Table 1 Yeast strains
Strain name
FY23
FY86
Ulp1 shuffle
ulp2
Smt3 shuffle strain
Nmd3 shuffle strain
RS453a
xpo1-LMB sensitive
Xpo1-LMB resistant
pse1-1
Genotype
MATa, ura3, trp1, leu2
Origin
derived fro
S288C
derived fro
S288C
MATa his3 leu2 lys2 ura3 ulp1::kanMX4 (ARS/CEN pURA3-ULP1)
this study
MATa his3 leu2 lys2 ura3 ulp2::kanMX4
Euroscarf
MATa his3 leu2 lys2 ura3 smt3::kanMX4 (ARS/CEN pURA3-SMT3) this study
MATa his3 leu2 lys2 ura3 nmd3::kanMX4 (ARS/CEN pURA3-NMD3) ref. 13
MATa, ade2, leu2, ura3, his3, trp1
ref. 29
MATa leu2 his3 trp1 ura3 xpo1::KAN (ARS/CEN
ref. 23
HIS3pDCCRM1T539C)
MATa leu2 his3 trp1 ura3 xpo1::KANr (ARS/CEN HIS3pDC-CRM1) ref. 23
MATa leu2∆2 his3 trp1∆63 ura3-52
ref. 32
Table 2 Plasmids and constructions
Plasmid
Ulp1-GFP
Ulp1N-GFP
Ulp1N(1-150)-GFP
Smt3-GFP
Ulp2-GFP
Arc1-GFP
Ulp1C-GFP-Arc1
Ulp1C-GFP-NES
cc-Ulp1C-GFP
Ulp1C-GFP-Nsp1C
Ulp1C-GFP-Nup42
Ulp1C-GFP-Nup60
nmd3NES-GFP-Ulp1N
Construction details
N-terminal GFP fusion, amplified as PstI/XhoI, ligated into pRS315NOP1::GFP
N-terminal GFP fusion, residues 1- 403 amplified as PstI/XhoI,
ligated into pRS315-NOP1::GFP
N-terminal GFP fusion, residues 1- 150 amplified as PstI/XhoI,
ligated into pRS315-NOP1::GFP
N-terminal GFP fusion, Smt3 amplified as PstI/XhoI, ligated into
pRS315-NOP1::GFP
N-terminal GFP fusion, Ulp2 amplified as PstI/XhoI, ligated into
pRS315-NOP1::GFP
N-terminal GFP fusion, Arc1 amplified as PstI/XhoI, ligated into
pRS315-NOP1::GFP
Ulp1C amplified as PstI/PstI and ligated into PstI digested Ulp1CGFP-Arc1
N-terminal GFP fusion, Ulp1C amplified as PstI/XhoI, PKI-NES
amplified with a specific primer set, ligated into pRS315-NOP1::GFP
N-terminal GFP fusion, residues 320-612 amplified as PstI/XhoI,
ligated into pRS315-NOP1::GFP
Nsp1C amplified as PstI/PstI and ligated into PstI digested Ulp1CGFP
Nup42 amplified as PstI/PstI and ligated into PstI digested Ulp1CGFP
Nup60 amplified as NsiI/NsiI and ligated into PstI digested Ulp1CGFP
N-domain of Ulp1 was amplified as BamHI/BamHI and ligated into
nmd3NES-GFP
nmd3NES-GFP-cc
Ulp1-ProtA
Ulp1N-ProtA
pGAL1::Ulp1C-GFP
pGAL1::Ulp1C-GFP-Nsp1C
pGAL1::Ulp1C-GFP-NLS
pRS316-Smt3
pRS316-Ulp1
Ulp1N(1-150)-ProtA
cc region of Ulp1 was amplified as BamHI/BamHI and ligated into
nmd3NES-GFP
N-terminal fusion, Ulp1 amplified as XmaI/XhoI and ligated into
pRS315NOP1::ProtA digested with XmaI/SalI
N-terminal fusion, residues 1 - 403 of Ulp1 amplified as XmaI/XhoI
and ligated into pRS315NOP1::ProtA digested with XmaI/SalI/XhoI
Ulp1C-GFP amplified as BamHI/NarI, ligated into YEP351GAL
Ulp1C-GFP-NSP1C amplified as BamHI/NarI, ligated into
YEP351GAL
Ulp1C-GFP-NLS amplified as BamHI/NarI, ligated into YEP351GAL
Smt3 +/-200 bp upstream and downstream amplified as
BamHI/HindIII, ligated into pRS316
Ulp1 +/-200 bp upstream and downstream amplified as
BamHI/HindIII, ligated into pRS316
N-terminal fusion, residues 1 – 150 of Ulp1 amplified as NdeI/XhoI
and ligated into pRS315NOP1::ProtA digested with NdeI /SalI
Ulp1N(150-621)-ProtA
Ulp1N(1-100)-GFP
Ulp1N(1-150)-ccUlp1C -GFP
Ulp1(150-621)-GFP
pGAL1::Ulp1C-GFP-Arc1
YCpGAL-YRB4N
N-terminal fusion, residues 150-621of Ulp1 amplified as NdeI/XhoI
and ligated into pRS315NOP1::ProtA digested with NdeI /SalI
N-terminal GFP fusion, residues 1- 100 amplified as PstI/XhoI,
ligated into pRS315-NOP1::GFP
N-terminal GFP fusion, residues 1- 150 amplified as PstI/ PstI, ligated
into PstI digested cc-Ulp1C-GFP
N-terminal GFP fusion, residues 150-621 amplified as PstI/XhoI,
ligated into pRS315-NOP1::GFP
Ulp1C-GFP-Arc1 amplified as BamHI/NarI, ligated into
YEP351GAL
ref. 21
Supplementary information
Fig. 1. Levels of Ulp1-GFP are not altered upon ovexpression of Yrb4N or in the
pse1-1 strain when shifted to restrictive temperature. Strains containing pGAL and
pGAL::Yrb4N were grown in raffinose-containing medium to an OD600 nm of 0.5
before Yrb4N expression was induced by addition of 2% galactose. The same amount
of cells corresponding to OD600 nm of 10 was used to prepare whole lysates after the cells
were harvested upon induction for 0, 1 and 4 hr. The pse1-1 temperature-sensitive mutant
strain was grown at 23°C and shifted for 3 hr to 37°C. Equivalent volumes of whole cell
lysates were separated on SDS-PAGE and analysed by Coomassie staining. Lanes 1,3
and 5 depict whole cell lysates derived from the pGAL strain prepared at 0, 1 and 4 hrs.
Lanes 2, 4, 6 depict whole cell lysates derived from the YRB4N strain prepared at 0, 1
and 4 hrs. Lanes 7 and 8 depict whole cell lysates of pse1-1 strain at 23 and shifted to
37C for 3 hr. Levels of Ulp1-GFP were examined by Western blot using anti-GFP
antibodies.