Sec61p cysteine mutants are proficient for ER protein export. (A) Wild-type and Sec61p mutant cells expressing the transmembrane ERAD substrate Deg1:Sec62^ProtA were pulse labeled for 5 min and chased for the indicated times. Deg1:Sec62^ProtA was immunoprecipitated, resolved by SDS-PAGE, and visualized by audioradiography. (B) Deg1:Sec62^ProtA decay was quantified by phosphorimager analysis and plotted as the sum of decay for the two species over time. The data in the plots reflect the mean of three independent experiments. Standard deviations for each time point were typically in the range of 4–8% but are not shown in the plots for ease of viewing. (C) Wild-type and Sec61p mutant cells expressing CPY* were pulse labeled for 15 min and chased for the indicated times. CPY* was immunoprecipitated from detergent-solubilized lysates, resolved by SDS-PAGE, and visualized by audioradiography. (D) CPY* decay was quantified and plotted as described for Deg1:Sec62^ProtA in B.

Deg1:Sec62^ProtA is modified by N-linked glycosylation. (A, left) Schematic representation of Deg1:Sec62^ProtA and its presumed membrane topology. The relative positions of the two cryptic N-glycan acceptor sites (Asn90 and Asn153) are depicted. (A, middle) Schematic representation of Deg1:Sec62^ProtA highlighting the amino acid boundaries of the individual segments of the hybrid protein. (A, right) Steady-state levels of Deg1:Sec62^ProtA from wild-type cells were immunoprecipitated with IgG Sepharose and either mock treated or treated with the deglycosylating enzyme endoglycosidase H. Samples were separated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-Flag M2 monoclonal antibodies to visualize the substrate protein. (B) Yeast cells carrying wild-type or the indicated Deg1:Sec62^ProtA mutants were pulse labeled for 5 min and hybrid protein was immunoprecipitated from cell lysates. Samples were separated on SDS-PAGE and visualized by audioradiography. The mobility of substrate forms without (0-CHO), with 1 (1-CHO), and with 2 (2-CHO) N-glycans is indicated. (C, left) BY4742 harboring Deg1:Sec62 1–149 was pulse labeled in the presence or absence of 10 μg/ml tunicamycin. Deg1:Sec62 1–149 was immunoprecipitated from detergent-solubilized lysates with anti-Flag antibodies. (C, right) BY4742 harboring proFactor:Sec62 1–149 were pulse labeled in the presence or absence of tunicamycin. After centrifugation, the supernatant and cell pellet fraction were subjected to detergent-solubilized immunoprecipitation with anti-Flag antibodies. Samples were separated on SDS-PAGE and visualized by audioradiography. Asterisks indicate the migration of glycosylated forms of proFactor:Sec62 from whole cell lysates. (D) Wild-type and glycosylation acceptor site mutants of Deg1:Sec62^ProtA were pulse labeled for 5 min and chased for the indicated times. Deg1:Sec62^ProtA was immunoprecipitated, resolved on SDS-PAGE, and visualized by audioradiography. (E) Wild-type and mutant Deg1:Sec62^ProtA decay were quantified and plotted as described in Fig. 1 B.

Sec61p cysteine mutants are proficient for ER protein import. Wild-type and Sec61p mutant cells were pulse labeled at 30°C for 15 min and model secretory proteins were immunoprecipitated from detergent-solubilized lysates. The immunoprecipitated proteins were resolved on SDS-PAGE and visualized by audioradiography. Where indicated, tunicamycin was present at 10 μg/ml. The positions of precursor forms (ppαf and pKar2), signal-cleaved unglycosylated proteins (pαf, proCPY, and Kar2), and signal-cleaved glycosylated forms (p1, p1, and mCPY) are indicated.

Glycosylation of Deg1:Sec62^ProtA occurs during degradation. (A) Wild-type and Δubc7 cells expressing Deg1:Sec62^ProtA were pulse-labeled for 2, 4, 6, or 8 min. Deg1:Sec62^ProtA was immunoprecipitated, resolved on SDS-PAGE, and visualized by audioradiography. (B) Phosphorimager quantitation of the results depicted in A. The data reflect the mean of at least three independent experiments with the SD of the mean indicated. The data are plotted as the ratio of the glycosylated to unglycosylated substrate protein present at the indicated time of the pulse label. (C) Wild-type and Deg1 region mutants of Deg1:Sec62^ProtA were pulse labeled for the indicated times. Deg1:Sec62^ProtA was immunoprecipitated, resolved on SDS-PAGE, and visualized by audioradiography. (D) Phosphorimager quantitation of lanes in C. Results are plotted as in B.

Glycosylation of Deg1:Sec62^ProtA is dependent on a cysteine residue within the substrate protein. (A) Wild-type and cysteine mutant alleles of Deg1:Sec62^ProtA were pulse labeled for 5 min. Deg1:Sec62^ProtA was immunoprecipitated, resolved by SDS-PAGE, and visualized by audioradiography. The position of unglycosylated (0-CHO) and glycosylated (1-CHO) substrate is indicated. (B) Degradation kinetics of Deg1:Sec62^ProtA cysteine mutants in a wild-type Sec61p background. Deg1Sec62^ProtA and cysteine mutant derivatives were subjected to a pulse-chase regimen and lysates were immunoprecipitated as described in Fig. 1 A. (C) Wild-type Deg1:Sec62^ProtA cysteine mutant decay was quantified and plotted as described in Fig. 1 B.

Two dimensional nonreducing/reducing electrophoresis. (A) Membranes from wild-type or Sec61p C373S cells expressing ΔCys4:Deg1:Sec62^ΔProtA were prepared for two-dimensional electrophoresis as described in Materials and methods. After electrophoresis, proteins were transferred to nitrocellulose and probed with anti-Flag M2 monoclonal antibodies to detect substrate protein. The migration of three spots representing the monomeric substrate and two disulfide-bonded complexes containing the substrate are marked A, B, and C, respectively. (B) Duplicate blots from the samples in A. In this case, the nitrocellulose membrane was probed with anti-Myc 9B11 monoclonal antibodies to detect Sec61p-myc. The migration of two spots representing monomeric Sec61p and a disulfide-bonded complex containing Sec61p are marked A and B, respectively.

Models for disulfide complex formation and substrate glycosylation. (A) A model depicting a role for Sec61p in the initial state of Deg1 translocation into the ER. In this model, the amphipathic Deg1 region mimicks a signal peptide and is recruited to the Sec61p channel (I and II). After inversion of this segment, the N-terminal cytosolic region is looped back through the channel and may either be actively translocated or passively backslide through the channel (III). Next, the cysteine residues of the substrate protein and Sec61p complex form a disulfide intermediate that provides for the extended exposure and glycosylation at Asn153 within the N-terminal tail of the substrate protein (IV and V). After this initial translocation step, the misfolded Deg-Sec62 may be transferred to another channel (cylindrical symbol) to execute retrotranslocation. (B) A model depicting a role for Sec61p in membrane protein retrotranslocation from the ER. Substrate is initially targeted to Sec61p, and its C-terminal transmembrane helix partitions into the channel through a lateral gate (I). Ubiquitination and the Cdc48p and/or 19S RP complex assist in membrane extraction of the substrate protein (II). The substrate's N-terminal transmembrane partitions into the channel where the extraction force at the C terminus results in the reorientation of the N-terminal transmembrane segment within the channel (III). The N-terminal segment inverts, resulting in a reorientation of the N-terminal cytoplasmic tail with regions exposed to the lumen of the ER. Next, the looped segment may enter the lumen further by backsliding within the channel (IV). Finally, the disulfide complex formation between Sec61p and substrate stalls the N terminus in the channel, permitting glycosylation of Asn153 (V).