C Control or LAPTM4B ‐ WT / 6RQ ‐ overexpressing MDA ‐ MB ‐ 231 cells were transfected with His ‐ tagged ubiquitin and myc ‐ tagged Hrs , starved , and stimulated with 100 ng / ml EGF for 30 min before whole ‐ cell lysate harvest . Total ubiquitinated proteins were purified by Ni ‐ NTA agarose and analyzed by Western blot . D Quantification of Hrs ubiquitination levels from the Western blot in ( C ) ( mean + SD ; n = 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Control or LAPTM4B‐WT/6RQ‐overexpressing MDA‐MB‐231 cells were transfected with His‐tagged ubiquitin and myc‐tagged Hrs, starved, and stimulated with 100 ng/ml EGF for 30 min before whole‐cell lysate harvest. Total ubiquitinated proteins were purified by Ni‐NTA agarose and analyzed by Western blot.D Quantification of Hrs ubiquitination levels from the Western blot in (C) (mean + SD; n = 5).


E The co ‐ IP of Myc ‐ Hrs with Flag ‐ LAPTM4B ‐ WT or 6RQ mutant in HEK293 cells .

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E The co‐IP of Myc‐Hrs with Flag‐LAPTM4B‐WT or 6RQ mutant in HEK293 cells.


F The co ‐ IP between Hrs and Nedd4 with Flag ‐ LAPTM4B ‐ WT or 6RQ mutant overexpression in HEK293 cells .

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F The co‐IP between Hrs and Nedd4 with Flag‐LAPTM4B‐WT or 6RQ mutant overexpression in HEK293 cells.


G The effects of LAPTM4B ‐ 6RQ mutant overexpression on EGF ‐ stimulated EGFR degradation and signaling . Flag ‐ LAPTM4B ‐ WT or 6RQ mutant overexpression was accomplished by lentivirus ‐ mediated infection approach . Cells with low levels of expression were selected as polyclonal pools for comparison . Control or overexpressing cells were starved and stimulated with 100 ng / ml EGF for indicated time periods and whole ‐ cell lysates were analyzed by Western blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G The effects of LAPTM4B‐6RQ mutant overexpression on EGF‐stimulated EGFR degradation and signaling. Flag‐LAPTM4B‐WT or 6RQ mutant overexpression was accomplished by lentivirus‐mediated infection approach. Cells with low levels of expression were selected as polyclonal pools for comparison. Control or overexpressing cells were starved and stimulated with 100 ng/ml EGF for indicated time periods and whole‐cell lysates were analyzed by Western blot.


H Quantification of EGFR and pAKT levels from the Western blot in ( G ) ( mean ± SD ; n = 5 ) .

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H Quantification of EGFR and pAKT levels from the Western blot in (G) (mean ± SD; n = 5).


I The PY motif mutant LAPTM4B ‐ 2PA loses interaction with Nedd4 but keeps interaction with Hrs in HEK293 cells cotransfected with the indicated proteins .

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I The PY motif mutant LAPTM4B‐2PA loses interaction with Nedd4 but keeps interaction with Hrs in HEK293 cells cotransfected with the indicated proteins.


J The effects of LAPTM4B ‐ 2PA mutant overexpression on EGF ‐ stimulated EGFR degradation and signaling . Overexpression of Flag ‐ LAPTM4B ‐ WT or 2PA mutant overexpression was accomplished by lentivirus ‐ mediated infection approach . Cells with high expression of Flag ‐ LAPTM4B were selected as polyclonal pools for comparison . Cells were starved and stimulated with 100 ng / ml EGF for the indicated time periods , and whole ‐ cell lysates were analyzed by Western blot . K Quantification of EGFR degradation from Western blot in ( J ) ( mean ± SD ; n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J The effects of LAPTM4B‐2PA mutant overexpression on EGF‐stimulated EGFR degradation and signaling. Overexpression of Flag‐LAPTM4B‐WT or 2PA mutant overexpression was accomplished by lentivirus‐mediated infection approach. Cells with high expression of Flag‐LAPTM4B were selected as polyclonal pools for comparison. Cells were starved and stimulated with 100 ng/ml EGF for the indicated time periods, and whole‐cell lysates were analyzed by Western blot.K Quantification of EGFR degradation from Western blot in (J) (mean ± SD; n = 3).


A - C Co ‐ immunoprecipitation ( co ‐ IP ) of wild ‐ type ( WT ) or C ‐ terminal deletion mutants of HA ‐ PIPKIγi5 with SNX5 ( A ) or LAPTM4B ( B and C ) in HEK293 cells cotransfected with indicated proteins . Δ675 indicates a deletion from amino acid 675 to the C ‐ terminus .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-C Co‐immunoprecipitation (co‐IP) of wild‐type (WT) or C‐terminal deletion mutants of HA‐PIPKIγi5 with SNX5 (A) or LAPTM4B (B and C) in HEK293 cells cotransfected with indicated proteins. Δ675 indicates a deletion from amino acid 675 to the C‐terminus.


D PIPKIγi5 promotes SNX5 association with LAPTM4B . HEK293 cells cotransfected with indicated proteins were harvested for co ‐ IP to assay the interaction between Myc ‐ SNX5 and Flag ‐ LAPTM4B .

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D PIPKIγi5 promotes SNX5 association with LAPTM4B. HEK293 cells cotransfected with indicated proteins were harvested for co‐IP to assay the interaction between Myc‐SNX5 and Flag‐LAPTM4B.


E SNX5 inhibits Hrs association with LAPTM4B . HEK293 cells cotransfected with indicated proteins were harvested for co ‐ IP to assay the interaction between Myc ‐ Hrs and Flag ‐ LAPTM4B .

E SNX5 inhibits Hrs association with LAPTM4B. HEK293 cells cotransfected with indicated proteins were harvested for co‐IP to assay the interaction between Myc‐Hrs and Flag‐LAPTM4B.


SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability . ( A , B , D , E ) Expression of ste11 + ( A , D ) using quantitative RT - PCR of RNA extracted from cells grown either in nutrient rich medium ( dark gray ) or shifted for 4 hours to starvation medium ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , gcn5Η , tsc1Η , gcn5Η tsc1Η , tsc2Η , gcn5Η tsc2Η , rhb1 - DA4 - a constitutively active ( CA ) rhb1 mutant [ 34 ] , gcn5Η rhb1 - DA4 , tor2 - L1310P - a CA tor2 mutant [ 33 ] , gcn5Η tor2 - L1310P , tor1Η , gcn5Η tor1Η , gad8Η , and gcn5Η gad8Η . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 4 ( A , B ) independent experiments , overlaid with individual data points and standard error ( SE ) bars . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 4 for A , B )

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability. (A,B,D,E) Expression of ste11+ (A,D) using quantitative RT-PCR of RNA extracted from cells grown either in nutrient rich medium (dark gray) or shifted for 4 hours to starvation medium (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, tsc1Η, gcn5Η tsc1Η, tsc2Η, gcn5Η tsc2Η, rhb1-DA4 - a constitutively active (CA) rhb1 mutant [34], gcn5Η rhb1-DA4, tor2-L1310P - a CA tor2 mutant [33], gcn5Η tor2-L1310P, tor1Η, gcn5Η tor1Η, gad8Η, and gcn5Η gad8Η. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 4 (A,B)independent experiments, overlaid with individual data points and standard error (SE) bars. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 4 for A,B)


SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability . ( A , B , D , E ) Expression of mei2 + ( B , E ) using quantitative RT - PCR of RNA extracted from cells grown either in nutrient rich medium ( dark gray ) or shifted for 4 hours to starvation medium ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , gcn5Η , tsc1Η , gcn5Η tsc1Η , tsc2Η , gcn5Η tsc2Η , rhb1 - DA4 - a constitutively active ( CA ) rhb1 mutant [ 34 ] , gcn5Η rhb1 - DA4 , tor2 - L1310P - a CA tor2 mutant [ 33 ] , gcn5Η tor2 - L1310P , tor1Η , gcn5Η tor1Η , gad8Η , and gcn5Η gad8Η . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 4 ( A , B ) independent experiments , overlaid with individual data points and standard error ( SE ) bars . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 4 for A , B )

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability. (A,B,D,E) Expression of mei2+ (B,E) using quantitative RT-PCR of RNA extracted from cells grown either in nutrient rich medium (dark gray) or shifted for 4 hours to starvation medium (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, tsc1Η, gcn5Η tsc1Η, tsc2Η, gcn5Η tsc2Η, rhb1-DA4 - a constitutively active (CA) rhb1 mutant [34], gcn5Η rhb1-DA4, tor2-L1310P - a CA tor2 mutant [33], gcn5Η tor2-L1310P, tor1Η, gcn5Η tor1Η, gad8Η, and gcn5Η gad8Η. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 4 (A,B) independent experiments, overlaid with individual data points and standard error (SE) bars. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 4 for A,B)


( C , F ) Cells were grown to mid - log phase either in rich medium or shifted for 8 hours to starvation medium . Zygotes and tetrads , which correspond to differentiated cells , were counted under a light microscope . Cells of the following genotypes were analyzed : WT isogenic controls , gcn5Η , tsc1Η , gcn5Η tsc1Η , rhb1 - DA4 , gcn5Η rhb1 - DA4 , gad8Η , and gcn5Η gad8Η . Each value represents the mean percentage and SE of differentiating cells to the total number of cells , averaged from 4 independent experiments . At least 200 cells from the indicated genotypes were counted in each experiment . White arrowheads indicate zygotes . Scale bar , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C,F) Cells were grown to mid-log phase either in rich medium or shifted for 8 hours to starvation medium. Zygotes and tetrads, which correspond to differentiated cells, were counted under a light microscope. Cells of the following genotypes were analyzed: WT isogenic controls, gcn5Η, tsc1Η, gcn5Η tsc1Η, rhb1-DA4, gcn5Η rhb1-DA4, gad8Η, and gcn5Η gad8Η. Each value represents the mean percentage and SE of differentiating cells to the total number of cells, averaged from 4 independent experiments. At least 200 cells from the indicated genotypes were counted in each experiment. White arrowheads indicate zygotes. Scale bar, 10 μm.


SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability . ( A , B , D , E ) Expression of ste11 + ( A , D ) using quantitative RT - PCR of RNA extracted from cells grown either in nutrient rich medium ( dark gray ) or shifted for 4 hours to starvation medium ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , gcn5Η , tsc1Η , gcn5Η tsc1Η , tsc2Η , gcn5Η tsc2Η , rhb1 - DA4 - a constitutively active ( CA ) rhb1 mutant [ 34 ] , gcn5Η rhb1 - DA4 , tor2 - L1310P - a CA tor2 mutant [ 33 ] , gcn5Η tor2 - L1310P , tor1Η , gcn5Η tor1Η , gad8Η , and gcn5Η gad8Η . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 3 ( D , E ) independent experiments , overlaid with individual data points and standard error ( SE ) bars . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 3 for D , E ) .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability. (A,B,D,E) Expression of ste11+ (A,D) using quantitative RT-PCR of RNA extracted from cells grown either in nutrient rich medium (dark gray) or shifted for 4 hours to starvation medium (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, tsc1Η, gcn5Η tsc1Η, tsc2Η, gcn5Η tsc2Η, rhb1-DA4 - a constitutively active (CA) rhb1 mutant [34], gcn5Η rhb1-DA4, tor2-L1310P - a CA tor2 mutant [33], gcn5Η tor2-L1310P, tor1Η, gcn5Η tor1Η, gad8Η, and gcn5Η gad8Η. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 3 (D,E) independent experiments, overlaid with individual data points and standard error (SE) bars. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 3 for D,E).


SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability . ( A , B , D , E ) Expression of mei2 + ( B , E ) using quantitative RT - PCR of RNA extracted from cells grown either in nutrient rich medium ( dark gray ) or shifted for 4 hours to starvation medium ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , gcn5Η , tsc1Η , gcn5Η tsc1Η , tsc2Η , gcn5Η tsc2Η , rhb1 - DA4 - a constitutively active ( CA ) rhb1 mutant [ 34 ] , gcn5Η rhb1 - DA4 , tor2 - L1310P - a CA tor2 mutant [ 33 ] , gcn5Η tor2 - L1310P , tor1Η , gcn5Η tor1Η , gad8Η , and gcn5Η gad8Η . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 3 ( D , E ) independent experiments , overlaid with individual data points and standard error ( SE ) bars . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 3 for D , E ) .

SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability. (A,B,D,E) Expression of mei2+ (B,E) using quantitative RT-PCR of RNA extracted from cells grown either in nutrient rich medium (dark gray) or shifted for 4 hours to starvation medium (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, tsc1Η, gcn5Η tsc1Η, tsc2Η, gcn5Η tsc2Η, rhb1-DA4 - a constitutively active (CA) rhb1 mutant [34], gcn5Η rhb1-DA4, tor2-L1310P - a CA tor2 mutant [33], gcn5Η tor2-L1310P, tor1Η, gcn5Η tor1Η, gad8Η, and gcn5Η gad8Η. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 3 (D,E) independent experiments, overlaid with individual data points and standard error (SE) bars. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 3 for D,E).


SAGA is phosphorylated in response to nutrient starvation . ( A , B ) 4 - 20 % gradient SDS - polyacrylamide gel electrophoresis analysis of SAGA purified from cells grown either in rich medium ( R ) or starved for 45 minutes ( S ) . SAGA was purified using endogenously TAP - tagged Ada1 . A fraction of each eluate was loaded and either stained with silver , to detect all proteins ( A ) , . A strain without any TAP tag is shown as a negative control for the purification . The graph on the left of the gel in B shows the fluorescence intensity of the phospho - specific stain , which was quantified along the left lane in blue ( rich ) and the middle lane in red ( starved ) , using ImageJ . The area of one peak , which corresponds to the bands marked with arrowheads and was coined p55 , is 1 . 8 - fold larger in SAGA purified from starved cells , as compared to rich conditions . Below is an anti - HA immuno - blot ( IB ) of each eluate , to reveal the amount of Ada1 - HA bait recovered . Shown are gels that are representative of 3 independent experiments . A . U . , arbitrary units .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

SAGA is phosphorylated in response to nutrient starvation. (A,B) 4-20% gradient SDS-polyacrylamide gel electrophoresis analysis of SAGA purified from cells grown either in rich medium (R) or starved for 45 minutes (S). SAGA was purified using endogenously TAP-tagged Ada1. A fraction of each eluate was loaded and either stained with silver, to detect all proteins (A),. A strain without any TAP tag is shown as a negative control for the purification. The graph on the left of the gel in B shows the fluorescence intensity of the phospho-specific stain, which was quantified along the left lane in blue (rich) and the middle lane in red (starved), using ImageJ. The area of one peak, which corresponds to the bands marked with arrowheads and was coined p55, is 1.8-fold larger in SAGA purified from starved cells, as compared to rich conditions. Below is an anti-HA immuno-blot (IB) of each eluate, to reveal the amount of Ada1-HA bait recovered. Shown are gels that are representative of 3 independent experiments. A.U., arbitrary units.


SAGA is phosphorylated in response to nutrient starvation . ( A , B ) 4 - 20 % gradient SDS - polyacrylamide gel electrophoresis analysis of SAGA purified from cells grown either in rich medium ( R ) or starved for 45 minutes ( S ) . SAGA was purified using endogenously TAP - tagged Ada1 . A fraction of each eluate was loaded and either stained with Pro - Q ® Diamond , which stains phosphorylated proteins ( B ) . A strain without any TAP tag is shown as a negative control for the purification . The graph on the left of the gel in B shows the fluorescence intensity of the phospho - specific stain , which was quantified along the left lane in blue ( rich ) and the middle lane in red ( starved ) , using ImageJ . The area of one peak , which corresponds to the bands marked with arrowheads and was coined p55 , is 1 . 8 - fold larger in SAGA purified from starved cells , as compared to rich conditions . Below is an anti - HA immuno - blot ( IB ) of each eluate , to reveal the amount of Ada1 - HA bait recovered . Shown are gels that are representative of 3 independent experiments . A . U . , arbitrary units .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

