Structural and mechanistic basis of the EMC-dependent biogenesis of distinct transmembrane clients
- Molecular, Cellular, and Computational Biophysics Graduate Program, University of California, San Francisco, United States
- Department of Biochemistry and Biophysics, University of California, San Francisco, United States
- Department of Biology, Whitehead Institute, MIT, United States
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, United States
- Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Germany
- Biomedical Sciences Graduate Program, University of California, San Francisco, United States
- Department of Pharmaceutical Chemistry, University of California, San Francisco, United States
- Howard Hughes Medical Institute, United States
Figures
Figure 1 with 9 supplements
Experimental strategy for the dissection of EMC function.
Schematic representation of the combined structural and mutational approach to dissect EMC function. (A) yEMC was purified either by overexpression of all subunits together and affinity pulldown …
Figure 1—figure supplement 1
Purification of yEMC.
(A) Size-exclusion chromatography (SEC) purification of overexpressed yEMC+FAb in DDM detergent micelles. (B) SEC purification of endogenous yEMC + FAb in MSP1D1 nanodisc. (C) Coomassie-stained …
Figure 1—figure supplement 2
Purification of recombinant hEMC.
(A) Size-exclusion chromatography (SEC) purification of hEMC in GDN detergent. (B) SEC purification of hEMC reconstituted in MSP1D1 nanodiscs. (C) Coomassie-stained SDS-PAGE analysis of the SEC …
Figure 1—figure supplement 3
Fluorescent reporter cell line generation.
Fluorescent reporter cell lines were created by introducing lentivirus containing the fluorescently-tagged client reporters into five K562 cells lines: (1) wild type, (2) hEMC1 knockout (3) hEMC2 …
Figure 1—figure supplement 4
Overview of functional assays.
(A) Into each K562 cell line (WT or knockout of respective subunit), fluorescent client reporters were introduced lentivirally. Subsequently, knockout phenotypes were rescued by re-introducing the …
Figure 1—figure supplement 5
Western blots for EMC1 and EMC2.
Western blots of endogenous human EMC subunits and client proteins functional assay to check for complex stability and endogenous protein levels. For each mutant, abundance of several hEMC subunits …
Figure 1—figure supplement 6
Western blots for EMC3 and EMC5.
Human functional assay to check for complex stability. For each mutant, abundance of several EMC subunits as well as several representative client proteins was tested. (A) Western blots for hEMC3 …
Figure 1—figure supplement 7
Amino acid conservation of EMC1.
(A) Yeast (yEMC sequence from uniprot entry for Saccharomyces cerevisiae EMC1, sequence identifier: P25574-1) to human (hEMC sequence from uniprot entry for Homo sapiens EMC1 sequence identifier: …
Figure 1—figure supplement 8
Amino acid conservation of EMC2, EMC3, EMC5.
Yeast to human sequence alignments. (A) Alignment of hEMC2 and yEMC2. Computed by t-Coffee PSI-Coffee homology extension online server. Colored by ClustalX coloring – Blue for hydrophobic (AILMFWV); …
Figure 1—figure supplement 9
Genotyping of 10 mutants.
(A) Sanger sequencing of mutant aligned to wild-type sequence for hEMC1 T82M. (B) Sanger sequencing of mutant aligned to wild-type sequence for hEMC3 E63K+D213K+E223K. (C) Sanger sequencing of …
Figure 2 with 9 supplements
Overall structures of yeast and human EMC.
(A) Cryo-EM structure of yEMC in nanodiscs. Three orthogonal views of the yEMC cryo-EM structure shown as surface rendering. Gray bars delineate the approximate ER membrane boundaries with the …
Figure 2—figure supplement 1
Cryo-EM reconstruction of yEMC.
(A) Representative motion-corrected micrograph for yEMC-FabE in DDM. Scale-bar = 500 Å. (B) Representative motion-corrected micrograph for yEMC-FabH in DDM. Scale-bar = 500 Å. (C–D) Gallery of 2D …
Figure 2—figure supplement 2
Cryo-EM reconstruction of hEMC.
(A) Representative motion-corrected micrograph for hEMC in nanodiscs. Scale-bar = 500 Å. (B) Gallery of 2D classes for the final consensus particle set of hEMC in nanodiscs. (C) Angular distribution …
Figure 2—figure supplement 3
Cryo-EM data processing workflow for yEMC.
(A) Schematic of cryoEM data processing workflow for yEMC+Fab in b-DDM detergent micelles. (B) Schematic of cryoEM data processing workflow for yEMC+Fab in lipid nanodiscs.
Figure 2—figure supplement 4
Cryo-EM data processing workflow for hEMC.
(A) Schematic of cryoEM data processing workflow for hEMC in GDN detergent micelles. (B) Schematic of cryoEM data processing workflow for hEMC in lipid nanodiscs.
Figure 2—figure supplement 5
yEMC cryo-EM map validation.
(A) Final model-to-map FSC curve shown for yEMC in nanodiscs (left) and detergent (right). (B) Consensus yEMC nanodisc density shown superposed on the final yEMC nanodisc model. Three cytoplasmic …
Figure 2—figure supplement 6
hEMC cryo-EM map validation.
(A) Final model-to-map FSC curves shown for hEMC in nanodiscs (left) and detergent (right). (B) Consensus hEMC nanodisc density shown superposed on the final hEMC nanodisc model. Three cytoplasmic …
Figure 2—figure supplement 7
Subunit-subunit correspondence between yEMC and hEMC.
(A–D) EMC models from both human (A,C) and yeast (B,D) in lipid nanodiscs are colored with the same subunit color code, shown in the middle. Both cartoon ribbons and surface rendering are shown in …
Figure 2—figure supplement 8
Comparison between individual yEMC and hEMC subunits.
(A–H) Each panel shows a side-by-side comparison of homologous yeast and human EMC subunits, colored from N- (blue) to C-terminus (red). Schematics above each panel depict domain organization for …
Figure 2—figure supplement 9
Pairwise superposition of EMC structures in the PDB.
Pairwise superpositions between hEMC and yEMC from this work and recently published EMC structures. Alignments were performed with the matchmaker command in ChimeraX, in each case aligning on the …
Figure 3 with 3 supplements
The EMC cytoplasmic domain contains conserved functional interfaces and may engage C-tail-anchored clients directly.
(A) Position of the hEMC cytoplasmic domain relative to the membrane and the rest of the complex. Shown is the surface rendered hEMC structure reconstituted in nanodiscs. (B) EMC2 nucleates a …
Figure 3—figure supplement 1
Flow cytometry for mutations in the EMC cytoplasmic domain.
(A) Mutant hEMC2K125E+R126D+K127E with TMEM97-mCherry, B1AR-mCherry, and mCherry-SQS378-410 cell lines. Image of hEMC ND model displaying the residues mutated. (B) Mutant hEMC2K18A+K21A with …
Figure 3—figure supplement 2
Additional flow cytometry for mutations in the EMC cytoplasmic domain.
(A) Mutant hEMC2R266A+Q269A+R273A with TMEM97-mCherry, B1AR-mCherry, and mCherry- SQS378-410 cell lines. Snapshot of hEMC ND model displaying the residues mutated. (B) Mutant hEMC2R80E+R81E+K90E+R112…
Figure 3—figure supplement 3
Both EMC8 and EMC9 can be fitted into the hEMC cryo-EM maps.
(A) Superposition of hEMC8 (cryo-EM model, this work) and hEMC9 (X-ray model, PDB code 6Y4L) reveals strong structural homology between the two paralogous hEMC subunits. (B) Central slide through …
Figure 4
The EMC houses two transmembrane cavities with conserved core structures and distinct accessibilities.
(A) Location and composition of the lipid-filled cavity. A zoom-in view on the cavity is shown below, which is composed of EMC1, EMC3, EMC5, and EMC6. Resolved lipid densities from the cryo-EM map …
Figure 5 with 5 supplements
EMC houses an insertase module centered on EMC3 in the gated membrane cavity.
(A) A transmembrane gate anchored in the cytosol and the lumen is a structural hallmark of the EMC gated cavity. Shown is a surface rendering of the hEMC model in lipid nanodiscs with an unresolved …
Figure 5—figure supplement 1
Flow cytometry of gated cavity mutants.
(A) Mutant hEMC1K951A+K957A with TMEM97-mCherry, B1AR-mCherry, and mCherry- SQS378-410 cell lines. Snapshot of hEMC ND model displaying the residues mutated. (B) Mutant hEMC3R147E with …
Figure 5—figure supplement 2
Additional flow cytometry of gated cavity mutants.
(A) Mutant hEMC3K42A+K43A with TMEM97-mCherry, B1AR-mCherry, and mCherry- SQS378-410 cell lines. Snapshot of hEMC ND model displaying the residues mutated. (B) Mutant hEMC3K244A+H247A+E249A with …
Figure 5—figure supplement 3
Comparison of EMC3 to YidC-family members.
Structure and sequence comparison between EMC3 and YidC-family proteins. (A) Side-by-side structure view of human EMC3, yeast EMC3, E. coli YidC, and M. jannaschii YidC-like protein. hEMC3 and yEMC …
Figure 5—figure supplement 4
Resolved lipid densities in hEMC and yEMC nanodisc maps.
(A) Resolved lipids in the hEMC gated cavity. Left: The hEMC model is shown as surface rendering, colored by molecular lipophilicity potential (mint = hydrophilic, gold = hydrophobic). Black mesh …
Figure 5—figure supplement 5
Comparison of gate conformations.
(A) Comparison of gate helix conformations across hEMC and yEMC structures. In each panel, the model of hEMC in nanodiscs is shown as ribbons (color-coded as elsewhere in the manuscript). Cryo-EM …
Figure 6 with 2 supplements
A lipid-filled cavity in the EMC transmembrane domain stabilizes disparate client proteins.
(A) An EMC1 amphipathic brace helix delineates the boundary of the lipid-filled transmembrane cavity and packs against EMC5. Shown is a surface rendering of the hEMC model in nanodiscs. EMC4, EMC5, …
Figure 6—figure supplement 1
Flow cytometry of lipid-filled cavity mutants.
(A) Mutant hEMC5K7E with TMEM97-mCherry, B1AR-mCherry, and mCherry- SQS378-410 cell lines. Snapshot of hEMC ND model displaying the residues mutated. (B) Mutant hEMC5K7A with TMEM97-mCherry, …
Figure 6—figure supplement 2
Additional flow cytometry of lipid-filled cavity mutants.
(A) Mutant hEMC5F22L with TMEM97-mCherry, B1AR-mCherry, and mCherry- SQS378-410 cell lines. Snapshot of hEMC ND model displaying the residues mutated. (B) Mutant hEMC1M483A+R487H+Q491N with …
Figure 7 with 3 supplements
The large EMC lumenal domain is the site for several annotated disease mutations.
(A) Two views of the hEMC nanodisc structure. Two beta propellers are present in EMC1, one proximal to the membrane and one distal. (B) EMC1 is the largest EMC subunit and differs in size between …
Figure 7—figure supplement 1
Conformational heterogeneity of the hEMC lumenal domain between detergent and nanodisc maps.
(A) Superposition of hEMC in detergent (green) and nanodiscs (color-coded). Models were aligned on hEMC2 (cytoplasm) and hEMC5 (transmembrane), revealing a rotation of the lumenal domain. (B) …
Figure 7—figure supplement 2
Flow cytometry of lumenal domain mutants.
(A) Mutant hEMC1G471R with TMEM97-mCherry, B1AR-mCherry, and mCherry- SQS378-410 cell lines. Snapshot of hEMC ND model displaying the residues mutated. (B) Mutant hEMC1A144T with TMEM97-mCherry, …
Figure 7—figure supplement 3
Additional flow cytometry of lumenal domain mutants.
(A) Mutant hEMC1T82A with TMEM97-mCherry, B1AR-mCherry, and mCherry- SQS378-410 cell lines. Snapshot of hEMC ND model displaying the residues mutated. (B) Mutant hEMC1R76D+K80D with TMEM97-mCherry, …
Figure 8
Model of coordinated EMC functions.
(A) Model of EMC insertase function for a C-lumenal tail-anchored client. Cytosolic factors bring post-translationally localized clients to the ER. Then the client engages the EMC cytoplasmic …
Scheme 1
Client reporters.
Scheme 2
hEMC subunit mutation construct design.
Videos
Video 1
hEMC mutagenesis displayed on hEMC structures.
Three identical copies of hEMC in nanodisc (colored blue) are displayed here. Subsequent labeling and coloring of mutated residues by flow cytometric measure of reporter abundance, grouped into …
Video 2
EMC transmembrane cavity gate conformations.
Overview of hEMC colored and labeled by subunit. Volume fades away to hEMC nanodisc model. hEMC nanodisc model remains constant as segmented maps of the unassigned gate helices are shown of hEMC …
Video 3
hEMC lumenal domain differences between nanodisc and detergent models.
Overview of hEMC nanodisc model colored and labeled by subunit. Structural landmarks are labeled. hEMC detergent model (colored gray) fades in and both models rotate. As the models rotate several …
Tables
Table 1
Cryo-EM data acquisition, reconstruction, and model refinement statistics.
| yEMC in detergent dataset 1 | yEMC in detergent dataset 2 | yEMC in nanodiscs | hEMC in detergent | hEMC in nanodiscs | |
|---|---|---|---|---|---|
| EMDB accession code | EMD-23033 | EMD-23003 | EMD-11733 | EMD-11732 | |
| PDB accession code | PDB-7KTX | PDB-7KRA | PDB-7ADP | PDB-7ADO | |
| Data collection and processing | |||||
| Microscope | FEI Technai Polara | FEI Titan Krios | FEI Titan Krios | FEI Titan Krios | FEI Titan Krios |
| Camera | Gatan K2 Summit | Gatan K2 Summit | Gatan K3 | Gatan K3 | Gatan K3 |
| Magnification | 31,000x | 22,500x | 105,000x | 81,000x | 105,000x |
| Voltage (kV) | 300 | 300 | 300 | 300 | 300 |
| Electron exposure (e-/Å2) | 56.8 | 58.3 | 67 | 62 | 72 |
| Defocus range (µm) | −1.0 to −3.0 | −1.0 to −3.0 | −0.8 to −2.5 | 0.7–2.8 | 0.7–2.8 |
| Pixel size (Å) | 1.22 | 1.31 | 0.853 | 1.094 | 0.8512 |
| Software | Relion 2.0, Relion 3.0, THUNDER | Relion 2.0, Relion 3.0, THUNDER | Relion 3.0, cryoSPARC v2 | Relion 3.0, cryoSPARC v2 | Relion 3.0, cryoSPARC v2 |
| Symmetry imposed | C1 | C1 | C1 | C1 | C1 |
| Initial particle images (no.) | 419,907 | 670,078 | 6,100,000 | 3,350,000 | 5,900,000 |
| Final particle images (no.) | 83,599 | 170,186 | 230,528 | 144,222 | 177,560 |
| Overall map resolution (Å) | 8 | 7 | |||
| FSC threshold 0.143 | 4.3 (combined) | 3.2 | 3.60 | 3.39 | |
| Local map resolution range (Å) | 3.6–6.4 | 2.6–6.4 | 2.8–6.0 | 3.0–7.2 | |
| Refinement | |||||
| Software | Phenix 1.18 real-space-refine | Phenix 1.18 real-space-refine | Phenix 1.18 real-space-refine | Phenix 1.18 real-space-refine | |
| Model resolution (Å) | |||||
| FSC threshold 0.5 | 4.5 | 3.5 | 3.9 | 3.6 | |
| Map sharpening B factor (Å2) | −125 | −75 | −115 | −126 | |
| Model composition | |||||
| Non-hydrogen atoms | 17,315 | 17,293 | 15,040 | 16,652 | |
| Protein residues | 2171 | 2164 | 1880 | 2086 | |
| Ligands | NAG: 6 | NAG: 5; PCW: 1 | NAG: 2 | NAG: 4; PCW: 5 | |
| B factors (Å2) | |||||
| Protein (mean) | 167 | 107 | 111 | 126 | |
| Ligand (mean) | 146 | 92 | 107 | 127 | |
| R.m.s. deviations | |||||
| Bond lengths (Å) | 0.006 | 0.005 | 0.005 | 0.005 | |
| Bond angles (°) | 0.988 | 0.804 | 0.718 | 0.782 | |
| Validation | |||||
| MolProbity score | 1.44 | 1.29 | 1.46 | 1.28 | |
| Clashscore | 8.1 | 3.5 | 9 | 6 | |
| Ramachandran plot | |||||
| Favored (%) | 98 | 97 | 97 | 97 | |
| Allowed (%) | 2 | 3 | 3 | 3 | |
| Disallowed (%) | 0 | 0 | 0 | 0 | |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information | |
|---|---|---|---|---|---|
| Gene (Homo sapiens) | hEMC1 | NIH Mammalian Gene Collection | NCBI: BC034589 | ||
| Gene (Homo sapiens) | hEMC2 | NIH Mammalian Gene Collection | NCBI: BC021667 | ||
| Gene (Homo sapiens) | hEMC3 | NIH Mammalian Gene Collection | NCBI: BC022807 | ||
| Gene (Homo sapiens) | hEMC4 | Genestrand (Eurofins, Germany) | Uniprot: Q5J8M3-1 | ||
| Gene (Homo sapiens) | hEMC5 | NIH Mammalian Gene Collection | NCBI: BC033588 | ||
| Gene (Homo sapiens) | hEMC6 | NIH Mammalian Gene Collection | NCBI: BC001409 | ||
| Gene (Homo sapiens) | hEMC7 | NIH Mammalian Gene Collection | NCBI: BC104936 | ||
| Gene (Homo sapiens) | hEMC8 | NIH Mammalian Gene Collection | NCBI: BC020250 | ||
| Gene (Homo sapiens) | hEMC9 | NIH Mammalian Gene Collection | NCBI: BC002491 | ||
| Gene (Homo sapiens) | hEMC10 | Genestrand (Eurofins, Germany) | Uniprot: Q5UCC4-1 | ||
| Gene (Saccharomyces cerevisiae) | yEMC1 | Uniprot | Uniprot: P25574 | ||
| Gene (Saccharomyces cerevisiae) | yEMC2 | Uniprot | Uniprot: P47133 | ||
| Gene (Saccharomyces cerevisiae) | yEMC3 | Uniprot | Uniprot: P36039 | ||
| Gene (Saccharomyces cerevisiae) | yEMC4 | Uniprot | Uniprot: P53073 | ||
| Gene (Saccharomyces cerevisiae) | yEMC5 | Uniprot | Uniprot: P40540 | ||
| Gene (Saccharomyces cerevisiae) | yEMC6 | Uniprot | Uniprot: Q12431 | ||
| Gene (Saccharomyces cerevisiae) | yEMC7 | Uniprot | Uniprot: P39543 | ||
| Gene (Saccharomyces cerevisiae) | yEMC10 | Uniprot | Uniprot: Q12025 | ||
| Recombinant DNA reagent | pX458 | Addgene | pX458 | ||
| Recombinant DNA reagent | pKDP041 | This study; available from the Weissman Lab | Cas9-sfGFP- EMC5 sgRNA3 | single guide KO system targeting EMC5 gene | |
| Recombinant DNA reagent | pKDP077 | This study; available from the Weissman Lab | Cas9-sfGFP-EMC1_ sgRNA3_sgRNA4 | dual guide KO system targeting EMC1 gene | |
| Recombinant DNA reagent | pKDP080 | This study; available from the Weissman Lab | Cas9-sfGFP-EMC2_ sgRNA4_sgRNA5 | dual guide KO system targeting EMC2 gene | |
| Recombinant DNA reagent | pKDP083 | This study; available from the Weissman Lab | Cas9-sfGFP-EMC3_ sgRNA1_sgRNA2 | dual guide KO system targeting EMC3 gene | |
| Recombinant DNA reagent | pKDP119 | This study; available from the Weissman Lab | SFFV-insert site- IRES-Puro-P2A-BFP | parental vector | |
| Recombinant DNA reagent | pKDP121 | This study; available from the Weissman Lab | pTwist+Lenti+SFFV+ EMC1+IRES+Puro+ P2A+BFP+WPRE | EMC1 covering plasmid | |
| Recombinant DNA reagent | pKDP122 | This study; available from the Weissman Lab | pTwist+Lenti+SFFV+ EMC3+IRES+Puro+ P2A+BFP+WPRE | EMC3 covering plasmid | |
| Recombinant DNA reagent | pKDP124 | This study; available from the Weissman Lab | pTwist+Lenti+SFFV+ EMC5+IRES+Puro+ P2A+BFP+WPRE | EMC5 covering plasmid | |
| Recombinant DNA reagent | pKDP125 | This study; available from the Weissman Lab | pTwist+Lenti+SFFV+ EMC2+IRES+Puro+ P2A+BFP+WPRE | EMC2 covering plasmid | |
| Recombinant DNA reagent | pKDP110 | This study; available from the Weissman Lab | bAR1_mCherry_ P2A_GFP | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP111 | This study; available from the Weissman Lab | TMEM97_mCherry_ P2A_GFP | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP136 | This study; available from the Weissman Lab | GFP_P2A_mCherry_ SQS_TMD_opsintag | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC1_ mut_D31K | Twist; available from the Weissman Lab | hsEMC1_mut_D31K | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC1_ mut_R69D | Twist; available from the Weissman Lab | hsEMC1_mut_R69D | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC1_ mut_G71S | Twist; available from the Weissman Lab | hsEMC1_mut_G71S | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_ hsEMC1_mut_ R76D_K80D | Twist; available from the Weissman Lab | hsEMC1_mut_ R76D_K80D | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC1_ mut_T82M | Twist; available from the Weissman Lab | hsEMC1_mut_T82M | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC1_ mut_T82A | Twist; available from the Weissman Lab | hsEMC1_mut_T82A | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC1_ mut_A144T | Twist; available from the Weissman Lab | hsEMC1_mut_A144T | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC1_ mut_H93D_E138D_N282K | Twist; available from the Weissman Lab | hsEMC1_mut_H93D_ E138D_N282K | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC1_ mut_R275E_R404E | Twist; available from the Weissman Lab | hsEMC1_mut_ R275E_R404E | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC1_ mut_G471R | Twist; available from the Weissman Lab | hsEMC1_mut_G471R | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC1_ mut_F473Y_R487K | Twist; available from the Weissman Lab | hsEMC1_mut_ F473Y_R487K | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC1_ mut_M483A_ R487H_Q491N | Twist; available from the Weissman Lab | hsEMC1_mut_M483A_ R487H_Q491N | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC1_ mut_G868R | Twist; available from the Weissman Lab | hsEMC1_mut_G868R | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC1_ mut_R881C | Twist; available from the Weissman Lab | hsEMC1_mut_R881C | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC1_ mut_K951A_K957A | Twist; available from the Weissman Lab | hsEMC1_mut_ K951A_K957A | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC2_ mut_K18A_K21A | Twist; available from the Weissman Lab | hsEMC2_mut_ K18A_K21A | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC2_ mut_R80A_R81A_ K90A_R112A | Twist; available from the Weissman Lab | hsEMC2_mut_R80A_ R81A_K90A_R112A | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC2_ mut_K125E_R126D_K127E | Twist; available from the Weissman Lab | hsEMC2_mut_K125E_ R126D_K127E | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC2_ mut_N137A_N167A | Twist; available from the Weissman Lab | hsEMC2_mut_ N137A_N167A | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC2_ mut_E146A_E149A_Q150A | Twist; available from the Weissman Lab | hsEMC2_mut_E146A_ E149A_Q150A | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC2_ mut_E168A_D170A_K173A | Twist; available from the Weissman Lab | hsEMC2_mut_E168A_ D170A_K173A | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC2_ mut_E206A_E209A_D252A | Twist; available from the Weissman Lab | hsEMC2_mut_E206A_ E209A_D252A | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC2_ mut_K248E_D252K_K255E | Twist; available from the Weissman Lab | hsEMC2_mut_K248E_ D252K_K255E | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC2_mut_ R266A_Q269A_R273A | Twist; available from the Weissman Lab | hsEMC2_mut_R266A_ Q269A_R273A | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC2_mut_ Q269A_E286A_E290A | Twist; available from the Weissman Lab | hsEMC2_mut_Q269A_ E286A_E290A | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC3_WT | Twist; available from the Weissman Lab | hsEMC3_WT | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC3_ mut_D9A | Twist; available from the Weissman Lab | hsEMC3_mut_D9A | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC3_ mut_R13E | Twist; available from the Weissman Lab | hsEMC3_mut_R13E | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC3_ mut_K42A_K43A | Twist; available from the Weissman Lab | hsEMC3_mut_ K42A_K43A | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC3_ mut_E63K_D213K_E223K | Twist; available from the Weissman Lab | hsEMC3_mut_E63K_ D213K_E223K | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC3_ mut_K70Y | Twist; available from the Weissman Lab | hsEMC3_mut_K70Y | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC3_ mut_V118A_I122A | Twist; available from the Weissman Lab | hsEMC3_mut_ V118A_I122A | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC3_ mut_N114D_N117D | Twist; available from the Weissman Lab | hsEMC3_mut_ N114D_N117D | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC3_ mut_R180A | Twist; available from the Weissman Lab | hsEMC3_mut_R180A | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC3_ mut_R59E_R62E_K216E | Twist; available from the Weissman Lab | hsEMC3_mut_R59E_ R62E_K216E | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC3_ mut_R147E | Twist; available from the Weissman Lab | hsEMC3_mut_R147E | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC3_ mut_F148L | Twist; available from the Weissman Lab | hsEMC3_mut_F148L | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC3_ mut_M151L | Twist; available from the Weissman Lab | hsEMC3_mut_M151L | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC3_ mut_I186V_I182V | Twist; available from the Weissman Lab | hsEMC3_mut_ I186V_I182V | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC3_ mut_K244A_ H247A_E249A | Twist; available from the Weissman Lab | hsEMC3_mut_K244A_ H247A_E249A | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC5_WT | Twist; available from the Weissman Lab | hsEMC5_WT | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC5_ mut_A18L | Twist; available from the Weissman Lab | hsEMC5_mut_A18L | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC5_ mut_D44K | Twist; available from the Weissman Lab | hsEMC5_mut_D44K | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC5_ mut_D82A_R85A | Twist; available from the Weissman Lab | hsEMC5_mut_ D82A_R85A | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC5_ mut_F22L | Twist; available from the Weissman Lab | hsEMC5_mut_F22L | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC5_ mut_E75A | Twist; available from the Weissman Lab | hsEMC5_mut_E75A | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC5_ mut_H19L_S23A_Q26L | Twist; available from the Weissman Lab | hsEMC5_mut_H19L_ S23A_Q26L | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC5_ mut_K7A | Twist; available from the Weissman Lab | hsEMC5_mut_K7A | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC5_ mut_K7E | Twist; available from the Weissman Lab | hsEMC5_mut_K7E | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC5_ mut_R28A_R32A | Twist; available from the Weissman Lab | hsEMC5_mut_ R28A_R32A | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC5_ mut_I63L | Twist; available from the Weissman Lab | hsEMC5_mut_I63L | See Supplementary file 5 for sequence | |
| Recombinant DNA reagent | pKDP119_hsEMC5_ mut_F90A | Twist; available from the Weissman Lab | hsEMC5_mut_F90A | See Supplementary file 5 for sequence | |
| Antibody | Mouse GAPDH Primary Antibody | Abcam | ab8245 | See Supplementary file 5 for sequence | |
| Antibody | Rabbit TMEM97 primary | ThermoFisher Scientific | PA-23003 | ||
| Antibody | Rabbit FDFT1 Primary Antibody | Abcam | ab195046 | ||
| Antibody | Rat BAP31 Primary Antibody | ThermoFisher Scientific | MA3-002 | ||
| Antibody | Rabbit (KIAA0090) EMC1 primary antibody | Abcam | ab242112 | ||
| Antibody | Rabbit TTC35 (EMC2) primary antibody | Proteintech | 25443–1-AP | ||
| Antibody | Rabbit TM111 (EMC3) primary antibody | ThermoFisher Scientific | #711771 | ||
| Antibody | Rabbit EMC4 primary antibody | Abcam | ab184544 | ||
| Antibody | Rabbit MMGT1 (EMC5) primary antibody | Bethyl Laboratories | A305-833A-M | ||
| Antibody | Rabbit (C19orf63) EMC10 primary antibody | Abcam | ab180148 | ||
| Antibody | IRDye 800CW Goat anti-Mouse IgG Secondary Antibody | LI-COR Biosciences | 925–32210 | ||
| Antibody | IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody | LI-COR Biosciences | 926–32211 | ||
| Peptide, recombinant protein | Fab DE4 | This study; available from the Weissman Lab | LMV83 | LFAIPLVVPFYSHSALDVVMTQSPLSLPV TPGEPASISCRSSQTLMNRNGNNFLDW YVQKPGQSPQLLIYLGSNRAPGVPDRFS GSGSGTDFTLKISRLEVEDVGVYYCMQA LQTPRTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC-- MAQVQLQQWGAGLLKPSETLSLTCAVYG GSFSGYYWSWIRQPPGKGLEWIGEINHS GSTNYNPSLKSRVTISVDTSKKQFSLKLS SVTAADTAVYYCARFSYYGSGIYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCAAAHHH HHHGAAEQKLISEEDLNGAA- | |
| Peptide, recombinant protein | Fab DH4 | This study; available from the Weissman Lab | LMV82 | LFAIPLVVPFYSHSALDVVMTQSPLSLPV TPGEPASISCRSSQTLMNRNGNNFLDW YLQKPGQSPQLLIYLGSNRAPGVPDRFS GSGSGTDFTLRISRVEPEDVGVYYCMQA LQTPSFGGGTKVEIRRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC-- MAQVQLQQWGAGLLKPSETLSLTCAVY GGSFSGYYWSWIRQPPGKGLEWIGEIN HSGSTNYNPSLKSRVTISVDTSKNQFSL KLSSVTAADTAVYYCARGLAGRGYYGSG SYLRWGQGTLVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCAAAHHHHHHGAAE QKLISEEDLNGAA- | |
| Commercial assay or kit | Superose 6, 10/300 GL | GE Healthcare | 17517201 | ||
| Commercial assay or kit | R1.2/1.3 200 and 300 mesh Cu holey carbon grids | Quantifoil | 1210627 | ||
| Commercial assay or kit | BL21 Gold Star competent cells | Invitrogen | C602003 | ||
| Commercial assay or kit | Anti-Flag agarose beads | Millipore | A2220 | ||
| Commercial assay or kit | EconoPac Chromatography Columns | Biorad | 7321010 | ||
| Commercial assay or kit | 100 KD MW | EMD Millipore | UFC810024 | ||
| Commercial assay or kit | Superose 6, 10/300 GL | Cytiva | 29-0915-96 | ||
| Commercial assay or kit | cOmplete EDTA-free Protease Inhibitor Cocktail | Roche | catalog No. 05056489001 | ||
| Commercial assay or kit | Bio-Beads | Biorad | 1523920 | ||
| Commercial assay or kit | R1.2/1.3 200 and 300 mesh Cu holey carbon grids | Quantifoil | 1210627 | ||
| Commercial assay or kit | Ultrathin Carbon Film on Lacey Carbon Support Film, 400 mesh, Copper | Ted Pella | #01824 | ||
| Chemical compound, drug | FuGENE HD transfection reagent | Promega | E2312 | ||
| Chemical compound, drug | 1-Palmitoyl-2-oleoyl-sn- glycero-3-PC (POPC) | Cayman Chemical | 15102 | ||
| Chemical compound, drug | Glyco-diosgenin (GDN) | Anatrace | GDN101 | ||
| Chemical compound, drug | yeast extract total | Avanti Polar Lipids | 190000 P-100mg | ||
| Chemical compound, drug | Cholesteryl Hemisuccinate Tris Salt | Anatrace | CH210 5 GM | ||
| Chemical compound, drug | b-DDM | Anatrace | D310 | ||
| Chemical compound, drug | IPTG | GoldBio | I2481C5 | ||
| Chemical compound, drug | EX-CELL 420 Serum-Free Medium | Sigma-Aldrich | 14420 C | ||
| Chemical compound, drug | FreeStyle 293 Expression Medium | Thermo fischer | 12338018 | ||
| Cell line (Homo sapiens) | HEK293S GnTI- | ATCC | CRL-3022 | Mycoplasma negative | |
| Cell line (Spodoptera frugiperda) | Sf9 | Thermo Fischer | 11496015 | ||
| Cell line (Homo sapiens) | K562 crispri | Gilbert et al., 2014 | K562 crispri | ||
| Strain, strain background Saccharomyces cerevisiae | Overexpressed EMC with yEMC5-linker- TEV-linker-3xFlag | This study; available from the Weissman Lab | LMV84 | BY4743 ---- MATa/alpha, his3∆0/his3∆0, leu2∆0/leu2∆0, LYS2/lys2∆0, met15∆0/MET15, ura3∆0/ura3∆0, emc1::NatMX:: TEF2pr-EMC1/EMC1, emc3:: KanMX::TEF2pr-EMC3/EMC3, emc4::his3(CG)::TEF2pr-EMC4/ EMC4, sop4::HphMx::TEF2pr- SOP4/SOP4, EMC2/emc2::NatMX:: TEF2pr-EMC2, emc5::EMC5-TEV- 3xFLAG::ura3(KL)/emc5::his3(CG):: TEF2pr-EMC5-TEV-3xFLAG::KanMX, EMC6/emc6::HphMX::TEF2pr-EMC6, YDR056c/ydr056c::leu2(CG):: TEF2pr-ydr056c | |
| Strain, strain background Saccharomyces cerevisiae | Endogenous yEMC5- linker-TEV-linker-3xFlag | This study; available from the Weissman Lab | LMV85 | W303 ---- EMC5-3xF:ura - Linker-TEV- linker-3xFlag (GGSGSGENLYFQSGSGS DYKDDDDKDYKDDDDKDYKDDDDK) | |
| Software, algorithm | CryoSPARC version 2.12.4. | Punjani et al., 2017 | RRID:SCR_016501 | ||
| Software, algorithm | UCSF ChimeraX Version 1.0 | Goddard et al., 2018 | RRID:SCR_015872 | ||
| Software, algorithm | PHENIX Version 1.17 | Adams et al., 2011; | RRID:SCR_014224 | ||
| Software, algorithm | Coot Version 0.8 | Emsley et al., 2010 | RRID:SCR_014222 | ||
| Software, algorithm | RELION 3.1 | Kimanius et al., 2016; Zivanov et al., 2018 | http://www2.mrclmb.cam.ac.uk/relion | ||
| Software, algorithm | SerialEM | Mastronarde, 2005 | RRID:SCR_017293 | ||
Additional files
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Supplementary file 1
Mass spectrometry analysis on purified hEMC.
SEC purified hEMC in detergent (sheet 1) or nanodiscs (sheet 2) were subjected to tryptic digestion and mass spectrometry. The tables list identified proteins sorted by iBAQ score (descending order). EMC subunits are highlighted in yellow.
- https://cdn.elifesciences.org/articles/62611/elife-62611-supp1-v3.xlsx
-
Supplementary file 2
Statistical significance values for flow cytometry data.
Table listing p-values for membrane controls (Sheet 1; relates to Figure 1—figure supplement 3) and flow cytometry for each of the three client reporters (Sheets 2, 3 and 4; relates to Main Figures 3 and 5–7 and figures supplements to those figures).
- https://cdn.elifesciences.org/articles/62611/elife-62611-supp2-v3.xlsx
-
Supplementary file 3
Comparison of EMC point mutant effects on client proteins.
Table listing point mutagenesis performed on hEMC and yEMC and assayed against different client types.
- https://cdn.elifesciences.org/articles/62611/elife-62611-supp3-v3.xlsx
-
Supplementary file 4
Uncropped western blots.
Blots provided here without cropping, related to Figure 1—figure supplements 5–6.
- https://cdn.elifesciences.org/articles/62611/elife-62611-supp4-v3.zip
-
Supplementary file 5
Plasmid sequences for hEMC mutants and reporters.
Table listing sequences of point mutagenesis plasmids used in the hEMC functional assay in this study.
- https://cdn.elifesciences.org/articles/62611/elife-62611-supp5-v3.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/62611/elife-62611-transrepform-v3.pdf
