Journal: Cell

Article Title: Cytosolic Protein Vms1 Links Ribosome Quality Control to Mitochondrial and Cellular Homeostasis.

doi: 10.1016/j.cell.2017.10.002

Figure Lengend Snippet: Figure 2. Deletion of Vms1 and Ltn1 Causes Respiratory Deficiency (A) Growth of wild-type (WT), ltn1D, vms1D, and vms1Dltn1D cells. Cells were grown in YPD for 8 to 10 hr at 30C (to log-phase) and spotted in 10-fold dilution steps on YPD (fermentable carbon source) or YPG plates (non-fermentable carbon source). YPD plates were incubated for 3 days (23C) or for 2 days (30C and 37C) and YPG plates for 4 days (23C) or for 3 days (30C and 37C). (B) Mitochondria were isolated from WT, ltn1D, vms1D, and vms1Dltn1D cells grown in YPD at 30C. Proteins were analyzed by SDS-PAGE and immunoblotting using the antibodies indicated. (C) Cell extracts prepared with SDS-containing buffer from WT or vms1Dltn1D cells grown in YPGal medium at 23C, 30C, or 37C were analyzed by SDS-PAGE and anti-Rip1 immunoblotting. i-Rip1, intermediate and m-Rip1, mature Rip1. Phosphoglycerate kinase (Pgk1) was analyzed as loading control. (D) Mitochondria were isolated from WT, ltn1D, vms1D, and vms1Dltn1D cells grown in YPGal at 37C. Mitochondria were lysed with 3% digitonin and analyzed by blue native PAGE and immunoblotting against cytochrome c1 (Cyt1) (left) and the b subunit of F1FO-ATP synthase (F1b) (right). III and IV, complexes III and IV of the respiratory chain. See also Figure S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-Myc Max-Planck-Institute of Biochemistry N/A Mouse anti-GFP Roche Cat. #11814460001 Mouse monoclonal anti-HA Santa Cruz biochemistry Cat. #sc-7392 Mouse monoclonal anti-Pgk1 Life Technologies Cat. #459250 Mouse monoclonal anti-ubiquitin Santa Cruz biochemistry Cat. #sc-8017 Rabbit monoclonal anti-FLAG Sigma-Aldrich Cat. #F7425 Rabbit polyclonal anti-Cytb This paper N/A Rabbit polyclonal anti-Cox2 This paper N/A Rabbit polyclonal anti-F1b This paper N/A Rabbit polyclonal anti-Rip1 This paper N/A Rabbit polyclonal anti-Tom40 This paper N/A Rabbit polyclonal anti-Tim50 This paper N/A Rabbit polyclonal anti-Tim23 This paper N/A Rabbit polyclonal anti-Mic60 This paper N/A Rabbit polyclonal anti-Mia40 This paper N/A Rabbit polyclonal anti-Cyt1 This paper N/A Rabbit polyclonal anti-Hsp60 Izawa et al., 2012 N/A Rabbit polyclonal anti-Ssc1 This paper N/A Rabbit polyclonal anti-Ssq1 This paper N/A Rabbit polyclonal anti-Ssa1 Takano et al., 2015 N/A Rabbit polyclonal anti-BiP This paper N/A Rabbit polyclonal anti-Sis1 Cosmo Bio Cat. #cop-080051 Rabbit polyclonal anti-Sis1 D. Cyr, UNC Chapel Hill N/A Rabbit anti-Cdc48 Richly et al., 2005 N/A IRDye 800CW Goat anti-Mouse IgG (H+L) LI-COR Cat. #926-32210 IRDye 800CW Goat anti-Rabbit IgG (H+L) LI-COR Cat. #926-32211 IRDye 680RD Goat anti-Mouse IgG (H+L) LI-COR Cat. #962-68070 Goat Anti-Rabbit IgG (H+L)-HRP Conjugate BIO-RAD Cat. #170-6515 Anti-Rabbit IgG (whole molecule)-Peroxidase antibody produced in goat Sigma-Aldrich Cat. #A6154 Goat anti-mouse IgG HRP conjugated Dako Cat. #P0447 Chemicals, Peptides, and Recombinant Proteins Complete protease inhibitor cocktail, EDTA-free Roche Cat. #05 056 489 001 Protein A Sepharose CL-4B GE healthcare Cat. #17-0963-03 Anti-HA Affinity Matrix Roche Cat. #11 815 016 001 Zymolyase 20T Amsbio Cat. #120491-1 Phusion High-Fidelity DNA Polymerase NEB Cat. #M0530S Q5 High-Fidelity DNA Polymerase NEB Cat. #M0491S Sequencing Grade Modified Trypsin Promega Cat. #V5111 Lysyl Endopeptidase, Mass Spectrometry Grade Wako Cat. #125-05061 Critical Commercial Assays mMACS GFP Isolation Kit Miltenyi Biotec Cat. #130-091-125 PD-10 Desalting columns GE healthcare Cat. #17-0851-01 (Continued on next page) Cell 171, 1–14.e1–e10, November 2, 2017 e1

