Sec61p is associated with components of the ERAD pathway. (A) Immunoprecipitation experiment using a plasmid encoded, N-terminally HA₃-tagged version of Hrd3p. Digitonin-solubilized microsomal proteins were prepared from wild type and Δhrd3+HA₃Hrd3p strains (input). Half of the cells were pre-treated with CHX for 2 h, whereas the other half was left untreated. HA₃Hrd3p and its interaction partners were isolated by immunoprecipitation with α-HA-antibodies and ProteinA-Sepharose (α-HA precipitates). The precipitates were analysed by immunoblotting with the indicated antibodies. Dpm1p serves as a negative control as it does not interact with Sec61p and was precipitated using specific α-Dpm1p antibodies. When detecting the CPY^* signal, the earlier signal of Der3p could still be seen in α-HA precipitates (indicated in grey letters). (B) Ratios of Der3p, Cdc48 and Sec61p to HA₃Hrd3p obtained by quantification of the corresponding immunoblot signals of the precipitates shown in (A) using the program ImageJ 1.41o. (C) ‘MIX' experiment to exclude formation of artificial Sec61p containing protein complexes. Cells from strains expressing either Sec61p-myc and endogenous Hrd3p or untagged Sec61p and HA₃Hrd3p were subjected to immunoprecipitation as described above. In the MIX, sample cells of these two strains were mixed before immunoprecipitation.

The pool of ubiquitinated CPY^* at the ER membrane increases with time, whereas Sec61p loses contact to CPY^*. (A) The immunoprecipitation experiment was carried out as described in Figure 2A using Δprc1 and Δprc1Δder3 strains expressing either CPY^* or CPY^*HA₃ (input). The cells were pre-treated for 0, 15 and 30 min with CHX. The immunoblot analysis included the detection of ubiquitinated proteins. The stars indicate unspecific cross-reactions. (B) Ratio of the ubiquitin, Sec61p, Cdc48 and Der3p signals to the CPY^*HA₃ signals in immunoprecipitates of Δprc1+CPY^*HA₃ shown in (A) using the program ImageJ 1.41o.

Fully glycosylated proteins are associated with Sec61p. (A) Schematic representation of carboxypeptidase Y (CPY), point mutated CPY G255R (CPY^*) and CPY^* including an additional glycan at the amino-acid position 423 (CPY^*G5). (B) Import model of CPY^*G5. The active site of the OST complex is located in a distance of ∼15 residues from the end of the hydrophobic transmembrane segments of Sec61p. (C) Immunoprecipitation experiment using a C-terminally HA₃-tagged version of Sec61p. Digitonin-solubilized microsomal proteins (input) were prepared from strains deleted for the coding sequence of CPY (Δprc1) and expressing either plasmid encoded CPY, CPY^* or CPY^*G5. Sec61pHA₃ was precipitated with α-HA antibodies and ProteinA–Sepharose (α-HA precipitates) and analysed by immunoblotting using specific α-CPY and α-Sec61p antibodies. The star indicates pre-pro-forms of CPY^* and CPY^*G5; h.c. indicates a cross-reaction with the heavy chains of the antibodies used for precipitation (D) Immunoprecipitation experiment carried out as described above. The cells were pre-treated with CHX for 30 min at 30°C. (E) Immunoprecipitation experiment using the strains SEC61HA₃, Δhrd3 SEC61HA₃ and Δder3 SEC61HA₃. All strains carry in addition a Δprc1 deletion and plasmid encoded CPY^*. The deletion of the core components of the Hrd–ubiquitin ligase complex, Der3p and Hrd3p, leads to an enhanced interaction of CPY^* with Sec61p. (F) Ratio of immunoblot signals of CPY^* to Sec61p signals of immunoprecipitates shown in (E).

Sec61p loses contact to misfolded proteins during the degradation process. (A) Immunoprecipitation experiment using CPY^*HA₃ as a bait. Digitonin-solubilized membrane proteins of isolated microsomes were prepared from Δprc1, Δprc1Δhrd3 and Δprc1Δder3 strains expressing either CPY^* or CPY^*HA₃ (input). The cells were pre-treated for 0 and 30 min with CHX. After precipitation using specific α-HA antibodies, the samples (α-HA precipitates) were analysed by immunoblotting with the indicated antibodies. The stars indicate unspecific cross-reactions. (B) Ratio of Sec61p to CPY^*HA₃ obtained by quantification of the corresponding immunoblot signals of the immunopreciptates shown in (A) using the program ImageJ 1.41o. (A). The error bars represent the s.e.m. of three independent experiments.

CTL^*myc interacts with Sec61p until the CPY^* moiety of CTL^*myc is degraded. Cells of the yeast strains Δprc1 and Δprc1Δhul5 expressing either plasmid encoded CTL^* or CTL^*myc were treated with CHX for 0 and 3.5 h. This enables the accumulation of the fragment truncCTL^*myc in a Δprc1Δhul5 strain (lane 7). Solubilized microsomal proteins were subjected to immunoprecipitation using α-myc antibodies (α-myc precipitates) and were analysed by immunoblot analysis with the indicated antibodies. Except for the interaction studies of Sec61p, this figure was earlier published in Kohlmann et al (2008). CTL^*myc interacts with Sec61p during its degradation (lanes 11 and 12) as well as the known components of the ERAD machinery, Der3p and Cdc48 (Kohlmann et al, 2008).