A screen for deficiencies in GPI‐anchorage of wall glycoproteins in yeast
Marlyn GonzalezNoël L. GoddardCharles B. HicksRafael OvalleJason M. RauceoChong K. JuePeter N. Lipke
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Abstract:
Many of the genes and enzymes critical for assembly and biogenesis of yeast cell walls remain unidentified or poorly characterized. Therefore, we designed a high throughput genomic screen for defects in anchoring of GPI-cell wall proteins (GPI-CWPs), based on quantification of a secreted GFP-Sag1p fusion protein. Saccharomyces cerevisiae diploid deletion strains were transformed with a plasmid expressing the fusion protein under a GPD promoter, then GFP fluorescence was determined in culture supernatants after mid-exponential growth. Variability in the amount of fluorescent marker secreted into the medium was reduced by growth at 18 degrees C in buffered defined medium in the presence of sorbitol. Secondary screens included immunoblotting for GFP, fluorescence emission spectra, cell surface fluorescence, and cell integrity. Of 167 mutants deleted for genes affecting cell wall biogenesis or structure, eight showed consistent hyper-secretion of GFP relative to parental strain BY4743: tdh3 (glyceraldehyde-3-phosphate dehydrogenase), gda1 (guanosine diphosphatase), gpi13 and mcd4 (both ethanolamine phosphate-GPI-transferases), kre5 and kre1 (involved in synthesis of beta1,6 glucan), dcw1(implicated in GPI-CWP cross-linking to cell wall glucan), and cwp1 (a major cell wall protein). In addition, deletion of a number of genes caused decreased secretion of GFP. These results elucidate specific roles for specific genes in cell wall biogenesis, including differentiating among paralogous genes.We have shown previously that an engineered form of Renilla luciferase (SRUC) can be secreted as a functional enzyme by mammalian cells, and that fusing wild-type Renilla luciferase with the green fluorescent protein from Aequorea victoria (GFP) yields a chimeric protein retaining light-emission properties similar to that of unfused Renilla luciferase and GFP. In the work presented here, SRUC was fused with GFP to determine whether it could be used to both visualize and quantify protein secretion in mammalian cells. Simian COS-7 and Chinese hamster ovary (CHO) cells were transiently transfected with gene constructs encoding a secreted or an intracellular version of a Renilla luciferase–GFP fusion protein. Renilla luciferase activity was measured from COS-7 cell lysates and culture media, and GFP activity was detected in CHO cells using fluorescence microscopy. Data indicated that the SRUC–GFP fusion protein was secreted as a chimeric protein that had both Renilla luciferase and GFP activity. This fusion protein could be a useful marker for the study of protein secretion in mammalian cells. Copyright © 2000 John Wiley & Sons, Ltd.
Aequorea victoria
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The SSU21/MCD4 gene encodes an essential component of the glycosylphosphatidylinositol (GPI)-anchor synthesis pathway in Saccharomyces cerevisiae. Here we demonstrate that the ssu21 mutation affected the transport and the incorporation into the cell wall of the major non-GPI yeast cross-linker – endoglucanase/glucanosyltransferase Bgl2p. This mutation also led to a decrease in the levels of both known types of cell wall mannoproteins, those covalently linked with glucan and SDS-extractable proteins. Our results indicate that the precision of the GPI-anchor synthesis is essential for cell wall assembly and suggest the strong interdependence of different groups of cell wall proteins during their incorporation into the cell wall.
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We found that YGR146C of Saccharomyces cerevisiae encodes a functional homolog of Ecl1 that is involved in the chronological lifespan of Schizosaccharomyces pombe. When YGR146C is overexpressed, it extends the viability of wild-type S. cerevisiae cells after entry into the stationary phase, as in the case of Ecl1. We propose that Ecl1 family proteins are novel regulatory factors involved in chronological lifespan among yeasts.
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Schizosaccharomyces
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The two model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe appear to have diverged 1000 million years ago. Here, we describe that S. pombe vectors can be propagated efficiently in S. cerevisiae as pUR19 derivatives, and the pREP and pJR vector series carrying the S. cerevisiae LEU2 or the S. pombe ura4(+) selection marker are maintained in S. cerevisiae cells. In addition, genes transcribed from the S. pombe nmt1(+) promoter and derivatives are expressed in budding yeast. Thus, S. pombe vectors can be used as shuttle vectors in S. cerevisiae and S. pombe. Our finding greatly facilitates the testing for functional orthologs of protein families and simplifies the cloning of new S. pombe plasmids by using the highly efficient in vivo homologous recombination activity of S. cerevisiae.
