Figure 8.

Asp924 causes persistent TM8 photocross-linking to Sec61α after puromycin release. (A) Photocross-linking to integration intermediates (residues 837–977) containing WT and mutant (D924V) TM8 revealed similar UV-dependent photoadducts (upward arrowheads) for truncations 957 and 967 that disappeared after puromycin treatment. Bands migrating at 67 kDa and higher were present in the absence of UV and represent nonspecific background. At truncations 977 and 987, photoadducts to WT TM8 increased in intensity and were peptidyl-tRNA independent (lanes 14 and 15 and 20 and 21), whereas photoadducts were absent for the D924V mutant (lanes 17, 18, 23, and 24). (B) Immunoprecipitation of photoadducts with Sec 61α antisera confirmed that the D924V mutation decreases Sec61α cross-linking at the same stage of synthesis when photocross-linking to WT TM8 becomes independent of the peptidyl-tRNA bond.