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.
Tretter et al. BMC Cell Biology 2013 14:56 doi:10.1186/1471-2121-14-56