Figure 6 ( B ‐ E ) DHAP , SBP and S7P levels in wild type , tkl1 / tkl2 ( B ) , tal1 / nqm1 ( C ) and pfk1 ( E ) strains upon carbon starvation , and wild ‐ type strain upon abruptly switching from glucose to no carbon ( WT ) versus to dihydroxyacetone ( WT + DHA ) as the sole carbon source ( D ) . The x axis represents hours after carbon starvation , and the logarithmic y axis represents absolute intracellular concentration ( mean ± range of N = 2 biological replicates ) .

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Figure 6(B‐E) DHAP, SBP and S7P levels in wild type, tkl1/tkl2 (B), tal1/nqm1 (C) and pfk1 (E) strains upon carbon starvation, and wild‐type strain upon abruptly switching from glucose to no carbon (WT) versus to dihydroxyacetone (WT+DHA) as the sole carbon source (D). The x axis represents hours after carbon starvation, and the logarithmic y axis represents absolute intracellular concentration (mean±range of N=2 biological replicates).


Figure 7 ( A ) Level of hallmark metabolites and energy charge in wild type , phm8 and pnp1 / urh1 strains in starvation . The x axis represents hours after starvation , and the logarithmic y axis represents energy charge ( [ ATP ] + 0 . 5 [ ADP ] / ( [ ATP ] + [ ADP ] + [ AMP ] ) or absolute intracellular concentration ( mean ± range of N = 2 biological replicates ) . ( B and C ) Growth and viability of wild type , phm8 and pnp1 / urh1 strains in starvation . ( D ) Growth of wild type , phm8 and pnp1 / urh1 strains in the switch from glucose to glycerol + ethanol . The x axis represents hours after starvation and the logarithmic y axis represents optical density ( A600 ) in ( B and D ) and percentage of live cells calculated by dividing the number of colonies formed after starvation by the number of colonies formed before starvation in ( C ) ( mean ± range of N = 2 biological replicates ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Level of hallmark metabolites and energy charge in wild type, phm8 and pnp1/urh1 strains in starvation. The x axis represents hours after starvation, and the logarithmic y axis represents energy charge ([ATP]+0.5[ADP]/([ATP]+[ADP]+[AMP]) or absolute intracellular concentration (mean±range of N=2 biological replicates).(B and C) Growth and viability of wild type, phm8 and pnp1/urh1 strains in starvation.(D) Growth of wild type, phm8 and pnp1/urh1 strains in the switch from glucose to glycerol+ethanol. The x axis represents hours after starvation and the logarithmic y axis represents optical density (A600) in (B and D) and percentage of live cells calculated by dividing the number of colonies formed after starvation by the number of colonies formed before starvation in (C) (mean±range of N=2 biological replicates).


Figure 8 ( C and D ) DHAP , S7P , G6P , and glutathione levels and NADPH / NADP + ratio in wild type , phm8 and pnp1 / urh1 strains in the experiment shown in ( A ) The x axis represents minutes after oxidative stress , and the y axis represents absolute intracellular concentration or ratio of intracellular concentration ( mean ± range of N = 2 biological replicates ) . ( E ) Viability of wild type , phm8 and pnp1 / urh1 strains in oxidative stress . The y axis represents percentage of survived cells calculated by dividing the number of colonies formed after oxidative stress by the number of colonies formed under the same starvation condition but without H2O2 treatment ( mean ± range of N = 2 biological replicates ) .

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Figure 8(C and D) DHAP, S7P, G6P, and glutathione levels and NADPH/NADP+ ratio in wild type, phm8 and pnp1/urh1 strains in the experiment shown in (A) The x axis represents minutes after oxidative stress, and the y axis represents absolute intracellular concentration or ratio of intracellular concentration (mean±range of N=2 biological replicates).(E) Viability of wild type, phm8 and pnp1/urh1 strains in oxidative stress. The y axis represents percentage of survived cells calculated by dividing the number of colonies formed after oxidative stress by the number of colonies formed under the same starvation condition but without H2O2 treatment (mean±range of N=2 biological replicates).


Figure 1Representative calcium signature enriched in P - EMT tumors ( comparing ECAD - vs . ECAD + tumor cells ) . NES , nominal P - value , and FDR are shown . Statistical analysis performed in GSEA . Representative image of tumor cells transduced with GCaMP6 ( Left ) . Yellow arrows ( mesenchymal cells ) , red arrows ( epithelial cells ) , scale bar = 50μm . GCaMP6 mean fluorescent intensity ( MFI ) in ECADHIGH or ECADLOW cells cultured in indicated medium ( 0 Ca2 + or 2 Ca2 + ) for 1h ( Right ) . Representative of 3 independent murine PDA cell lines run in triplicate . Statistical analysis by Student ' s unpaired t - test ( * , p < 0 . 05 ; non - significant ( NS ) ; ± SD ) . Flow cytometry analysis of ECADHIGH or ECADLOW cells loaded with Indo - 1 and cultured in indicated medium ( 0 Ca2 + or 2 Ca2 + ) for 1h . Representative of 2 independent cell lines ( n indicates the number of cells analyzed in each group , ECADHIGH 0 Ca2 + n = 4963 , ECADLOW 0 Ca2 + n = 14 , ECADHIGH 2 Ca2 + n = 6575 , ECADLOW 2 Ca2 + n = 29 ) . Representative of 2 independent murine PDA cell lines run in triplicate . Statistical analysis by Student ' s unpaired t - test ( * * * * , p < 0 . 0001 ; non - significant ( NS ) ; ± SD ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Representative calcium signature enriched in P-EMT tumors (comparing ECAD- vs. ECAD+ tumor cells). NES, nominal P-value, and FDR are shown. Statistical analysis performed in GSEA.Representative image of tumor cells transduced with GCaMP6 (Left). Yellow arrows (mesenchymal cells), red arrows (epithelial cells), scale bar = 50μm. GCaMP6 mean fluorescent intensity (MFI) in ECADHIGH or ECADLOW cells cultured in indicated medium (0 Ca2+ or 2 Ca2+) for 1h (Right). Representative of 3 independent murine PDA cell lines run in triplicate. Statistical analysis by Student's unpaired t-test (*, p<0.05; non-significant (NS); ±SD).Flow cytometry analysis of ECADHIGH or ECADLOW cells loaded with Indo-1 and cultured in indicated medium (0 Ca2+ or 2 Ca2+) for 1h. Representative of 2 independent cell lines (n indicates the number of cells analyzed in each group, ECADHIGH 0 Ca2+ n=4963, ECADLOW 0 Ca2+ n=14, ECADHIGH 2 Ca2+ n=6575, ECADLOW 2 Ca2+ n=29). Representative of 2 independent murine PDA cell lines run in triplicate. Statistical analysis by Student's unpaired t-test (****, p<0.0001; non-significant (NS); ±SD).


Figure 2Representative 20X brightfield and fluorescent images of murine YFP + tumor cells treated for 72h with vehicle control ( DMSO ) , 2 . 5μM Ionomycin , or 10ng / ml TGFβ and co - stained for E - cadherin ( Red ) and DAPI ( Blue ) . Scale bar is 100μm . Representative flow cytometry analysis of surface ECAD ( Left ) , MFI of surface ECAD ( Middle ) , and percentage of surface ECAD negative cells ( Right ) in murine PDA cells , after treatment with DMSO , 2 . 5μM Ionomycin ( IONO ) , and 10ng / ml TGFβ for 72h . Representative of 3 independent murine PDA cell lines run in triplicate . Statistical analysis by ANOVA ( * * * * , p < 0 . 0001 ; non - significant ( NS ) ; ± SD ) . Top : Relative mRNA expression of Ecad after treatment with 2 . 5μM ionomycin or 10ng / ml TGFβ for denoted time . Bottom : Western blot analysis of total E - cadherin and Vimentin protein for denoted time . Experiment was run in duplicate with three different murine PDA cell lines . Statistical analysis by ANOVA ( * * * , p < 0 . 001 ; non - significant ( NS ) ; ± SEM ) . Normalized MFI of surface E - cadherin in PDA cells treated with DMSO , 2 . 5μM Ionomycin ( IONO ) , and 10ng / ml TGFβ for 7 days , followed by withdrawal of Ionomycin or TGFβ for 7 days as indicated on the x - axis . Treated cells were analyzed at day 2 , 3 , and 7 . Withdrawn cells were analyzed on days 1 , 2 , and 7 . Experiment was performed in duplicate in two different murine PDA cell lines . Error bars indicate SD . Quantification of live cellular migration over 4h after treatment with DMSO , 2 . 5μM Ionomycin ( IONO ) , or 10ng / ml TGFβ for 48hrs . ( n = 16 cells per condition . Experiment was run in triplicate in two murine PDA different cell lines ) ( Right ) . Quantification of transwell migration after treatment with DMSO , 2 . 5μM Ionomycin ( IONO ) , or 10ng / ml TGFβ for 72h ( n = 2 cell lines , 3 replicates per cell line with 3 20X images taken per transwell ) ( Left ) . Statistical analysis by ANOVA ( * , p < 0 . 05 ; * * * * , p < 0 . 0001 ; non - significant ( NS ) ; ± SD ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Representative 20X brightfield and fluorescent images of murine YFP+ tumor cells treated for 72h with vehicle control (DMSO), 2.5μM Ionomycin, or 10ng/ml TGFβ and co-stained for E-cadherin (Red) and DAPI (Blue). Scale bar is 100μm.Representative flow cytometry analysis of surface ECAD (Left), MFI of surface ECAD (Middle), and percentage of surface ECAD negative cells (Right) in murine PDA cells, after treatment with DMSO, 2.5μM Ionomycin (IONO), and 10ng/ml TGFβ for 72h. Representative of 3 independent murine PDA cell lines run in triplicate. Statistical analysis by ANOVA (****, p<0.0001; non-significant (NS); ±SD).Top: Relative mRNA expression of Ecad after treatment with 2.5μM ionomycin or 10ng/ml TGFβ for denoted time. Bottom: Western blot analysis of total E-cadherin and Vimentin protein for denoted time. Experiment was run in duplicate with three different murine PDA cell lines. Statistical analysis by ANOVA (***, p<0.001; non-significant (NS); ±SEM).Normalized MFI of surface E-cadherin in PDA cells treated with DMSO, 2.5μM Ionomycin (IONO), and 10ng/ml TGFβ for 7 days, followed by withdrawal of Ionomycin or TGFβ for 7 days as indicated on the x-axis. Treated cells were analyzed at day 2, 3, and 7. Withdrawn cells were analyzed on days 1, 2, and 7. Experiment was performed in duplicate in two different murine PDA cell lines. Error bars indicate SD.Quantification of live cellular migration over 4h after treatment with DMSO, 2.5μM Ionomycin (IONO), or 10ng/ml TGFβ for 48hrs. (n=16 cells per condition. Experiment was run in triplicate in two murine PDA different cell lines) (Right). Quantification of transwell migration after treatment with DMSO, 2.5μM Ionomycin (IONO), or 10ng/ml TGFβ for 72h (n= 2 cell lines, 3 replicates per cell line with 3 20X images taken per transwell) (Left). Statistical analysis by ANOVA (*, p<0.05; ****, p<0.0001; non-significant (NS); ±SD).


Figure 3Schematic for RNA sequencing after EMT induction for 48h . Heatmap illustration of EMT related genes in three independent murine PDA cell lines , sequenced in duplicate , treated with DMSO , 2 . 5μM Ionomycin , or 10ng / ml TGFβ . Scale indicates Z - score . GSEA of ionomycin versus DMSO ( Left ) or TGFβ versus DMSO ( Right ) for EMT signatures . NES , nominal P - value , and FDR are shown . Statistical analysis performed in GSEA . GSEA of ionomycin versus DMSO ( Left ) or TGFβ versus DMSO ( Right ) for calcium signatures . NES , nominal P - value , and FDR are shown . Statistical analysis performed in GSEA . Leading edge plots showing enrichment of C - EMT gene signatures in TGFβ treated cells ( Left ) or P - EMT gene signatures in ionomycin ( IONO ) treated cells ( Right ) , based on GSEA . Enrichment scores ( ES ) , NES , nominal P - value , and FDR are shown . Statistical analysis performed in GSEA .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Schematic for RNA sequencing after EMT induction for 48h. Heatmap illustration of EMT related genes in three independent murine PDA cell lines, sequenced in duplicate, treated with DMSO, 2.5μM Ionomycin, or 10ng/ml TGFβ. Scale indicates Z-score.GSEA of ionomycin versus DMSO (Left) or TGFβ versus DMSO (Right) for EMT signatures. NES, nominal P-value, and FDR are shown. Statistical analysis performed in GSEA.GSEA of ionomycin versus DMSO (Left) or TGFβ versus DMSO (Right) for calcium signatures. NES, nominal P-value, and FDR are shown. Statistical analysis performed in GSEA.Leading edge plots showing enrichment of C-EMT gene signatures in TGFβ treated cells (Left) or P-EMT gene signatures in ionomycin (IONO) treated cells (Right), based on GSEA. Enrichment scores (ES), NES, nominal P-value, and FDR are shown. Statistical analysis performed in GSEA.


Figure 4Fura - 2 Ca2 + measurements after addition of 5μM CNO ( Top ) or 10μM ATP ( Bottom ) in murine PDA cells expressing a Gαq - DREADD . Blue or red line indicates average of n = 22 cells . ± SD . Tracings are representative of experiments performed twice in 2 independent cell lines . Representative 20X brightfield images of Gαq - DREADD - expressing cells 48h after addition of CNO ( Left ) . Scale bar = 100μm . MFI of surface E - cadherin in cells expressing a Gαq - DREADD after treatment with DMSO , 5μM CNO , or 2 . 5μM ionomycin ( Right ) . Both experiments performed in triplicate in 2 independent cell lines . Statistical analysis by ANOVA ( * * , p < 0 . 01 ; ± SD ) . MFI of surface E - cadherin in CnB knockout lines after 48h treatment with DMSO , 2 . 5μM ionomycin , or 10ng / ml TGFβ . Experiment was repeated in two different murine PDA cell lines in triplicate . Statistical analysis by ANOVA ( * * * , p < 0 . 001 ; * * * * , p < 0 . 00001 ; ± SD ) . MFI of surface E - cadherin of murine PDA cells pretreated with 20μM W7 for 24h and then DMSO , 2 . 5μM ionomycin , or 20μM W7 with 2 . 5μM ionomycin for 24h . Experiment was repeated in two different murine PDA cell lines in triplicate . Statistical analysis by ANOVA ( * * , p < 0 . 01 ; * * * , p < 0 . 001 ; non - significant ( NS ) ; ± SD ) . MFI of surface E - cadherin of shRNA non - targeting ( shNT ) ( Left ) or shCAMK2B ( Right ) after 48h treatment with DMSO , 2 . 5μM ionomycin , or 10ng / ml TGFβ . Experiment was repeated in two different murine PDA cell lines . Statistical analysis by ANOVA ( * * * , p < 0 . 001 ; * * * * , p < 0 . 00001 ; ± SD ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Fura-2 Ca2+ measurements after addition of 5μM CNO (Top) or 10μM ATP (Bottom) in murine PDA cells expressing a Gαq-DREADD. Blue or red line indicates average of n=22 cells. ±SD. Tracings are representative of experiments performed twice in 2 independent cell lines.Representative 20X brightfield images of Gαq-DREADD-expressing cells 48h after addition of CNO (Left). Scale bar = 100μm. MFI of surface E-cadherin in cells expressing a Gαq-DREADD after treatment with DMSO, 5μM CNO, or 2.5μM ionomycin (Right). Both experiments performed in triplicate in 2 independent cell lines. Statistical analysis by ANOVA (**, p<0.01; ±SD).MFI of surface E-cadherin in CnB knockout lines after 48h treatment with DMSO, 2.5μM ionomycin, or 10ng/ml TGFβ. Experiment was repeated in two different murine PDA cell lines in triplicate. Statistical analysis by ANOVA (***, p<0.001; ****, p<0.00001; ±SD).MFI of surface E-cadherin of murine PDA cells pretreated with 20μM W7 for 24h and then DMSO, 2.5μM ionomycin, or 20μM W7 with 2.5μM ionomycin for 24h. Experiment was repeated in two different murine PDA cell lines in triplicate. Statistical analysis by ANOVA (**, p<0.01; ***, p<0.001; non-significant (NS); ±SD).MFI of surface E-cadherin of shRNA non-targeting (shNT) (Left) or shCAMK2B (Right) after 48h treatment with DMSO, 2.5μM ionomycin, or 10ng/ml TGFβ. Experiment was repeated in two different murine PDA cell lines. Statistical analysis by ANOVA (***, p<0.001; ****, p<0.00001; ±SD).


Figure 1 ( A ) Schematic representation of the SILAC - based quantitative proteomics experiment in B . subtilis . Cells overexpressing active YqgP ( BS50 , Table EV2 ) or its catalytically dead mutant YqgP . S288A ( BS51 ) , both auxotrophic for lysine , were grown in parallel in " heavy " ( containing 13C615N2 - Lysine isotope ) or " light " ( containing stable 12C614N2 - Lysine ) M9 minimal medium , respectively . After mixing the cell cultures in the 1 : 1 ratio ( based on OD600 ) , cell suspension was lysed and the fraction enriched for transmembrane proteome was analysed in a GeLC - MS / MS experiment . Using bioinformatic analysis of the MS data , highest - scoring candidate substrates were further evaluated . ( E ) Results of MS analyses of affinity co - purification experiments in wild - type B . subtilis control ( strain BTM2 , Table EV2 ) and B . subtilis deficient in endogenous YqgP expressing the wild - type YqgP - sfGFP bait ( strain BTM84 , Table EV2 ) or the proteolytically inactive YqgP . S288A - sfGFP bait ( strain BBM1 , Table EV2 ) . Proteins were considered as potential interactors of YqgP if they were identified only in both positive co - purifications with a minimum of 3 weighted spectral counts or enriched at least 5 times in positive bait samples compared to control ones based on weighted spectral counts . The protein highlighted in red was the only overlapping hit between the two proteomics approaches .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Schematic representation of the SILAC-based quantitative proteomics experiment in B. subtilis. Cells overexpressing active YqgP (BS50, Table EV2) or its catalytically dead mutant YqgP.S288A (BS51), both auxotrophic for lysine, were grown in parallel in "heavy" (containing 13C615N2- Lysine isotope) or "light" (containing stable 12C614N2- Lysine) M9 minimal medium, respectively. After mixing the cell cultures in the 1:1 ratio (based on OD600), cell suspension was lysed and the fraction enriched for transmembrane proteome was analysed in a GeLC-MS/MS experiment. Using bioinformatic analysis of the MS data, highest-scoring candidate substrates were further evaluated.(E) Results of MS analyses of affinity co-purification experiments in wild-type B. subtilis control (strain BTM2, Table EV2) and B. subtilis deficient in endogenous YqgP expressing the wild-type YqgP-sfGFP bait (strain BTM84, Table EV2) or the proteolytically inactive YqgP.S288A-sfGFP bait (strain BBM1, Table EV2). Proteins were considered as potential interactors of YqgP if they were identified only in both positive co-purifications with a minimum of 3 weighted spectral counts or enriched at least 5 times in positive bait samples compared to control ones based on weighted spectral counts. The protein highlighted in red was the only overlapping hit between the two proteomics approaches.


Figure 2 ( A ) Steady - state cleavage profile of endogenous MgtE processed by endogenous and ectopically overexpressed YqgP from the inducible Phyperspank promoter in living B . subtilis ( BTM2 and BTM501 , respectively , Table EV2 ) , in minimal medium at low magnesium concentration ( 10 µM ) . Strain lacking YqgP ( ΔyqgP , BTM78 , Table EV2 ) was used as a control . Proteins were detected by immunoblotting with chemiluminescence detection . Data information : In all panels , endogenous full - length MgtE is indicated by a black arrow , and N - terminal cleavage product by red arrows . Endogenous MgtE was visualised by anti - MgtE ( 2 - 275 ) ( α - MgtE ) ( B ) The cleavage is efficiently inhibited by 1 μM STS736 , a specific peptidyl ketoamide rhomboid inhibitor Ticha et al . , 2017b ( ) . Data information : In all panels , endogenous full - length MgtE is indicated by a black arrow , and N - terminal cleavage product by red arrows . Endogenous MgtE was visualised by anti - MgtE ( 2 - 275 ) ( α - MgtE ) and ectopic YqgP by anti - YqgP antibodies . ( C ) To map the cleavage site region within endogenous MgtE ( second lane from the left , from strain BTM501 ) , MgtE - derived reference fragments encoding first 300 , 315 , 330 amino acids as well as full - length MgtE ( black arrow ) , were in vitro transcribed and translated . Mobility of the N - terminal cleavage product ( red arrow ) of MgtE on SDS PAGE was compared to the mobilities of the translated reference fragments . Data information : In all panels , endogenous full - length MgtE is indicated by a black arrow , and N - terminal cleavage product by red arrows . Endogenous MgtE was visualised by anti - MgtE ( 2 - 275 ) ( α - MgtE ) and ectopic YqgP by anti - YqgP antibodies .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Steady-state cleavage profile of endogenous MgtE processed by endogenous and ectopically overexpressed YqgP from the inducible Phyperspank promoter in living B. subtilis (BTM2 and BTM501, respectively, Table EV2), in minimal medium at low magnesium concentration (10 µM). Strain lacking YqgP (ΔyqgP, BTM78, Table EV2) was used as a control. Proteins were detected by immunoblotting with chemiluminescence detection. Data information: In all panels, endogenous full-length MgtE is indicated by a black arrow, and N-terminal cleavage product by red arrows. Endogenous MgtE was visualised by anti-MgtE(2-275) (α-MgtE)(B) The cleavage is efficiently inhibited by 1 μM STS736, a specific peptidyl ketoamide rhomboid inhibitor Ticha et al., 2017b(). Data information: In all panels, endogenous full-length MgtE is indicated by a black arrow, and N-terminal cleavage product by red arrows. Endogenous MgtE was visualised by anti-MgtE(2-275) (α-MgtE) and ectopic YqgP by anti-YqgP antibodies.(C) To map the cleavage site region within endogenous MgtE (second lane from the left, from strain BTM501), MgtE-derived reference fragments encoding first 300, 315, 330 amino acids as well as full-length MgtE (black arrow), were in vitro transcribed and translated. Mobility of the N-terminal cleavage product (red arrow) of MgtE on SDS PAGE was compared to the mobilities of the translated reference fragments. Data information: In all panels, endogenous full-length MgtE is indicated by a black arrow, and N-terminal cleavage product by red arrows. Endogenous MgtE was visualised by anti-MgtE(2-275) (α-MgtE) and ectopic YqgP by anti-YqgP antibodies.


Figure 3 ( A ) Detection and quantification of the cleavage of endogenous MgtE by YqgP in living B . subtilis cells ( BS72 , Table EV2 ) depending on the concentrations of magnesium and manganese ions . Cells were cultivated in glucose M9 minimal medium with limiting ( 0 . 01 mM ) or high ( 1 mM ) concentration of MgSO4 , in the presence or absence of 100 μM MnCl2 , and analysed by western blotting with near - infrared detection ( upper panel ) . Black arrow denotes full - length MgtE , and red arrow denotes its N - terminal cleavage product formed by YqgP . The corresponding fluorescence signals were quantified by densitometry , and are displayed as relative specific activity , which is substrate conversion normalized to enzyme expression level ( lower panel ) . ( B ) Growth curves of wild - type ( BTM843 , Table EV2 ) , yqgP deficient ( BTM844 , Table EV2 ) and rescue ( BTM845 , Table EV2 ) strains of B . subtilis in M9 minimal medium with limiting magnesium ( 0 . 01 mM MgSO4 ) , exposed to manganese stress elicited by adding 75 µM MnSO4 in mid - exponential phase ( stress - phase denoted by blueish background ) . All strains further contain a deletion in the putative manganese efflux pump MntP ( ΔywlD , Table EV2 ) . Bottom panel shows that manganese is more toxic in the yqgP deficient strain than in the wild type strain , and that reintroduction of YqgP rescues fitness during manganese stress to above - wild - type level . Top panel shows no difference between the strains in the absence of manganese stress . Data are shown as individual datapoints from three independent experiments overlaid with dashed line connecting average values from each time - point , which illustrates the reproducibility of the assay . ( C ) Top panel : growth curves of wild - type ( BTM843 ) , yqgP deficient ( BTM844 ) and rescue ( BTM845 ) strains of B . subtilis in M9 minimal medium with limiting magnesium ( 0 . 01 mM MgSO4 ) , exposed to manganese shock elicited by adding 75 µM MnSO4 in mid - exponential phase . Bottom panel : manganese toxicity is prevented by further adding 5 mM magnesium ( MgSO4 ) in otherwise identical conditions . ( D ) Inhibition of YqgP by a rhomboid - specific peptidyl ketoamide inhibitor ( STS736 , i . e . compound 9 from Ticha et al . , 2017b ( ) ) abolishes the YqgP induced fitness of B . subtilis under manganese stress , in a dose - dependent manner , both with endogenous YqgP ( top panel ) and overexpressed YqgP ( bottom panel ) . Media and growth conditions were identical to those used in panel ( B ) . ( E ) Overexpression of heterologous MgtE inhibits growth of yqgP deficient strain ( BTM610 , Table EV2 ) in rich LB medium supplemented with 75 μM MnSO4 . Cell fitness is improved by overexpression of YqgP ( BTM611 , Table EV2 ) or its catalytically dead mutant YqgP . S288A ( BTM612 , Table EV2 ) . For clarity , for panels C - E , representative experiments of 2 - 3 independent biological replicates are shown .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Detection and quantification of the cleavage of endogenous MgtE by YqgP in living B. subtilis cells (BS72, Table EV2) depending on the concentrations of magnesium and manganese ions. Cells were cultivated in glucose M9 minimal medium with limiting (0.01 mM) or high (1 mM) concentration of MgSO4, in the presence or absence of 100 μM MnCl2, and analysed by western blotting with near-infrared detection (upper panel). Black arrow denotes full-length MgtE, and red arrow denotes its N-terminal cleavage product formed by YqgP. The corresponding fluorescence signals were quantified by densitometry, and are displayed as relative specific activity, which is substrate conversion normalized to enzyme expression level (lower panel).(B) Growth curves of wild-type (BTM843, Table EV2), yqgP deficient (BTM844, Table EV2) and rescue (BTM845, Table EV2) strains of B. subtilis in M9 minimal medium with limiting magnesium (0.01 mM MgSO4), exposed to manganese stress elicited by adding 75 µM MnSO4 in mid-exponential phase (stress-phase denoted by blueish background). All strains further contain a deletion in the putative manganese efflux pump MntP (ΔywlD, Table EV2). Bottom panel shows that manganese is more toxic in the yqgP deficient strain than in the wild type strain, and that reintroduction of YqgP rescues fitness during manganese stress to above-wild-type level. Top panel shows no difference between the strains in the absence of manganese stress. Data are shown as individual datapoints from three independent experiments overlaid with dashed line connecting average values from each time-point, which illustrates the reproducibility of the assay.(C) Top panel: growth curves of wild-type (BTM843), yqgP deficient (BTM844) and rescue (BTM845) strains of B. subtilis in M9 minimal medium with limiting magnesium (0.01 mM MgSO4), exposed to manganese shock elicited by adding 75 µM MnSO4 in mid-exponential phase. Bottom panel: manganese toxicity is prevented by further adding 5 mM magnesium (MgSO4) in otherwise identical conditions.(D) Inhibition of YqgP by a rhomboid-specific peptidyl ketoamide inhibitor (STS736, i.e. compound 9 from Ticha et al., 2017b()) abolishes the YqgP induced fitness of B. subtilis under manganese stress, in a dose-dependent manner, both with endogenous YqgP (top panel) and overexpressed YqgP (bottom panel). Media and growth conditions were identical to those used in panel (B).(E) Overexpression of heterologous MgtE inhibits growth of yqgP deficient strain (BTM610, Table EV2) in rich LB medium supplemented with 75 μM MnSO4. Cell fitness is improved by overexpression of YqgP (BTM611, Table EV2) or its catalytically dead mutant YqgP.S288A (BTM612, Table EV2). For clarity, for panels C - E, representative experiments of 2-3 independent biological replicates are shown.


Figure 4 ( B ) Detection ( left panel ) and quantification ( right panel ) of steady - state conversion of endogenous MgtE and model chimeric substrate derived from Providencia stuartii TatA Stevenson et al . , 2007Ticha et al . , 2017aTicha et al . , 2017b ( , , ) ( schemes in the middle ) by ectopically expressed YqgP and its N - and C - terminally truncated variants ( strains BS184 - 187 , Table EV2 ) in living B . subtilis grown in rich LB medium . Black arrows indicate full - length substrates and red arrows indicate cleavage products . ( C ) Similar analysis of the same strains but grown in minimal M9 medium ( with 10 µM MgSO4 ) supplemented with increasing concentrations of MnCl 2 . Black arrows denote full - length substrate ( MgtE ) , red arrows denote the cleavage products generated by YqgP . ( D ) Western blots of four independent experiments shown in panel ( C ) were quantified by near - infrared fluorescence detection , quantified by densitometry and the results are displayed as fold activation of MgtE cleavage at 100 µM MnCl 2 relative to 1 µM MnCl 2 activity . Average values and all fours datapoints are plotted for each indicated YqgP variant .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) Detection (left panel) and quantification (right panel) of steady-state conversion of endogenous MgtE and model chimeric substrate derived from Providencia stuartii TatA Stevenson et al., 2007Ticha et al., 2017aTicha et al., 2017b(, , ) (schemes in the middle) by ectopically expressed YqgP and its N- and C-terminally truncated variants (strains BS184-187, Table EV2) in living B. subtilis grown in rich LB medium. Black arrows indicate full-length substrates and red arrows indicate cleavage products.(C) Similar analysis of the same strains but grown in minimal M9 medium (with 10 µM MgSO4) supplemented with increasing concentrations of MnCl2. Black arrows denote full-length substrate (MgtE), red arrows denote the cleavage products generated by YqgP. (D) Western blots of four independent experiments shown in panel (C) were quantified by near-infrared fluorescence detection, quantified by densitometry and the results are displayed as fold activation of MgtE cleavage at 100 µM MnCl2 relative to 1 µM MnCl2 activity. Average values and all fours datapoints are plotted for each indicated YqgP variant.


Figure 5 ( A ) Detection ( left panel ) and quantification ( right panel ) of steady - state conversion of endogenous MgtE by YqgP ( strain BS72 , Table EV2 ) grown in minimal M9 medium containing 10 μM MgSO4 and 1 μM each of MnCl2 , ZnCl2 , CoCl2 , NiCl2 and CaCl2 ( reference conditions ) with the additions of 10 µM or 100 μM of a given divalent cation salt solution ( MnCl2 , ZnCl2 , CoCl2 , or NiCl2 ) , or 50 µM and 500 μM for CaCl2 . Western blots were quantified by near - infrared fluorescence detection ( left ) , quantified by densitometry and displayed as relative specific activity ( graph on the right ) , which is substrate conversion normalized to enzyme expression level . Black arrow denotes full - length substrate ( MgtE ) , and red arrow denotes the N - terminal cleavage product ( s ) generated by YqgP . All bands originate from the same western blot and identical treatment series . ( B ) Isothermal titration calorimetry of purified recombinant YqgPNTD and selected divalent cations . ( C ) Cation toxicity assays and their relationship to YqgP activity . Wild type B . subtilis ( BTM2 , Table EV2 ) , its variant lacking YqgP ( ΔyqgP , BTM78 , Table EV2 ) , and the rescue strain ectopically expressing YqgP ( BTM501 , Table EV2 ) were cultivated in minimal M9 medium containing 10 μM MgSO4 and 1 μM each of MnCl2 , ZnCl2 , CoCl2 , NiCl2 and CaCl2 ( i . e . reference conditions ) , and in mid - exponential phase were stressed by the addition of either 70 µM MnCl2 , 500 µM ZnCl2 , 25 µM CoSO4 or 400 µM NiCl2 ( pale blue area ) . YqgP activity specifically improved cell fitness during manganese and zinc stress , while it had no effect on growth of cells cultivated in the presence of high cobalt and nickel concentrations . Representative experiments of 2 - 3 independent replicates are shown .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Detection (left panel) and quantification (right panel) of steady-state conversion of endogenous MgtE by YqgP (strain BS72, Table EV2) grown in minimal M9 medium containing 10 μM MgSO4 and 1 μM each of MnCl2, ZnCl2, CoCl2, NiCl2 and CaCl2 (reference conditions) with the additions of 10 µM or 100 μM of a given divalent cation salt solution (MnCl2, ZnCl2, CoCl2, or NiCl2), or 50 µM and 500 μM for CaCl2. Western blots were quantified by near-infrared fluorescence detection (left), quantified by densitometry and displayed as relative specific activity (graph on the right), which is substrate conversion normalized to enzyme expression level. Black arrow denotes full-length substrate (MgtE), and red arrow denotes the N-terminal cleavage product(s) generated by YqgP. All bands originate from the same western blot and identical treatment series.(B) Isothermal titration calorimetry of purified recombinant YqgPNTD and selected divalent cations.(C) Cation toxicity assays and their relationship to YqgP activity. Wild type B. subtilis (BTM2, Table EV2), its variant lacking YqgP (ΔyqgP, BTM78, Table EV2), and the rescue strain ectopically expressing YqgP (BTM501, Table EV2) were cultivated in minimal M9 medium containing 10 μM MgSO4 and 1 μM each of MnCl2, ZnCl2, CoCl2, NiCl2 and CaCl2 (i.e. reference conditions), and in mid-exponential phase were stressed by the addition of either 70 µM MnCl2, 500 µM ZnCl2, 25 µM CoSO4 or 400 µM NiCl2 (pale blue area). YqgP activity specifically improved cell fitness during manganese and zinc stress, while it had no effect on growth of cells cultivated in the presence of high cobalt and nickel concentrations. Representative experiments of 2-3 independent replicates are shown.


Figure 6 ( E Detection of steady - state conversions of endogenous MgtE by YqgP variants bearing single - point mutations in putative Mn - binding region ( BS184 ; 187 ; 196 - 203 , Table EV2 ) , cultivated in modified M9 minimal medium supplemented by low ( 1 μM ) or high ( 100 μM ) MnCl2 . Black arrow marks full - length MgtE , and red arrow marks its N - terminal cleavage product by YqgP . F ) quantification of steady - state conversions of endogenous MgtE by YqgP variants bearing single - point mutations in putative Mn - binding region ( BS184 ; 187 ; 196 - 203 , Table EV2 ) , cultivated in modified M9 minimal medium supplemented by low ( 1 μM ) or high ( 100 μM ) MnCl2 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(E Detection of steady-state conversions of endogenous MgtE by YqgP variants bearing single-point mutations in putative Mn-binding region (BS184; 187; 196-203, Table EV2), cultivated in modified M9 minimal medium supplemented by low (1 μM) or high (100 μM) MnCl2. Black arrow marks full-length MgtE, and red arrow marks its N-terminal cleavage product by YqgP.F) quantification of steady-state conversions of endogenous MgtE by YqgP variants bearing single-point mutations in putative Mn-binding region (BS184; 187; 196-203, Table EV2), cultivated in modified M9 minimal medium supplemented by low (1 μM) or high (100 μM) MnCl2.


Figure 7 ( A ) The proteolytically dead mutant YqgP . S288A behanes as a pseudoprotease and acts as a substrate adaptor of FtsH . Pattern of C - terminally truncated forms of endogenous MgtE formed in vivo in the presence of ectopically expressed active YqgP ( strain BS72 , Table EV2 ) or its inactive S288A mutant ( BS73 , Table EV2 ) in rich LB medium in the presence or absence of endogenous FtsH . Black arrow denotes the full - length MgtE substrate , and red arrows denote its cleavage products generated by YqgP . While the presence of the faster migrating YqgP - dependent products ( full red arrows ) do not depend on the presence of FtsH , the slower migrating product formed in the presence of YqgP . S288A ( open red arrow ) does , indicating that FtsH protease is responsible for the alternative cleavage of MgtE . The same arrow symbolics are valid throughout this figure . ( B ) Mapping of the boundary of the FtsH generated proteolytic product of MgtE . Immunoblot comparing the SDS PAGE mobility of the N - terminal fragment of endogenous MgtE generated by FtsH in the presence of the pseudoprotease version of YqgP ( catalytically dead YqgP . S288A , strains BTM78 , BTM502 , Table EV2 ) to the mobilities of in vitro translated reference fragments corresponding to MgtE 1 - 340 , - 355 and - 370 shows that YqgP . S288A acts as an FtsH adaptor , and that FtsH processing of MgtE in the presence of YqgP . S288A stops at around amino acid 355 , which is near or at the cytoplasmic end of TMH 3 of MgtE . ( D ) In vivo cleavage of endogenous MgtE in the presence of ectopically expressed full - length YqgP . S288A ( strain BS73 ) , YqgPΔNTD . S288A ( BS185 , Table EV2 ) or YqgPΔCTD . S288A ( BS186 , Table EV2 ) , in rich LB medium shows that YqgPNTD is necessary for the FtsH - dependent processing of MgtE . ( E ) Analysis of the in vivo kinetics of formation and fate of MgtE cleavage products . B . subtilis strains expressing YqgP or its S288A mutant ( strains BTM78 ; 501 and 502 , Table EV2 ) in the presence or absence of endogenous FtsH protease ( strains BTM795 , BTM796 and BTM797 , Table EV2 ) were cultured in LB medium and at early exponential phase , after having been expressing YqgP variants for 30 min , were either left grown untreated ( as a control with ongoing translation ) or treated with 20 μg / mL tetracycline to stop proteosynthesis ( translation shut - off ) . At given time intervals afterwards , all cultures were analysed by α - MgtE western blotting with near infrared detection . Equal cell number ( judged by OD600 ) was loaded into each lane . Black arrow marks full - length MgtE , red full arrows mark YqgP - dependent and red open arrows mark YqgP . S288A - dependent cleavage product of MgtE .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) The proteolytically dead mutant YqgP.S288A behanes as a pseudoprotease and acts as a substrate adaptor of FtsH. Pattern of C-terminally truncated forms of endogenous MgtE formed in vivo in the presence of ectopically expressed active YqgP (strain BS72, Table EV2) or its inactive S288A mutant (BS73, Table EV2) in rich LB medium in the presence or absence of endogenous FtsH. Black arrow denotes the full-length MgtE substrate, and red arrows denote its cleavage products generated by YqgP. While the presence of the faster migrating YqgP-dependent products (full red arrows) do not depend on the presence of FtsH, the slower migrating product formed in the presence of YqgP.S288A (open red arrow) does, indicating that FtsH protease is responsible for the alternative cleavage of MgtE. The same arrow symbolics are valid throughout this figure.(B) Mapping of the boundary of the FtsH generated proteolytic product of MgtE. Immunoblot comparing the SDS PAGE mobility of the N-terminal fragment of endogenous MgtE generated by FtsH in the presence of the pseudoprotease version of YqgP (catalytically dead YqgP.S288A, strains BTM78, BTM502, Table EV2) to the mobilities of in vitro translated reference fragments corresponding to MgtE 1-340, -355 and -370 shows that YqgP.S288A acts as an FtsH adaptor, and that FtsH processing of MgtE in the presence of YqgP.S288A stops at around amino acid 355, which is near or at the cytoplasmic end of TMH 3 of MgtE.(D) In vivo cleavage of endogenous MgtE in the presence of ectopically expressed full-length YqgP.S288A (strain BS73), YqgPΔNTD.S288A (BS185, Table EV2) or YqgPΔCTD.S288A (BS186, Table EV2), in rich LB medium shows that YqgPNTD is necessary for the FtsH-dependent processing of MgtE.(E) Analysis of the in vivo kinetics of formation and fate of MgtE cleavage products. B. subtilis strains expressing YqgP or its S288A mutant (strains BTM78; 501 and 502, Table EV2) in the presence or absence of endogenous FtsH protease (strains BTM795, BTM796 and BTM797, Table EV2) were cultured in LB medium and at early exponential phase, after having been expressing YqgP variants for 30 min, were either left grown untreated (as a control with ongoing translation) or treated with 20 μg/mL tetracycline to stop proteosynthesis (translation shut-off). At given time intervals afterwards, all cultures were analysed by α-MgtE western blotting with near infrared detection. Equal cell number (judged by OD600) was loaded into each lane. Black arrow marks full-length MgtE, red full arrows mark YqgP-dependent and red open arrows mark YqgP.S288A-dependent cleavage product of MgtE.


Figure 1A , B In - gel kinase assay of OST1 activity in ABA receptor mutants under cold stress . Ten - day - old wild type , ost1 - 3 , pyr1 pyl1 , 4 , pyr1 pyl1 , 2 , 4 , pyr1 pyl4 , 5 , 8 and pyr1 pyl1 , 4 , 5 , 8 mutants were treated at 4 ° C for 2 h . Total protein extracts were prepared and separated on SDS - PAGE gel containing 0 . 1 mg / mL GST - ∆ ABF2 as a substrate , and incubated with 60 μCi of [ γ - 32P ] ATP . Representative pictures are shown in ( A ) and relative kinase activity is shown in ( B ) . Data information : In ( A , Top , gel autoradiograph ; bottom , Coomassie Brilliant Blue ( CBB ) staining of RuBisCO large subunit was used as a loading control . The ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1 . 00 . In ( B , each bar represents the mean ± SEM of three biological repeats . * P < 0 . 05 ( two - tailed t - test ) . C , D In - gel kinase assay of OST1 activity in ABI1 - OE14 and abi1 abi2 hab1 mutants under cold stress . Representative pictures are shown in ( C ) and relative kinase activity is shown in ( D ) . Data information : In C ) , Top , gel autoradiograph ; bottom , Coomassie Brilliant Blue ( CBB ) staining of RuBisCO large subunit was used as a loading control . The ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1 . 00 . In D ) , each bar represents the mean ± SEM of three biological repeats . * P < 0 . 05 ( two - tailed t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A, B In-gel kinase assay of OST1 activity in ABA receptor mutants under cold stress. Ten-day-old wild type, ost1-3, pyr1 pyl1,4, pyr1 pyl1,2,4, pyr1 pyl4,5,8 and pyr1 pyl1,4,5,8 mutants were treated at 4°C for 2 h. Total protein extracts were prepared and separated on SDS-PAGE gel containing 0.1 mg/mL GST-∆ABF2 as a substrate, and incubated with 60 μCi of [γ-32P]ATP. Representative pictures are shown in (A) and relative kinase activity is shown in (B). Data information: In (A , Top, gel autoradiograph; bottom, Coomassie Brilliant Blue (CBB) staining of RuBisCO large subunit was used as a loading control. The ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In (B , each bar represents the mean ± SEM of three biological repeats. *P < 0.05 (two-tailed t-test).C, D In-gel kinase assay of OST1 activity in ABI1-OE14 and abi1 abi2 hab1 mutants under cold stress. Representative pictures are shown in (C) and relative kinase activity is shown in (D). Data information: In C), Top, gel autoradiograph; bottom, Coomassie Brilliant Blue (CBB) staining of RuBisCO large subunit was used as a loading control. The ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In D), each bar represents the mean ± SEM of three biological repeats. *P < 0.05 (two-tailed t-test).


Figure 2A Interaction of OST1 and EGR2 in yeast . Yeast cells harboring different vector combinations were grown on SC / − Leu / − Trp medium for 3 d or SC / − Leu / − Trp / − His / − Ade medium for 6 d . OST1 - BD / AD and BD / EGR2 - AD were used as negative controls . B OST1 interacts with EGR2 in vitro . Purified recombinant MBP - His - EGR2 or MBP - His proteins from E . coli were immunoprecipitated with MBP beads , and then incubated with purified recombinant GST - OST1 . Precipitated proteins were detected with anti - His and anti - GST antibodies . C Co - IP assay of OST1 with EGR2 in vivo . 35S : HF - OST1 / Super : EGR2 - Myc or 35S : HF - OST1 / Super : Myc was expressed in N . benthamiana leaves . Total proteins were immunoprecipitated with anti - Myc agarose beads , and the co - immunoprecipitation products were subjected to immunoblot analysis . EGR2 - Myc and HF - OST1 were detected with anti - Myc and anti - HA antibodies , respectively . D Bimolecular fluorescence complementation ( BiFC ) assay . Wild - type Arabidopsis protoplasts transformed with OST1 - YFPC and EGR2 - YFPN proteins were incubated for 18 h . The interaction signal was detected by confocal microcopy . The combinations of OST1 - YFPC / GUS - YFPN and GUS - YFPC / EGR2 - YFPN were used as negative controls . Scale bars : 100 μm . E EGR2 inhibits OST1 kinase activity in vitro . Proteins were incubated with 1 μCi [ γ - 32P ] ATP in kinase reaction buffer for 30 min at 30 ° C , and then separated by SDS - PAGE . Top , autoradiograph ; bottom , CBB staining . F , G In - gel assay of OST1 activity in 10 - d - old wild type , egr1 and egr2 single mutants , and the egr1 egr2 double mutant under cold stress . Data information : the ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1 . 00 . In ( F representative pictures are shown : Top , autoradiograph ; bottom , CBB staining . In ( G relative kinase activity is shown . Each bar represents the mean ± SEM of three biological repeats . * * P < 0 . 01 ( two - tailed t - test ) . H , I In - gel assay of OST1 activity in 10 - d - old wild - type and EGR2 - overexpressing plants under cold stress . Data information the ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1 . 00 . In H ) , representative pictures are shown : Top , autoradiograph ; bottom , CBB staining . In I ) , relative kinase activity is shown . Each bar represents the mean ± SEM of three biological repeats . * * P < 0 . 01 ( two - tailed t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Interaction of OST1 and EGR2 in yeast. Yeast cells harboring different vector combinations were grown on SC/−Leu/−Trp medium for 3 d or SC/−Leu/−Trp/−His/−Ade medium for 6 d. OST1-BD/AD and BD/EGR2-AD were used as negative controls.B OST1 interacts with EGR2 in vitro. Purified recombinant MBP-His-EGR2 or MBP-His proteins from E. coli were immunoprecipitated with MBP beads, and then incubated with purified recombinant GST-OST1. Precipitated proteins were detected with anti-His and anti-GST antibodies.C Co-IP assay of OST1 with EGR2 in vivo. 35S:HF-OST1/Super:EGR2-Myc or 35S:HF-OST1/Super:Myc was expressed in N. benthamiana leaves. Total proteins were immunoprecipitated with anti-Myc agarose beads, and the co-immunoprecipitation products were subjected to immunoblot analysis. EGR2-Myc and HF-OST1 were detected with anti-Myc and anti-HA antibodies, respectively.D Bimolecular fluorescence complementation (BiFC) assay. Wild-type Arabidopsis protoplasts transformed with OST1-YFPC and EGR2-YFPN proteins were incubated for 18 h. The interaction signal was detected by confocal microcopy. The combinations of OST1-YFPC/GUS-YFPN and GUS-YFPC/EGR2-YFPN were used as negative controls. Scale bars: 100 μm.E EGR2 inhibits OST1 kinase activity in vitro. Proteins were incubated with 1 μCi [γ-32P]ATP in kinase reaction buffer for 30 min at 30°C, and then separated by SDS-PAGE. Top, autoradiograph; bottom, CBB staining.F, G In-gel assay of OST1 activity in 10-d-old wild type, egr1 and egr2 single mutants, and the egr1 egr2 double mutant under cold stress. Data information: the ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In (F representative pictures are shown: Top, autoradiograph; bottom, CBB staining. In (G relative kinase activity is shown. Each bar represents the mean ± SEM of three biological repeats. **P < 0.01 (two-tailed t-test).H, I In-gel assay of OST1 activity in 10-d-old wild-type and EGR2-overexpressing plants under cold stress. Data information the ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In H), representative pictures are shown: Top, autoradiograph; bottom, CBB staining. In I), relative kinase activity is shown. Each bar represents the mean ± SEM of three biological repeats. **P < 0.01 (two-tailed t-test).


Figure 3Freezing phenotype ( A ) of the egr2 - 3 mutant and egr2 / EGR2 : EGR2 - Myc complementation lines under non - acclimated ( NA ) and acclimated ( CA ; 4 d at 4 ° C ) conditions . Two - week - old seedlings grown on MS medium containing 0 . 8 % agar were treated at − 5 ° C for 0 . 5 h ( NA ) or − 8 ° C for 0 . 5 h ( CA ) . survival rate ( B ) of the egr2 - 3 mutant and egr2 / EGR2 : EGR2 - Myc complementation lines under non - acclimated ( NA ) and acclimated ( CA ; 4 d at 4 ° C ) conditions . Two - week - old seedlings grown on MS medium containing 0 . 8 % agar were treated at − 5 ° C for 0 . 5 h ( NA ) or − 8 ° C for 0 . 5 h ( CA ) . Data information each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) . ion leakage ( C ) of the egr2 - 3 mutant and egr2 / EGR2 : EGR2 - Myc complementation lines under non - acclimated ( NA ) and acclimated ( CA ; 4 d at 4 ° C ) conditions . Two - week - old seedlings grown on MS medium containing 0 . 8 % agar were treated at − 5 ° C for 0 . 5 h ( NA ) or − 8 ° C for 0 . 5 h ( CA ) . Data information each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) . Freezing phenotype of EGR2 - overexpressing plants . Two - week - old Super : EGR2 - Myc plantssurvival rate ( E ) of EGR2 - overexpressing plants . Two - week - old Super : EGR2 - Myc plants Data information : each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) . ion leakage ( F ) of EGR2 - overexpressing plants . Two - week - old Super : EGR2 - Myc plants Data information : each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) . Expression of CBFs ( G in egr2 - 3 mutant plants . Two - week - old seedlings were treated at 4 ° C for the indicated period and subjected to qRT - PCR analysis . Data information : each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) . Expression of CBFs target genes ( H in egr2 - 3 mutant plants . Two - week - old seedlings were treated at 4 ° C for the indicated period and subjected to qRT - PCR analysis . Data information : each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) . Expression of CBFs in EGR2 - overexpressing plants . Two - week - old seedlings were treated at 4 ° C for the indicated period and subjected to qRT - PCR analysis . Data information : each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) . Expression of CBFs target genes J ) in EGR2 - overexpressing plants . Two - week - old seedlings were treated at 4 ° C for the indicated period and subjected to qRT - PCR analysis . Data information : each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Freezing phenotype (A) of the egr2-3 mutant and egr2/EGR2:EGR2-Myc complementation lines under non-acclimated (NA) and acclimated (CA; 4 d at 4°C) conditions. Two-week-old seedlings grown on MS medium containing 0.8% agar were treated at −5°C for 0.5 h (NA) or −8°C for 0.5 h (CA).survival rate (B) of the egr2-3 mutant and egr2/EGR2:EGR2-Myc complementation lines under non-acclimated (NA) and acclimated (CA; 4 d at 4°C) conditions. Two-week-old seedlings grown on MS medium containing 0.8% agar were treated at −5°C for 0.5 h (NA) or −8°C for 0.5 h (CA). Data information each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).ion leakage (C) of the egr2-3 mutant and egr2/EGR2:EGR2-Myc complementation lines under non-acclimated (NA) and acclimated (CA; 4 d at 4°C) conditions. Two-week-old seedlings grown on MS medium containing 0.8% agar were treated at −5°C for 0.5 h (NA) or −8°C for 0.5 h (CA). Data information each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).Freezing phenotype of EGR2-overexpressing plants. Two-week-old Super:EGR2-Myc plantssurvival rate (E) of EGR2-overexpressing plants. Two-week-old Super:EGR2-Myc plants Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).ion leakage (F) of EGR2-overexpressing plants. Two-week-old Super:EGR2-Myc plants Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).Expression of CBFs (G in egr2-3 mutant plants. Two-week-old seedlings were treated at 4°C for the indicated period and subjected to qRT-PCR analysis. Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).Expression of CBFs target genes (H in egr2-3 mutant plants. Two-week-old seedlings were treated at 4°C for the indicated period and subjected to qRT-PCR analysis. Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).Expression of CBFs in EGR2-overexpressing plants. Two-week-old seedlings were treated at 4°C for the indicated period and subjected to qRT-PCR analysis. Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).Expression of CBFs target genes J) in EGR2-overexpressing plants. Two-week-old seedlings were treated at 4°C for the indicated period and subjected to qRT-PCR analysis. Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).


Figure 4N - myristoylation of EGR2 and EGR2G2A in N . benthamiana leaves . Protein extracts prepared from N . benthamiana leaves expressing EGR2 - Myc or EGR2G2A - Myc were immunoprecipated by anti - Myc agarose beads . Precipitated proteins were detected by anti - Myc and anti - myristic acid antibodies . D Immunoblot analysis showing that the total level of myristoylated proteins in wild type plants . Total proteins were prepared from two - week - old wild type seedlings treated with or without 80 μM myristic acid alkyne at 22 ° C for 72 h and then subjected to click - reaction with the biotin azide . The reaction was protected from light at room temperature for 3 h . The products were detected by anti - streptavidin - HRP antibody . E N - myristoylation of EGR2 in plants . Total proteins were prepared from two - week - old EGR2 - Myc seedlings The products were immunoprecipated by anti - Myc agarose beads and detected by anti - streptavidin - HRP antibody . EGR2 - Myc detected by anti - Myc antibody was used as a loading control . F Immunoblot assay of myristoylated EGR2 under cold stress . Total proteins were prepared form EGR2 - Myc plants treated with 4 ° C for the indicated period , and immunoprecipated by anti - Myc agarose beads . Precipitated proteins were detected by anti - Myc and anti - myristic acid antibodies . The ratio of band intensity detected by anti - myristic acid antibody to anti - Myc antibody without cold treatment was set to 1 . 00 . Representative pictures are shown ( left ) and quantitative analysis is shown ( right ) . Each bar represents the mean ± SEM of three biological repeats . * * P < 0 . 01 ( two - tailed t - test ) . G Expression of EGR2 in egr2 - 3 EGR2 : EGR2G2A - Myc complementation transgenic plants . Total RNAs were extracted from 2 - week - old seedlings and subjected to RT - PCR analysis . EF1α was used as a loading control . Freezing tolerance assays of the egr2 - 3 mutant and EGR2 : EGR2G2A - Myc transgenic plants in egr2 - 3 mutant . Two - week - old seedlings grown on MS medium containing 0 . 8 % agar at 22 ° C were treated at − 5 ° C for 0 - 1 . 5 h ( NA ) or − 8 ° C for 0 - 1 . 5 h ( CA ) . Representative photographs ( - 5oC , 0 . 5 h ; - 8oC , 0 . 5 h ) are shown in ( H ) Freezing tolerance assays of the egr2 - 3 mutant and EGR2 : EGR2G2A - Myc transgenic plants in egr2 - 3 mutant . Two - week - old seedlings grown on MS medium containing 0 . 8 % agar at 22 ° C were treated at − 5 ° C for 0 - 1 . 5 h ( NA ) or − 8 ° C for 0 - 1 . 5 h ( CA ) . survival rate is shown in ( I ) . Data shown in ( I ) are mean values ± SEM of three biological replicates , each of which has three technical replicates . Asterisks indicate significant differences compared with wild type for the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) ; n . s . , not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4N-myristoylation of EGR2 and EGR2G2A in N. benthamiana leaves. Protein extracts prepared from N. benthamiana leaves expressing EGR2-Myc or EGR2G2A-Myc were immunoprecipated by anti-Myc agarose beads. Precipitated proteins were detected by anti-Myc and anti-myristic acid antibodies.D Immunoblot analysis showing that the total level of myristoylated proteins in wild type plants. Total proteins were prepared from two-week-old wild type seedlings treated with or without 80 μM myristic acid alkyne at 22°C for 72 h and then subjected to click-reaction with the biotin azide. The reaction was protected from light at room temperature for 3 h. The products were detected by anti-streptavidin-HRP antibody.E N-myristoylation of EGR2 in plants. Total proteins were prepared from two-week-old EGR2-Myc seedlings The products were immunoprecipated by anti-Myc agarose beads and detected by anti-streptavidin-HRP antibody. EGR2-Myc detected by anti-Myc antibody was used as a loading control.F Immunoblot assay of myristoylated EGR2 under cold stress. Total proteins were prepared form EGR2-Myc plants treated with 4°C for the indicated period, and immunoprecipated by anti-Myc agarose beads. Precipitated proteins were detected by anti-Myc and anti-myristic acid antibodies. The ratio of band intensity detected by anti-myristic acid antibody to anti-Myc antibody without cold treatment was set to 1.00. Representative pictures are shown (left) and quantitative analysis is shown (right). Each bar represents the mean ± SEM of three biological repeats. **P < 0.01 (two-tailed t-test).G Expression of EGR2 in egr2-3 EGR2:EGR2G2A-Myc complementation transgenic plants. Total RNAs were extracted from 2-week-old seedlings and subjected to RT-PCR analysis. EF1α was used as a loading control.Freezing tolerance assays of the egr2-3 mutant and EGR2:EGR2G2A-Myc transgenic plants in egr2-3 mutant. Two-week-old seedlings grown on MS medium containing 0.8% agar at 22°C were treated at −5°C for 0-1.5 h (NA) or −8°C for 0-1.5 h (CA). Representative photographs (-5oC, 0.5 h; -8oC, 0.5 h) are shown in (H)Freezing tolerance assays of the egr2-3 mutant and EGR2:EGR2G2A-Myc transgenic plants in egr2-3 mutant. Two-week-old seedlings grown on MS medium containing 0.8% agar at 22°C were treated at −5°C for 0-1.5 h (NA) or −8°C for 0-1.5 h (CA). survival rate is shown in (I). Data shown in (I) are mean values ± SEM of three biological replicates, each of which has three technical replicates. Asterisks indicate significant differences compared with wild type for the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test); n. s., not significant.


Figure 5A In vitro pull - down assay showing the interaction of NMT1 and EGR2 . Recombinant GST - NMT1 was incubated with immunoprecipated MBP - His - EGR2 or MBP - His at 4 ° C for 2 h . Anti - GST and anti - MBP were used to detect NMT1 , EGR2 and MBP - His , respectively . Co - localization of NMT1 and EGR2 in N . benthamiana leaves . Scale bar : 50 μmC , D Effect of cold stress on the interaction of EGR2 and NMT1 determined by co - IP assay . Arabidopsis protoplasts expressing HF / NMT1 - Myc and HF - EGR2 / NMT1 - Myc were treated with or without 4 ° C for 2 h . Total proteins were immunoprecipated by anti - HA agarose beads . Anti - Myc and anti - HA were used to detect NMT1 - Myc and HF - EGR2 , respectively . In ( C ) , relative band intensity is shown below the co - IP blot . In ( D ) , each bar represents the mean ± SEM of three biological experiments ( * * P < 0 . 01 , two - tailed t - test ) . E In vitro pull - down assay showing decreased interaction intensity of EGR2 ( 4 ° C ) and NMT1 compared with that of EGR2 ( 22 ° C ) and NMT1 . Total proteins prepared from two - week - old EGR2 - Myc plants grown at 4 ° C for 0 h and 6 h were immunoprecipated by anti - Myc agarose beads and incubated with GST - NMT1 in pull down buffer for 2 h at 4 ° C . Anti - Myc and anti - GST antibodies were used to detect EGR2 and NMT1 proteins . Relative band intensity is shown below the output blot . F Expression of NMT1 in NMT1 - Myc overexpression plants . EF1α was used as a loading control . Freezing phenotype ( G ) of NMT1 - Myc overexpression transgenic plants . Two - week - old Super : NMT1 - Myc plants were treated at − 5 ° C ( NA ) or − 8 ° C for 0 . 5 h ( CA ) . survival rate ( H ) of NMT1 - Myc overexpression transgenic plants . Two - week - old Super : NMT1 - Myc plants were treated at − 5 ° C ( NA ) or − 8 ° C for 0 . 5 h ( CA ) . In ( H ) , each bar represents the mean ± SEM of three biological experiments , and asterisks indicate significant differences compared with the wild type for the same treatment ( * P < 0 . 05 , two tailed t - test ) . OST1 kinase activity in NMT1 - Myc overexpression plants under cold stress . Top , autoradiograph ; bottom , CBB staining . Representative pictures are shown in ( I ) In ( I ) : the ratio of band intensity of OST1 to RuBisCO in wild type with cold treatment for 2 h was set to 1 . 00 . OST1 kinase activity in NMT1 - Myc overexpression plants under cold stress . the relative kinase activity is shown in ( J ) . In ( J ) : each bar represents the mean ± SEM of three independent experiments ( * * P < 0 . 01 , two tailed t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A In vitro pull-down assay showing the interaction of NMT1 and EGR2. Recombinant GST-NMT1 was incubated with immunoprecipated MBP-His-EGR2 or MBP-His at 4°C for 2 h. Anti-GST and anti-MBP were used to detect NMT1, EGR2 and MBP-His, respectively.Co-localization of NMT1 and EGR2 in N. benthamiana leaves. Scale bar: 50 μmC, D Effect of cold stress on the interaction of EGR2 and NMT1 determined by co-IP assay. Arabidopsis protoplasts expressing HF/NMT1-Myc and HF-EGR2/NMT1-Myc were treated with or without 4°C for 2 h. Total proteins were immunoprecipated by anti-HA agarose beads. Anti-Myc and anti-HA were used to detect NMT1-Myc and HF-EGR2, respectively. In (C), relative band intensity is shown below the co-IP blot. In (D), each bar represents the mean ± SEM of three biological experiments (**P < 0.01, two-tailed t-test).E In vitro pull-down assay showing decreased interaction intensity of EGR2 (4°C) and NMT1 compared with that of EGR2 (22°C) and NMT1. Total proteins prepared from two-week-old EGR2-Myc plants grown at 4°C for 0 h and 6 h were immunoprecipated by anti-Myc agarose beads and incubated with GST-NMT1 in pull down buffer for 2 h at 4°C. Anti-Myc and anti-GST antibodies were used to detect EGR2 and NMT1 proteins. Relative band intensity is shown below the output blot.F Expression of NMT1 in NMT1-Myc overexpression plants. EF1α was used as a loading control.Freezing phenotype (G) of NMT1-Myc overexpression transgenic plants. Two-week-old Super:NMT1-Myc plants were treated at −5°C (NA) or −8°C for 0.5 h (CA).survival rate (H) of NMT1-Myc overexpression transgenic plants. Two-week-old Super:NMT1-Myc plants were treated at −5°C (NA) or −8°C for 0.5 h (CA). In (H), each bar represents the mean ± SEM of three biological experiments, and asterisks indicate significant differences compared with the wild type for the same treatment (*P < 0.05, two tailed t-test).OST1 kinase activity in NMT1-Myc overexpression plants under cold stress. Top, autoradiograph; bottom, CBB staining. Representative pictures are shown in (I) In (I): the ratio of band intensity of OST1 to RuBisCO in wild type with cold treatment for 2 h was set to 1.00.OST1 kinase activity in NMT1-Myc overexpression plants under cold stress. the relative kinase activity is shown in (J). In (J): each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two tailed t-test).