SAGA is phosphorylated in response to nutrient starvation. (A,B) 4-20% gradient SDS-polyacrylamide gel electrophoresis analysis of SAGA purified from cells grown either in rich medium (R) or starved for 45 minutes (S). SAGA was purified using endogenously TAP-tagged Ada1. A fraction of each eluate was loaded and either stained with Pro-Q® Diamond, which stains phosphorylated proteins (B). A strain without any TAP tag is shown as a negative control for the purification. The graph on the left of the gel in B shows the fluorescence intensity of the phospho-specific stain, which was quantified along the left lane in blue (rich) and the middle lane in red (starved), using ImageJ. The area of one peak, which corresponds to the bands marked with arrowheads and was coined p55, is 1.8-fold larger in SAGA purified from starved cells, as compared to rich conditions. Below is an anti-HA immuno-blot (IB) of each eluate, to reveal the amount of Ada1-HA bait recovered. Shown are gels that are representative of 3 independent experiments. A.U., arbitrary units.


( D ) FLAG - tagged Taf12 was purified from cells grown in rich medium ( R ) or shifted for 45 minutes to starvation medium ( S ) . Anti - FLAG immuno - precipitations ( IP ) were incubated with and without λ - phosphatase or its inhibitor and immuno - blotted ( IB ) , together with 1 % of whole cell extracts ( WCE ) , using an anti - FLAG antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) FLAG-tagged Taf12 was purified from cells grown in rich medium (R) or shifted for 45 minutes to starvation medium (S). Anti-FLAG immuno-precipitations (IP) were incubated with and without λ-phosphatase or its inhibitor and immuno-blotted (IB), together with 1% of whole cell extracts (WCE), using an anti-FLAG antibody.


( E ) Phospho - Taf12 ( P - Taf12 ) , total Taf12 , and tubulin levels were quantified from cells grown in rich medium ( R ) or starved for 45 minutes ( S ) . P - Taf12 levels were normalized to those of total Taf12 , while total Taf12 levels were normalized to those of tubulin . Data points were individually plotted on the graph and overlaid with the mean and SE . Signal intensities were quantified from IBs of 7 independent experiments . Statistical significance was determined using a t - test ( n = 7 , unpaired , two - tailed ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Phospho-Taf12 (P-Taf12), total Taf12, and tubulin levels were quantified from cells grown in rich medium (R) or starved for 45 minutes (S). P-Taf12 levels were normalized to those of total Taf12, while total Taf12 levels were normalized to those of tubulin. Data points were individually plotted on the graph and overlaid with the mean and SE. Signal intensities were quantified from IBs of 7 independent experiments. Statistical significance was determined using a t-test (n = 7, unpaired, two-tailed).


( F ) P - Taf12 and total Taf12 were followed in WT , taf12 - T283A and taf12 - 5A mutants , grown in rich medium ( R ) or starved for 45 minutes ( S ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) P-Taf12 and total Taf12 were followed in WT, taf12-T283A and taf12-5A mutants, grown in rich medium (R) or starved for 45 minutes (S).


( G , H ) SAGA and TFIID were tandem affinity - purified using endogenously TAP - tagged Spt7 ( G ) from strains containing FLAG - tagged Taf12 , grown in rich medium or starved for 45 minutes . TAP - tagged Spt7 and Taf4 were eluted using the TEV protease , releasing a shorter form of each bait ( Spt7 - CBP or Taf4 - CBP ) . Eluates were loaded and immuno - blotted ( IB ) using anti - FLAG or anti - CBP antibodies , together with 1 % of either whole cell extracts ( WCE ) or IgG - sepharose flow - through ( FT ) . Shown are IBs that are representative of 2 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G,H) SAGA and TFIID were tandem affinity-purified using endogenously TAP-tagged Spt7 (G) from strains containing FLAG-tagged Taf12, grown in rich medium or starved for 45 minutes. TAP-tagged Spt7 and Taf4 were eluted using the TEV protease, releasing a shorter form of each bait (Spt7-CBP or Taf4-CBP). Eluates were loaded and immuno-blotted (IB) using anti-FLAG or anti-CBP antibodies, together with 1% of either whole cell extracts (WCE) or IgG-sepharose flow-through (FT). Shown are IBs that are representative of 2 independent experiments.


( G , H ) SAGA and TFIID were tandem affinity - purified using endogenously TAP - tagged Taf4 ( H ) , from strains containing FLAG - tagged Taf12 , grown in rich medium or starved for 45 minutes . TAP - tagged Spt7 and Taf4 were eluted using the TEV protease , releasing a shorter form of each bait ( Spt7 - CBP or Taf4 - CBP ) . Eluates were loaded and immuno - blotted ( IB ) using anti - FLAG or anti - CBP antibodies , together with 1 % of either whole cell extracts ( WCE ) or IgG - sepharose flow - through ( FT ) . Shown are IBs that are representative of 2 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G,H) SAGA and TFIID were tandem affinity-purified using endogenously TAP-tagged Taf4 (H), from strains containing FLAG-tagged Taf12, grown in rich medium or starved for 45 minutes. TAP-tagged Spt7 and Taf4 were eluted using the TEV protease, releasing a shorter form of each bait (Spt7-CBP or Taf4-CBP). Eluates were loaded and immuno-blotted (IB) using anti-FLAG or anti-CBP antibodies, together with 1% of either whole cell extracts (WCE) or IgG-sepharose flow-through (FT). Shown are IBs that are representative of 2 independent experiments.


