1. Volume 50, Issue 4, Pages 528-539 (May 2013)
Ubiquitin Binding by a CUE Domain Regulates Ubiquitin Chain Formation by ERAD E3 Ligases 
Katrin Bagola, Maximilian von Delbrück, Gunnar Dittmar, Martin Scheffner, Inbal Ziv, Michael H. Glickman, Aaron Ciechanover, Thomas Sommer 
Molecular Cell 
Volume 50, Issue 4, Pages (May 2013)
DOI: /j.molcel
Copyright © 2013 Elsevier Inc. Terms and Conditions

2. Figure 1
Cue1p Stimulates the Ubiquitylation Activity of Ubc7p at Both ERAD Ligases
(A) Schematic representation of Ubc7p, Cue1p, Hrd1p, and Doa10p. Full-length Ubc7p and soluble parts of Cue1p, Hrd1p, and Doa10p, indicated by brackets, were used for heterologous expression in E. coli and the following in vitro ubiquitylation assays.
(B and C) In vitro ubiquitylation reactions with Ubc7p, Cue1p, and the ERAD ligases Hrd1p or Doa10p. Equal amounts (3.5 μM) of the indicated proteins were mixed together with 7.2 μM Flag epitope-tagged ubiquitin, E1 enzyme, and ATP in a buffer system followed by a 15 min incubation in a 30°C water bath. Ubiquitin was detected by a monoclonal anti-Flag antibody. Small amounts of diubiquitin were detected in all reactions due to preformed Flag-ubiquitin conjugates. Presence of Ubc7p, Cue1p, Hrd1p, or Doa10p was verified by protein-specific immunoblotting. (B) Reactions were performed with the soluble C-terminal part of Hrd1p (amino acids 325–552) containing the RING motif. (C) Reactions with the N-terminal soluble part of Doa10p (amino acids 1–125) which comprises the RING.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
K48-Linked Ubiquitin Chain Formation by the HRD Ligase Complex Facilitates Degradation of ERAD Substrates
(A and B) Degradation of the ERAD model substrate CPY* (mutant carboxypeptidase Y, left panel) was analyzed in a cycloheximide (CHX) decay assay. (A) Subsequent to 4 hr CuSO42--induced overexpression of plasmids encoding wild-type ubiquitin or the different indicated K-R ubiquitin mutants, cells were analyzed after 0, 40, 80, or 120 min. The ER membrane-bound protein Sec61p (right panel) served as loading control. (B) Cells with constitutively overexpressed N-terminally RGS-8xHis-tagged wild-type ubiquitin or K-only ubiquitin mutants were lysed at indicated time points.
(C) Quantitative analysis of ubiquitin linkages by mass spectrometry. Different in vitro ubiquitylation reactions with Ubc7p (U7), Cue1p (C1), or Hrd1p (H1) and bovine ubiquitin (ub) were measured in selected reaction monitoring (SRM) in comparison to human ubiquitin standards (ub with identical amino acid sequence). The reaction with only E1 enzyme and ubiquitin served as negative control. Standard error bars were obtained from three technical replicates. Error bars indicate the standard deviation of three technical replicates.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Enhanced Polyubiquitylation Activity of Ubc7p Is Obtained through Complex Formation with Cue1p and Hrd1p
(A) In vitro ubiquitylation activity of Ubc7p was examined over a time period of 15 min. Incubation of Ubc7p with Alexa Fluor 488-labeled ubiquitin in the absence or presence of Hrd1p, Cue1p, or the short Ubc7p-binding region (sU7BR, amino acids 147–203) of Cue1p. Sample analysis was performed by SDS-PAGE, followed by fluorescence scan of the gels.
(B) Time course of in vitro ubiquitin chain formation. Different N- or C-terminally truncated Cue1p variants, wild-type Cue1p, and the mutant form Cue1pRGA were added to reactions with Ubc7p, Hrd1p, and Flag epitope-tagged ubiquitin, and chain formation was analyzed at indicated time points (0, 8, 16, or 24 min). Mono- and oligoubiquitin (upper panel, 18% SDS gel) as well as higher-molecular-weight ubiquitin chains (lower panel, 9% SDS gel) were visualized by immunoblotting using an anti-Flag antibody. Sample aliquots from time point 0 were also analyzed by western blotting to visualize the amounts of the added Cue1p variants. Since the anti-Cue1-peptide antibody recognizes the C terminus of Cue1p, this antibody was not able to bind to the Cue1pΔC variant. Amounts of the Cue1p variants can therefore be also compared from a Coomassie-stained gel (Figure S3A).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Cue1p Binds Ubiquitin through Its Extended CUE Domain
(A) Alignment of amino acid sequences of CUE domains from various yeast or mammalian (gp78) proteins. Numbers indicate the respective residues. The sequence of the Cue1p CUE domain is written in bold, and the LAP motif used for mutagenesis is framed by a black box. Highly conserved amino acids are highlighted in yellow, and those known to be absolutely required for ubiquitin binding are highlighted in red.
