Figure 4.

RI₃₃₂ interacts with calnexin only in the absence of castanospermine. HeLa-RI₃₃₂ (lanes a–f) and HeLa-RI₃₃₂-Thr (lanes g and h) cells were preincubated, pulse labeled for 10 min, and chased for the times indicated in the absence (lanes a, c, e, and g) or in the presence (lanes b, d, f, and h) of castanospermine (CST; 1 mM). Cells were lysed in buffer containing CHAPS (2%), and anti-calnexin immunoprecipitations were performed under nonstringent conditions in the presence of 1% of the same detergent. The second steps of the sequential immunoprecipitations were carried out under stringent conditions in the presence of SDS (0.6%) and Triton X-100 (1%). Only ribophorin I (RI) and RI₃₃₂ or RI₃₃₂-Thr reprecipitated under stringent conditions from anti-calnexin immunoprecipitations obtained under nonstringent conditions are shown for each chase time point. As a control, RI₃₃₂ and RI₃₃₂-Thr were immunoprecipitated under stringent conditions from cell lysates of HeLa-RI₃₃₂ and HeLa-RI₃₃₂-Thr cells metabolically labeled for 10 min (lanes i and j, respectively). All samples were analyzed by SDS-PAGE and fluorography. Exposure times: lanes a and b, 7 d; lanes c, d, and f–h, 10 d; lane e, 30 d; lanes i and j, 1 d.