Techniques: Incubation, Isolation, SDS Page, Western Blot, Control, Blue Native PAGE

Journal: bioRxiv

Article Title: Cex1 is a new component of the COPI Golgi-to-vacuole intracellular trafficking machinery

doi: 10.1101/414367

Figure Lengend Snippet: A Summary Table displaying 11 interesting partners identified by CoIP using a cex1 Δ strain expressing the fusion protein Cex1-HA expressed from its own promoter. B Comparison of yeast (Cex1) and mammalian (SCYL1) interactome. Proteins from COPI vesicles are shown in orange, Golgi proteins in blue, proteins from plasmic membrane are in pink and proteins from the nuclear pore or from the bud neck are in grey and light grey respectively. Genes genetically linked to CEX1 based on the work of Costanzo and colleagues, Science. 2006 are circled. C Cellular components analysis of Cex1 interactants in respiration. Enriched GO terms relative to “microtubule organizing center part”, “cytoplasmic vesicle membrane” and “integral component of organelle membrane” were highlighted in grey. D Cex1 physical and genetic interactants from the literature (BioGrid and SGD) were compared to our Co-IP data. Fifteen physical partners were identified so far for Cex1, of which 4 were retrieved in our experiments. None of these interactants belongs to intracellular traffic (Upper scheme). Among the 90 genes shown to be genetically linked to CEX1 , 37 are related to the Golgi and intracellular trafficking, and 15 of them are also Cex1 physical partners based on our experiments (Lower scheme). E Co-immunoprecipitation performed with a cex1 Δ strain expressing the fusion protein Cex1-HA together with a Myc-tagged version of Sec27. Immunoprecipitations were done against HA, and a control IP was performed on non-coated beads. Immunoblotting against HA and MYC revealed respectively the presence of Cex1 and Sec27. Anti-Pgk1 was used as control to assess non-specific adsorption on HA or MYC-coated beads. Total protein loading for each lane was revealed by the stain free technology. Arrows indicate the protein of interest.

Article Snippet: Presence of Pgk1 was probed with a mouse monoclonal anti-Pgk1 antibody (1:5000; Abcam), Cex1-HA proteins were identified using a mouse monoclonal anti-HA antibody (1:10000).

Techniques: Expressing, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Adsorption, Staining

Journal: bioRxiv

Article Title: Cex1 is a new component of the COPI Golgi-to-vacuole intracellular trafficking machinery

doi: 10.1101/414367

Figure Lengend Snippet: A-B Subcellular fractionation of the WT SEY6210 strain, the control cex1 Δ strain, compared to the vps27 Δ, sec27-1, sec28 Δ and sec33-1 strains expressing the fusion protein Cex1-HA, and grown in fermentation (SC-Glc; A ) or in in semi-respiratory medium (SC-Gal; B ). Localization of Cex1 in each fraction was verified by immunodetection using anti-HA antibodies. Protein loading of each fraction was controlled by stain free gel. Contamination by cytosolic proteins was assessed by detecting the soluble Pgk1 protein, and presence of membrane fraction was evaluated by detection of the ER-to-Golgi and endosomal protein Emp47. ns: non-specific.

Article Snippet: Presence of Pgk1 was probed with a mouse monoclonal anti-Pgk1 antibody (1:5000; Abcam), Cex1-HA proteins were identified using a mouse monoclonal anti-HA antibody (1:10000).

Techniques: Fractionation, Expressing, Immunodetection, Staining

Journal: bioRxiv

Article Title: Cex1 is a new component of the COPI Golgi-to-vacuole intracellular trafficking machinery

doi: 10.1101/414367

Figure Lengend Snippet: A Localization of Cex1-GFP in VPS10 -deficient cells by epifluorescence microscopy in fermentation and respiration. In both conditions vacuoles and endosomes were stained by FM4-64 for 15 min. B Cex1-GFP localization in vps10Δ cells visualized after 3D reconstruction of Z-stack images. Two panels were taken in fermentation (1-2) and in respiration (3-4). For each panel (1-2-3-4), one section of the Z-stack was used to show GFP localization and the corresponding DIC image. Scale bar 2 µm. C Subcellular fractionation of vps10Δ cells expressing Cex1 coupled to GFP. Cex1 sublocalization was followed by immunodetection using anti-GFP antibodies. Anti-Pgk1 and anti-Emp47 antibodies were respectively used as cytosolic and ER/Golgi marker, to ascertained proper fractionation.

Article Snippet: Presence of Pgk1 was probed with a mouse monoclonal anti-Pgk1 antibody (1:5000; Abcam), Cex1-HA proteins were identified using a mouse monoclonal anti-HA antibody (1:10000).

Techniques: Epifluorescence Microscopy, Staining, Fractionation, Expressing, Immunodetection, Marker

Journal: bioRxiv

Article Title: Lipid bilayer thinning near a ubiquitin ligase selects ER membrane proteins for degradation

doi: 10.1101/2025.10.31.685944

Figure Lengend Snippet: (a) Reporter constructs coding for cytosolic mCherry (mCh), a 2A ribosome skip site, and a type-I ER membrane protein were expressed in S. cerevisiae . The membrane protein contains a signal sequence (ss) that places mNeonGreen (mNG) and a HA tag into the ER lumen, followed by a trans-membrane (TM) segment and a cytosolic tail containing an ER retention signal (KKXX) (scheme on the right). (b) An idealized hydrophobic TM segment (LA) was placed into the reporter construct shown in (a). Cycloheximide-chase experiments were performed with wild-type cells (wt), or cells lacking Der1 or Hrd1, and the samples were analyzed by SDS-PAGE and immunoblotting with anti-HA antibodies. Immunoblotting for phosphoglyceratekinase (Pgk1) and mCh served as loading controls. (c) Yeast cells expressing the LA construct were analyzed by fluorescence microscopy. (d) Left: As in (b), but with a TM in which Ala at position 10 was replaced by Asp. The degradation of this D10 construct was analyzed in the indicated yeast strains. Right: Flow cytometry analysis of the indicated cells transformed with the D10 construct. (e) As in (b), but with the indicated amino acids at position 10. Loading control for P10 construct is shown.

Article Snippet: The lysates were supplemented with SDS loading buffer, incubated at 65 °C for 10 min, spun in a table-top centrifuge (15,000 rpm, 1 min), and subjected to SDS-PAGE and immunoblotting with anti-HA antibody (clone 3F10 from rat, Roche, diluted 1:3000), anti-FLAG antibody (from rabbit, F7425, Sigma, 1:2000), anti-Pgk1 antibody (mouse monoclonal [22C5D8], #ab113687, AbCam, 1:5000), anti-mCherry polyclonal antibody (from rabbit,#26765-1-AP, Proteintech, 1:2000), anti-Myc antibody (rabbit polyclonal, #ab9106, AbCam, 1:2000), or anti-SBP antibody (mouse monoclonal, clone 20, #MAB10764, Millipore Sigma, 1:2000).

Techniques: Construct, Membrane, Sequencing, SDS Page, Western Blot, Expressing, Fluorescence, Microscopy, Flow Cytometry, Transformation Assay, Control

Journal: bioRxiv

Article Title: Lipid bilayer thinning near a ubiquitin ligase selects ER membrane proteins for degradation

doi: 10.1101/2025.10.31.685944

Figure Lengend Snippet: (a) The membrane localization of HA-tagged LA and D10 constructs was tested by cell fractionation. Both constructs were expressed in Hrd1-lacking cells to prevent their degradation. Cells were either analyzed directly (input) or fractionated into a supernatant (SN1) and membrane pellet (P1). The membrane pellet was resuspended and again subjected to centrifugation to yield a supernatant (SN2) and pellet (P2). All samples were subjected to SDS-PAGE and immunoblotting with anti-HA antibodies. Immunoblotting for phosphoglyceratekinase (Pgk1) and dolichol-phosphate mannosyltransferase (Dpm1) served as cytosolic and ER membrane markers, respectively. (b) The HA-tagged D10 construct was expressed in cells lacking the indicated components. The degradation of D10 was followed by cycloheximide-chase experiments. The samples were analyzed by SDS-PAGE and immunoblotting with anti-HA antibodies. Immunoblotting for phosphoglyceratekinase (Pgk1) served as a loading control. (c) The HA-tagged LA and D10 constructs were expressed in cdc48-3 cells that contain a temperature-sensitive Cdc48 mutation. The cells were incubated at either the permissive (25 °C) or non-permissive (37 °C) temperature and subjected to cycloheximide-chase experiments. The samples were analyzed as in (b). Immunoblotting for phosphoglyceratekinase (Pgk1) and mCh served as loading controls. (d) Constructs with the hydrophobic TM (LA) or with a TM containing the indicated amino acids at position 10 were expressed in wt cells or cells lacking the indicated components. Cycloheximide-chase experiments were performed and the samples analyzed as in (c). Dotted lines indicate data shown in ; the loading control for the P10 construct is shown.

Article Snippet: The lysates were supplemented with SDS loading buffer, incubated at 65 °C for 10 min, spun in a table-top centrifuge (15,000 rpm, 1 min), and subjected to SDS-PAGE and immunoblotting with anti-HA antibody (clone 3F10 from rat, Roche, diluted 1:3000), anti-FLAG antibody (from rabbit, F7425, Sigma, 1:2000), anti-Pgk1 antibody (mouse monoclonal [22C5D8], #ab113687, AbCam, 1:5000), anti-mCherry polyclonal antibody (from rabbit,#26765-1-AP, Proteintech, 1:2000), anti-Myc antibody (rabbit polyclonal, #ab9106, AbCam, 1:2000), or anti-SBP antibody (mouse monoclonal, clone 20, #MAB10764, Millipore Sigma, 1:2000).

Techniques: Membrane, Construct, Cell Fractionation, Centrifugation, SDS Page, Western Blot, Control, Mutagenesis, Incubation

Journal: bioRxiv

Article Title: Lipid bilayer thinning near a ubiquitin ligase selects ER membrane proteins for degradation

doi: 10.1101/2025.10.31.685944

Figure Lengend Snippet: (a) HA-tagged constructs containing the hydrophobic LA segment or TMs with Asp residues at the indicated positions were tested for their degradation by cycloheximide-chase experiments. The samples were subjected to SDS-PAGE and immunoblotting with anti-HA antibodies. Immunoblotting for phosphoglyceratekinase (Pgk1) and mCh served as loading controls. (b) Scheme illustrating that a TM with a hydrophilic residue (in blue) can slide into the lumen and pull instead residues from the cytosol (red dot) into the membrane. The minimal distance from the lumenal side required for N-glycosylation by oligosaccharyl transferase (OST) is 12 residues (red cross: no modification; green check sign, modification). HA-tagged LA, D1, or D4 constructs with the indicated N-glycosylation sites at different luminal positions were expressed in cells lacking Hrd1 and Asi1. The attachment of a glycan was determined by the mobility shift in immunoblots (right panel). The red arrows point to minimal linker length at which glycosylation was observed. (c) As in (a), but where indicated, the constructs contained the RPRR sequence instead of the GPGG linker at the cytosolic end of the TM to prevent sliding of the TM into the ER lumen. (d) As in (a), but with Asp at position 10 (in red) and replacement of some Ala residues with Leu. Note that increasing the hydrophobicity in the cytosolic leaflet slows degradation.

Article Snippet: The lysates were supplemented with SDS loading buffer, incubated at 65 °C for 10 min, spun in a table-top centrifuge (15,000 rpm, 1 min), and subjected to SDS-PAGE and immunoblotting with anti-HA antibody (clone 3F10 from rat, Roche, diluted 1:3000), anti-FLAG antibody (from rabbit, F7425, Sigma, 1:2000), anti-Pgk1 antibody (mouse monoclonal [22C5D8], #ab113687, AbCam, 1:5000), anti-mCherry polyclonal antibody (from rabbit,#26765-1-AP, Proteintech, 1:2000), anti-Myc antibody (rabbit polyclonal, #ab9106, AbCam, 1:2000), or anti-SBP antibody (mouse monoclonal, clone 20, #MAB10764, Millipore Sigma, 1:2000).

Techniques: Construct, SDS Page, Western Blot, Residue, Membrane, Glycoproteomics, Modification, Mobility Shift, Sequencing

Journal: bioRxiv

Article Title: Lipid bilayer thinning near a ubiquitin ligase selects ER membrane proteins for degradation

doi: 10.1101/2025.10.31.685944

Figure Lengend Snippet: (a) HA-tagged constructs containing the hydrophobic LA segment or TMs with Asp residues at the indicated positions were tested in wild-type (wt) cells or cells lacking Der1 for their degradation by cycloheximide-chase experiments. The samples were subjected to SDS-PAGE and immunoblotting with anti-HA antibodies. Immunoblotting for phosphoglyceratekinase (Pgk1) and mCh served as loading controls. (b) HA-tagged LA or D4 constructs with GPGG (-G) or RPRR (-R) linkers at the cytosolic end of the TM and N-glycosylation sites at different luminal positions were expressed in cells lacking Hrd1 and Asi1. The attachment of a glycan was determined by the mobility shift in anti-HA antibody immunoblots. Immunoblotting for phosphoglyceratekinase (Pgk1) served as a loading control. (c) The degradation of LA constructs with GPGG (-G) or RPRR (-R) linkers at the cytosolic end of the TM was tested by cycloheximide-chase experiments as in (a). (d) A TM of the multi-pass membrane protein Pdr5 was placed into the reporter construct. The TM contains an Asn residue (in blue) that was mutated to Leu for the experiments on the right. The constructs were expressed in wild-type (wt) cells or cells lacking the indicated components, and cycloheximide-chase experiments were performed as in (a). (e) As in (d), but with the entirely hydrophobic TM of the Wsc1 protein. (f) To test whether the TMs serve as a stop-transfer sequences, a N-glycosylation site was introduced into the cytosolic tail of the LA construct or into constructs containing Asp residues at the indicated positions (the glycosylation site NST is 10 residues downstream of the last TM residue). Cycloheximide-chase experiments were performed as in (a). Glycosylated species have an increased molecular weight (red arrowheads). (g) The RPRR linker was placed at the cytosolic end of the indicated TMs to prevent sliding of the TM towards the ER lumen. Cycloheximide-chase experiments were performed with wt cells or cells lacking Der1 as in (a).

Article Snippet: The lysates were supplemented with SDS loading buffer, incubated at 65 °C for 10 min, spun in a table-top centrifuge (15,000 rpm, 1 min), and subjected to SDS-PAGE and immunoblotting with anti-HA antibody (clone 3F10 from rat, Roche, diluted 1:3000), anti-FLAG antibody (from rabbit, F7425, Sigma, 1:2000), anti-Pgk1 antibody (mouse monoclonal [22C5D8], #ab113687, AbCam, 1:5000), anti-mCherry polyclonal antibody (from rabbit,#26765-1-AP, Proteintech, 1:2000), anti-Myc antibody (rabbit polyclonal, #ab9106, AbCam, 1:2000), or anti-SBP antibody (mouse monoclonal, clone 20, #MAB10764, Millipore Sigma, 1:2000).

Techniques: Construct, SDS Page, Western Blot, Glycoproteomics, Mobility Shift, Control, Membrane, Residue, Molecular Weight

Journal: bioRxiv

Article Title: Lipid bilayer thinning near a ubiquitin ligase selects ER membrane proteins for degradation

doi: 10.1101/2025.10.31.685944

Figure Lengend Snippet: (a) Left: The HA-tagged D10 construct was placed under the control of the tef1 promoter and tetracycline aptamers to allow selective shut-off of expression. The construct was expressed in wild-type (wt) cells or cells lacking Hrd1. After addition of tetracyclin, samples were taken at different time points and analyzed by SDS-PAGE and immunoblotting with anti-HA antibodies. Immunoblotting for phosphoglyceratekinase (Pgk1) and mCh served as loading controls. Right: The degradation of D10 was followed after addition of cycloheximide to stop the expression of all proteins. (b) The TM stability score (TMSS), calculated as the ratio of the relative depletion in the high mNG/mCh population over the enrichment in the low mNG/mCh population, was plotted against ΔG water-octanol , the free energy required to move an amino acid from water into octanol .

