Glycosyl-phosphatidylinositol anchor attachment in a yeast in vitro system.
The yeast mating pheromone precursor prepro-alpha factor was fused to C-terminal signals for glycosyl-phosphatidylinositol (GPI) anchor attachment, based on the sequence of the Saccharomyces cerevisiae protein Gas1p. Maturation of fusion proteins expressed in vivo required the presence of both a functional GPI attachment site and the synthesis of GPI precursors. Constructs were translated in vitro for use in cell-free studies of glycolipid attachment. The radiolabelled polypeptides were post-translationally translocated into yeast microsomes, where at least one third of the molecules received a GPI anchor. This approach offers distinct advantages over anchor attachment reactions that require co-translational translocation of secretory peptide substrates.
Doering
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TL
Department of Molecular and Cell Biology, Barker Hall, Howard Hughes Research Institute, University of California, Berkeley, CA 94720-3202, USA.
Schekman
R
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D001692:Biological Transport; D002474:Cell-Free System; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012107:Research Design; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D005656:Fungal Proteins; C066709:GAS1 protein, S cerevisiae; D017261:Glycosylphosphatidylinositols; C048632:MF(ALPHA)1 protein, S cerevisiae; D008562:Membrane Glycoproteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins