Figure 3

ER translocation defects in sec61∆L7 cells. A) Yeast were grown o.n. at 30°C to early log-phase and shifted for 3 h to the indicated temperatures. Equal amounts of cells were lysed, proteins resolved on 10% SDS-gels, transferred to nitrocellulose, and detected with specific antibodies. To detect cotranslationally translocated DPAPB, cells were grown to early log phase and pulse labelled for 5 min prior to cell lysis and immunoprecipitation with specific antibodies. B) Yeast were grown to an OD₆₀₀ = 1 and translation was inhibited by adding cycloheximide, extracts were prepared by bead-beating, and samples were resolved by SDS-PAGE. Proteins were transferred to nitrocellulose and detected with antibodies against CPY, Sec61p and Sec62p. Quantitation of pCPY* and CPY* are shown in the graph. C) Wildtype and sec61∆L7 yeast expressing CPY* were labelled with [³⁵S]-Met/Cys for 5 min and chased for the indicated times, cells were lysed, and pCPY* and CPY* immunoprecipitated. Quantitation of CPY* is shown in the graph. D) Cells were grown to OD₆₀₀ = 1 and cycloheximide was added to inhibit translation. Whole cell extracts at 0′, 20′, 40′ and 60′ min were analysed by gel electrophoresis and immunoblotting for Sec61p, CPY* and Sec62p, which served as a loading control. Quantitation of CPY* is shown in the graph. E) Wildtype and sec61∆L7 yeast expressing transmembrane ERAD substrate KWW, or its soluble counterpart KHN were labelled with [³⁵S]-Met/Cys for 5 min and chased for the indicated times; cells were lysed, proteins immunoprecipitated and detected by autoradiography. Degradation of the polytopic transmembrane ERAD substrate Deg1:Sec62p was detected in a cycloheximide chase as described in B). Experiments were repeated once (KHN) or twice (KWW, Deg1:Sec62p).