title: Defining the boundaries of species specificity for the Saccharomyces cerevisiae glycosylphosphatidylinositol transamidase using a quantitative in vivo assay.
abstract: In eukaryotes, GPI (glycosylphosphatidylinositol) lipid anchoring of proteins is an abundant post-translational modification. The attachment of the GPI anchor is mediated by GPI-T (GPI transamidase), a multimeric, membrane-bound enzyme located in the ER (endoplasmic reticulum). Upon modification, GPI-anchored proteins enter the secretory pathway and ultimately become tethered to the cell surface by association with the plasma membrane and, in yeast, by covalent attachment to the outer glucan layer. This work demonstrates a novel in vivo assay for GPI-T. Saccharomyces cerevisiae INV (invertase), a soluble secreted protein, was converted into a substrate for GPI-T by appending the C-terminal 21 amino acid GPI-T signal sequence from the S. cerevisiae Yapsin 2 [Mkc7p (Y21)] on to the C-terminus of INV. Using a colorimetric assay and biochemical partitioning, extracellular presentation of GPI-anchored INV was shown. Two human GPI-T signal sequences were also tested and each showed diminished extracellular INV activity, consistent with lower levels of GPI anchoring and species specificity. Human/fungal chimaeric signal sequences identified a small region of five amino acids that was predominantly responsible for this species specificity.
authors.affiliation: Department of Chemistry, Remsen Hall, Johns Hopkins University, Baltimore, MD 21218, U.S.A.
authors.forename: Rachel
authors.initials: R
authors.lastname: Morissette
authors.forename: Yug
authors.initials: Y
authors.lastname: Varma
authors.forename: Tamara L
authors.initials: TL
authors.lastname: Hendrickson
mesh_terms: D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000032:analysis / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods; D017261:Glycosylphosphatidylinositols / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000166:cytology / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D013045:Species Specificity; D043324:beta-Fructofuranosidase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D017261:Glycosylphosphatidylinositols; D021382:Protein Sorting Signals; D029701:Saccharomyces cerevisiae Proteins; D019881:Aminoacyltransferases; C023175:transamidases; D043324:beta-Fructofuranosidase; D016282:Aspartic Acid Endopeptidases; C119545:MKC7 protein, S cerevisiae