title: A screen for deficiencies in GPI-anchorage of wall glycoproteins in yeast.
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.
authors.affiliation: Department of Biology, Brooklyn College of the City University of New York, USA.
authors.forename: Marlyn
authors.initials: M
authors.lastname: Gonzalez
authors.forename: Noel
authors.initials: N
authors.lastname: Goddard
authors.forename: Charles
authors.initials: C
authors.lastname: Hicks
authors.forename: Rafael
authors.initials: R
authors.lastname: Ovalle
authors.forename: Jason M
authors.initials: JM
authors.lastname: Rauceo
authors.forename: Chong K
authors.initials: CK
authors.lastname: Jue
authors.forename: Peter N
authors.initials: PN
authors.lastname: Lipke
mesh_terms: D019677:Artificial Gene Fusion; D002473:Cell Wall / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D006023:Glycoproteins / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000172:deficiency* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D006023:Glycoproteins; D017261:Glycosylphosphatidylinositols; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins