FIGURE 3.
Structural and functional features of LPH deletion mutants in COS-1 cells. A, assessment of the quaternary structure. Transiently transfected COS-1 cells were biosynthetically labeled and solubilized in 6 mm dodecyl-β-m-maltoside. Cell lysates were layered on a sucrose density gradient. After centrifugation for 18 h at 100,000 × g, fractions were collected, immunoprecipitated, and analyzed on SDS-PAGE. WT, wild type. B, transport kinetics of wild type LPH and mutant proteins. Transfected COS-1 cells were pulse-labeled for 1.5 h with [35S]methionine and chased for the indicated periods of time with cold methionine. The immunoprecipitates were treated with endo H or not treated and analyzed by SDS-PAGE on 6% slab gels. C, trypsin sensitivity assay of wild type and LPH mutants. Transiently transfected COS-1 cells were biosynthetically labeled followed by immunoprecipitation of LPH proteins from the cell lysates. The immunoprecipitates were treated with trypsin for different times and analyzed by SDS-PAGE on 7% slab gels.