Figure 9.

Sequence-specific control of TM8-Sec61α photocross-linking. (A) Photocross-linking to CFTR integration intermediates (residues 837–977) containing the ε-ANB-Lys probe at residue 913 in which Asp924 was mutated to glutamine (E), arginine (R), or valine (V). Samples were treated with puromycin and UV irradiation as indicated. (B) Immunoprecipitation of WT and mutant TM8 photoadducts with Sec61α antiserum revealed that both photocross-linking efficiency and persistence of cross-linking after puromycin treatment were highly favored by acidic residues, with aspartate greater than glutamate. Equivalent amounts of read-through translation products were used for immunoprecipitation. (C) Integration intermediates containing the ε-ANB-Lys probe at residues 912, 913, or 914 were truncated at residue 977, photolyzed, and immunoprecipitated with Sec61α antisera. A923D, D924V mutations decreased Sec61α photocross-linking (to residue 913) after puromycin treatment (compare lane 4 with lane 10) but significantly increased the peptidyl-tRNA–independent photocross-linking to residue 912 (lanes 2 and 8). Thus, TM8 release from Sec61α is highly sensitive to the presence as well as orientation of acidic residues. Immunoprecipitations used equivalent amounts of read-through products.