrolyzed several synthetic and natural substrates (p-nitrophenyl-α-d-galactopyranoside, stachyose, melibiose, raffinose, soybean meal, locust bean gum, and guar gum).     

Biochemical Changes Associated with Brassica juncea Seed Development, II: Glycosidases

Changes in α- and β-galactosidase, glucosidase, and α-mannosidase and β-xylosidase activities were analyzed in developing mustard (Brassica juncea) seed. A cubic polynomial described the seed dry weight data adequately. A close parallelism between the water content and dry matter accumulation was shown (r= 0.991). Glycosidase activities were detected in both cytoplasmic and wall fractions. The trend was similar in both of the fractions, but the activity of α-mannosidase and α-galactosidase was considerably greater in the wall-bound fraction. The water content and the activity of glycosidic enzymes showed a significant linear correlation (p < 0.001). The results suggest that glycosidases have an important role in cell wall loosening during mustard seed development. Received July 10, 1997; accepted January 28, 1998     

Molecular dissection of the large mechanosensitive ion channel (MscL) of E. coli: Mutants with altered channel gating and pressure sensitivity

In the search for the essential functional domains of the large mechanosensitive ion channel (MscL) of E. coli, we have cloned several mutants of the mscL gene into a glutathione S-transferase fusion protein expression system. The resulting mutated MscL proteins had either amino acid additions, substitutions or deletions in the amphipathic N-terminal region, and/or deletions in the amphipathic central or hydrophilic C-terminal regions. Proteolytic digestion of the isolated fusion proteins by thrombin yielded virtually pure recombinant MscL proteins that were reconstituted into artificial liposomes and examined for function by the patch-clamp technique. The addition of amino acid residues to the N-terminus of the MscL did not affect channel activity, whereas N-terminal deletions or changes to the N-terminal amino acid sequence were poorly tolerated and resulted in channels exhibiting altered pressure sensitivity and gating. Deletion of 27 amino acids from the C-terminus resulted in MscL protein that formed channels similar to the wild-type, while deletion of 33 C-terminal amino acids extinguished channel activity. Similarly, deletion of the internal amphipathic region of the MscL abolished activity. In accordance with a recently proposed spatial model of the MscL, our results suggest that (i) the N-terminal portion participates in the channel activation by pressure, and (ii) the essential channel functions are associated with both, the putative central amphipathic α-helical portion of the protein and the six C-terminal residues RKKEEP forming a charge cluster following the putative M2 membrane spanning α-helix. Received: 25 September 1996/Revised: 21 November 1996     

A novel protease-resistant α-galactosidase with high hydrolytic activity from <Emphasis Type="Italic">Gibberella</Emphasis> sp. F75: gene cloning,expression, and enzymatic characterization

A novel α-galactosidase gene (aga-F75) from Gibberella sp. F75 was cloned and expressed in Escherichia coli. The gene codes for a protein of 744 amino acids with a 24-residue putative signal peptide and a calculated molecular mass of 82.94 kDa. The native structure of the recombinant Aga-F75 was estimated to be a trimer or tetramer. The deduced amino acid sequence showed highest identity (69%) with an α-galactosidase from Hypocrea jecorina (Trichoderma reesei), a member of the glycoside hydrolase family 36. Purified recombinant Aga-F75 was optimally active at 60°C and pH 4.0 and was stable at pH 3.0–12.0. The enzyme exhibited broad substrate specificity and substantial resistance to neutral and alkaline proteases. The enzyme K _m values using pNPG, melibiose, stachyose, and raffinose as substrates were 1.06, 1.75, 54.26, and 8.23 mM, respectively. Compared with the commercial α-galactosidase (Aga-A) from Aspergillus niger var. AETL and a protease-resistant α-galactosidase (Aga-F78) from Rhizopus sp. F78, Aga-F75 released 1.4- and 4.9-fold more galactose from soybean meal alone, respectively, and 292.5- and 8.6-fold more galactose from soybean meal in the presence of trypsin, respectively. The pH and thermal stability and hydrolytic activity of Aga-F75 make it potentially useful in the food and feed industries.     

