Glycosyl-phosphatidylinositol anchor attachment in a yeast in vitro system

Tamara L. Doering, Randy Schekman

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

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 Gaslp. 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 radiolabeIled 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.

Original languageEnglish
Pages (from-to)669-675
Number of pages7
JournalBiochemical Journal
Volume328
Issue number2
DOIs
StatePublished - Dec 1 1997

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