An internally positioned signal can direct attachment of a glycophospholipid membrane anchor.
All known glycophosphatidylinositol (GPI)-anchored membrane proteins contain a COOH-terminal hydrophobic domain necessary for signalling anchor attachment. To examine the requirement that this signal be at the COOH terminus of the protein, we constructed a chimeric protein, DAFhGH, in which human growth hormone (hGH) was fused to the COOH terminus of decay accelerating factor (DAF) (a GPI-anchored protein), thereby placing the GPI signal in the middle of the chimeric protein. We show that the fusion protein appears to be processed at the normal DAF processing site in COS cells, producing GPI-anchored DAF on the cell surface. This result indicates that the GPI signal does not have to be at the COOH terminus to direct anchor addition, suggesting that the absence of a hydrophilic COOH-terminal extension (beyond the hydrophobic domain) is not a necessary requirement for GPI anchoring. A similar DAFhGH fusion, containing an internal GPI signal in which the DAF hydrophobic domain was replaced with the signal peptide of hGH, also produced GPI-anchored cell surface DAF. The signal for GPI attachment thus exhibits neither position specificity nor sequence specificity. In addition, mutant DAF or DAFhGH constructs lacking an NH2-terminal signal peptide failed to produce GPI-anchored protein, suggesting that membrane translocation is necessary for anchor addition.
Caras
I W
IW
Department of Immunobiology, Genentech, Inc., South San Francisco, California 94080.
D000595:Amino Acid Sequence; D000818:Animals; D018958:CD55 Antigens; D002451:Cell Compartmentation; D002460:Cell Line; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis; D019856:Ethanolamine; D004983:Ethanolamines / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006017:Glycolipids / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D014162:Transfection
D018958:CD55 Antigens; D004983:Ethanolamines; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D010716:Phosphatidylinositols; D011993:Recombinant Fusion Proteins; D019856:Ethanolamine; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase