An amphipathic helix in Brl1 is required for nuclear pore complex biogenesis in S. cerevisiae
- Institute of Biochemistry, Department of Biology, ETH Zurich, Switzerland
- Department of Biochemistry, University of Zurich, Switzerland
- Department of Plant and Environmental Sciences, Weizmann Institute of Science, Israel
- Institute of Biochemistry and Molecular Cell Biology, Medical School, RWTH Aachen University, Germany
- Department of Biological Sciences, University of Bergen, Norway
Figures
Figure 1 with 1 supplement
Brl1 preferentially binds young nuclear pore complexes (NPCs).
(A) Scheme of the NPC architecture. The colors indicate the assembly order as found in Onischenko et al., 2020. Nucleoporins (NUPs) that were reproducibly identified in Brl1 affinity purifications …
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Figure 1—source data 1
Label-free Brl1 intensities with 10 nucleoporin (NUP) baits from Onischenko et al., 2020; related to Figure 1C.
- https://cdn.elifesciences.org/articles/78385/elife-78385-fig1-data1-v2.xlsx
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Figure 1—source data 2
Pearson’s correlation coefficients for Brl1-mCherry and new or old Nup170-yeGFP.
- https://cdn.elifesciences.org/articles/78385/elife-78385-fig1-data2-v2.xlsx
Figure 1—figure supplement 1
Proteomic characterization of Brl1 nuclear pore complex (NPC) interactions.
(A) All 1500 proteins co-purified in affinity pulldowns with 10 different nucleoporin (NUP) baits (Onischenko et al., 2020) were ranked by their fold enrichment difference between early and late …
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Figure 1—figure supplement 1—source data 1
Label-free nucleoporin/nuclear transport receptor (NUP/NTR) intensities with Brl1 bait (related to Figure 1—figure supplement 1B).
- https://cdn.elifesciences.org/articles/78385/elife-78385-fig1-figsupp1-data1-v2.xlsx
Figure 2
Mapping Brl1 association with nuclear pore complex (NPC) assembly intermediates using KARMA (Kinetic Analysis of Incorporation Rates in Macromolecular Assemblies).
(A) Principles of KARMA: newly synthesized proteins are pulse-labeled followed by the affinity purification of the nucleoporin (NUP) complexes through a tagged NPC-binding protein. The extent of …
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Figure 2—source data 1
Fractional labeling of nucleoporins/nuclear transport receptors (NUPs/NTRs) in Brl1 KARMA (Kinetic Analysis of Incorporation Rates in Macromolecular Assemblies) assays (related to Figure 2 and Figure 1—figure supplement 1).
- https://cdn.elifesciences.org/articles/78385/elife-78385-fig2-data1-v2.xlsx
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Figure 2—source data 2
Inaccessible pools as estimated by the three-step kinetic state model (KSM) (Onischenko et al., 2020) for Brl1 and NUP baits (related to Figure 2C).
- https://cdn.elifesciences.org/articles/78385/elife-78385-fig2-data2-v2.xlsx
Figure 3 with 5 supplements
Brl1 degradation interferes with nuclear pore complex (NPC) assembly.
(A) Tomographic slices of focused ion beam (FIB)-milled, 4–4.5 hr auxin-treated Brl1-AID cells showing the structures quantified in (B). Image frames colored according to the color code used in (B). …
Figure 3—figure supplement 1
Characterization and subtomogram analysis of Brl1 depletion.
(A) Auxin-inducible degradation of Brl1 (Nishimura et al., 2009). Interaction between degron-tagged Brl1 and the E3 ubiquitin ligase SCF is mediated by the auxin-binding receptor OsTir1. (B) …
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Figure 3—figure supplement 1—source data 1
Uncropped Western blots (related to Figure 3—figure supplement 1B, Figure 5E and F).
- https://cdn.elifesciences.org/articles/78385/elife-78385-fig3-figsupp1-data1-v2.pdf
Figure 3—video 1
Sequential sections of a cryo-tomogram from Brl1-depleted cells.
The tomogram is 6× binned; pixel size: 2.1 nm; scale bar: 100 nm.
Figure 3—video 2
Sequential sections of a cryo-tomogram from Brl1- depleted cells.
The tomogram is 6x binned; pixelsize: 2.1nm; scalebar: 100nm.
Figure 3—video 3
Sequential sections of a cryo-tomogram from non-depleted Brl1 control cells.
