Biosynthesis of glycosylphosphatidylinositol (GPI)-anchored membrane proteins in intact cells: specific amino acid requirements adjacent to the site of cleavage and GPI attachment.
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.
Kodukula
K
K
Department of Neurosciences, Roche Research Center, Nutley, New Jersey 07110.
Gerber
L D
LD
Amthauer
R
R
Brink
L
L
Udenfriend
S
S
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
D000596:Amino Acids; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase