Kim and Rose 1
Supplemental Material
Inventory
- 7 Figures
1. Supplemental Fig. S1, related to Fig. 1
2. Supplemental Fig. S2, related to Fig. 2
3. Supplemental Fig. S3, related to Figs. 3 and 4
4. Supplemental Fig. S4, related to Discussion
5. Supplemental Fig. S5, related to Discussion
6. Supplemental Fig. S6, related to Discussion
7. Supplemental Fig. S7, related to Discussion
8. Supplemental Fig. S8, related to Discussion
- 2 Tables
1. Supplemental Table S1. Strains used in this study
2. Supplemental Table S2. Plasmids used in this study
Supplemental Figure Legends
Supplemental Figure S1. Fus2p localization.
(A) The localization of Fus2p truncations in mitotic cells. Fus2p truncations (pMR6011, 6009, 6149, 6159, 6151, 6152, and 6148) were expressed in fus2D (MY10174) under the GAL1 promoter for 90 min. Cells were fixed and stained with DAPI. Bar, 5 mm.
(B) The localization of Fus2p truncations in pheromone-treated cells. Fus2p truncations (pMR6009, 6150, 6151, and 6152) were expressed in fus2D (MY10174) under the GAL1 promoter for 90 min, after which glucose and a-factor were added for 2 h. Cells were fixed and stained with DAPI. Bar, 5 mm.
(C) Fus2p localization in importin mutants. Fus2p54-99-GFP3 was expressed from the GAL1 promoter for 90 min either 25°C or 37°C in indicated mutant cells (MY12415, 12136, 12135, 12155, 12151, 12132, 12152, 12153, 12154, and 10542). GFP fluorescence was observed in living cells. Bar, 5 mm.
Supplemental Figure S2. Fus2p phosphorylation in fus3Dkss1D cells.
Fus2p54-104 (pMR6009) were expressed in FUS3 (MY10174), fus3Dcln3D (MY10273), fus3Dkss1Dcln3D cells (MY12179) under the GAL1 promoter for 90 min during after a-factor arrest for 2 h. Samples were run on 50 mM phos-tag gels.
Supplemental Figure S3. The regulation of Fus2p nuclear transport by phosphorylation.
(A) Fus2p54-83 mutants (pMR6152, 6153, and 6154) were expressed in fus2D (MY10174) under the GAL1 promoter for 90 min.
(B) Fus2p54-85 mutants (pMR6151, 6155, and 6156) were expressed in fus2D (MY10174) under the GAL1 promoter for 90 min.
(C) Fus2p54-99 mutants (pMR6159, 6218, and 6160) were expressed in fus2D (MY10174) under the GAL1 promoter for 90 min, after which glucose and a-factor were added for 2 h.
(D) Fus2p54-104 mutants (pMR6184, 6185, 6169, and 6177) were expressed in fus2D (MY10174) under the GAL1 promoter for 90 min. SSA and SSE refer to residues 67, 84, and 100.
(A-D) GFP fluorescence was observed in living cells. Bar, 5 mm.
Supplemental Figure S4. Nuclear accumulation of Fus2p in cells with a defect in export.
Box and whisker plot. GFP fluorescence was observed in living cells and its intensity of the nucleus relative to the cytoplasm was quantified as described in Materials and methods. The line inside the box indicates the median. Error bars indicate the entire range. (left) Indicated Fus2p truncated mutant (pMR6159) was expressed from the GAL1 promoter at 37°C for 90 min in CRM1 (MY10239) or crm1-1 (MY10240) cells. N/C ratio is 3.14±0.74 (n=29) for CRM1 and 4.76±0.88 (n=30) for crm1-1 cells. (middle) Indicated Fus2p truncated mutants (pMR6159 and pMR6151) were expressed from the GAL1 promoter at 30°C for 90 min in fus2D (MY10174). Fus2p54-85-GFP3 (pMR6151) lacks the NES sequence (Fig. 1A). N/C ratio is 3.48±0.83 (n=55) for +NES and 4.50±1.07 (n=64) for -NES. (right) Indicated Fus2p truncated mutants (pMR6009 and pMR6180) were expressed from the GAL1 promoter at 30°C for 90 min in fus2D (MY10174). Fus2p54-104-NES6A-GFP3 (pMR6180) contains the NES mutations in which six hydrophobic residues important for nuclear exit were substituted for alanines (Fig. 1D). N/C ratio is 3.37±0.86 (n=70) for wild-type NES and 5.32±1.28 (n=27) for NES6A mutant.
Supplemental Figure S5. Suppression of S67 phosphorylation-dependent Fus2p import by S84 phosphorylation.
Box and whisker plot. The NES activity-defective Fus2p54-85-GFP3 mutants (pMR6216, 6156, 6157, and 6158) were expressed from the GAL1 promoter at 30°C for 90 min in fus2D (MY10174). GFP fluorescence was observed in living cells and its intensity of the nucleus relative to the cytoplasm was quantified as described in Materials and methods. The line inside the box indicates the median. Error bars indicate the entire range. ES, SE, AE, and EE refer to the specific amino acids at residues 67 and 84.
If S84 phosphorylation blocks S67 phosphorylation, S84 phosphomimetic mutant, SE will show the similar N/C ratio to S67 phosphorylation defective mutant, AE. However, this is not the case. N/C ratio of SE (3.13±0.73; n=47) is higher than AE (1.36±0.20; n=36), but instead similar to EE (2.99±0.48; n=73), suggesting that S67 phosphorylation is independent of S84 phosphorylation. On the other hand, N/C ratio of ES (4.54±1.03; n=46) was decreased by S84 phospho-mimetic mutation in EE mutant, suggesting that S84 phosphorylation suppresses the ability of S67 phosphorylation to facilitate Fus2p nuclear import.
Supplemental Figure S6. Co-immunoprecipitation of Fus2p mutants with Srp1p or Crm1p.
(A and B) Indicated Fus2p54-104 truncated mutants fused with GFP3 (pMR6170 and 6237) were expressed in (A) SRP1-3HA (MY12172) or (B) CRM1-3HA (MY12170) cells under the GAL1 promoter for 90 min, and then cells were harvested. Extracts were prepared, and Srp1p or Crm1p was precipitated with anti-HA as described in Materials and methods. Fus2p and Srp1p or Crm1p were detected with anti-GFP and anti-HA, respectively. The intensity of co-purified Fus2p was normalized by purified Srp1p or Crm1p and expressed relative to AAA. Error bars represent the means ± SEM of two independent experiments. AAA and EEE refer to the specific amino acids at residues 67, 84, and 100.
Supplemental Figure S7. S67, S84, and S100 phosphorylations by the NLS mutation.
Samples were run on gels containing 50 mM phos-tag and detected by anti-GFP immunoblot. (A) Indicated Fus2p54-104 mutants fused with GFP3 (pMR6009, 6226, and 6231) were expressed in fus2D cells (MY10174) under the GAL1 promoter for 90 min. Arrow indicates the S67 phosphorylated species. (B) fus2D cells (MY10174) were arrested with a-factor for 2h and then indicated Fus2p54-104 mutants fused with GFP3 (pMR6226 and 6231) were expressed under the GAL1 promoter for 90 min.
Supplemental Figure S8. Failure of S67 phospho-mimetic mutation in overriding the defect of 63KR64 mutation in Fus2p nuclear import.
Indicated plasmid (pMR6310) was expressed from the GAL1 promoter for 3 h in fus2D (MY10174). GFP fluorescence was observed in living cells. Bar, 5 mm.
Supplemental Table S1. Strains used in this study
BY4741 / MATa ura3-D0 his3-D200 leu2-D0 met15-D0
MY06457 / MATa srp1-31 ade2-1 ura3-1 his3-11,15 trp1-Δ63 leu2-3,112 / G. R. Fink
MY10174 / MATa fus2Δ::HIS3 CDC3-mcherry::KanMX ura3-D0 his3-D200 leu2-D0 met15-D0 / C. A. Ydenberg
MY10239 / MATa crm1D::LEU2 [HIS3, CRM1] ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112
MY10240 / MATa crm1D::LEU2 [HIS3, crm1-1] ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112
MY10273 / MATa fus2Δ::HIS3 CDC3-mcherry::KanMX fus3D::NatMX cln3::ura3::LEU2 ura3-D0 his3-D200 leu2-D0 met15-D0 / C. A. Ydenberg
MY10323 / MATa fus2Δ::HIS3 CDC3-mcherry::KanMX cdc28as-1 ura3-D0 his3-D200 leu2-D0 met15-D0 / C. A. Ydenberg
MY10469 / MATa fus2Δ::HIS3 CDC3-mcherry::KanMX cdc28as-1 ste5-8a ura3-D0 his3-D200 leu2-D0 met15-D0 / C. A. Ydenberg
MY10542 / MATa msn5Δ::KanMX Δfus2::HIS3 ura3-Δ0 leu2-Δ0 his3-Δ1 met15-Δ0 / C. A. Ydenberg
MY12131 / MATa rsl1-4 ura3-52 leu2-1 trp1-63 / P. A. Silver
MY12132 / MATa nmd5Δ::HIS3 / P. A. Silver
MY12135 / MATa pse1-1 ura3-52 trp1-63 leu2-1 / P. A. Silver
MY12136 / MATa mtr10D::HIS3 ade2-1 his3-11,15 ura3-52 leu2-3,112 trp1-1 can1-100 / Ed C. Hurt
MY12151 / MATa kap114Δ::KanMX ura3-D0 his3-D200 leu2-D0 met15-D0 / Open Biosystems
MY12152 / MATa kap120Δ::KanMX ura3-D0 his3-D200 leu2-D0 met15-D0 / Open Biosystems
MY12153 / MATa pdr6Δ::KanMX ura3-D0 his3-D200 leu2-D0 met15-D0 / Open Biosystems
MY12154 / MATa kap123Δ::KanMX ura3-D0 his3-D200 leu2-D0 met15-D0 / Open Biosystems
MY12155 / MATa sxm1Δ::KanMX ura3-D0 his3-D200 leu2-D0 met15-D0 / Open Biosystems
MY12156 / MATa cla4Δ::KanMX ura3-D0 his3-D200 leu2-D0 met15-D0 / Open Biosystems
MY12170 / MATa CRM1-HA3::KanMX Δfus3::HIS2 ura3-D0 his3-D200 leu2-D0 met15-D0 / This study
MY12172 / MATa SRP1-HA3::KanMX Δfus3::HIS2 ura3-D0 his3-D200 leu2-D0 met15-D0 / This study
MY12179 / MATa cln3Δ::KanMX fus3Δ::HIS3 Kss1Δ::TRP1 Gal+ his3-11,15 ade2-1 ura3-1 leu2 can1 / This study
MY12398 / MATa srp1-31 crm1T539C::LEU2 ura3-52 leu2D1 trp1 / This study
MY12415 / Kap104D::ura::HIS [pKap104-16] / J. D. Aitchison
MY12594 / MATa srp1-31 ura3-52 leu2D1 trp1 / A. H. Corbett
Supplemental Table S2. Plasmids used in this study
Plasmid / Marker / Source*pMR0727 / CEN URA3 fus1-LacZ / G. R. Fink
pMR1863 / LEU2 pRS405
pMR5469 / CEN URA3 PGAL-FUS2::GFP104 / J. M. Paterson
pMR5630 / Ptet-6×HN-Fus2(1-328) Cmr / C. A. Ydenberg
pMR5738 / CEN LEU2 PGAL-FUS2::GFP104 / C. A. Ydenberg
pMR6009 / CEN URA3 PGAL-Fus2(54-104)-GFP3 / C. A. Ydenberg
pMR6011 / CEN URA3 PGAL-Fus2(1-53)-GFP3 / C. A. Ydenberg
pMR6148 / CEN URA3 PGAL-Fus2(54-79)-GFP3
pMR6149 / CEN URA3 PGAL-Fus2(65-104)-GFP3
pMR6150 / CEN URA3 PGAL-Fus2(54-95)-GFP3
pMR6151 / CEN URA3 PGAL-Fus2(54-85)-GFP3
pMR6152 / CEN URA3 PGAL-Fus2(54-83)-GFP3
pMR6153 / CEN URA3 PGAL-Fus2(54-83)-S67A-GFP3
pMR6154 / CEN URA3 PGAL-Fus2(54-83)-S67E-GFP3
pMR6155 / CEN URA3 PGAL-Fus2(54-85)-S84A-GFP3
pMR6156 / CEN URA3 PGAL-Fus2(54-85)-S84E-GFP3
pMR6157 / CEN URA3 PGAL-Fus2(54-85)-AE-GFP3 (S67A S84E)
pMR6158 / CEN URA3 PGAL-Fus2(54-85)-EE-GFP3 (S67E S84E)
pMR6159 / CEN URA3 PGAL-Fus2(54-99)-GFP3
pMR6160 / CEN URA3 PGAL-Fus2(54-99)-SE-GFP3 (S84E)
pMR6161 / CEN URA3 PGAL-Fus2(54-99)-AS-GFP3 (S67A)
pMR6162 / CEN URA3 PGAL-Fus2(54-99)-AA-GFP3 (S67A S84A)
pMR6163 / CEN URA3 PGAL-Fus2(54-99)-AE-GFP3 (S67A S84E)
pMR6165 / CEN URA3 PGAL-Fus2(54-104)-SAS-GFP3 (S84A)
pMR6166 / CEN URA3 PGAL-Fus2(54-104)-ASS-GFP3 (S67A)
pMR6168 / CEN URA3 PGAL-Fus2(54-104)-ESS-GFP3 (S67E)
pMR6169 / CEN URA3 PGAL-Fus2(54-104)-SSA-GFP3 (S100A)
pMR6170 / CEN URA3 PGAL-Fus2(54-104)-AAA-GFP3 (S67A S84A S100A)
pMR6173 / CEN URA3 PGAL-Fus2(54-104)-EAA-GFP3 (S67E S84A S100A)
pMR6174 / CEN URA3 PGAL-Fus2(54-104)-SEE-GFP3 (S84E S100E)
pMR6175 / CEN URA3 PGAL-Fus2(54-104)-SAE-GFP3 (S84A S100E)
pMR6176 / CEN URA3 PGAL-Fus2(54-104)-SEA-GFP3 (S84E S100A)
pMR6177 / CEN URA3 PGAL-Fus2(54-104)-SSE-GFP3 (S100E)
pMR6178 / CEN URA3 PGAL-Fus2(54-104)-AEA-GFP3 (S67A S84E S100A)
pMR6179 / CEN URA3 PGAL-Fus2(54-104)-AAE-GFP3 (S67A S84A S100E)
pMR6180 / CEN URA3 PGAL-Fus2(54-104)-NES6A-GFP3
pMR6181 / Ptet-6×HN-Fus2(1-328)-S67A Cmr
pMR6182 / CEN URA3 PGAL-Fus2(54-104)-NES6A-AAA-GFP3 (S67A S84A S100A)
pMR6183 / CEN URA3 PGAL-Fus2(54-104)-NES6A-AAE-GFP3 (S67A S84A S100E)
pMR6184 / CEN URA3 PGAL-Fus2(54-104)-NES6A-SSA-GFP3 (S100A)
pMR6185 / CEN URA3 PGAL-Fus2(54-104)-NES6A-SSE-GFP3 (S100E)
pMR6186 / CEN URA3 PGAL-Fus2-K54A-GFP
pMR6187 / CEN URA3 PGAL-Fus2-KK58AA-GFP
pMR6188 / CEN URA3 PGAL-Fus2-KR63AA--GFP
pMR6189 / CEN URA3 PGAL-Fus2-KK80AA-GFP
pMR6190 / CEN URA3 PGAL-Fus2-LNL97ANA-GFP
pMR6191 / CEN URA3 PGAL-Fus2-VV94AA-GFP
pMR6192 / CEN URA3 PGAL-Fus2-IF90AA-GFP
pMR6193 / CEN URA3 PGAL-Fus2-NES6A-GFP
pMR6194 / CEN URA3 Amp PGAL-myc-Cla4 / J. Thorner
pMR6195 / CEN URA3 Amp PGAL-myc-Cla4KD (K594A) / J. Thorner
pMR6196 / GST-Cla4 Ampr / J. Thorner
pMR6197 / GST-Cla4KD Ampr / J. Thorner
pMR6214 / CEN URA3 PGAL-Fus2(54-85)-AS-GFP3 (S67A)
pMR6215 / CEN URA3 PGAL-Fus2(54-85)-AA-GFP3 (S67A S84A)
pMR6216 / CEN URA3 PGAL-Fus2(54-85)-ES-GFP3 (S67E)
pMR6218 / CEN URA3 PGAL-Fus2(54-99)-SA-GFP3 (S84A)
pMR6219 / CEN URA3 PGAL-Fus2(54-99)-ES-GFP3 (S67E)
pMR6226 / CEN URA3 PGAL-Fus2(54-104)-KR63AA-GFP3
pMR6231 / CEN URA3 PGAL-Fus2(54-104)-KK80AA-GFP3
pMR6237 / CEN URA3 PGAL-Fus2(54-104)-EEE-GFP3 (S67E S84E S100E)
pMR6254 / CEN LEU2 PGAL-Fus2(54-99)-GFP3
pMR6310 / CEN URA3 PGAL-Fus2::GFP104 (KR63AA S67E)
pMR6337 / LEU2 CRM1329-1085-HA3
pMR6338 / LEU2 CRM1(T539C)329-1085-HA3
pMR6388 / CEN URA3 PGAL-Fus2(54-104)-NES6A-ASS-GFP3 (S67A)
pMR6389 / CEN URA3 PGAL-Fus2(54-104)-NES6A-ESS-GFP3 (S67E)
*All plasmids without source were constructed in this study.