COOH-terminal requirements for the correct processing of a phosphatidylinositol-glycan anchored membrane protein.
Placental alkaline phosphatase (PLAP) is anchored to the plasma membrane by a phosphatidylinositol-glycan (PI-G) moiety. During processing of nascent PLAP, a 29-residue COOH-terminal peptide is cleaved out and the PI-G moiety is attached to the newly created COOH terminus of the mature protein. To investigate the structural requirements of the COOH terminus of the nascent protein for PI-G tailing and anchoring to the plasma membrane, we have transfected COS cells with wild type and mutant forms of cDNA encoding human prepro-PLAP. Utilizing a series of COOH-terminal deletion mutants of prepro-PLAP, it was found that to be PI-G-tailed the newly synthesized protein must possess an uncharged, predominantly hydrophobic amino acid sequence of a minimal length in the COOH-terminal peptide. While forms of prepro-PLAP with 17 consecutive hydrophobic residues in the terminal sequence yielded PI-G-tailed and membrane-bound products, prepro-PLAP mutants with 13 or fewer of such residues yielded hydrophilic proteins that were no longer PI-G-tailed but efficiently secreted into the medium. Studies using cassette mutants demonstrated that the precise amino sequence of the COOH-terminal region could be altered as long as minimal hydrophobicity and length was maintained.
Berger
J
J
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
Howard
A D
AD
Brink
L
L
Gerber
L
L
Hauber
J
J
Cullen
B R
BR
Udenfriend
S
S
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002461:Cell Line, Transformed; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D017261:Glycosylphosphatidylinositols; D006801:Humans; D007163:Immunosorbent Techniques; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology; D011134:Polysaccharides / Q000378:metabolism*; D011247:Pregnancy; D014162:Transfection
D004792:Enzyme Precursors; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D009227:Myristic Acids; D010169:Palmitic Acids; D010716:Phosphatidylinositols; D011134:Polysaccharides; D019814:Myristic Acid; D019308:Palmitic Acid; D004247:DNA; D000469:Alkaline Phosphatase