The tomogram is 6× binned; pixel size: 2.1 nm; scale bar: 100 nm.
Figure 3—video 4
Sequential sections of a cryo-tomogram from non-depleted Brl1 control cells.
The tomogram is 6x binned; pixelsize: 2.1nm; scalebar: 100nm.
Figure 4 with 1 supplement
Proteomic characterization of nuclear pore complex (NPC) assembly intermediates induced by Brl1 depletion.
(A) Depiction of the metabolic labeling assays to examine NPC assembly effects that occur upon Brl1 degradation. Newly synthesized proteins are pulse-labeled simultaneously with the auxin-induced …
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Figure 4—source data 1
Fractional labeling in Brl1-AID Nup170 affinity pulldowns (APs) with and without auxin treatment (related to Figure 4).
- https://cdn.elifesciences.org/articles/78385/elife-78385-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Exchange rates of nuclear pore complex (NPC) assembly intermediates in Brl1-depleted cells.
(A) Correlation between normalized nucleoporin (NUP) fractional labeling observed in Brl1 KARMA (Kinetic Analysis of Incorporation Rates in Macromolecular Assemblies) assays with the normalized …
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Figure 4—figure supplement 1—source data 1
Fractional labeling in the lysis intermixing tests for Nup170 bait (related to Figure 4—figure supplement 1).
- https://cdn.elifesciences.org/articles/78385/elife-78385-fig4-figsupp1-data1-v2.xlsx
Figure 5 with 2 supplements
A conserved luminal amphipathic helix binds to membranes and is essential for Brl1 function.
(A) AlphaFold prediction for Brl1 (Jumper et al., 2021). Unstructured termini are not shown; blue: N-terminus; red: C-terminus. Transmembrane domain highlighted by the lipid bilayer. (B) Predicted …
Figure 5—figure supplement 1
AlphaFold structure prediction for Brl1.
(A) Predicted structure for full-length Brl1 using AlphaFold2, colored based on the predicted local distance difference test score (pLDDT). (B) pLDDT score for Brl1. High-confidence region shown in …
Figure 5—figure supplement 2
Conservation and structure prediction of Brl1 homologues in different fungi.
(A) Sequence alignment of full-length Brl1 homologues, black box indicates the zoom-in in panel. (B) Hydrophobic: blue, negative: magenta, red: positive polar: green, glycine: orange, proline: …
Figure 6 with 1 supplement
Overexpression of Brl1(I395D) with an impaired amphipathic helix interferes with nuclear pore complex (NPC) assembly.
(A) Spotting assay of wildtype cells expressing Brl1, Brl1Δah, or Brl1(I395D) from the GAL1 promoter in glucose or galactose-containing medium. (B) Localization of yEGFP-tagged Brl1, Brl1Δah, or …
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Figure 6—source code 1
Fluorescence recovery after photobleaching (FRAP) analysis.
- https://cdn.elifesciences.org/articles/78385/elife-78385-fig6-code1-v2.zip
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Figure 6—source data 1
Line plots.
- https://cdn.elifesciences.org/articles/78385/elife-78385-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Luminal amphipathic helix (AH) of Brl1 is involved in nuclear pore complex (NPC) biogenesis.
(A) Spotting assay of fivefold serial dilutions of nup116ΔGLFG PMET3-NUP188 cells expressing various ahBrl1 domain mutants from the GAL1 promoter. (B) Co-localization of mCherry-tagged Brl1 or …
Figure 7 with 4 supplements
Brl1(I395D) overexpression leads to the formation of multilayered nuclear envelope (NE) herniations.
(A) Tomographic slices of the nuclear pore complex (NPC)-like structures quantified in (B), observed in focused ion beam (FIB)-milled cells overexpressing Brl1(I395D), scale bar: 100 nm. N: nucleus, …
Figure 7—video 1
Sequential sections of a cryo-tomogram from Brl1(I395D)-overexpressing cells.
The tomogram is 6× binned; pixel size: 2.1 nm; scale bar: 100 nm.
Figure 7—video 2
Sequential sections of a cryo-tomogram from Brl1(I395D)-overexpressing cells.
The tomogram is 6x binned; pixelsize: 2.1nm; scalebar: 100nm.
Figure 7—video 3
Sequential sections of a cryo-tomogram from Brl1-overexpressing cells.
The tomogram is 6× binned; pixel size: 2.1 nm; scale bar: 100 nm.
Figure 7—video 4
Sequential sections of a cryo-tomogram from Brl1-overexpressing cells.
The tomogram is 6x binned; pixelsize: 2.1nm; scalebar: 100nm.
Figure 8
The role of Brl1 during nuclear pore complex (NPC) assembly.
(A) Brl1 (red) enriches on the inside of NPC maturation intermediates and promotes inner nuclear membrane–outer nuclear membrane (INM-ONM) fusion through the membrane-binding amphipathic helix (AH) …
Appendix 1—figure 1
Control of protein labeling in KARMA (Kinetic Analysis of Incorporation Rates in Macromolecular Assemblies) assays.
(A) Fractional labeling of nucleoporins (NUPs) and nuclear transport receptors (NTRs) in KARMA assays with Brl1 bait and the respective source lysate, 90 min after labeling onset. Median ± SD of …
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Appendix 1—figure 1—source data 1
Fractional labeling in the source lysate (parallel reaction monitoring [PRM] assay, related to Appendix 1—figure 1A).
- https://cdn.elifesciences.org/articles/78385/elife-78385-app1-fig1-data1-v2.xlsx
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Appendix 1—figure 1—source data 2
Fractional labeling in the lysis intermixing tests with Brl1 bait (related to Appendix 1—figure 1B–D).
- https://cdn.elifesciences.org/articles/78385/elife-78385-app1-fig1-data2-v2.xlsx
Appendix 1—figure 2
Potential maturation processes of onion-like herniations.
(A) Tomographic slice of an onion-like herniation and an example line plot with fitted Gaussians measured at the indicated red line. Brackets indicate how the distances were classified for the plot …
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Appendix 1—figure 2—source data 1
Distance measurements.
- https://cdn.elifesciences.org/articles/78385/elife-78385-app1-fig2-data1-v2.xlsx
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Chemical compound, drug | IP6 | Sigma-Aldrich | P5681 | AID experiments |
| Chemical compound, drug | Auxin | Sigma-Aldrich | I2886 | AID experiments |
| Chemical compound, drug | β-Estradiol | Sigma-Aldrich | E8875 | RITE experiments |
| Chemical compound, drug | Hygromycin B | Roche | 10843555001 | RITE experiments |
| Chemical compound, drug | Concanavalin A (ConA) | Sigma-Aldrich | C2010 | Fluorescence microscopy |
| Chemical compound, drug | IPTG | AppliChem | A10080025 | Protein expression |
| Chemical compound, drug | Protease inhibitor cocktail | Sigma-Aldrich | P8215 | MS assays |
| Chemical compound, drug | Purified IgG protein from rabbit serum | Sigma-Aldrich | I5006 | MS assays |
| Chemical compound, drug | Coomassie Brilliant Blue R-250 | Bio-Rad | 161-0400 | MS assays |
| Chemical compound, drug | Sequencing grade porcine trypsin | Promega | V5113 | MS assays |
| Chemical compound, drug | L-Lysine:2HCL (13C6, 99%; 15N2, 99%) | Cambridge Isotope Laboratories | CNLM-291-H-0.25 | MS assays |
| Chemical compound, drug | Iodoacetamide | Sigma-Aldrich | I1149 | MS assays |
| Chemical compound, drug | Ammonium bicarbonate | Sigma-Aldrich | 09832 | MS assays |
| Chemical compound, drug | Formic acid 99–100% | VWR Chemicals | 20318.297 | MS assays |
| Chemical compound, drug | iRT Kit | Biognosis | Ki-3002-1 | MS assays |
| Chemical compound, drug | BioPureSPN MINI Columns Silica C18 | The Nest Group, Inc | HUM S18V | MS assays |
