Key steps in ERAD of luminal ER proteins reconstituted with purified components

Abstract

Misfolded proteins of the endoplasmic reticulum (ER) are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome, a process called ER-associated protein degradation (ERAD). Here, we use purified components from Saccharomyces cerevisiae to analyze the mechanism of retrotranslocation of luminal substrates (ERAD-L), recapitulating key steps in a basic process in which the ubiquitin ligase Hrd1p is the only required membrane protein. We show that Hrd1p interacts with substrate through its membrane-spanning domain and discriminates misfolded from folded polypeptides. Both Hrd1p and substrate are polyubiquitinated, resulting in the binding of Cdc48p ATPase complex. Subsequently, ATP hydrolysis by Cdc48p releases substrate from Hrd1p. Finally, ubiquitin chains are trimmed by the deubiquitinating enzyme Otu1p, which is recruited and activated by the Cdc48p complex. Cdc48p-dependent membrane extraction of polyubiquitinated proteins can be reproduced with reconstituted proteoliposomes. Our results suggest a model for retrotranslocation in which Hrd1p forms a membrane conduit for misfolded proteins.

Figure 1. Substrate Interaction with Hrd1p

(A) Fluorescently labeled CPY* (10 nM) was incubated with increasing concentrations of bead-immobilized SBP-tagged Hrd1p (Hrd1p). The bound and unbound fractions were analyzed by SDS-PAGE and fluorescence scanning. (B) Quantification of four different experiments as in (A). Fitting of the data points gives an apparent dissociation constant of 30 nM. Also shown are experiments with wild type CPY, either purified as a native protein or after unfolding and refolding, as done with CPY*. (C) As in (A), but with sCPY* (100 nM). (D) Quantification of three different experiments as in (C). The apparent dissociation constant is ∼300 nM. (E) Quantification of binding experiments of wild type Hrd1p with fluorescently labeled sCPY*-DHFR (10 nM), sCPY*-GFP (100 nM), DHFR (100 nM), or GFP (100 nM). (F) As in (C), but sCPY* was incubated with either wild type Hrd1p, a fusion of the TMs of Hrd1p with GFP (Hrd1p-TM-GFP), the cytoplasmic domain of Hrd1p (Hrd1p-c), or GFP. (G) sCPY*-DHFR (200 nM) labeled with DyLight800 was incubated with a mixture of unlabeled Hrd1p (20 μM) and Hrd1p (200 nM) labeled with DyLight680. The sample was subjected to gel filtration in a buffer containing 120 μM DMNG and fractions were analyzed in two fluorescence channels. A control was performed with labeled sCPY*-DHFR alone. The arrows indicate the void volume, and the retention volume of size standards. See also Figure S1.

Figure 2. Poly-ubiquitination by Hrd1p

(A) Time-course of ubiquitination of CPY* labeled with DyLight800. Some reactions were analyzed after 60 min with the indicated components omitted. Where indicated, wild type Hrd1p (100 nM) was replaced with 100 nM of an inactive Hrd1 p mutant (C399S) or 1 μM of the cytoplasmic domain of Hrd1 p (Hrd1p-c). (B) Time-course of auto-ubiquitination of Hrd1p labeled with DyLight680. (C) The time-course of auto- and substrate-ubiquitination was determined in parallel. The concentration of labeled Hrd1p was kept constant, while that of unlabeled Hrd1p was varied. (D) The time-course of auto-ubiquitination was determined with 50 nM of labeled wild type (WT) Hrd1p or C399S mutant in the absence or presence of a 10-fold excess of unlabeled WT Hrd1p (solid and broken curves, respectively). See also Figure S2.

Figure 3. Recruitment of Cdc48p to Ubiquitinated Hrd1p

(A) Bead-immobilized Hrd1p (1 μM) was ubiquitinated and incubated at different salt conditions with 2 μM of the indicated components (UN, Ufd1p/Npl4p) in the presence of 250 μM ATPγS. The bound material was analyzed by SDS-PAGE and staining with IRDye Blue. (B) As in (A), but bead-immobilized Hrd1p was modified with methylated ubiquitin. (C) As in (A), but with non-ubiquitinated Hrd1p. See also Figure S3.

Figure 4. Substrate Release from Hrd1p by Cdc48p ATPase

(A) A bead-immobilized complex of 500 nM SBP-tagged Hrd1p and 100 nM fluorescently labeled CPY* was incubated with the ubiquitination machinery. The beads were washed and incubated with 100 nM Cdc48p and ATP in the absence or presence of 100 nM Ufd1/Npl4p (UN) complex, or with UN alone. Where indicated, ATP was depleted with hexokinase/glucose (Hk/G). The released material was analyzed by SDS-PAGE and fluorescence scanning. The total releasable amount of CPY* was determined by incubating the beads with biotin. (B) Quantification (means and standard deviations) of released poly-ubiquitinated CPY* determined from four experiments as shown in (A). The released fraction is expressed relative to the total releasable material. (C) Bead-immobilized SBP-tagged Hrd1p was ubiquitinated (Ub-Hrd1p) for 1 h. The beads were washed, incubated with fluorescently labeled CPY*, and treated as in (A). The lower panel shows the same experiment with non-ubiquitinated Hrd1p (non-Ub-Hrd1p). (D) Quantification (means and standard deviations) of released non-ubiquitinated CPY* determined from four experiments as shown in (C) (released from Ub-Hrd1p, black columns; released from non-Ub-Hrd1p, grey columns). (E) Immobilized Hrd1p mutants with Lys to Arg mutations in three different regions were ubiquitinated for 30 min, and Cdc48p-dependent release of unmodified CPY* was tested as in (C). Shown are means and standard deviation of three experiments. (F) Bead-immobilized Hrd1p was incubated with untagged, fluorescently labeled Hrd1p. After ubiquitination and washing, the beads were incubated with the indicated components. The material released from the beads was analyzed by SDS-PAGE and fluorescence scanning. (G) Quantification of three experiments performed as in (E) (means and standard deviations). See also Figure S4.

Figure 5. Cdc48p-dependent function of Otu1p in vivo

(A) The degradation of a fusion of sCPY* with DHFR and a hemagglutinin (HA) tag (sCPY*-DHFR-HA) was tested in S. cerevisiae. The cells were transformed with an empty vector or plasmids expressing FLAG-tagged wild type or mutant Otu1p (Otu1p (C120S)) from a Gal1 promoter. Where indicated, Otu1p variants lacking their Ubx domains were expressed instead. The samples were analyzed at different time points after addition of cycloheximide (chx) by SDS-PAGE and immunoblotting with anti-HA and anti-FLAG antibodies. Loading controls were performed with Kar2p antibodies. (B) Quantification of two experiments as in (A) (means and standard deviations). (C) As in (A), but following simultaneously the degradation of Erg1p and Deg1-LacZ with antibodies to the endogenous protein and to LacZ, respectively. (D) Quantification of three experiments as in (C) (means and standard deviations). (E) The degradation of sCPY*-DHFR-HA was analyzed in cells lacking Otu1p and wild type (wt) cells. Cells lacking Hrd1p were analyzed in parallel. (F) Quantification of the experiment in (E). See also Figure S5.

Figure 6. Cdc48p-dependent in vitro de-ubiquitination by Otu1p

(A) Bead-immobilized fluorescently labeled Hrd1p-SBP was incubated with the ubiquitination machinery. After washing, Hrd1p was eluted from the beads with biotin and incubated with the indicated components (Ufd1/Npl4p; UN) for different time periods in the presence of ATP. Hrd1p was in a 30-fold excess over Otu1p, whereas all other components were about equimolar to Hrd1p. The samples were analyzed by SDS-PAGE and fluorescence-scanning. (B) Quantification of experiments performed as in (A). The disappearance of the longest ubiquitin chains was quantified under different conditions (means and standard deviation of three experiments). ATP was depleted with hexokinase/glucose (Hk/G). Where indicated, an ATPase-defective Cdc48p mutant (Cdc48p E588Q) or an Otu1p mutant lacking the Ubx domain were used. (C) Bead-immobilized complexes of Hrd1p-SBP and CPY*, labeled with different fluorescent dyes, were treated as in (A). The de-ubiquitination of modified Hrd1p and CPY* was followed in parallel (solid and broken lines, respectively). Shown are the means and standard deviations of three experiments. (D) Bead-immobilized ubiquitinated Hrd1-SBP was incubated with Cdc48p complex in the presence of ATPγS. After washing, the beads were incubated for 1 h with the indicated components. Supernatants and beads were analyzed by SDS-PAGE and Coomassie staining. See also Figure S6.

Figure 7. Membrane Extraction of Poly-ubiquitinated Proteins by Cdc48p

(A) Proteoliposomes containing fluorescently labeled Hrd1p-SBP and Texas-red labeled phosphatidyl ethanolamine (TR-PE) were subjected to flotation in a Nycodenz gradient. Fractions were collected from the top, and analyzed by SDS-PAGE and fluorescence scanning of the gel. The lipid content of the fractions was determined by absorbance at 590 nm. (B) Proteoliposomes containing Hrd1p-SBP with a TEV-cleavage site at the C-terminus were treated with TEV protease in the absence or presence of DMNG. The samples were analyzed by SDS-PAGE and staining with Coomassie blue. (C) Fluorescently labeled CPY* was incubated with either protein-free liposomes or proteoliposomes containing Hrd1p, or it was co-reconstituted with Hrd1p into vesicles (black, light blue and red bars, respectively). The samples were subjected to flotation in a Nycodenz gradient and fractions were analyzed by SDS-PAGE and fluorescence scanning. Wild type CPY was used as a control. (D) Proteoliposomes containing Hrd1p and labeled CPY* were incubated with trypsin in the absence or presence of DMNG. The samples were analyzed by SDS-PAGE and fluorescence scanning. (E) Proteoliposomes were generated by co-reconstitution of Hrd1p with either CPY* or wild type CPY. Hrd1p and substrate were labeled with different fluorophores. The vesicles were incubated with the ubiquitination machinery for different time periods, and samples were analyzed by SDS-PAGE and fluorescence scanning. For Hrd1p, the gel was cropped to only show the disappearance of unmodified protein. (F) The disappearance of unmodified protein in (D) was quantitated. Solid and broken lines show the modification of substrate and Hrd1p, respectively. (G) Fluorescently labeled Hrd1p and CPY* were co-reconstituted into proteoliposomes. The vesicles were incubated with the ubiquitination machinery, followed by incubation in the absence or presence of the Cdc48p complex (Cdc48p/UN). The vesicles were floated in a Nycodenz gradient and fractions analyzed by SDS-PAGE and fluorescence scanning. (H) Experiments as in (G) were quantified by determining the total fluorescence in the bottom two fractions (material released from the vesicles) as a fraction of the total fluorescence in the gradient (mean and standard deviation of at least three experiments). Where indicated, ATP was depleted with hexokinase/glucose (Hk/G) or an ATPase-deficient Cdc48p mutant (Cdc48p E588Q) was used. (I) As in (G), but with Hrd1p mutants carrying Lys to Arg mutations in three different regions. Quantification of three experiments was done as in (H). See also Figure S7.