Placental alkaline phosphatase: a model for studying COOH-terminal processing of phosphatidylinositol-glycan-anchored membrane proteins.
Placental alkaline phosphatase (PLAP) has been used as a model for studying the biosynthesis of the phosphatidylinositol-glycan (PI-G)-protein linkage in intact cells and in cell-free systems. However, for the study of processing in cell-free systems, a small protein devoid of glycosylation sites is preferable. A PLAP-derived cDNA was engineered that codes for a nascent protein (mini-PLAP) of 28 kDa in which the NH2- and COOH-termini are retained but most of the interior of PLAP is deleted. In vitro translation of mini-PLAP mRNA in the presence of rough microsomal membranes yields mature PI-G-tailed mini-PLAP. Processing of nascent mutant proteins occurs only when a small amino acid is located at the site of cleavage and PI-G attachment (omega site). Mutations adjacent and COOH-terminal to the omega site have revealed that the omega + 1 site is promiscuous in its requirements but that only glycine and alanine are effective at the omega + 2 site. Rough microsomal membranes from T cells deficient in PI-G biosynthesis do not support processing of mini-PLAP; addition of exogenous PI-G restores activity. Translocation of the proprotein, most likely requiring ATP and GTP, precedes COOH-terminal processing.
Amthauer
R
R
Department of Neurosciences, Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110.
Kodukula
K
K
Udenfriend
S
S
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology*; D004247:DNA / Q000235:genetics; D005260:Female; D058851:GPI-Linked Proteins; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology*; D015202:Protein Engineering
D058851:GPI-Linked Proteins; D017261:Glycosylphosphatidylinositols; D007527:Isoenzymes; D010446:Peptide Fragments; D004247:DNA; D000469:Alkaline Phosphatase; C019839:alkaline phosphatase, placental