| Chemical compound, drug | BioPureSPN MACRO Columns Silica C18 | The Nest Group, Inc | HMM S18V | MS assays |
| Peptide, recombinant protein | DNase I | Roche | 10104159001 | Protein purification |
| Chemical compound, drug | cOmplete Protease Inhibitor Cocktail | Roche | 05053489001 | Protein purification |
| Other | Ni-NTA Agarose | QIAGEN | 30210 | Protein purification |
| Antibody | α-V5 (mouse monoclonal) | Invitrogen | R960-25 | Western blotting, 1:2000 |
| Antibody | α-Hexokinase (rabbit monoclonal) | US Biologicals | H2035-01 | Western blotting, 1:3000 |
| Antibody | α-Mouse IgG Alexa Fluor 680 (goat polyclonal) | Thermo Fisher Scientific | A-21057 | Western blotting, 1:10,000 |
| Antibody | α-Rabbit IgG IRDye800CW (goat polyclonal) | Li-COR Biosciences | 926-32211 | Western blotting, 1:10,000 |
| Antibody | α-EGFP (mouse monoclonal) | Roche | 11814460001 | Liposome experiments, 1:2000 |
| Antibody | α-Mouse IgG peroxidase conjugate (goat polyclonal) | Calbiochem | 401215 | Liposome experiments, 1:5000 |
| Other | Zymolyase 100T | ICN | 320932 | Tetrad dissection |
| Other | E. coli polar lipids | Avanti Polar Lipids | 100600C | Liposome experiments |
| Other | 18:1 Liss Rhodamine PE | Avanti Polar Lipids | 810150C | Liposome experiments |
| Software, algorithm | NuRim | Rajoo et al., 2018; Vallotton et al., 2019 | Data analysis | |
| Software, algorithm | ImageJ/Fiji | Schindelin et al., 2012 | Data acquisition | |
| Software, algorithm | NIS Elements | Nikon | Data acquisition | |
| Software, algorithm | MATLAB | MathWorks | Data analysis | |
| Software, algorithm | IMOD | https://bio3d.colorado.edu/imod/ | Data analysis | |
| Software, algorithm | SerialEM | Mastronarde, 2003 | Data acquisition | |
| Software, algorithm | UCSF Chimera | Pettersen et al., 2004 | Data visualization | |
| Software, algorithm | SBGrid | Morin et al., 2013 | Data processing | |
| Software, algorithm | TOM toolbox | Nickell et al., 2005; Förster et al., 2005, https://www.biochem.mpg.de/6348566/tom_e | Data processing | |
| Software, algorithm | AlphaFold2.1.1 | Jumper et al., 2021 | Data analysis | |
| Software, algorithm | AlphaFold visualization | https://colab.research.google.com/drive/1CizC7zmYvFkav5qfBbWxhgUHrOxwym2w | Data analysis | |
| Software, algorithm | COBALT | https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi | Sequence alignment | |
| Software, algorithm | Jalview | Waterhouse et al., 2009 | Sequence alignment | |
| Software, algorithm | Excel | Microsoft | Data analysis | |
| Software, algorithm | R v. 4.1.2 | R Project | Data analysis | |
| Software, algorithm | Prism 7 Prism 9 | GraphPad | Visualization | |
| Software, algorithm | Inkscape 1.1 | https://inkscape.org/ | Visualization | |
| Software, algorithm | Illustrator v. 26.0.3 | Adobe | Visualization |
Table 1
Plasmids used in this study.
| ID | Description | Details | Source |
|---|---|---|---|
| pKW4689 | pRS306-pGAL1-yEGFP | Integration in ura3 locus, expression of yEGFP | This study |
| pKW4558 | pRS306-pGAL1-ahBrl1-yEGFP | Integration in ura3 locus, expression of ahBrl1-yEGFP | This study |
| pKW4578 | pRS306-pGAL1-MRATSK | Integration in ura3 locus, overexpression of small peptide MRATSK | This study |
| pKW4568 | pRS306-pGAL1-BRL1 | Integration in ura3 locus, overexpression of Brl1 | This study |
