title: Soluble GPI8 restores glycosylphosphatidylinositol anchoring in a trypanosome cell-free system depleted of lumenal endoplasmic reticulum proteins.
abstract: We previously established an in vitro assay for glycosylphosphatidylinositol (GPI) anchoring of proteins using trypanosome membranes. We now show that GPI anchoring is lost when the membranes are washed at high pH and restored 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 for GPI anchoring. We reconstituted the high-pH extract with high-pH-treated membranes and demonstrated restoration of activity. Size fractionation of the high-pH 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 restore activity to iodoacetamide-treated membranes. Antibodies raised against L. mexicana GPI8 detected a protein of approx. 38 kDa in an immunoblot of the high-pH extract of trypanosome membranes. Our data indicate (1) that trypanosome GPI8 is a soluble lumenal protein, (2) that the interaction between GPI8 and other putative components of the transamidase may be dynamic, and (3) that GPI anchoring can be biochemically reconstituted using an isolated transamidase component.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA.
authors.forename: D K
authors.initials: DK
authors.lastname: Sharma
authors.forename: J D
authors.initials: JD
authors.lastname: Hilley
authors.forename: J D
authors.initials: JD
authors.lastname: Bangs
authors.forename: G H
authors.initials: GH
authors.lastname: Coombs
authors.forename: J C
authors.initials: JC
authors.lastname: Mottram
authors.forename: A K
authors.initials: AK
authors.lastname: Menon
mesh_terms: D019881:Aminoacyltransferases / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002474:Cell-Free System; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007894:Leishmania mexicana / Q000378:metabolism*; D015800:Protozoan Proteins / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D012995:Solubility
chemical_list: D015815:Cell Adhesion Molecules; D017931:DNA Primers; D017261:Glycosylphosphatidylinositols; D015800:Protozoan Proteins; D029701:Saccharomyces cerevisiae Proteins; D019881:Aminoacyltransferases; C103133:GPI8 protein, S cerevisiae; C023175:transamidases