Figure 8.

Effects of N-terminal sequence on TM3 membrane insertion. (A) Translation of CF1,2-P3R. CF1,2-P3R RNA was translated in RRL in the presence of RM, and the membrane-associated proteins (P) (lane 3) were separated from soluble fractions (S) (lane 2) by centrifugation. Lane 1 represents the total (T) reaction before separation by centrifugation. Lane 4 is membrane-associated proteins subjected to endoglycosidase treatment. (B) Proteinase K digestion of membrane-associated CF1,2-P3R. Proteinase K digestion was performed in the absence (lanes 1 and 2) or presence (lane 3) of Triton X-100 or further treated with endoglycosidase PNGase F (lane 4). The symbols (←) and (*) indicate glycosylated and deglycosylated proteins, respectively. (C) Two possible orientations of CF1,2-P3R in the membrane. The branched symbol indicates oligosaccharide chains and the arrow indicates the site of fusion between CFTR and Pgp.