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. 2009 Jan 15;20(2):685–698. doi: 10.1091/mbc.E08-09-0902

Figure 5.

Figure 5.

TM8 release from Sec61α is ATP dependent. CFTR integration intermediates (residues 837–977) containing ε-ANB-Lys at residue 913 were translated in the presence of NYT for 30 min, incubated in puromycin for indicated time and analyzed by SDS-PAGE. (A) In the absence of puromycin (Puro) most of the read-through polypeptide remains bound to tRNA (downward arrow, lane 1). Peptidyl-tRNA release was essentially complete 30 s after addition of 1 mM puromycin (lanes 2–6), but peptidyl tRNA-cleaved chains continue to cross-link Sec61α upon UV exposure (upward arrowheads, lanes 7–11). (B) Translation was carried out as in A, but cycloheximide (20 μg/ml) was added at the end of translation. In the absence of puromycin, peptidyl-tRNA bond remains intact in RRL at 24°C for at least 2 h. (C) Translation products (generated as described in A) were incubated in puromycin for times indicated and then exposed to UV irradiation. TM8-Sec61α cross-linking was stable for 2 h in the absence of puromycin (lanes 3–6) but gradually decreased within 60 min after puromycin addition (lanes 7–10). ATP depletion (apyrase) stabilized TM8-Sec61α cross-linking (lanes 11–14). (D) Results from similar experiments shown in C were quantitated, and intensity of the photoadduct was plotted at each time point up to 60 min (n = 4 ± SEM). (E) Translation reactions were incubated as indicated before photocross-linking as in C (lanes 1–6). For lanes 7–10, microsomes were pelleted after translation, resuspended in a mock translation reaction containing RRL and UV irradiated after 10- or 30-min incubation with puromycin. (F) Photocross-linking was carried out as in C under conditions indicated (lanes 1–5). TM8 integration intermediates treated with puromycin were released from the translocon after 45 min (lanes 4–5). When these microsomes were isolated before UV and photocross-linking was subsequently performed in fresh RRL ± ATP, no TM8 photocross-links were observed (lanes 6 and 7). All translations were performed in the presence of the NYT tripeptide.

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