title: Biosynthesis of glycosylphosphatidylinositol (GPI)-anchored membrane proteins in intact cells: specific amino acid requirements adjacent to the site of cleavage and GPI attachment.
abstract: Mutational studies were previously carried out at the omega site intact cells (Micanovic, R., L. Gerber, J. Berger, K. Kodukula, and S. Udenfriend. 1990. Proc. Natl. Acad. Sci. USA. 87:157-161; Micanovic R., K. Kodukula, L. Gerber, and S. Udenfriend. 1990. Proc. Natl. Acad. Sci. USA: 87:7939-7943) and at the omega + 1 and omega + 2 sites in a cell-free system (Gerber, L., K. Kodukula, and S. Udenfriend. 1992. J. Biol. Chem. 267:12168-12173) of nascent proteins destined to be processed to a glycosylphosphatidyl-inositol (GPI)-anchored form. We have now mutated the omega + 1 and omega + 2 sites in placental alkaline phosphatase (PLAP) cDNA and transfected the wild-type and mutant cDNAs into COS 7 cells. Only glycine at the omega + 2 site yielded enzymatically active GPI membrane-anchored PLAP in amounts comparable to the wild type (alanine). Serine was less active and threonine and valine yielded very low but significant activity. By contrast the omega + 1 site was promiscuous, with only proline being inactive. These and the previous studies indicate that the omega and omega + 2 sites of a nascent protein are key determinants for recognition by COOH-terminal signal transamidase. Comparisons have been made to specific requirements for substitution at the -1, -3 sites of amino terminal signal peptides for recognition by NH2-terminal signal peptidase and the mechanisms of NH2 and COOH-terminal signaling are compared.
authors.affiliation: Department of Neurosciences, Roche Research Center, Nutley, New Jersey 07110.
authors.forename: K
authors.initials: K
authors.lastname: Kodukula
authors.forename: L D
authors.initials: LD
authors.lastname: Gerber
authors.forename: R
authors.initials: R
authors.lastname: Amthauer
authors.forename: L
authors.initials: L
authors.lastname: Brink
authors.forename: S
authors.initials: S
authors.lastname: Udenfriend
mesh_terms: D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002474:Cell-Free System; D005260:Female; D005455:Fluorescent Antibody Technique; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010920:Placenta / Q000201:enzymology; D010957:Plasmids; D011247:Pregnancy; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014162:Transfection
chemical_list: D000596:Amino Acids; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase