HA becomes incorporated into lipid rafts during biosynthetic transport to the plasma membrane in T cells.
(A) After 3 hours of infection with influenza virus, Jurkat cells metabolically labeled with a 5-minute pulse of (35S)methionine/cysteine were incubated for the indicated times in normal medium lacking radioactive precursors. Equivalent aliquots from the soluble (S) and the insoluble lipid raft (I) fractions were subjected to SDS-PAGE, and HA was detected by autoradiography. The position of the core-glycosylated and endoglycosidase H-resistant forms of HA are indicated by solid and empty circles, respectively. (B) Infected cells were metabolically labeled as in panel A. At the end of each chase period, surface proteins were biotinylated, and the lipid raft fraction was immunoprecipitated with streptavidin-agarose. The arrival of radiolabeled HA at the cell surface was monitored by autoradiography of the immunoprecipitates. HA was easily identified due to the profound shut-off of host protein synthesis induced by influenza virus infection. (C) T lymphoblasts were infected for 4 hours, and then surface proteins were biotinylated. After centrifugation to equilibrium, the soluble (S) and floating raft (I) fractions, and the cytoskeleton-associated rafts (P) were subjected to immunoprecipitation with antibodies specific to the indicated proteins and immunoblotted with streptavidin-peroxidase or analyzed with cholera toxin B subunit coupled to peroxidase to detect GM1.