Article Snippet: The lysates were supplemented with SDS loading buffer, incubated at 65 °C for 10 min, spun in a table-top centrifuge (15,000 rpm, 1 min), and subjected to SDS-PAGE and immunoblotting with anti-HA antibody (clone 3F10 from rat, Roche, diluted 1:3000), anti-FLAG antibody (from rabbit, F7425, Sigma, 1:2000), anti-Pgk1 antibody (mouse monoclonal [22C5D8], #ab113687, AbCam, 1:5000), anti-mCherry polyclonal antibody (from rabbit,#26765-1-AP, Proteintech, 1:2000), anti-Myc antibody (rabbit polyclonal, #ab9106, AbCam, 1:2000), or anti-SBP antibody (mouse monoclonal, clone 20, #MAB10764, Millipore Sigma, 1:2000).

Techniques: Construct, Control, Expressing, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Lipid bilayer thinning near a ubiquitin ligase selects ER membrane proteins for degradation

doi: 10.1101/2025.10.31.685944

Figure Lengend Snippet: (a) Domain structure of the HA-tagged single-pass protein Erv25. The construct contains a cleavable signal sequence (SS; cleavage site indicated by a scissor), an HA tag, a luminal GOLD domain, a TM segment, and an ER retention signal. The predicted TM has two hydrophilic residues (in blue) close to its cytosolic end. (b) HA-Erv25 was expressed in wild-type (wt) cells or cells lacking the indicated components. Cycloheximide-chase experiments were performed and the samples analyzed by SDS-PAGE and blotting with anti-HA antibodies. The time-dependent mobility shift is caused by mannosylation of HA-Erv25 in the Golgi. Immunoblotting for phosphoglyceratekinase (Pgk1) served as a loading control. (c) Left: domain structure of Emp24, the binding partner of Erv25. Right: Cycloheximide-chase experiments of HA-Erv25 expressed in cells lacking Emp24. The cells also contained either an empty vector (e.v.) or plasmids encoding full-length (f.l.) Emp24, Emp24 lacking its TM (Emp24ΔTM), or Emp24 in which the TM was replaced with an HDEL ER-retention signal (Emp24ΔTM HDEL). (d) The two hydrophilic residues in the TM of HA-Erv25 were replaced by Ala. The stability of this protein was analyzed as in (c). (e) Hydrophilic residues in the TM of Erv25 (in blue) are shielded in the Emp24-Erv25 dimer and exposed in orphaned Erv25.

Article Snippet: The lysates were supplemented with SDS loading buffer, incubated at 65 °C for 10 min, spun in a table-top centrifuge (15,000 rpm, 1 min), and subjected to SDS-PAGE and immunoblotting with anti-HA antibody (clone 3F10 from rat, Roche, diluted 1:3000), anti-FLAG antibody (from rabbit, F7425, Sigma, 1:2000), anti-Pgk1 antibody (mouse monoclonal [22C5D8], #ab113687, AbCam, 1:5000), anti-mCherry polyclonal antibody (from rabbit,#26765-1-AP, Proteintech, 1:2000), anti-Myc antibody (rabbit polyclonal, #ab9106, AbCam, 1:2000), or anti-SBP antibody (mouse monoclonal, clone 20, #MAB10764, Millipore Sigma, 1:2000).

Techniques: Construct, Sequencing, SDS Page, Mobility Shift, Western Blot, Control, Binding Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: Lipid bilayer thinning near a ubiquitin ligase selects ER membrane proteins for degradation

doi: 10.1101/2025.10.31.685944

Figure Lengend Snippet: (a) HA-Emp24 was expressed from a CEN plasmid under the endogenous promoter in wild-type (wt) cells or cells lacking the indicated components. Cycloheximide-chase experiments were performed and the samples analyzed by SDS-PAGE and blotting with anti-HA antibodies. Immunoblotting for phosphoglyceratekinase (Pgk1) served as loading control. (b) Model for the partitioning of a TM with a hydrophilic residue (in blue) into the aqueous environment of Hrd1’s cytosolic cavity. (c) Membranes from cells containing FLAG-tagged wild-type (wt) Hrd1 or Hrd1 with Cys residues introduced at different positions were incubated with 5 kDa maleimide-polyethyleneglycol (mal-PEG) in the absence or presence of detergent, as indicated. Unreacted mal-PEG was quenched with cysteine followed by detergent solubilization and immunoprecipitation with anti-FLAG beads. The samples were analyzed by SDS-PAGE and blotting with anti-FLAG antibodies. The red arrows point to Hrd1 modified by mal-PEG in the absence of detergent. (d) Residues tested for mal-PEG modification are shown in a cartoon model of Hrd1 (pdbID: 6VJY), viewed from the ER lumen (top panel) and from the side (lower panel). Residues modified by mal-PEG are highlighted in green, residues resistant to modification in blue, and endogenous cysteines in yellow. The lateral gate is indicated by an asterisk.

Article Snippet: The lysates were supplemented with SDS loading buffer, incubated at 65 °C for 10 min, spun in a table-top centrifuge (15,000 rpm, 1 min), and subjected to SDS-PAGE and immunoblotting with anti-HA antibody (clone 3F10 from rat, Roche, diluted 1:3000), anti-FLAG antibody (from rabbit, F7425, Sigma, 1:2000), anti-Pgk1 antibody (mouse monoclonal [22C5D8], #ab113687, AbCam, 1:5000), anti-mCherry polyclonal antibody (from rabbit,#26765-1-AP, Proteintech, 1:2000), anti-Myc antibody (rabbit polyclonal, #ab9106, AbCam, 1:2000), or anti-SBP antibody (mouse monoclonal, clone 20, #MAB10764, Millipore Sigma, 1:2000).

Techniques: Plasmid Preparation, SDS Page, Western Blot, Control, Residue, Incubation, Immunoprecipitation, Modification

Journal: bioRxiv

Article Title: Lipid bilayer thinning near a ubiquitin ligase selects ER membrane proteins for degradation

doi: 10.1101/2025.10.31.685944

Figure Lengend Snippet: (a) The hydrophobic TM of Hrd3 has a short residence time near Hrd1 and therefore does not prevent loop insertion of the ERAD-L substrate CPY* into the Hrd1 complex, allowing CPY* polyubiquitination and degradation (first two panels). TMs with hydrophilic residues (blue) partition into Hrd1’s hydrophilic cavity and therefore have a long residence time, blocking CPY* loop insertion (last panel). (b) The degradation of HA-CPY* was followed by cycloheximide-chase experiments in Δhrd3 cells containing an empty vector (e.v.) or plasmids coding for full-length (f.l.) Hrd3, Hrd3 lacking its cytoplasmic tail (Δtail) or both its TM and tail (ΔTM+tail), or Hrd3 in which the endogenous TM was replaced by TMs from Spt23 (MLLFFWIPLTLVLLLCFTLS N LG) or Pdr5 (NYGIFICYIAF N YIAGVFFYWLA). Note the hydrophilic Asn residues in both TMs. Immunoblotting for phosphoglyceratekinase (Pgk1) served as a loading control. (c) As in (b), but with the endogenous TM of Hrd3 replaced with idealized TMs. (d) Disulfide crosslinking of the TM of Hrd3 with the lateral gate of Hrd1. HA-tagged Hrd3 with endogenous Cys773 in the TM (LVTMG C ILGIFLLSILMSTLAA) was expressed together with FLAG-tagged Hrd1 containing cysteines at the indicated positions in cells lacking Hrd3 and Hrd1. Disulfide bond formation was induced with an oxidant, and a detergent extract subjected to immunoprecipitation with FLAG antibodies. The samples were analyzed by non-reducing SDS-PAGE followed by blotting with HA- and FLAG-antibodies. (e) Space-filling model of Hrd1 with disulfide-crosslinking positions in red or pink, and non-crosslinking positions in blue.

Article Snippet: The lysates were supplemented with SDS loading buffer, incubated at 65 °C for 10 min, spun in a table-top centrifuge (15,000 rpm, 1 min), and subjected to SDS-PAGE and immunoblotting with anti-HA antibody (clone 3F10 from rat, Roche, diluted 1:3000), anti-FLAG antibody (from rabbit, F7425, Sigma, 1:2000), anti-Pgk1 antibody (mouse monoclonal [22C5D8], #ab113687, AbCam, 1:5000), anti-mCherry polyclonal antibody (from rabbit,#26765-1-AP, Proteintech, 1:2000), anti-Myc antibody (rabbit polyclonal, #ab9106, AbCam, 1:2000), or anti-SBP antibody (mouse monoclonal, clone 20, #MAB10764, Millipore Sigma, 1:2000).

