|
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 Fluor™ 647 C2-maleimide |
ThermoFisher Scientific |
A20347 |
| Alexa Fluor™ 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 |