Development of an arming yeast strain for efficient utilization of starch by co-display of sequential amylolytic enzymes on the cell surface

The construction of a whole-cell biocatalyst with its sequential reaction has been performed by the genetic immobilization of two amylolytic enzymes on the yeast cell surface. A recombinant strain of Saccharomyces cerevisiae that displays glucoamylase and α-amylase on its cell surface was constructed and its starch-utilizing ability was evaluated. The gene encoding Rhizopus oryzae glucoamylase, with its own secretion signal peptide, and a truncated fragment of the α-amylase gene from Bacillus stearothermophilus with the prepro secretion signal sequence of the yeast α factor, respectively, were fused with the gene encoding the C-terminal half of the yeast α-agglutinin. The constructed fusion genes were introduced into the different loci of chromosomes of S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The glucoamylase and α-amylase activities were not detected in the culture medium, but in the cell pellet fraction. The transformant strain co-displaying glucoamylase and α-amylase could grow faster on starch as the sole carbon source than the transformant strain displaying only glucoamylase. Received: 16 June 1998 / Received last revision: 21 August 1998 / Accepted: 3 September 1998     

Screening for Glycosylphosphatidylinositol-Modified Cell Wall Proteins in Pichia pastoris and Their Recombinant Expression on the Cell Surface

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different uses in vitro. In the present study, the genome of Pichia pastoris GS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and mature Candida antarctica lipase B (CALB). The expression of fusion proteins on the cell surface of P. pastoris GS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by β-1,3-glucanase digestion. CALB displayed on the cell surface of P. pastoris GS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis of p-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins in P. pastoris GS115, which can be used to display heterologous proteins on the yeast cell surface.     

Secretion, purification, and characterisation of barley α-amylase produced by heterologous gene expression in Aspergillus niger

Efficient production of recombinant barley α-amylase has been achieved in Aspergillus niger. The cDNA encoding α-amylase isozyme 1 (AMY1) and its signal peptide was placed under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter and the A. nidulans trpC gene terminator. Secretion yields up to 60 mg/l were obtained in media optimised for α-amylase activity and low protease activity. The recombinant AMY1 (reAMY1) was purified to homogeneity and found to be identical to native barley AMY1 with respect to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the endogenous plant signal peptide is correctly processed in A. niger. Electrospray ionisation/mass spectrometry gave a molecular mass for the dominant form of 44 960 Da, in accordance with the loss of the LQRS C-terminal residues; glycosylation apparently did not occur. The activities of recombinant and native barley α-amylases are very similar towards insoluble and soluble starch as well as 2-chloro-4-nitrophenol β-d-maltoheptaoside and amylose (degree of polymerisation = 17). Barley α-amylase is the first plant protein efficiently secreted and correctly processed by A. niger using its own signal sequence. Received: 22 August 1997 / Received revision: 21 November 1997 / Accepted: 29 November 1997     

Construction of a Novel <Emphasis Type="Italic">Pichia pastoris</Emphasis> Cell-Surface Display System Based on the Cell Wall Protein Pir1

A novel system based on Pir1 from Saccharomyces cerevisiae was developed for cell-surface display of heterologous proteins in Pichia pastoris with the alpha-factor secretion signal sequence. As a model protein, enhanced green fluorescence protein (EGFP) was fused to the N-terminal of the mature peptide of Pir1 (Pir1-a). The expression of fusion protein EGFP-Pir1-a was irregular throughout the P. pastoris cell surface per detection by confocal laser scanning microscopy. A truncated sequence containing only the internal repetitive sequences of Pir1-a (Pir1-b) was used as a new anchor protein in further study. The fusion protein EGFP-Pir1-b was expressed uniformly on the cell surface. The fluorescence intensity of the whole yeast was measured by spectrofluorometer. Western blot confirmed that the fusion proteins were released from cell walls after mild alkaline treatment. The results indicate that a Pir1-based system can express proteins on the surface of P. pastoris and that the fusion proteins do not affect the manner in which Pir1 attaches to the cell wall. The repetitive sequences of Pir1 are required for cell wall retention, and the C-terminal sequence contributes to the irregular distribution of fusion proteins in P. pastoris.     

The YSIRK-G/S motif of staphylococcal protein A and its role in efficiency of signal peptide processing

Many surface proteins of pathogenic gram-positive bacteria are linked to the cell wall envelope by a mechanism requiring a C-terminal sorting signal with an LPXTG motif. Surface proteins of Streptococcus pneumoniae harbor another motif, YSIRK-G/S, which is positioned within signal peptides. The signal peptides of some, but not all, of the 20 surface proteins of Staphylococcus aureus carry a YSIRK-G/S motif, whereas those of surface proteins of Listeria monocytogenes and Bacillus anthracis do not. To determine whether the YSIRK-G/S motif is required for the secretion or cell wall anchoring of surface proteins, we analyzed variants of staphylococcal protein A, an immunoglobulin binding protein with an LPXTG sorting signal. Deletion of the YSIR sequence or replacement of G or S significantly reduced the rate of signal peptide processing of protein A precursors. In contrast, cell wall anchoring or the functional display of protein A was not affected. The fusion of cell wall sorting signals to reporter proteins bearing N-terminal signal peptides with or without the YSIRK-G/S motif resulted in hybrid proteins that were anchored in a manner similar to that of wild-type protein A. The requirement of the YSIRK-G/S motif for efficient secretion implies the existence of a specialized mode of substrate recognition by the secretion pathway of gram-positive cocci. It seems, however, that this mechanism is not essential for surface protein anchoring to the cell wall envelope.     

