Folding and assembly in rhodopsin - Effect of mutations in the sixth transmembrane helm on the conformation of the third cytoplasmic loop

Previous studies on bovine opsin folding and assembly have identified an am ino-terminal fragment, EF(1-232), which folds and inserts into a membrane o nly after coexpression with its complementary carboxyl-terminal fragment, E F(233-348), To further characterize this interaction, EF(1-232) production was examined upon coexpression with carboxyl-terminal fragments of varying length and/or amino acid composition. These included fragments with increme ntal deletions of the third cytoplasmic loop (TH(241-348) and EF(249-348)), a fragment composed of the third cytoplasmic loop and sixth transmembrane helix (HF(233-280)), a fragment composed of the sixth and seventh transmemb rane helices (FG(249-312)), and EF(233-348) and TH(241-348) fragments with Pro-267 or Trp-265 mutations. Although EF(1-232) production was independent of the third cytoplasmic loop and carboxyl-terminal tail, both the sixth a nd seventh transmembrane helices were essential. The effects of mutations i n the sixth transmembrane helix on EF(1-232) expression were dependent on t he length of the third cytoplasmic loop. Although Pro-267 mutations in EF(2 33-348) failed to stabilize EF(1-232) expression, their introduction into T H(241-348) was without discernible effects. However, Trp-265 substitutions in the EF(233-348) and TH(241-348) fragments conferred significant EF(1-232 ) production. Therefore, key residues in the transmembrane helices may exer t their effects on opsin folding, assembly, and/or function by influencing the conformation of the connecting loops.