| pKW4649 | pRS306-pGAL1-BRL1-yEGFP | Integration in ura3 locus, overexpression of Brl1-yEGFP | This study |
| pKW4651 | pRS306-pGAL1-BRL1Δah-yEGFP | Integration in ura3 locus, overexpression of BRL1Δah-yEGFP | This study |
| pKW4712 | pRS306-pGAL1-BRL1(I395D)-yEGFP | Integration in ura3 locus, overexpression of Brl1(I395D)-yEGFP | This study |
| pKW589 | pRS305 | Integration in leu2 locus | Sikorski and Hieter, 1989 |
| pKW4915 | pRS305-pGAL1-BRL1-mCherry | Integration in leu2 locus, overexpression of Brl1-mCherry | This study |
| pKW4919 | pRS305-pGAL1-BRL1(I395D)-mCherry | Integration in leu2 locus, overexpression of Brl1(I395D)-mCherry | This study |
| pKW1358 | Ylplac204-pTPI1-dsRED-HDEL | Integration in trp1 locus, ER marker | Bevis and Glick, 2002 |
| pKW1964 | pFA6A-link-yEGFP-CaURA3 | PCR template for endogenous tagging with yEGFP | Sheff and Thorn, 2004 |
| pKW3359 | pFA6a-NH605-3V5-IAA7-KanMX6 | PCR template for endogenous tagging with V5-IAA7 | Derrer et al., 2019 |
| pKW2874 | pNH603-pGPD1-osTIR1-HIS3MX | Integration in his3 locus, OsTIR1 expression | Derrer et al., 2019 |
| pKW2830 | pNH603-pGPD1-osTIR1-LEU2 | Integration in leu2 locus, OsTIR1 expression | Chan et al., 2018 |
| pKW354 | pRS426 | 2µ–URA3 plasmid | Christianson et al., 1992 |
| pKW4468 | pRS426-BRL1 | 2µ–URA3 plasmid containing BRL1 locus | This study |
| pKW4659 | pSV272-6xHis-MBP-TEV-yEGFP | Bacterial expression of 6xHis-MBP-TEV-yEGFP | This study |
| pKW4660 | pSV272-6xHis-MBP-TEV-ahBrl1-yEGFP | Bacterial expression of 6xHis-MBP-TEV-ahBrl1-yEGFP | This study |
| pKW4683 | pSV272-6xHis-MBP-TEV-ahBrl1(F391D)-yEGFP | Bacterial expression of 6xHis-MBP-TEV-ahBrl1(F391D)-yEGFP | This study |
| pKW4684 | pSV272-6xHis-MBP-TEV-ahBrl1(I395D)-yEGFP | Bacterial expression of 6xHis-MBP-TEV-ahBrl1(I395D)-yEGFP | This study |
| pKW4612 | pRS306-pGAL1-BRL1(F391D) | Integration in ura3 locus, overexpression of Brl1(F391D) | This study |
| pKW4613 | pRS306-pGAL1-BRL1(I395D) | Integration in ura3 locus, overexpression of Brl1(I395D) | This study |
| pKW4614 | pRS306-pGAL1-BRL1(F398D) | Integration in ura3 locus, overexpression of Brl1(F398D) | This study |
| pKW4615 | pRS306-pGAL1-BRL1(L402D) | Integration in ura3 locus, overexpression of Brl1(L402D) | This study |
| pKW4616 | pRS306-pGAL1-BRL1(D393A) | Integration in ura3 locus, overexpression of Brl1(D393A) | This study |
| pKW4617 | pRS306-pGAL1-BRL1(D400A) | Integration in ura3 locus, overexpression of Brl1(D400A) | This study |
| pKW3061 | pRS303-GPD-CRE-EBD78 | Integration in his3 locus CRE-EBD78 expression | Terweij et al., 2013 |
| pKW804 | pFA6-S-TEV-ZZ-KanMX | PCR template for endogenous tagging with S-TEV-ZZ | Onischenko et al., 2020 |
| pKW953 | pFA6-S-TEV-ZZ-URA3 | PCR template for endogenous tagging with S-TEV-ZZ | This study |
| pKW4746 | pFa6a-loxP-HA-mCherry-HygMX-loxP-GFP | PCR template for endogenous tagging with loxP-HA-mCherry-HygMX-loxP-GFP | Colombi et al., 2013 |
| pKW2407 | pFA6a-mCherry-NAT | PCR template for endogenous tagging with mCherry | Onischenko et al., 2020 |
| pKW4945 | pFA6a-loxP-yEGFP_URA3-loxP | PCR template for endogenous tagging with loxP-yEGFP_URA3-loxP | This study |
| pKW4946 | pFA6a-loxP-URA3-loxP_yEGFP | PCR template for endogenous tagging with loxP-URA3-loxP_yEGFP | This study |
Table 2
Yeast strains used in this study.
| ID | Genotype | Source |
|---|---|---|
| KWY165 | haploid, MATa leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 [phi+] | W303 haploid |
| KWY166 | diploid, leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 [phi+] | W303 diploid |
| KWY1602 | MAT(alpha) his3-1 leu2-0 lys2-0 ura3-0 | BY4742 |
| KWY9200 | KWY165, BRL1::BRL1-V5-IAA7-KanMX trp1::pADH1-dsRED-HDEL-TRP1 | This study |
| KWY9204 | KWY165, BRL1::BRL1-V5-IAA7-KanMX his3::pGPD1-OsTIR1 trp1::pADH1-dsRED-HDEL-TRP1 | This study |
| KWY9268 | KWY165, BRL1::BRL1-V5-IAA7-KanMX trp1::pADH1-dsRED-HDEL-TRP1 NUP82::NUP82-yEGFP-URA3 | This study |
| KWY9269 | BRL1::BRL1-V5-IAA7-KanMX his3::pGPD1-OsTIR1 trp1::pADH1-dsRED-HDEL-TRP1 NUP82::NUP82-yEGFP-URA3 | This study |
| KWY9270 | KWY165, BRL1::BRL1-V5-IAA7-KanMX trp1::pADH1-dsRED-HDEL-TRP1 NUP116::NUP116-yEGFP-URA3 | This study |
| KWY9271 | KWY165, BRL1::BRL1-V5-IAA7-KanMX his3::pGPD1-OsTIR1 trp1::pADH1-dsRED-HDEL-TRP1 NUP116::NUP116-yEGFP-URA3 | This study |
| KWY9272 | KWY165, BRL1::BRL1-V5-IAA7-KanMX trp1::pADH1-dsRED-HDEL-TRP1 NUP100::NUP100-yEGFP-URA3 | This study |
| KWY9273 | KWY165, BRL1::BRL1-V5-IAA7-KanMX his3::pGPD1-OsTIR1 trp1::pADH1-dsRED-HDEL-TRP1 NUP100::NUP100-yEGFP-URA3 | This study |
| KWY9274 | KWY165, BRL1::BRL1-V5-IAA7-KanMX trp1::pADH1-dsRED-HDEL-TRP1 NUP133::NUP133-yEGFP-URA3 | This study |
| KWY9275 | KWY165, BRL1::BRL1-V5-IAA7-KanMX his3::pGPD1-OsTIR1 trp1::pADH1-dsRED-HDEL-TRP1 NUP133::NUP133-yEGFP-URA3 | This study |
| KWY9276 | KWY165, BRL1::BRL1-V5-IAA7-KanMX trp1::pADH1-dsRED-HDEL-TRP1 NUP170::NUP170-yEGFP-URA3 | This study |
| KWY9277 | KWY165, BRL1::BRL1-V5-IAA7-KanMX his3::pGPD1-OsTIR1 trp1::pADH1-dsRED-HDEL-TRP1 NUP170::NUP170-yEGFP-URA3 | This study |
| KWY9908 | KWY165, BRL1::BRL1-V5-IAA7 KanMX trp1::ADH1-dsRED-HDEL-TRP1 NUP60::NUP60-yEGFP-URA3 | This study |
| KWY10108 | KWY165, BRL1::BRL1-V5-IAA7 KanMX his3::pGPD1-OsTIR1 trp1::pADH1-dsRED-HDEL-TRP1 NUP60::NUP60-yEGFP-URA3 | This study |
| KWY9909 | KWY165, BRL1::BRL1-V5-IAA7 KanMX trp1::pADH1-dsRED-HDEL-TRP1 NUP159::NUP159-yEGFP-URA3 | This study |
| KWY10109 | KWY165, BRL1::BRL1-V5-IAA7 KanMX his3::pGPD1-OsTIR1 trp1::pADH1-dsRED-HDEL-TRP1 NUP159::NUP159-yEGFP-URA3 | This study |
| KWY9911 | KWY165, BRL1::BRL1-V5-IAA7 KanMX trp1::pADH1-dsRED-HDEL-TRP1 MLP1::MLP1-yEGFP-URA3 | This study |
| KWY10110 | KWY165, BRL1::BRL1-V5-IAA7 KanMX his3::pGPD1-OsTIR1 trp1::pADH1-dsRED-HDEL-TRP1 MLP1::MLP1-yEGFP-URA3 | This study |
| KWY9939 | KWY165, BRL1::BRL1-V5-IAA7 KanMX trp1::pADH1-dsRED-HDEL-TRP1 NUP85::NUP85-yEGFP-URA3 | This study |
| KWY10107 | KWY165, BRL1::BRL1-V5-IAA7 KanMX his3::pGPD1-OsTIR1 trp1::pADH1-dsRED-HDEL-TRP1 NUP85::NUP85-yEGFP-URA3 | This study |
| KWY10029 | KWY165, BRL1::BRL1-V5-IAA7 KanMX trp1::pADH1-dsRED-HDEL-TRP1 NUP192::NUP192-yEGFP-URA3 | This study |
| KWY10111 | KWY165, BRL1::BRL1-V5-IAA7 KanMX his3::pGPD1-OsTIR1 trp1::pADH1-dsRED-HDEL-TRP1 NUP192::NUP192-yEGFP-URA3 | This study |
| KWY5540 | KWY165, nup116ΔGLFG NUP188::HIS3MX-pMet3-3xHA-Nup188 | Onischenko et al., 2017 |
| KWY8891 | KWY165, ura3::pGAL1-BRL1-yeGFP-CaURA3 | This study |
| KWY8893 | KWY165, ura3::pGAL1-BRL1Δah-yeGFP-CaURA3 | This study |
| KWY9070 | KWY165, ura3::pGAL1-BRL1(I395D)-yeGFP-CaURA3 | This study |
| KWY8894 | KWY165, ura3::pGAL1-MRATSK-CaURA3 | This study |
| KWY8895 | KWY165, ura3::pGAL1-BRL1-CaURA3 | This study |
| KWY8896 | KWY165, ura3::pGAL1-BRL1Δah-CaURA3 | This study |
| KWY8898 | KWY165, ura3::pGAL1-BRL1(I395D)-CaURA3 | This study |
| KWY10154 | KWY165, NUP82::NUP82-yeGFP-CaURA3 leu2::pRS305-LEU2 | This study |
| KWY10155 | KWY165, NUP82::NUP82-yeGFP-CaURA3 leu2::pGAL1-BRL1-mCherry-LEU2 | This study |
| KWY10159 | KWY165, NUP82::NUP82-yeGFP-CaURA3 leu2::pGAL1-BRL1(I395D)-mCherry-LEU2 | This study |
| KWY10161 | KWY165, NUP116::NUP116-yeGFP-CaURA3 leu2::pRS305-LEU2 | This study |
| KWY10162 | KWY165, NUP116::NUP116-yeGFP-CaURA3 leu2::pGAL1-BRL1-mCherry-LEU2 | This study |
| KWY10166 | KWY165, NUP116::NUP116-yeGFP-CaURA3 leu2::pGAL1-BRL1(I395D)-mCherry-LEU2 | This study |
| KWY10168 | KWY165, NUP133::NUP133-yeGFP-CaURA3 leu2::pRS305-LEU2 | This study |
| KWY10169 | KWY165, NUP133::NUP133-yeGFP-CaURA3 leu2::pGAL1-BRL1-mCherry-LEU2 | This study |
| KWY10173 | KWY165, NUP133::NUP133-yeGFP-CaURA3 leu2::pGAL1-BRL1(I395D)-mCherry-LEU2 | This study |
| KWY10175 | KWY165, NUP170::NUP170-yeGFP-CaURA3 leu2::pRS305-LEU2 | This study |
| KWY10176 | KWY165, NUP170::NUP170-yeGFP-CaURA3 leu2::pGAL1-BRL1-mCherry-LEU2 | This study |
| KWY10180 | KWY165, NUP170::NUP170-yeGFP-CaURA3 leu2::pGAL1-BRL1(I395D)-mCherry-LEU2 | This study |
| KWY10260 | KWY165, SEC61::SEC61-yEGFP-CaURA3 leu2-3::pRS305-LEU2 | This study |
| KWY10261 | KWY165, SEC61::SEC61-yEGFP-CaURA3 leu2-3::BRL1-mCherry-LEU2 | This study |
| KWY10262 | KWY165, SEC61::SEC61-yEGFP-CaURA3 leu2-3::BRL1(I395D)-mCherry-LEU2 | This study |
| KWY8876 | KWY165, nup116ΔGLFG NUP188::HIS3MX-pMet3-3xHA-Nup188 ura3::pGAL1-BRL1Δah-URA3 | This study |
| KWY8874 | KWY165, nup116ΔGLFG NUP188::HIS3MX-pMet3-3xHA-Nup188 ura3::pGAL1-BRL1-URA3 | This study |
| KWY8877 | KWY165, nup116ΔGLFG NUP188::HIS3MX-pMet3-3xHA-Nup188 ura3::pGAL1-MRATSK-URA3 | This study |
| KWY8882 | KWY165, nup116ΔGLFG NUP188::HIS3MX-pMet3-3xHA-Nup188 ura3::pGAL1-BRL1(F391D)-URA3 | This study |
| KWY8883 | KWY165, nup116ΔGLFG NUP188::HIS3MX-pMet3-3xHA-Nup188 ura3::pGAL1-BRL1(I395D)-URA3 | This study |
| KWY8884 | KWY165, nup116ΔGLFG NUP188::HIS3MX-pMet3-3xHA-Nup188 ura3::pGAL1-BRL1(F398D)-URA3 | This study |
| KWY8885 | KWY165, nup116ΔGLFG NUP188::HIS3MX-pMet3-3xHA-Nup188 ura3::pGAL1-BRL1(L402D)-URA3 | This study |
| KWY8886 | KWY165, nup116ΔGLFG NUP188::HIS3MX-pMet3-3xHA-Nup188 ura3::pGAL1-BRL1(D393A)-URA3 | This study |
| KWY8887 | KWY165, nup116ΔGLFG NUP188::HIS3MX-pMet3-3xHA-Nup188 ura3::pGAL1-BRL1(D400A)-URA3 | This study |
| KWY9079 | KWY165, trp1::dsRed-HDEL-TRP1 ura3::pGAL1-ahBRL1-yEGFP-CaURA3 | This study |
| KWY9075 | KWY165, trp1::dsRed-HDEL-TRP1 ura3::pGAL1-yEGFP-CaURA3 | This study |
| KWY10418 | KWY165, Brl1-mCherry::Nat his3::CRE-EBD78 NUP170::NUP170-loxP-GFP_URA3-loxP_STOP | This study |
| KWY10419 | KWY165, Brl1-mCherry::Nat his3::CRE-EBD78 NUP170::NUP170-loxP-STOP_URA3-loxP_GFP | This study |
| KWY9964 | KWY1602, BRL1::BRL1-S-TEV-ZZ-KanMX | This study |
| KWY10453 | KWY1602, BRL1::BRL1-V5-IAA7-KanMX his3::pGPD1-OsTIR1 NUP170::NUP170-S-TEV-ZZ-URA3 | This study |
| KWY10241 | KWY1602, BRL1::BRL1-V5-IAA7-KanMX his3::pGPD1-OsTIR1 | This study |
| KWY10697 | KWY1602, BRL1::BRL1-V5-IAA7-KanMX his3::pGPD1-OsTIR1 ura3::Nup82-yEGFP leu2-3::BRL1(I395D)-mCherry-LEU2 | This study |
| KWY10445 | KWY1602, BRL1::BRL1-V5-IAA7-KanMX leu2::pGPD1-OsTIR1 MLP1::MLP1-V5-loxP-mCherry_HygMX-loxP_GFP his3::CRE-EBD78 | This study |
| KWY10450 | KWY1602, BRL1::BRL1-V5-IAA7-KanMX leu2::pGPD1-OsTIR1 NUP82::NUP82-V5-loxP-mCherry_HygMX-loxP_GFP his3::CRE-EBD78 | This study |
| KWY10451 | KWY1602, BRL1::BRL1-V5-IAA7-KanMX leu2::pGPD1-OsTIR1 NUP188::NUP188-V5-loxP-mCherry_HygMX-loxP_GFP his3::CRE-EBD78 | This study |
| KWY10452 | KWY1602, BRL1::BRL1-V5-IAA7-KanMX leu2::pGPD1-OsTIR1 NUP133::NUP133-V5-loxP-mCherry_HygMX-loxP_GFP his3::CRE-EBD78 | This study |
| KWY9485 | KWY166, BRL1/brl1Δah | This study |
| KWY9489 | KWY166, BRL1/brl1(I395D) | This study |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/78385/elife-78385-mdarchecklist1-v2.pdf
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/78385/elife-78385-transrepform1-v2.pdf
-
Appendix 1—figure 1—source data 1
Fractional labeling in the source lysate (parallel reaction monitoring [PRM] assay, related to Appendix 1—figure 1A).
- https://cdn.elifesciences.org/articles/78385/elife-78385-app1-fig1-data1-v2.xlsx
-
Appendix 1—figure 1—source data 2
Fractional labeling in the lysis intermixing tests with Brl1 bait (related to Appendix 1—figure 1B–D).
- https://cdn.elifesciences.org/articles/78385/elife-78385-app1-fig1-data2-v2.xlsx
-
Appendix 1—figure 2—source data 1
Distance measurements.
- https://cdn.elifesciences.org/articles/78385/elife-78385-app1-fig2-data1-v2.xlsx
