IN-VITRO INCORPORATION OF GPI-ANCHORED PROTEINS INTO HUMAN ERYTHROCYTES AND THEIR FATE IN THE MEMBRANE

In many different cells, glycosylphosphatidylinositol (GPI)-anchored m olecules are clustered in membrane microdomains that resist extraction by detergents at 4 degrees C. In this report, we identified the prese nce of such domains in human erythrocytes and examined the ability of exogenously-added GPI-anchored molecules to colocalize with the endoge nous GPI-anchored proteins in these detergent-insoluble complexes. We found that the addition to human erythrocytes of three purified GPI-an chored proteins having different GPI lipid moieties resulted in their efficient and correct incorporation into the membrane. The extent of m embrane insertion was dependent on the intactness of the GPI lipid moi ety. However, unlike the endogenous GPI-anchored proteins, the in vitr o incorporated GPI molecules were not resistant to membrane extraction by Triton X-100 at 4 degrees C. In addition, in contrast to the endog enous GPI-anchored proteins, they were not preferentially released fro m erythrocytes during vesiculation induced by calcium loading of the c ells, These results suggest that in vitro incorporated GPI-linked mole cules are excluded from pre-existing GPI-enriched membrane areas in hu man erythrocytes and that these microdomains may represent the sites o f membrane vesicle formation. (C) 1998 by The American Society of Hema tology.