PMC

Homology models of wildtype Sec61p and Sec61∆L7p in the ER membrane. Side view of homology model of wildtype Sec61p (left) and Sec61∆L7p mutant (right). TM7, TM8 and their connecting loop (Loop 7) are highlighted in green (wildtype) and red (∆L7). Parts of the protein that remain unchanged are shown in shades of yellow. The membrane is indicated in light gray.

Identification of a sec61 mutant lacking L7. A) Topology model of Sec61p including positions of introduced restriction sites. B) Yeast (SEC61, sec61∆L7, DER1 and ∆der1) were grown as described in Materials & Methods, transferred to nitrocellulose, lysed, and intracellular CPY* detected by immunoblotting. Note that in the strain used for the SEC61 mutagenesis there was higher background accumulation of CPY* compared to the DER1 strain (compare SEC61 and DER1). C) Amino acid sequences of L7 and adjacent regions (grey) in wildtype Sec61p and Sec61∆L7p.

Proteasome 19S particle binding to wildtype, sec61Y345H and sec61∆L7 proteoliposomes. Microsomes from wildtype or sec61 mutant yeast were prepared, stripped of ribosomes, solubilized, and total protein reconstituted into proteoliposomes. Membranes were incubated with purified 19S particles in the presence of 5 mM ATP, and samples analyzed by flotation in 1.8 M sucrose for 1 h at 200,000 g. Fractions were collected from the top and analyzed by SDS-PAGE and immunoblotting for Sec61p and the 19S subunit Rpn12p. Rpn12p in each fraction was quantified. Note that 19S particles in the absence of membranes (green) remain at the bottom of the gradient. YTX69 is the standard wildtype yeast strain used in the lab for proteasome and ribosome binding experiments; SEC61, sec61Y345H, and sec61∆L7 were all in the KRY461 background.

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.

Viability and growth of sec61∆L7 yeast under different stress conditions. A) Growth of SEC61, sec61∆L7 and sec61-32 cells was analysed on YPD at 37°C, 30°C (3 d) and at 20°C (7 d). B) Tunicamycin sensitivity was examined on plates containing no (left), 0.25 μg/ml (center) or 0.5 μg/ml tunicamycin (right) at 30°C for 6 d. C) UPR induction in SEC61 wildtype and the indicated sec61 mutant strains was measured as beta-galactosidase activity after transformation of the strains with a UPRE-LacZ plasmid (31) or a plasmid encoding LacZ alone (30). Wildtype cells treated with 2 μg/ml tunicamycin for 1 h and sec61-3 cells shifted to 20°C for 1.5 hrs were used as positive controls. Samples were measured in duplicate and the experiment performed 3 times.

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).