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. 1997 Dec 1;328(Pt 2):669–675. doi: 10.1042/bj3280669

Glycosyl-phosphatidylinositol anchor attachment in a yeast in vitro system.

T L Doering 1, R Schekman 1
PMCID: PMC1218970  PMID: 9371730

Abstract

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.

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Selected References

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