Selectivity at the cleavage/attachment site of phosphatidylinositol-glycan anchored membrane proteins is enzymatically determined.

Abstract

Nascent precursors of phosphatidylinositol-glycan (PI-G)-linked membrane proteins contain a hydrophobic COOH-terminal sequence of 15-30 residues that is eliminated during processing to yield a newly exposed COOH terminus to which the PI-G moiety is added. There is no consensus as to the primary structure of the terminal peptide but there is a specific requirement for the amino acid destined to become the COOH terminus. In nascent human placental alkaline phosphatase (PLAP), the PI-G tail is attached to Asp-484. Site-directed mutants with glycine, alanine, cysteine, serine, or asparagine (category I) at residue 484 become PI-G tailed, appear in the plasma membrane, and are enzymatically active when expressed in COS cells. Although mutants with glutamic acid, glutamine, proline, tryptophan, leucine, valine, phenylalanine, threonine, methionine, and tyrosine (category II) are expressed equally well, only small amounts appear on the plasma membrane. Furthermore, they are not PI-G tailed and have little alkaline phosphatase activity. Studies with truncated PLAP-489 rule out nonspecific conformational changes in category II mutant proteins as a reason for their failure to be processed in COS cells and point to a specific COOH-terminal processing enzyme. Direct evidence that the selectivity for category I amino acids is enzymatically determined was obtained in a cell-free translation/processing system by using rabbit reticulocyte lysate and CHO cell rough microsomal membranes. In this in vitro system, both category I and category II mutants of PLAP-513 were translated, glycosylated, and cleaved by NH2-terminal signal peptidase. However, an additional and selective cleavage at residue 484 was observed only with category I mutants.