Figure 6A , B Pull - down assay . Total proteins prepared from EGR2 - Myc or EGR2G2A - Myc transgenic plants was immunoprecipated with anti - Myc agarose beads , and then incubated with GST - OST1 . Anti - Myc and anti - GST antibody were used to detect GST - OST1 and EGE2 - Myc . Representative pictures are shown in ( A ) . Relative interaction is shown in ( B ) . Data information : In ( A Relative band intensities are shown . In ( B each bar represents the mean ± SEM of three independent experiments ( * * P < 0 . 01 , two - tailed t - test ) . C LCI of EGR2 and EGR2G2A with OST1 in N . benthamiana leaves . EGR2 - cLuc / OST1 - nLuc and EGR2G2A - cLuc / OST1 - nLuc were expressed in N . benthamiana leaves for 48 h . EGR2 - cLuc / nLuc , EGR2G2A - cLuc / nLuc , and cLuc / OST1 - nLuc were used as negative controls . Scale bar : 2 cm . D , E Co - IP assay . EGR2 - Myc / HF - OST1 , EGR2G2A - Myc / HF - OST1 , HF / EGR2 - Myc and HF / EGR2G2A - Myc were expressed in N . benthamiana leaves . Total proteins were immunoprecipated by anti - HA agarose beads . Anti - HA and anti - Myc antibodies were used to detect HF - OST1 , EGR2 - Myc and EGR2G2A - Myc , respectively . Representative pictures are shown in ( D ) , and relative interaction is shown in ( E ) . Data information : In : Relative band intensities are shown . In each bar represents the mean ± SEM of three independent experiments ( * * P < 0 . 01 , two - tailed t - test ) . F , G Effect of EGR2G2A on the interaction of OST1 and EGR2 in Arabidopsis protoplasts expressing HF - OST1 / EGR2 - GFP / EGR2 - Myc , HF - OST1 / EGR2 - GFP / EGR2G2A - Myc , or HF - OST1 / EGR2 - GFP . Total proteins were immunoprecipated by anti - HA agarose beads . Anti - Myc , anti - GFP , and anti - HA antibodies were used to detect EGR2 - Myc , EGR2 - GFP , and HF - OST1 , respectively . Representative pictures are shown in ( F ) , and relative interaction is shown in ( G ) . Data information In Relative band intensities are shown . In each bar represents the mean ± SEM of three independent experiments ( * * P < 0 . 01 , two - tailed t - test ) . H , I Effect of cold stress on the interaction of OST1 and EGR2 . Arabidopsis protoplasts expressing EGR2 - Myc / HF - OST1 and EGR2 - Myc / HF were treated at 4 ° C for 0 and 2 h . Total proteins were immunoprecipated by anti - HA agarose beads . The co - immunoprecipitated products were detected by anti - HA and anti - Myc antibodies , respectively . Representative pictures are shown in ( H ) , and relative interaction is shown in ( I ) . Data information : In H ) Relative band intensities are shown . In I ) : each bar represents the mean ± SEM of three independent experiments ( * * P < 0 . 01 , two - tailed t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A, B Pull-down assay. Total proteins prepared from EGR2-Myc or EGR2G2A-Myc transgenic plants was immunoprecipated with anti-Myc agarose beads, and then incubated with GST-OST1. Anti-Myc and anti-GST antibody were used to detect GST-OST1 and EGE2-Myc. Representative pictures are shown in (A). Relative interaction is shown in (B). Data information: In (A Relative band intensities are shown. In (B each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two-tailed t-test).C LCI of EGR2 and EGR2G2A with OST1 in N. benthamiana leaves. EGR2-cLuc/OST1-nLuc and EGR2G2A-cLuc/OST1-nLuc were expressed in N. benthamiana leaves for 48 h. EGR2-cLuc/nLuc, EGR2G2A-cLuc/nLuc, and cLuc/OST1-nLuc were used as negative controls. Scale bar: 2 cm.D, E Co-IP assay. EGR2-Myc/HF-OST1, EGR2G2A-Myc/HF-OST1, HF/EGR2-Myc and HF/EGR2G2A-Myc were expressed in N. benthamiana leaves. Total proteins were immunoprecipated by anti-HA agarose beads. Anti-HA and anti-Myc antibodies were used to detect HF-OST1, EGR2-Myc and EGR2G2A-Myc, respectively. Representative pictures are shown in (D), and relative interaction is shown in (E). Data information: In : Relative band intensities are shown. In each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two-tailed t-test).F, G Effect of EGR2G2A on the interaction of OST1 and EGR2 in Arabidopsis protoplasts expressing HF-OST1/EGR2-GFP/EGR2-Myc, HF-OST1/EGR2-GFP/EGR2G2A-Myc, or HF-OST1/EGR2-GFP. Total proteins were immunoprecipated by anti-HA agarose beads. Anti-Myc, anti-GFP, and anti-HA antibodies were used to detect EGR2-Myc, EGR2-GFP, and HF-OST1, respectively. Representative pictures are shown in (F), and relative interaction is shown in (G). Data information In Relative band intensities are shown. In each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two-tailed t-test).H, I Effect of cold stress on the interaction of OST1 and EGR2. Arabidopsis protoplasts expressing EGR2-Myc/HF-OST1 and EGR2-Myc/HF were treated at 4°C for 0 and 2 h. Total proteins were immunoprecipated by anti-HA agarose beads. The co-immunoprecipitated products were detected by anti-HA and anti-Myc antibodies, respectively. Representative pictures are shown in (H), and relative interaction is shown in (I). Data information: In H) Relative band intensities are shown. In I): each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two-tailed t-test).


Figure 7Freezing phenotype ( A ) of wild type , ost1 - 3 , egr2 - 3 , and ost1 egr2 mutants . Two - week - old seedlings grown on MS medium were treated at − 5 ° C ( NA ) or − 8 ° C for 0 . 5 h ( CA ) . survival rate and ion leakage ( B ) of wild type , ost1 - 3 , egr2 - 3 , and ost1 egr2 mutants . Two - week - old seedlings grown on MS medium were treated at − 5 ° C ( NA ) or − 8 ° C for 0 . 5 h ( CA ) . Data information : each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) . C Expression of CBFs in wild type , ost1 - 3 , egr2 - 3 , and ost1 egr2 - 3 plants under cold stress . Two - week - old seedlings grown at 22 ° C were treated at 4 ° C for 0 h and 3 h . Data information : each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Freezing phenotype (A) of wild type, ost1-3, egr2-3, and ost1 egr2 mutants. Two-week-old seedlings grown on MS medium were treated at −5°C (NA) or −8°C for 0.5 h (CA).survival rate and ion leakage (B) of wild type, ost1-3, egr2-3, and ost1 egr2 mutants. Two-week-old seedlings grown on MS medium were treated at −5°C (NA) or −8°C for 0.5 h (CA). Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).C Expression of CBFs in wild type, ost1-3, egr2-3, and ost1 egr2-3 plants under cold stress. Two-week-old seedlings grown at 22°C were treated at 4°C for 0 h and 3 h. Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).


Figure 1Fluorescence polarization ( FP ) competition derived affinity assessment of consecutive truncations of the DAT C13 C - terminus as indicated by the sequences . Bars indicate the mean fold change relative to C13 ( Ki = 1 . 69 µM ) . Data is shown as mean with error - bars as SEM with n = 6 . Representative binding curves are shown in EV1A . FP competition binding of C5 and Tat - C5 to PICK1 with 5FAM - Tat - C5 ( 20 nM ) as tracer . Saturation binding curve is shown in EV1B . Backbone amide chemical shift changes ( ΔδHN ) of PICK1 - PDZ - C10 after addition of Tat - C5 and addition of protease to cleave C10 from the PDZ domain and allow exchange . Numbers refer to residue number in PICK1 ( Uniprot : Q9EP80 ) . Red bars indicate a chemical shift larger than mean + SD . 1H - 15N - HSQC 2D spectra of PICK1 - PDZ - C10 ( orange ) following addition of Tat - C5 ( green ) and subsequent addition of protease ( red ) to allow for exchange between C10 and Tat - C5 . Selectivity screen for Tat - C5 against a selection of 42 purified PDZ domains , performed with a fixed concentration of Tat - C5 ( 10 µM ) in competition with PDZ domains and their respective fluorescent ligands . Data are normalized to binding in absence of peptide ( full line ) . Dashed line represents the level of competition obtained for PICK1 . Screen for C5 can be seen in EV1C . Affinity gain of bivalent C5 peptides ( Pn - ( C5 ) 2 ) N - terminally conjugated with different length PEG linkers as indicated . Bars indicate the mean fold change relative to C5 ( Ki = 1 . 69 µM ) . Data is shown as mean with error - bars as SEM with n = 6 . Representative binding curves are shown in EV1C . FP competition binding of C5 ( also used in C ) and Tat - P4 - ( C5 ) 2 to PICK1 with 5FAM - Tat - C5 ( 20 nM ) as tracer .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Fluorescence polarization (FP) competition derived affinity assessment of consecutive truncations of the DAT C13 C-terminus as indicated by the sequences. Bars indicate the mean fold change relative to C13 (Ki = 1.69 µM). Data is shown as mean with error-bars as SEM with n = 6. Representative binding curves are shown in EV1A.FP competition binding of C5 and Tat-C5 to PICK1 with 5FAM-Tat-C5 (20 nM) as tracer. Saturation binding curve is shown in EV1B.Backbone amide chemical shift changes (ΔδHN) of PICK1-PDZ-C10 after addition of Tat-C5 and addition of protease to cleave C10 from the PDZ domain and allow exchange. Numbers refer to residue number in PICK1 (Uniprot: Q9EP80). Red bars indicate a chemical shift larger than mean+SD.1H-15N-HSQC 2D spectra of PICK1-PDZ-C10 (orange) following addition of Tat-C5 (green) and subsequent addition of protease (red) to allow for exchange between C10 and Tat-C5.Selectivity screen for Tat-C5 against a selection of 42 purified PDZ domains, performed with a fixed concentration of Tat-C5 (10 µM) in competition with PDZ domains and their respective fluorescent ligands. Data are normalized to binding in absence of peptide (full line). Dashed line represents the level of competition obtained for PICK1. Screen for C5 can be seen in EV1C.Affinity gain of bivalent C5 peptides (Pn-(C5)2) N-terminally conjugated with different length PEG linkers as indicated. Bars indicate the mean fold change relative to C5 (Ki = 1.69 µM). Data is shown as mean with error-bars as SEM with n = 6. Representative binding curves are shown in EV1C.FP competition binding of C5 (also used in C) and Tat-P4-(C5)2 to PICK1 with 5FAM-Tat-C5 (20 nM) as tracer.


Figure 2Representative confocal images of SCMS expressing Tac - YFP - DATC24 , incubated with pre - complexed fluorescently labelled PICK1 and unlabelled peptides as indicated ( 10 nM ) , scale bar 10 µm . Full concentration curves shown in Fig S4 and include the representative images shown here . SCMS derived competitive binding curves for peptides as indicated , red line indicates 10 nM as shown in representative images . Representative confocal images of SCMS expressing Tac - YFP - DATC24 , incubated with fluorescently labelled PICK1 and subsequently unlabelled peptide as indicated ( 10µM ) . Scale bar 10 µm . Full concentration curves shown in Fig EV2 and include the representative images shown here . Efficacy of the peptides as indicated to facilitate dissociation of PICK1 from SCMS , red line indicates 10 µM as shown in representative images .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Representative confocal images of SCMS expressing Tac-YFP-DATC24, incubated with pre-complexed fluorescently labelled PICK1 and unlabelled peptides as indicated (10 nM), scale bar 10 µm. Full concentration curves shown in Fig S4 and include the representative images shown here.   SCMS derived competitive binding curves for peptides as indicated, red line indicates 10 nM as shown in representative images.   Representative confocal images of SCMS expressing Tac-YFP-DATC24, incubated with fluorescently labelled PICK1 and subsequently unlabelled peptide as indicated (10µM). Scale bar 10 µm. Full concentration curves shown in Fig EV2 and include the representative images shown here. Efficacy of the peptides as indicated to facilitate dissociation of PICK1 from SCMS, red line indicates 10 µM as shown in representative images.