The carboxyl terminus of Dictyostelium discoideum protein 1I encodes a functional glycosyl-phosphatidylinositol signal sequence.
The 1I gene is expressed in the prespore cells of culminating Dictyostelium discoideum. The open reading frame of 1I cDNA encodes a protein of 155 amino acids with hydrophobic segments at both its NH(2)- and COOH-termini that are indicative of a glycosyl-phosphatidylinositol (GPI)-anchored protein. A hexaHis-tagged form of 1I expressed in D. discoideum cells appeared on Western blot analysis as a doublet of 27 and 24 kDa, with a minor polypeptide of 22 kDa. None of the polypeptides were released from the cell surface with bacterial phosphatidylinositol-specific phospholipase C, although all three were released upon nitrous acid treatment, indicating the presence of a phospholipase-resistant GPI anchor. Further evidence for the C-terminal sequence of 1I acting as a GPI attachment signal was obtained by replacing the GPI anchor signal sequence of porcine membrane dipeptidase with that from 1I. Two constructs of dipeptidase with the 1I GPI signal sequence were constructed, one of which included an additional six amino acids in the hydrophilic spacer. Both of the resultant constructs were targeted to the surface of COS cells and were GPI-anchored as shown by digestion with phospholipase C, indicating that the Dictyostelium GPI signal sequence is functional in mammalian cells. Site-specific antibodies recognising epitopes either side of the expected GPI anchor attachment site were used to determine the site of GPI anchor attachment in the constructs. These parallel approaches show that the C-terminal signal sequence of 1I can direct the addition of a GPI anchor.
Stevens
B A
BA
School of Biochemistry and Molecular Biology, University of Leeds, Woodhouse Lane, LS2 9JT, Leeds, UK.
White
I J
IJ
Hames
B D
BD
Hooper
N M
NM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D004023:Dictyostelium / Q000737:chemistry / Q000235:genetics*; D004150:Dipeptidases / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics; D017261:Glycosylphosphatidylinositols / Q000737:chemistry / Q000235:genetics*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection
D005656:Fungal Proteins; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D004150:Dipeptidases; C038937:dipeptidase