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Schizosaccharomyces
Cloning (programming)
Cyclin-dependent kinase 7
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Although Saccharomyces cerevisiae is unable to produce siderophores, this fungal organism can assimilate iron bound to the hydroxamate-type siderophore ferrichrome (Fc) produced and secreted by other microbes. Fc can enter S. cerevisiae cells via Arn1. Unlike S. cerevisiae, Schizosaccharomyces pombe synthesizes and secretes Fc. The sib1+ and sib2+ genes encode, respectively, a Fc synthetase and an ornithine-N5-oxygenase, which are required for Fc production. When both genes were expressed in S. pombe, cross-feeding experiments revealed that S. cerevisiae fet3Δ arn1-4Δ cells expressing Arn1 could grow in the vicinity of S. pombe under low-iron conditions. In contrast, deletion of sib1+ and sib2+ produced a defect in the ability of S. pombe to keep S. cerevisiae cells alive when Fc is used as the sole source of iron. Further analysis identified a gene designated sib3+ that encodes an N5-transacetylase required for Fc production in S. pombe. The sib3Δ mutant strain exhibited a severe growth defect in iron-poor media, and it was unable to promote Fc-dependent growth of S. cerevisiae cells. Microscopic analyses of S. pombe cells expressing a functional Sib3-GFP protein revealed that Sib3 was localized throughout the cells, with a proportion of Sib3 being colocalized with Sib1 and Sib2 within the cytosol. Collectively, these results describe the first example of a one-way cross-feeding interaction, with S. pombe providing Fc that enables S. cerevisiae to grow when Fc is used as the sole source of iron.
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Schizosaccharomyces
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Objective To study the expression and localization of s-lap/GFP fusion protein in Hela cell lines. Methods According to the coding area sequence of s-lap gene, stop codon located at 3′terminus of s-lap gene was mutated by PCR and fused with GFP gene. Then the s-lap/GFP fusion gene was transfected into Hela cells. The expression of s-lap/GFP fusion protein was observed under fluorescence microscope. Results DNA sequencing demonstrated that s-lap/GFP recombinant vector was constructed successfully and its fusion protein was expressed in Hela cells. The observation under fluorescence microscope showed that s-lap/GFP fusion protein was spread in entire cells, but expressed highly in the nucleus. Conclusion The s-lap/GFP fusion protein can express in Hela cells and locate in entire cells, but express highly in the nucleus.
HeLa
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The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe: models for cell biology research.
Yeast species provide excellent models for fundamental biological research. In this review, I will describe characteristics of the two most common laboratory systems: the fission yeast Schizosaccharomyces pombe, and the budding yeast Saccharomyces cerevisiae. They have substantial similarities that make them powerful as research tools, and also striking biological differences that make them complementary experimental models. Each provides unique tools for understanding environmental effects on cellular systems.
Budding yeast
Schizosaccharomyces
Eukaryotic cell
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The SSU21/MCD4 gene encodes an essential component of the glycosylphosphatidylinositol (GPI)-anchor synthesis pathway in Saccharomyces cerevisiae. Here we demonstrate that the ssu21 mutation affected the transport and the incorporation into the cell wall of the major non-GPI yeast cross-linker - endoglucanase/glucanosyltransferase Bgl2p. This mutation also led to a decrease in the levels of both known types of cell wall mannoproteins, those covalently linked with glucan and SDS-extractable proteins. Our results indicate that the precision of the GPI-anchor synthesis is essential for cell wall assembly and suggest the strong interdependence of different groups of cell wall proteins during their incorporation into the cell wall.
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Objective:To study expression and localization of GFP-HPV18 E2 and its TAD or DBD fusion proteins in macrophages.Methods:The plasmid of pEGFP-C1/ E2,pEGFP-C1/TAD,pEGFP-C1/DBD or pEGFP-C1 was transfected into macrophages,respectively.Their expression and localization were observed with fluorescent microscope.Western blot was used to analyze GFP or its fusion proteins by the antiGFP antibody as the first antibody.Results:Fluorescence could be observed in the whole cells transfected by pEGFP-C1,mainly in the nuclei of the cells transfected by pEGFP-C1/E2,but only in the nuclei of the cells transfected by pEGFP-C1/DBD while only in the cytoplasma of the cells transfected by pEGFP-C1/TAD.Western blot showed that the expression of GFP,GFP-E2,GFP-DBD,GFP-TAD fusion proteins.Conclusion:GFP-HPV18 E2 and its mutants could express in macrophages.GFP-E2 fusion protein locate mainly in nuclei,while GFP-DBD fusion protein locate completely in nuclei and GFP-TAD fusion protein locate completely in cytoplasma.
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Objective To find out the role of GFP-tag on the localization of overexpressed MDA-7/IL-24 protein.Methods Recombinant plasmids pEGFP-C1-mda-7/IL-24 with GFP-tag at NH4-terminal and pEGFP-N3-mda-7/IL-24 with GFP-tag at COOH-terminal were constructed.After the recombinant plasmids were transfected transiently into monkey embryonic kidney cell of Cos7 for 48 hours,nucleus of Cos7 cells were dyed with Hoechst 33258.Distribution of the fusion proteins GFP-tag-MDA-7/IL-24 was observed under the fluorescence microscope.Results Sequential analysis proved that recombinant plasmids pEGFP-C1-mda-7/IL-24 and pEGFP-N3-mda-7/IL-24 were constructed successfully.In the positive cells for MDA-7/IL-24 fusion protein with GFP-tag at NH4-terminal,the GFP was found to be distributed equally both in the cytoplasm and the nucleus.The GFP in the positive cells for MDA-7/IL-24 fusion protein with GFP-tag at COOH-terminal was found to be localized around the outer part of the nuclear membrane and the cytoplasm.Conclusion The different position of GFP-tag in the fusion protein influenced the distribution of overexpressed MDA-7/IL-24 markedly.
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