Protein secretion in phosphate-limited cultures of Bacillus subtilis 168

The secretion of proteins from Bacillus subtilis was studied under physiologically well-defined conditions in continuous cultures at a range of specific growth rates. The kinetics of secretion was analysed by using pulse-chase and immunoprecipitation techniques that allowed both processing and release to be monitored. Growth conditions were selected that were known to lead to significant changes in the anionic polymer composition of the cell wall. Under magnesium limitation only low levels of native proteins were released into the growth medium. In contrast, much higher amounts of released protein were observed under phosphate limitation. Although synthesis of native secretory proteins appeared to be highly regulated, only minor changes in the secretion of heterologous proteins were detected. Comparable kinetics of protein release of cells grown under different conditions indicated similar cell wall permeabilities. The large changes in the amounts of released proteins were not reflected in the production of chaperones and components required for protein secretion. The data suggest that the capacity of the secretion machinery is not a major limiting step in the export of native secretory proteins. Received: 23 September 1997 / Received revision: 10 November 1997 / Accepted: 16 November 1997     

The C-terminal region of α′ subunit of soybean β-conglycinin contains two types of vacuolar sorting determinants

In maturing seed cells, proteins that accumulate in the protein storage vacuoles (PSVs) are synthesized on the endoplasmic reticulum (ER) and transported by vesicles to the PSVs. Vacuolar sorting determinants (VSDs) which are usually amino acid sequences of short or moderate length direct the proteins to this pathway. VSDs identified so far are classified into two types: sequence specific VSDs (ssVSDs) and C-terminal VSDs (ctVSDs). We previously demonstrated that VSDs of α′ and β subunits of β-conglycinin, one of major storage proteins of soybean (Glycine max), reside in the C-terminal ten amino acids. Here we show that both types of VSDs coexist within this region of the α′ subunit. Although ctVSDs can function only at the very C-termini of proteins, the C-terminal ten amino acids of α′ subunit directed green fluorescent protein (GFP) to the PSVs even when they were placed at the N-terminus of GFP, indicating that an ssVSD resides in the sequence. By mutation analysis, it was found that the core sequence of the ssVSD is Ser-Ile-Leu (fifth to seventh residues counted from the C-terminus) which is conserved in the α and β subunits and some vicilin-like proteins. On the other hand, the sequence composed of the C-terminal three amino acids (AFY) directed GFP to the PSVs when it was placed at the C-terminus of GFP, though the function as a VSD was disrupted at the N-terminus of GFP, indicating that the AFY sequence is a ctVSD.     

Construction of an engineered yeast with glucose-inducible emission of green fluorescence from the cell surface

An engineered yeast with emission of fluorescence from the cell surface was constructed. Cell surface engineering was applied to display a visible reporter molecule, green fluorescent protein (GFP). A glucose-inducible promoter GAPDH as a model promoter was selected to control the expression of the reporter gene in response to environmental changes. The GFP gene was fused with the gene encoding the C-terminal half of α-agglutinin of Saccharomyces cerevisiae having a glycosylphosphatidylinositol anchor attachment signal sequence. A secretion signal sequence of the fungal glucoamylase precursor protein was connected to the N-terminal of GFP. This designed gene was integrated into the TRP1 locus of the chromosome of S. cerevisiae with homologous recombination. Fluorescence microscopy demonstrated that the transformant cells emitted green fluorescence derived from functionally expressed GFP involved in the fusion molecule. The surface display of GFP was further verified by immunofluorescence labeling with a polyclonal antibody (raised in rabbits) against GFP as the first antibody and Rhodamine Red-X-conjugated goat anti-rabbit IgG as the second antibody which cannot penetrate into the cell membrane. The display of GFP on the cell surface was confirmed using a confocal laser scanning microscope and by measuring fluorescence in each cell fraction obtained after the subcellular fractionation. As GFP was proved to be displayed as an active form on the cell surface, selection of promoters will endow yeast cells with abilities to respond to changes in environmental conditions, including nutrient concentrations in the media, through the emission of fluorescence. Received: 23 August 1999 / Received revision: 16 November 1999 / Accepted: 29 November 1999