Techniques: Blocking Assay, Plasmid Preparation, Western Blot, Control, Immunoprecipitation, SDS Page

Journal: bioRxiv

Article Title: Lipid bilayer thinning near a ubiquitin ligase selects ER membrane proteins for degradation

doi: 10.1101/2025.10.31.685944

Figure Lengend Snippet: (a) Asn residues were introduced into Sec61 at the indicated positions. The side chains are expected to face phospholipids inside the membrane. (b) HA-tagged wild-type (WT) Sec61 or mutants with Asn at the indicated positions were tested for degradation in wild-type (wt) cells by cycloheximide-chase experiments. The samples were analyzed by SDS-PAGE followed by immunoblotting with HA antibodies. Immunoblotting for phosphoglyceratekinase (Pgk1) served as a loading control. (c) As in (b), but for HA-Sec61 carrying the V375N mutation in cells lacking the indicated components. (d) As in (b) and (c), but for HA-Sec61 carrying either the V375N or V375D mutation. (e) CryoEM structure of a disulfide-crosslinked Hrd1 dimer complex in nanodiscs. Shown is a side view of the density map (in dark blue) with cartoon models for the two Hrd1 molecules (light blue and orange). The nanodisc density is shown in yellow. (f) Magnified views from the cytosol and lumen of the crosslinked TM3’s of the two Hrd1 molecules. (g) View of the Hrd1-containing nanodisc from the cytosol. Note the deep cavities. (h) View as in (g) with the Hrd1 molecules shown as cartoon models embedded in a semi-transparent space-filling model. The stars indicate the lateral gates formed between TMs 3 and 8.

Article Snippet: The lysates were supplemented with SDS loading buffer, incubated at 65 °C for 10 min, spun in a table-top centrifuge (15,000 rpm, 1 min), and subjected to SDS-PAGE and immunoblotting with anti-HA antibody (clone 3F10 from rat, Roche, diluted 1:3000), anti-FLAG antibody (from rabbit, F7425, Sigma, 1:2000), anti-Pgk1 antibody (mouse monoclonal [22C5D8], #ab113687, AbCam, 1:5000), anti-mCherry polyclonal antibody (from rabbit,#26765-1-AP, Proteintech, 1:2000), anti-Myc antibody (rabbit polyclonal, #ab9106, AbCam, 1:2000), or anti-SBP antibody (mouse monoclonal, clone 20, #MAB10764, Millipore Sigma, 1:2000).

Techniques: Membrane, SDS Page, Western Blot, Control, Mutagenesis

Journal: bioRxiv

Article Title: Lipid bilayer thinning near a ubiquitin ligase selects ER membrane proteins for degradation

doi: 10.1101/2025.10.31.685944

Figure Lengend Snippet: (a) HA-tagged wild-type (WT) Sec61 or mutants with Asn at the indicated positions were tested for degradation in cells lacking Hrd1 by cycloheximide-chase experiments. The samples were analyzed by SDS-PAGE followed by immunoblotting with anti-HA antibodies. Immunoblotting for phosphoglyceratekinase (Pgk1) served as a loading control. Note that the mutants are stable, whereas they are degraded in wild-type cells . (b) HA-Sec61 with position 375 mutated to either Asn or Asp was tested for degradation in wild-type (wt) cells or cells lacking the indicated components.

Article Snippet: The lysates were supplemented with SDS loading buffer, incubated at 65 °C for 10 min, spun in a table-top centrifuge (15,000 rpm, 1 min), and subjected to SDS-PAGE and immunoblotting with anti-HA antibody (clone 3F10 from rat, Roche, diluted 1:3000), anti-FLAG antibody (from rabbit, F7425, Sigma, 1:2000), anti-Pgk1 antibody (mouse monoclonal [22C5D8], #ab113687, AbCam, 1:5000), anti-mCherry polyclonal antibody (from rabbit,#26765-1-AP, Proteintech, 1:2000), anti-Myc antibody (rabbit polyclonal, #ab9106, AbCam, 1:2000), or anti-SBP antibody (mouse monoclonal, clone 20, #MAB10764, Millipore Sigma, 1:2000).

Techniques: SDS Page, Western Blot, Control