FIG. 7.
Transport of HA-GPI to the plasma membranes of HeLa cells is unaffected by myriocin treatment. (A) Schematic representation of HA-GPI. The protein is synthesized as a GPI-anchored polypeptide (HA0, corresponding to the ectodomain of influenza virus hemagglutinin protein) containing a disulfide bond as shown. Upon trypsin treatment, HA0 yields two disulfide-linked fragments, HA2 and HA1. (B) HeLa cells expressing HA-GPI were preincubated for 30 min with myriocin (125 μM) or BFA (5 μg/ml) as indicated and were then pulse-labeled with 200 μCi/ml [35S]methionine-cysteine for 20 min. The cells were chased for 2 h in the presence of drugs before being trypsinized to convert cell surface HA-GPI molecules into HA2 and HA1. The extent of conversion of radiolabeled protein to HA2 and HA1 was determined after immunoprecipitation and SDS-PAGE. A scan of a typical fluorography is presented. No HA2 is seen in the BFA-treated cells, whereas radiolabeled HA2 is readily observed in both control (−) and myriocin-treated cells.