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A screen for deficiencies in GPI‐anchorage of wall glycoproteins in yeast
Author(s) -
Gonzalez Marlyn,
Goddard Noel,
Hicks Charles,
Ovalle Rafael,
Rauceo Jason M.,
Jue Chong K.,
Lipke Peter N.
Publication year - 2010
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.1797
Subject(s) - biology , green fluorescent protein , saccharomyces cerevisiae , gene , fusion protein , schizosaccharomyces pombe , secretion , microbiology and biotechnology , cell wall , biochemistry , recombinant dna
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 °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 β1,6 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. Copyright © 2010 John Wiley & Sons, Ltd.