Soluble GP18 restores glycosylphosphatidylinositol anchoring in a trypanosome cell-free system depleted of lumenal endoplasmic reticulum proteins

We previously established an in vitro assay for glycosylphosphatidylinosito l (GPI) anchoring of proteins using trypanosome membranes. We now show that GPI anchoring is lost when the membranes are washed at high pH and restore d to physiological pH prior to assay. We show that soluble component(s) of the endoplasmic reticulum that are lost in the high-pH wash are required fo r GPI anchoring. We reconstituted the high-pH extract with high-pH-treated membranes and demonstrated restoration of activity. Size fractionation of t he high-DH extract indicated that the active component(s) was 30 50 kDa in size and was inactivated by iodoacetamide. Activity could also be restored by reconstituting the inactivated membranes with Escherichia coli-expressed , polyhistidine-tagged Leishmania mexicana GPI8 (GPI8-His; L. mexicana GPI8 is a soluble homologue of yeast and mammalian Gpi8p). No activity was seen when iodoacetamide-treated GPI8-His was used; however, GPI8-His could rest ore activity to iodoacetamide-treated membranes. Antibodies raised against L. mexicana GPI8 detected a protein of approx. 38 kDa in an immunoblot of t he high-pH extract of trypanosome membranes. Our data indicate (1) that try panosome GPI8 is a soluble lumenal protein, (2) that the interaction betwee n GPI8 and other putative components of the transamidase may be dynamic, an d (3) that GPI anchoring can be biochemically reconstituted using an isolat ed transamidase component.