KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Monoclonal antibody mouse Anti-NSP1 | Abcam | ab4641 |
| Monoclonal antibody mouse anti-GFP | Santa Cruz | sc-9996 |
| Monoclonal antibody anti-mouse m-IgGκ BP-HRP | Santa Cruz | sc-516102 |
| Chemicals, Peptides, and Recombinant Proteins | ||
| Albumin bovine serum (BSA) | Sigma-Aldrich | A9647 |
| Kap95 yeast Bscript BamHl/Xhol | In house | N/A |
| Drop out media aminoacids | Sigma-Aldrich | N/A |
| D-Glucose anhydrous | Fisher Chemical™ | 10141520 |
| D-Raffinose pentahydrate | Thermo Scientific | 195675000 |
| D-Galactose | Acros Organics | 150610010 |
| 1,6-Hexanediol | Sigma-Aldrich | 240117 |
| Phosphase buffered saline | Sigma-Aldrich | P4417 |
| Tris base | Fisher Scientific™ | BP152–1 |
| HEPES | PanReac Application | 7365-45-9 |
| HEPES | Fisher Scientific™ | BP310–500 |
| Sodium chloride | Acros Organics | 207790010 |
| Sodium chloride | Sigma-Aldrich | S2889 |
| Tween20 | MP Biomedicals | TWEEN201 |
| Magnesium chloride hexahydrate | Sigma-Aldrich | M2393 |
| Magnesium chloride hexahydrate | Sigma-Aldrich | M9272 |
| 1,4-Dithiothreitol (DTT) | Roche | 10708984001 |
| Glycerol | Sigma-Aldrich | G5516 |
| Phenylmethanesulfonyl fluoride (PMSF) | Sigma-Aldrich | P7626 |
| cOmplete ULTRA tablets, Mini EDTA-free | Roche | 05892791001 |
| Albumin bovine serum (BSA) | Acros Organics | 268131000 |
| Glass beads | BioSpec Products | 11079105 |
| PierceTM BCA Protein Assay Kit | Fisher Scientific™ | 23225 |
| ECL Prime Western Blotting Detection Reagent | Amersham | RPN2232 |
| TGX Stain-Free™ FastCast™ Acrylamide Kit, 10% | BioRad | 1610183 |
| Alexa FluorTM 647 C2-maleimide | ThermoFisher Scientific | A20347 |
| Alexa FluorTM 568 C5-maleimide | ThermoFisher Scientific | A20341 |
| Critical Commercial Assays | ||
| Deposited Data | ||
| Custom code used for HS-AFM analysis | This paper | |
| Experimental Models: Saccharomyces cerevisiae Strains | ||
| MATα ade2–1 ura3–1 his3–11,15 trp1–1 leu2–3,112 can1–100 | Euroscarf | W303α (parental) |
| MATα ade2–1 ura3–1 his3–11,15 trp1–1 leu2–3,112 can1–100 MLP1-PPX-ProteinA::HIS5 | (Kim et al., 2018) | Mlp1ppxPrA |
| trp1–1 lys2 ura3 leu2 his3 Flag-LoxP-nsp1DFGDFxFG T7-LoxP-Nup1DFxFG myc-LoxP-nup2DFxFG myc-LoxP-nup60DFxF HA-LoxP-Nup42DFG my-LoxP-Nup159DFG | (Strawn et al., 2004) | SWY3062 |
| trp1–1 lys2 ura3 leu2 his3 Flag-LoxP-nsp1DFGDFxFG T7-LoxP-Nup1DFxFG myc-LoxP-nup2DFxFG myc-LoxP-nup60DFxF HA-LoxP-Nup42DFG my-LoxP-Nup159DFG MLP1-PPX-ProteinA::HIS5 | This study | DeltaFG, Mlp1-PPX-PrA |
| MATα ade2–1 ura3–1 his3–11,15 trp1–1 leu2–3,112 can1–100 nsp1::NatMX4 pRS416-NSP1 | This study | DeltaNsp1 |
| MATα ade2–1 ura3–1 his3–11,15 trp1–1 leu2–3,112 can1–100 nsp1::NatMX4 p410-ADH1pr-NSP1(1–591)-NSP1(2–565)-NSP1(592–824)::KanMX MLP1-PPX-ProteinA::HIS5 | This study | Nsp1FGx2, Mlp1-PPX-PrA |
| MATα ade2–1 ura3–1 his3–11,15 trp1–1 leu2–3,112 can1–100 KAP95-link-ymNeongreen::URA3 | This study | W303, Kap95-mNeongreen |
| MATα ade2–1 ura3–1 his3–11,15 trp1–1 leu2–3,112 can1–100 MLP1-PPX-ProteinA::HIS5 KAP95-link-ymNeongreen::URA3 | This study | Mlp1ppxPrA, Kap95-mNeongreen |
| MATα ade2–1 ura3–1 his3–11,15 trp1–1 leu2–3,112 can1–100 nsp1::NatMX4 p410-ADH1pr-NSP1(1–591)-NSP1(2–565)-NSP1(592–824)::KanMX MLP1-PPX-ProteinA::HIS5 KAP95-link-ymNeongreen::URA3 | This study | Nsp1FGx2, Mlp1-PPX-PrA, Kap95-mNeongreen |
| MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pGAL1-GFP-Nup100FG | This study | GFP-Nup100FG |
| MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pGAL1-GFP-Nup153FG | This study | GFP-Nup153FG |
| MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pGAL1-GFP-Nup116FG | This study | GFP-Nup116FG |
| MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pGAL1-GFP-Nup159SFG | This study | GFP-Nup159SFG |
| MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pGAL1-GFP-Nsp1FG | This study | GFP-Nsp1FG |
| MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pGAL1-GFP-Nup60FG | This study | GFP-Nup60FG |
| MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 nup100-GFP::His3MX6 | ThermoFisher | Nup100-GFP |
| MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 nup133-GFP::His3MX6 | ThermoFisher | Nup133-GFP |
| MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 nup159-GFP::His3MX6 | ThermoFisher | Nup159-GFP |
| MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 nsp1-GFP::His3MX6 | ThermoFisher | Nsp1-GFP |
| MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 nup60-GFP::His3MX6 | ThermoFisher | Nup60-GFP |
| MATα ade2–1 ura3–1 his3–11,15 trp1–1 leu2–3,112 can1–100 TEFF-mCherry-WALP-HDEL::Leu2 pGAL1-GFP-Nup100FG | This study | mCherry-WALP-HDEL, GFP-Nup100FG |
| MATα ade2–1 ura3–1 his3–11,15 trp1–1 leu2–3,112 can1–100 MLP1-PPX-ProteinA::HIS5 TEFF-mCherry-WALP-HDEL::Leu2 pGAL1-GFP-Nup100FG | This study | Mlp1-PPX-PrA, mCherry-WALP-HDEL, GFP-Nup100FG, |
| MATα ade2–1 ura3–1 his3–11,15 trp1–1 leu2–3,112 can1–100 nsp1::natMX4 p410-ADH1pr-NSP1(1–591)-NSP1(2–565)-NSP1(592–824)::KanMX MLP1-PPX-ProteinA::HIS5 TEFF-mCherry-WALP-HDEL::Leu2 pGAL1-GFP-Nup100FG | This study | Nsp1FGx2, Mlp1-PPX-PrA, mCherry-WALP-HDEL, GFP-Nup100FG |
| trp1–1 lys2 ura3 leu2 his3 Flag-LoxP-nsp1DeltaFGDeltaFxFG T7-LoxP-Nup1DeltaFxFG myc-LoxP-nup2DeltaFxFG myc-LoxP-nup60DeltaFxF HA-LoxP-Nup42DeltaFG my-LoxP-Nup159DeltaFG MLP1-PPX-ProteinA::HIS5 TEFF-mCherry-WALP-HDEL::Leu2 pGAL1-GFP-Nup100FG | This study | DeltaFG, Mlp1-PPX-PrA, mCherry-WALP-HDEL, GFP-Nup100FG |
| MATα ade2–1 ura3–1 his3–11,15 trp1–1 leu2–3,112 can1–100 pGAL1-mCherry-L-TM pGAL1-MG4 | This study | W303, mCherry-L-TM, MG4 |
| MATα ade2–1 ura3–1 his3–11,15 trp1–1 leu2–3,112 can1–100 nsp1::natMX4 p410-ADH1pr-NSP1(1–591)-NSP1(2–565)-NSP1(592–824)::KanMX pGAL1-mCherry-L-TM pGAL1-MG4 | This study | Nsp1FGx2, mCherry-L-TM, MG4 |
| trp1–1 lys2 ura3 leu2 his3 Flag-LoxP-nsp1DeltaFGDeltaFxFG T7-LoxP-Nup1DeltaFxFG myc-LoxP-nup2DeltaFxFG myc-LoxP-nup60DeltaFxF HA-LoxP-Nup42DeltaFG my-LoxP-Nup159DeltaFG pGAL1-mCherry-L-TM pGAL1-MG4 | This study | DeltaFG, mCherry-L-TM, MG4 |
| Recombinant DNA: List of Plasmids Used in this Study | ||
| pAG25 | Euroscarf | dominant marker gene deletion cassette: pAgTEF1-natMX4-tAgTEF1 |
| PLPC19 | (Colombi et al., 2013) | pRS416-NSP1 |
| pYX242–2xGFP | (Timney et al., 2016) | N/A |
| pYX242-SV40NLS-GFP-PrA | (Leslie et al., 2006) | #202 (http://ncdir.org) |
| p410 Adh1 Nsp1FGx2 | This study | p410 plasmid with NSP1(1–591)-(2–565)-(592–824) under control of yeast Adh1 promoter and terminator |
| pGAL1-GFP-Nup100FG | This study | Adapted from pACM021 (Meinema et al., 2011), GAL1 promoter, N-term GFP tag Nup100FG domain, His3+ selection marker |
| pGAL1-GFP-Nup116FG | This study | As above, Nup116FG domain |
| pGAL1-GFP-Nup153FG | This study | As above, Nup153FG domain |
| pGAL1-GFP-Nup159SFG | This study | As above, Nup159SFG domain |
| pGAL1-GFP-Nsp1FG | This study | As above, Nsp1FG domain |
| pGAL1-GFP-Nup60FG | This study | As above, Nup60FG domain |
| pGAL1-GFP-Nup100FG | This study | Adapted from pACM021, GAL1 promoter, N-term GFP tag Nup100FG domain, Ura3+ selection marker |
| pTEFF-mCherry-WALP-HDEL::Leu | This study | mCherry N-term tagged NE/ER marker protein under constitutive TEFF promoter, Leu2 selection marker |
| pGAL1-mCherry-L-TM | (Meinema et al., 2011) | pUG36, mCherry-linker(180)-TM under GAL1 promoter URA selection marker |
| pGAL1-MG4 | (Popken et al., 2015) | MBP-4xGFP under GAL1 promoter His selection marker |
| pFA6a-link-ymNeongreen-URA3 | Addgene (Botman et al., 2019) | Addgene #168059 |
| Sequence-Based Reagents | ||
| Software and Algorithms | ||
| Python-based HS-AFM program | (Sakiyama et al., 2016) | N/A |
| ImageJ | (Schindelin et al., 2012) | https://imagej.nih.gov/ij/ |
| UCSF ChimeraX, version 1.5 | (Pettersen et al., 2004) | https://www.rbvi.ucsf.edu/chimerax/ |
| SpectroMine version 2.8.210609.47784 | Biognosys AG | https://biognosys.com/software/spectromine/ |
| Igor Pro 8.0 | WaveMetrics | https://www.wavemetrics.com/ |
| LAMMPS Molecular Dynamcis Simulator | (Thompson et al., 2022) | https://www.lammps.org |
| cryoSPARCv4 | (Punjani et al., 2017) | https://structura.bio/ |
| Relion v.3.1.2 | (Zivanov et al., 2018) | https://relion.readthedocs.io/en/release-3.1/ |
| easyFRAP | (Rapsomaniki et al., 2012) | N/A |
| softWoRx | Cytiva | N/A |
| Other | ||
| HS-AFM 1.0 system | RIBM | SS-NEX -Ando model- |
| Cantilever | Nanotools GmbH | QUANTUM-AC10-SuperSharp |
| TEM | ThermoFisher Scientific | Talos L120C TEM |
| TEM | FEI Company | Tecnai G2 Spirit TEM |
| Point scanning confocal LSM880 Inverted Microscope with Airyscan detector built on a Zeiss Axio Observer stand | Zeiss | N/A |
| Mass Spectrometer | Thermo Fisher Scientific | Orbitrap Exploris |
| DeltaVision Deconvolution Microscope | Applied Precision (GE) | N/A |