(B) Schematic depiction of Cue1p constructs used in the in vitro ubiquitylation assay or in vitro binding studies. All variants were purified from E. coli through an N-terminal GST tag that was removed by Prescission Protease cleavage for ubiquitylation experiments. The C-terminal His6 tag of some constructs was added to prevent expression and purification of degradation products.
(C) Binding of ubiquitin chains to immobilized GST-fusion proteins. GST, GST-Cue1p constructs, and GST-UBA (Dsk2) were purified from E. coli, immobilized by binding to glutathione Sepharose, and incubated with equal amounts of ubiquitin chains. After binding, TCA-precipitated supernatant (S) and material sedimented with the Sepharose (B) were loaded to 18% SDS gels.
(C) Binding of preformed K48-linked ubiquitin chains (ub2-8) was analyzed by anti-ubiquitin immunoblotting. The lower panel visualizes the amount of GST fusion protein for each binding sample.
(D) In vitro deubiquitylation of K48-linked ubiquitin chains by Otubain-1 in a 4 min progress. Equal amounts of ubiquitin chains were mixed with either BSA, Cue1p, or Cue1pRGA. Time point 0 shows the ubiquitin chains before addition of Otubain-1. Incubation with Otubain-1 for 360 s on ice. Aliquots were taken at indicated time points.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
A Functional CUE Domain Increases the Efficiency of Polyubiquitin Chain Formation
(A and B) Analysis of ubiquitin chain formation by Ubc7p, Flag-tagged ubiquitin, and various Cue1p constructs monitoring the formation of diubiquitin (lower panel, 18% SDS gel) and high-molecular-weight ubiquitin chains (upper panel, 9% SDS gel). Flag-tagged ubiquitin conjugates were detected by anti-Flag immunoblotting. (A) Twenty-four minute time course showing polyubiquitylation reactions with Hrd1p in the presence of Cue1p or different CUE domain mutants. The anti-Cue1 blot shows the amount of added Cue1 proteins at time point t = 0. (B) In vitro ubiquitylation reactions with Cue1p or Cue1pRGA and in the absence or presence of either Hrd1p or Doa10p after 15 min incubation.
(C and D) Quantification of a typical in vitro ubiquitylation reaction of Ubc7p, Hrd1p, and different Cue1p variants with Alexa Fluor 488-labeled ubiquitin using ImageQuant software. SDS-PAGE of samples taken at the indicated time points followed by measurement of fluorescence intensities. (C) Synthesis rates of all ubiquitin conjugations (ub2 to ubn) were analyzed from 18% SDS gels. Time point t = 0 of each reaction was used for normalization of fluorescence intensities measured throughout the time course. (D) Diagrams illustrating the formation of di-, tri-, or tetraubiquitin in reactions with different Cue1p variants, each over a time period of 25 min. Fluorescence intensities (counts) were normalized by subtraction of values measured at time point t = 0 of the appropriate reaction.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
The CUE Domain Allows the Formation of High-Molecular-Weight Ubiquitin Chains
(A and B) Quantification of a typical in vitro ubiquitylation reaction of Ubc7p, Hrd1p, and different Cue1p variants with Alexa Fluor 488-labeled ubiquitin (see above). (A) Formation rates of ubiquitin chains with a molecular weight above 175 kDa were quantified from 9% SDS gels. Fluorescence intensity values of the different time points were normalized to time point t = 0 min of the appropriate reaction. (B) Distribution of ubiquitin chains of different molecular weight range in reactions with the indicated Cue1p constructs after 25 min incubation. Relevant molecular weight sizes were estimated by protein standards.
(C) Ubiquitin chain formation of Ubc7p and different indicated Cue1p constructs in the presence of Hrd1p and Flag-tagged ubiquitin after 15 min incubation at 30°C (upper panel, 9% SDS gel; lower panel, 18% SDS gel).
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Mutations within the CUE Domain Affect the Degradation of Certain ERAD Substrates In Vivo
(A) Graphical description of Cue1p proteins that were examined in in vivo studies. The corresponding genes were expressed from ARS/CEN plasmids in yeast strains.
(B) Turnover of Ubc6p in the indicated cue1 mutants within 3 hr after cycloheximide treatment of the yeast cells (left panel). The stable ER membrane-bound protein Sec61p (right panel) served as loading control. Samples were analyzed by SDS-PAGE and immunoblotting using anti-Ubc6p and anti Sec61p antibodies.
(C) Quantification of CPY* turnover in cue1 mutants from three independent pulse-chase experiments. Values measured for the indicated time points were normalized (time point 0 = 100%; error bars indicate standard deviation).
(D) Separate cycloheximide decay assays of the cytosolic Deg1-GFP-GFP or the membrane anchored Deg1-Flag-Vma12-ProtA fusion protein in cue1 mutants. The remaining amount of each protein was analyzed 0, 20, 40, and 60 min after addition of cycloheximide to the cells. Proteins were detected by immunoblotting with anti-GFP or anti-Flag antibody.
(E) Immunoprecipitation of 13xmyc-tagged Doa10p from yeast cells by anti-myc antibody coupled to protein A Sepharose. Analysis of coprecipitated proteins by SDS-PAGE and immunoblotting with protein-specific antibodies (anti-Ubx2, anti-Cdc48; Neuber et al., 2005).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions