Figure 4

Stability of Sec61 complex and Sec complex in sec61∆L7 membranes. A) The Sec61 complex is unstable in sec61∆L7 yeast. Upper: Microsomes from SEC61 and sec61∆L7 yeast were solubilised in Triton-X100 and layered onto a 0-15% sucrose gradient. After centrifugation, fractions were collected from the top and proteins resolved by SDS-PAGE. Sec61p, Sss1p and Sbh1p were detected by immunoblotting. Lower: Wildtype and mutant microsomes were treated with 5 mg/ml DSS for 20 min at 20°C. After quenching the crosslinked proteins were resolved by SDS-PAGE and Sec61p- and Sec61∆L7p-containing crosslinks were detected by immunoblotting with anti-Sec61p antibodies or anti-Sss1p antibodies. The asterisk marks a background band that is independent of crosslinking and migrates slightly slower than the Sec61pxSss1p band. B) Microsomes were solubilized in digitonin and centrifuged at high speed to remove ribosome-bound Sec61 complex. From the cleared lysate, the heptameric Sec complec was precipitated with ConcanavalinA-Sepharose, and Sec61p and Sec62 in supernatant and precipitates detected by immunoblotting. Note that the gel is overexposed to show the substantial fraction of Sec61∆L7p found in SDS-resistant aggregates at the top of the gel. Ratios of Sec61p to Sec62p in wildtype and mutant Sec complexes are shown in the graph.