The Engagement of Sec61p in the ER Dislocation Process Mingyue Zhou, Randy Schekman  Molecular Cell  Volume 4, Issue 6, Pages 925-934 (December 1999) DOI: 10.1016/S1097-2765(00)80222-1

Figure 1 Yeast Strains Defective in Dislocation Exhibit Elevated UPR Wild-type yeast strains WT-1(YPH499), WT-2(YPH500), WCG4a, and strains harboring Δubc6, Δubc7, and Δcue1 mutations were transformed with a reporter plasmid (2μ/URA) carrying the UPRE-lacZ construct (pMCZ-Y, Kawarata et al., 1994). (A) UPR measured by β-galactosidase assay: Individual transformants were grown at 30°C in SC-ura medium to mid–log phase, harvested, and incubated with fresh medium in the absence or presence of tunicamycin (Tm, 10 μg/ml) at 30°C for 3 hr. β-galactosidase activities were measured by the standard assay with units normalized to the OD600 units of cells used for the assay. Data represent means of three independent samples. (B) UPR exhibited on an X-gal plate. Yeast cells were grown as patches and replica-plated onto an X-gal plate without tunicamycin. The plate was scanned after incubation at 30°C for 3 days. Molecular Cell 1999 4, 925-934DOI: (10.1016/S1097-2765(00)80222-1)

Figure 2 sec61-R Mutant Alleles Exhibit Elevated UPR (A) UPR exhibited on an X-gal plate: Newly isolated sec61 mutant alleles (sec61-R1, sec61-R2, sec61-R3, and sec61-R4) and two isogenic wild-type control strains (WT-1 and WT-2) that were created as described in the Experimental Procedures were grown on SC-trp-leu plates as patches and replica-plated onto X-gal plates (SC-trp-leu) without tunicamycin. The plates were scanned 4 days after replica plating. (B) UPR measured with β-galactosidase assays. sec61 mutant alleles and one of the isogenic wild-type control strain (WT) were grown in SC-trp-leu medium. Treatment with tunicamycin (Tm) was performed as described in Figure 1A. Molecular Cell 1999 4, 925-934DOI: (10.1016/S1097-2765(00)80222-1)

Figure 3 sec61-R Mutant Alleles Are Proficient in Translocation of α Factor and Kar2p Precursors into the ER Previously isolated yeast mutant strains (sec61-2, sec61-3, sec61-41, sec62, and sec63), newly isolated sec61-R alleles (sec61-R1, sec61-R2, sec61-R3, and sec61-R4), and the isogenic wild-type control strain (WT) from this study were pulse labeled with [35S]-methionine at 30°C for 12 min as described. Lane 2 represents WT cells treated with tunicamycin (+Tm, 10 μg/ml) during the pulse labeling. Cell lysates were immunoprecipitated either with anti-α factor precursor antibody (upper panel) or anti-Kar2p antibody (lower panel). The positions of signal peptide–carrying precursor forms (ppαF, pKar2p), signal peptide-cleaved, unglycosylated α factor precursor (pαF), and signal peptide–cleaved Kar2p (Kar2p) are indicated. Molecular Cell 1999 4, 925-934DOI: (10.1016/S1097-2765(00)80222-1)

Figure 4 sec61-R Mutations Impair Degradation of an ER-Luminal and an Integrated Membrane Substrate (A) Stabilization of an unglycosylated, ER form of α factor precursor. sec61-R mutant alleles and two isogenic wild-type strains (WT-1 and WT-2) were grown in SC-trp-leu medium. Pulse–chase radiolabeling was performed in the presence of tunicamycin (10 μg/ml) at 30°C for the indicated periods of time. Cell lysates were immunoprecipitated with anti-α factor precursor antibody. The bands on the left panel represent the unglycosylated, ER form of α factor precursor. The curves on the right panel represent data quantified on a PhosphorImager. (B) Stabilization of an ER membrane–integrated model protein. sec61-R1 and the isogenic wild-type strain (WT) were transformed with a plasmid (2μ/URA) carrying the Deg1-SEC62-protA fusion construct under the control of the Gal1 promoter. Transformants were grown in SC-trp-leu-ura medium containing 2% raffinose and 0.1% glucose at 30°C to log phase and shifted to SC-trp-leu-ura medium containing 2% galactose for 2 hr at 30°C. A cycloheximide chase (0.1 mg/ml) was performed at 30°C for the indicated times. Equal aliquots of total cell proteins were separated with SDS-PAGE and analyzed by immunoblotting using protein A–reactive antibody conjugated with horseradish peroxidase. Molecular Cell 1999 4, 925-934DOI: (10.1016/S1097-2765(00)80222-1)

Figure 5 sec61-R Mutant Microsomes Are Proficient in Polypeptide Import but Deficient in Protein Export (A) In vitro translocation reactions with radiolabeled wild-type α factor precursor. Radiolabeled α factor precursor was produced by in vitro transcription and translation. Aliquots of 1.5 × 106 cpm of the marker protein were incubated with 20 μl of microsomes OD280 = 30 and the translocation reactions were performed with an ATP-regenerating system at 20°C for 50 min in the presence of 0.8 M urea. Reactions were divided and either immediately terminated with TCA or treated with trypsin in the absence or presence of TX-100 on ice for another 20 min before TCA termination. (B) In vitro ERAD assays with radiolabeled ΔgppαF. Radiolabeled ΔgppαF was produced in vitro and introduced by translocation into the microsomes as described above. Loaded microsomes were washed twice with buffer 88, and export reactions were performed at 30°C for 0, 20, or 40 min in the presence of wild-type cytosol (final concentration of 6 μg protein/μl of reaction) and an ATP-regenerating system. The right panel represents PhosphorImager-quantified data. Data represent the means of five independent experiments. Molecular Cell 1999 4, 925-934DOI: (10.1016/S1097-2765(00)80222-1)

Figure 6 The UPR of SEC61 Heterozygotes and the Stability of Sec61p (A) UPR measured by β-galactosidase assay. sec61-R mutant strains and an isogenic wild-type strain (WT) were transformed in parallel either with a vector (pRS316) or with the plasmid-carrying wild-type SEC61 that harbors a 6His tag at the N terminus (Pilon et al. 1998). Transformants were grown on SC-trp-leu-ura medium. β-galactosidase activities were measured as described in Figure 1. (B) Sec61p levels measured by Immunoblot. Total cell extracts were prepared from isogenic wild-type and mutant sec61 alleles that had been transformed with vector or SEC61 plasmid, as described above, and were grown to mid–log phase in SC-leu-trp-ura medium at 30°C. Equal amounts of total cell proteins were separated by 10% SDS-PAGE and analyzed by immunoblot using affinity-purified rabbit anti-Sec61p antibody. Note that the presence of the 6His tag retards the mobility of Sec61p, making the Sec61p forms in heterozygotes migrate in a broader band. (C) Sec61p levels before and after heat shock treatment. The isogenic wild-type and mutant sec61-R alleles growing at mid–log phase at 30°C were subjected to heat shock treatment (HS) at 37°C for 2 hr. Protein extracts were prepared as above; 10% SDS-PAGE was performed with sample amounts that were normalized to the levels of Sec12p. After transfer, the same membrane was cut and blotted with anti-Sec61p and anti-Sec12p antibodies, respectively. Molecular Cell 1999 4, 925-934DOI: (10.1016/S1097-2765(00)80222-1)