( I ) P - Taf12 , total Taf12 , and Ser546 - phosphorylated Gad8AKT were followed in WT cells , grown in rich conditions or over a time - course after a shift to starvation medium . Gad8AKT phosphorylation at Ser546 is a proxy of TORC2 activity in S . pombe .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) P-Taf12, total Taf12, and Ser546-phosphorylated Gad8AKT were followed in WT cells, grown in rich conditions or over a time-course after a shift to starvation medium. Gad8AKT phosphorylation at Ser546 is a proxy of TORC2 activity in S. pombe.


( J ) P - Taf12 and total Taf12 were followed in WT cells , grown in rich conditions or shifted to different starvation media for 30 minutes . These include both the removal of the nitrogen source , ammonium chloride ( NH4Cl ) , and the reduction of the carbon source , glucose , from a concentration of 2 % to 0 . 5 % . Alternatively , cells were either only deprived of NH4Cl or only exposed to reduced glucose levels ( 2 % to 0 . 5 % ) .

O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O

(J) P-Taf12 and total Taf12 were followed in WT cells, grown in rich conditions or shifted to different starvation media for 30 minutes. These include both the removal of the nitrogen source, ammonium chloride (NH4Cl), and the reduction of the carbon source, glucose, from a concentration of 2% to 0.5%. Alternatively, cells were either only deprived of NH4Cl or only exposed to reduced glucose levels (2% to 0.5%).


( K ) P - Taf12 and total Taf12 were followed in WT cells , grown in rich conditions or deprived of NH4Cl for one hour . Then , NH4Cl was added back to the medium for 30 or 60 minutes . Both short and long exposures of the FLAG IBs are shown to detect total Taf12 and P - Taf12 , respectively , within the linear range of the chemi - luminescence signal . Anti - tubulin IBs are shown as controls for loading between samples . Shown are star that are representative of at least 3 independent experiments . The number sign ( # ) symbol identifies antibody heavy chains and the star ( * ) symbol labels an unspecific band detected by the anti - FLAG antibody in S . pombe .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) P-Taf12 and total Taf12 were followed in WT cells, grown in rich conditions or deprived of NH4Cl for one hour. Then, NH4Cl was added back to the medium for 30 or 60 minutes. Both short and long exposures of the FLAG IBs are shown to detect total Taf12 and P-Taf12, respectively, within the linear range of the chemi-luminescence signal. Anti-tubulin IBs are shown as controls for loading between samples. Shown are star that are representative of at least 3 independent experiments. The number sign (#) symbol identifies antibody heavy chains and the star (*) symbol labels an unspecific band detected by the anti-FLAG antibody in S. pombe.


( A , B ) Expression of ste11 + ( A ) using quantitative RT - PCR of RNA extracted from cells grown either in rich medium ( dark gray ) or starved for 4 hours ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , ppa2Η , par1Η , pab1Η , tsc1Η , and pab1Η tsc1Η . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 4 independent experiments , overlaid with individual data points and SE . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A,B) Expression of ste11+ (A) using quantitative RT-PCR of RNA extracted from cells grown either in rich medium (dark gray) or starved for 4 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), ppa2Η, par1Η, pab1Η, tsc1Η, and pab1Η tsc1Η. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 4 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 4).


( A , B ) Expression of mei2 + ( B ) using quantitative RT - PCR of RNA extracted from cells grown either in rich medium ( dark gray ) or starved for 4 hours ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , ppa2Η , par1Η , pab1Η , tsc1Η , and pab1Η tsc1Η . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 4 independent experiments , overlaid with individual data points and SE . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 4 ) .

(A,B) Expression of mei2+ (B) using quantitative RT-PCR of RNA extracted from cells grown either in rich medium (dark gray) or starved for 4 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), ppa2Η, par1Η, pab1Η, tsc1Η, and pab1Η tsc1Η. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 4 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 4).


( C ) Cells were grown to mid - log phase either in rich medium or starved for 8 hours . Zygotes and tetrads , which correspond to differentiated cells , were counted under a light microscope . Each value represents the mean percentage and SE of differentiating cells to the total number of cells , averaged from 4 independent experiments . At least 200 cells from the indicated genotypes were counted in each experiment . White arrowheads indicate zygotes . Scale bar , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Cells were grown to mid-log phase either in rich medium or starved for 8 hours. Zygotes and tetrads, which correspond to differentiated cells, were counted under a light microscope. Each value represents the mean percentage and SE of differentiating cells to the total number of cells, averaged from 4 independent experiments. At least 200 cells from the indicated genotypes were counted in each experiment. White arrowheads indicate zygotes. Scale bar, 10 μm.


( D ) P - Taf12 was followed by anti - FLAG IB of protein extracts from WT and pab1Η strains , grown in rich conditions ( R ) or starved for 45 minutes ( S ) . An anti - Rpb1 IB is shown as a control for loading . The signal intensities of P - Taf12 and total Taf12 were quantified in each strain and condition . Ratios of P - Taf12 to total Taf12 were calculated from 6 independent experiments and individually plotted in a graph below the IBs , together with the mean and SE . Averaged values from all WT controls grown in rich medium were set at 1 to allow comparisons across culture conditions and strains . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 6 ; * p < 0 . 01 ; # p < 0 . 05 ) . A short and a long exposure of the FLAG IB are shown to detect total Taf12 and P - Taf12 , respectively , within the linear range of the chemi - luminescence signal .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) P-Taf12 was followed by anti-FLAG IB of protein extracts from WT and pab1Η strains, grown in rich conditions (R) or starved for 45 minutes (S). An anti-Rpb1 IB is shown as a control for loading. The signal intensities of P-Taf12 and total Taf12 were quantified in each strain and condition. Ratios of P-Taf12 to total Taf12 were calculated from 6 independent experiments and individually plotted in a graph below the IBs, together with the mean and SE. Averaged values from all WT controls grown in rich medium were set at 1 to allow comparisons across culture conditions and strains. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 6; *p < 0.01; #p < 0.05). A short and a long exposure of the FLAG IB are shown to detect total Taf12 and P-Taf12, respectively, within the linear range of the chemi-luminescence signal.


( E ) FLAG - tagged Taf12 and TAP - tagged Pab1 were affinity purified separately from cells grown in rich conditions ( upper panels ) . TAP - tagged Pab1 was cleaved off from beads using the Tobacco Etch Virus ( TEV ) protease , releasing a shorter form of Pab1 ( CBP - Pab1 ) in the eluate ( E ) . CBP - Pab1 was then mixed with beads containing FLAG - Taf12 IPs and incubated in a phosphatase buffer , with and without 0 . 5 µM microcystin . Each reaction was then analyzed by IB and simultaneously probed with anti - FLAG or anti - CBP antibodies ( lower panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

(E) FLAG-tagged Taf12 and TAP-tagged Pab1 were affinity purified separately from cells grown in rich conditions (upper panels). TAP-tagged Pab1 was cleaved off from beads using the Tobacco Etch Virus (TEV) protease, releasing a shorter form of Pab1 (CBP-Pab1) in the eluate (E). CBP-Pab1 was then mixed with beads containing FLAG-Taf12 IPs and incubated in a phosphatase buffer, with and without 0.5 µM microcystin. Each reaction was then analyzed by IB and simultaneously probed with anti-FLAG or anti-CBP antibodies (lower panel).


( F ) Exponentially growing cells were treated for 1 hour with Torin1 , which was added at increasing concentrations , 7 and 21 µM , to rich medium . Dimethylsulphoxide ( DMSO ) was used as the vehicle and added as a negative control .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Exponentially growing cells were treated for 1 hour with Torin1, which was added at increasing concentrations, 7 and 21 µM, to rich medium. Dimethylsulphoxide (DMSO) was used as the vehicle and added as a negative control.


( G ) P - Taf12 was followed by anti - FLAG IB of protein extracts from WT and tor2 - ts10 strains , grown in rich conditions at 25 ° C or shifted to 30 ° C for 4 and 6 hours . ( F , G ) Anti - tubulin IBs are shown as a control for loading between samples . Shown are IBs that are representative of 3 independent experiments . The star ( * ) symbol labels unspecific bands detected by the anti - FLAG or anti - CBP antibodies in S . pombe .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) P-Taf12 was followed by anti-FLAG IB of protein extracts from WT and tor2-ts10 strains, grown in rich conditions at 25°C or shifted to 30°C for 4 and 6 hours. (F,G) Anti-tubulin IBs are shown as a control for loading between samples. Shown are IBs that are representative of 3 independent experiments. The star (*) symbol labels unspecific bands detected by the anti-FLAG or anti-CBP antibodies in S. pombe.


( A ) Igo1 phosphorylation ( P - Igo1 ) was followed by anti - MYC IB of protein extracts from WT , ppk18Η , and igo1 - S64A strains , grown in rich conditions ( R ) or starved for 45 minutes ( S ) . Igo1 migration was analyzed by electrophoresis of both a 12 % SDS - polyacrylamide gel containing the Phos - tag ™ molecule ( right panel ) and a standard 10 % SDS - polyacrylamide gel ( left panel ) . Arrowheads indicate the various phosphorylated isoforms of Igo1 - MYC in each strain and condition . Shown is an IB representative of 2 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Igo1 phosphorylation (P-Igo1) was followed by anti-MYC IB of protein extracts from WT, ppk18Η, and igo1-S64A strains, grown in rich conditions (R) or starved for 45 minutes (S). Igo1 migration was analyzed by electrophoresis of both a 12% SDS-polyacrylamide gel containing the Phos-tag™ molecule (right panel) and a standard 10% SDS-polyacrylamide gel (left panel). Arrowheads indicate the various phosphorylated isoforms of Igo1-MYC in each strain and condition. Shown is an IB representative of 2 independent experiments.


( B , C ) Expression of ste11 + ( C ) using quantitative RT - PCR of RNA extracted from cells grown either in rich medium ( dark gray ) or starved for 4 hours ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , gcn5Η , pab1Η , gcn5Η ppk18Η , pab1Η , pab1Η ppk18Η , igo1Η , igo1 - S64A , gcn5Η igo1 - S64A and pab1Η igo1 - S64A . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 4 independent experiments , overlaid with individual data points and SE . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,C) Expression of ste11+ (C) using quantitative RT-PCR of RNA extracted from cells grown either in rich medium (dark gray) or starved for 4 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, pab1Η, gcn5Η ppk18Η, pab1Η, pab1Η ppk18Η, igo1Η, igo1-S64A, gcn5Η igo1-S64A and pab1Η igo1-S64A. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 4 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 4).


( B , C ) Expression of mei2 + ( D ) using igo1 of RNA extracted from cells grown either in rich medium ( dark gray ) or starved for 4 hours ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , gcn5Η , ppk18Η , gcn5Η ppk18Η , pab1Η , pab1Η ppk18Η , igo1Η , igo1 - S64A , gcn5Η igo1 - S64A and pab1Η igo1 - S64A . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 4 independent experiments , overlaid with individual data points and SE . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,C) Expression of mei2+ (D) using igo1 of RNA extracted from cells grown either in rich medium (dark gray) or starved for 4 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, ppk18Η, gcn5Η ppk18Η, pab1Η, pab1Η ppk18Η, igo1Η, igo1-S64A, gcn5Η igo1-S64A and pab1Η igo1-S64A. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 4 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 4).


( D ) P - Taf12 was followed by anti - FLAG IB of protein extracts from WT , S . pombeΗ , and igo1Η strains , grown in rich conditions ( R ) or starved for 45 minutes ( S ) . An anti - MYC IB is shown as a control for loading . ( D , E ) The signal intensities of P - Taf12 and total Taf12 were quantified in each strain and condition . Shown below each blot are average measurements of 3 independent experiments ( n = 3 ) . A short and a long exposure of the FLAG IB are shown to detect total Taf12 and P - Taf12 , respectively , within the linear range of the chemi - luminescence signal . The star ( * ) symbol labels an unspecific band detected by the FLAG antibody in S . pombe .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) P-Taf12 was followed by anti-FLAG IB of protein extracts from WT, S. pombeΗ, and igo1Η strains, grown in rich conditions (R) or starved for 45 minutes (S). An anti-MYC IB is shown as a control for loading. (D,E) The signal intensities of P-Taf12 and total Taf12 were quantified in each strain and condition. Shown below each blot are average measurements of 3 independent experiments (n = 3). A short and a long exposure of the FLAG IB are shown to detect total Taf12 and P-Taf12, respectively, within the linear range of the chemi-luminescence signal. The star (*) symbol labels an unspecific band detected by the FLAG antibody in S. pombe.


( E ) P - Taf12 was followed in WT , igoS64A , pab1Η , and pab1Η igoS64A strains , grown in rich conditions ( R ) or starved for 45 minutes ( S ) . An anti - Rpb1 IB is shown as a control for loading . ( D , E ) The signal intensities of P - Taf12 and total Taf12 were quantified in each strain and condition . Shown below each blot are average measurements of 3 independent experiments ( n = 3 ) . A short and a long exposure of the FLAG IB are shown to detect total Taf12 and P - Taf12 , respectively , within the linear range of the chemi - luminescence signal . The star ( * ) symbol labels an unspecific band detected by the S . pombe antibody in S . pombe .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) P-Taf12 was followed in WT, igoS64A, pab1Η, and pab1Η igoS64A strains, grown in rich conditions (R) or starved for 45 minutes (S). An anti-Rpb1 IB is shown as a control for loading. (D,E) The signal intensities of P-Taf12 and total Taf12 were quantified in each strain and condition. Shown below each blot are average measurements of 3 independent experiments (n = 3). A short and a long exposure of the FLAG IB are shown to detect total Taf12 and P-Taf12, respectively, within the linear range of the chemi-luminescence signal. The star (*) symbol labels an unspecific band detected by the S. pombe antibody in S. pombe.


( A ) P - Taf12 was followed by anti - FLAG IB of protein extracts from WT , tor1Η , and gad8Η strains , grown in rich conditions ( R ) or starved for 45 minutes ( S ) . An anti - tubulin IB is shown as a control for loading . The signal intensities of P - Taf12 and total Taf12 were quantified in each strain and condition . P - Taf12 to Taf2 ratios were calculated from 3 independent experiments and individually plotted in a graph below the IBs , together with the mean and SE . Averaged values from all WT controls grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) P-Taf12 was followed by anti-FLAG IB of protein extracts from WT, tor1Η, and gad8Η strains, grown in rich conditions (R) or starved for 45 minutes (S). An anti-tubulin IB is shown as a control for loading. The signal intensities of P-Taf12 and total Taf12 were quantified in each strain and condition. P-Taf12 to Taf2 ratios were calculated from 3 independent experiments and individually plotted in a graph below the IBs, together with the mean and SE. Averaged values from all WT controls grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 3).


( B ) P - Taf12 was followed by anti - FLAG IB of protein extracts from sty1Η cells ( left panel ) or ssp2Η cells ( right panel ) , grown in rich conditions ( R ) or starved for 45 minutes ( S ) . An anti - tubulin IB is shown as a control for loading . The star ( * ) symbols label an unspecific band detected by the anti - FLAG antibody in S . pombe . Shown are IBs that are representative of 2 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) P-Taf12 was followed by anti-FLAG IB of protein extracts from sty1Η cells (left panel) or ssp2Η cells (right panel), grown in rich conditions (R) or starved for 45 minutes (S). An anti-tubulin IB is shown as a control for loading. The star (*) symbols label an unspecific band detected by the anti-FLAG antibody in S. pombe. Shown are IBs that are representative of 2 independent experiments.


( C ) TAP - tagged Gad8 was immuno - precipitated using IgG - sepharose beads ( IP ) from strains containing FLAG - tagged Taf12 , grown either in rich medium ( left panels ) or starved for 45 minutes ( right panels ) . TAP - tagged Gad8 was eluted from beads using the TEV protease , releasing a shorter form of Gad8 ( CBP - Gad8 ) . Eluates were loaded and immuno - blotted ( IB ) using anti - FLAG or anti - CBP antibodies , together with 1 % of whole cell extracts ( WCE ) , to detect Taf12 co - precipitation with Gad8 . A strain which does not contain any TAP tag is shown as a negative control for the IP . Shown is an IB representative of two independent experiments . ( A , C ) The star ( * ) symbols label unspecific bands detected by the FLAG or the CBP antibodies in S . pombe .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) TAP-tagged Gad8 was immuno-precipitated using IgG-sepharose beads (IP) from strains containing FLAG-tagged Taf12, grown either in rich medium (left panels) or starved for 45 minutes (right panels). TAP-tagged Gad8 was eluted from beads using the TEV protease, releasing a shorter form of Gad8 (CBP-Gad8). Eluates were loaded and immuno-blotted (IB) using anti-FLAG or anti-CBP antibodies, together with 1% of whole cell extracts (WCE), to detect Taf12 co-precipitation with Gad8. A strain which does not contain any TAP tag is shown as a negative control for the IP. Shown is an IB representative of two independent experiments. (A,C) The star (*) symbols label unspecific bands detected by the FLAG or the CBP antibodies in S. pombe.


( D ) Endogenous Gad8 - HA was affinity purified from starved cells , in order to activate its kinase activity ( left and middle panels ) , and mixed with recombinant , purified GST - Taf12 , GST - Taf12 - 5A , or GST - Fkh2 proteins ( right panels ) in a kinase assay buffer . Each assay was then analyzed by IB and probed with an anti - phospho - AKT substrate antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Endogenous Gad8-HA was affinity purified from starved cells, in order to activate its kinase activity (left and middle panels), and mixed with recombinant, purified GST-Taf12, GST-Taf12-5A, or GST-Fkh2 proteins (right panels) in a kinase assay buffer. Each assay was then analyzed by IB and probed with an anti-phospho-AKT substrate antibody.


( A , B ) Expression of quantitative RT - PCR + ( A ) using quantitative RT - PCR of RNA extracted from cells grown either in rich medium ( dark gray ) or starved for 2 hours ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) and taf12 - 5A mutants . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 6 independent experiments , overlaid with individual data points and SE . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 6 ) .

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(A,B) Expression of quantitative RT-PCR+ (A) using quantitative RT-PCR of RNA extracted from cells grown either in rich medium (dark gray) or starved for 2 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT) and taf12-5A mutants. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 6 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 6).


mei2 + ( B ) using quantitative RT - PCR of RNA extracted from cells grown either in rich medium ( dark gray ) or starved for 2 hours ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) and taf12 - 5A mutants . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 6 independent experiments , overlaid with individual data points and SE . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 6 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

mei2+ (B) using quantitative RT-PCR of RNA extracted from cells grown either in rich medium (dark gray) or starved for 2 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT) and taf12-5A mutants. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 6 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 6).


( C , E ) Zygotes and tetrads , which correspond to differentiated cells , were counted from cultures of homothallic cells grown to mid - log phase in rich medium ( t0 ) and shifted to starvation medium for up to 24 hours ( C ) Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , taf12 - 5A , taf12 - 5DE , tor1 - 1972A , and taf12 - 5A tor1 - 1972A . Each value represents the mean percentage and SE of differentiating cells to the total number of cells , averaged from 6 independent experiments . At least 200 cells from the indicated genotypes were counted in each experiment . Statistical significance was determined by 2 - way ANOVA followed by Tukey ' s multiple comparison tests ( n = 6 ) .

(C,E) Zygotes and tetrads, which correspond to differentiated cells, were counted from cultures of homothallic cells grown to mid-log phase in rich medium (t0) and shifted to starvation medium for up to 24 hours (C) Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), taf12-5A, taf12-5DE, tor1-1972A, and taf12-5A tor1-1972A. Each value represents the mean percentage and SE of differentiating cells to the total number of cells, averaged from 6 independent experiments. At least 200 cells from the indicated genotypes were counted in each experiment. Statistical significance was determined by 2-way ANOVA followed by Tukey's multiple comparison tests (n = 6).


( D ) P - Taf12 was followed by anti - FLAG IB of protein extracts from WT and tor1 - 1972A strains , grown in rich medium and shifted to starved conditions for 60 minutes . An anti - Tubulin IB is shown as a control for loading . Shown is an IB which is representative of 2 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) P-Taf12 was followed by anti-FLAG IB of protein extracts from WT and tor1-1972A strains, grown in rich medium and shifted to starved conditions for 60 minutes. An anti-Tubulin IB is shown as a control for loading. Shown is an IB which is representative of 2 independent experiments.


( C , E ) taf12 and tetrads , which correspond to differentiated cells , were counted from heterothallic cells mixed and incubated for 2 days on mating medium ( E ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , taf12 - 5A , taf12 - 5DE , tor1 - 1972A , and taf12 - 5A tor1 - 1972A . Each value represents the mean percentage and SE of differentiating cells to the total number of cells , averaged from 6 independent experiments . At least 200 cells from the indicated genotypes were counted in each experiment . Statistical significance was determined by 2 - way ANOVA followed by Tukey ' s multiple comparison tests ( n = 6 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C,E) taf12 and tetrads, which correspond to differentiated cells, were counted from heterothallic cells mixed and incubated for 2 days on mating medium (E). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), taf12-5A, taf12-5DE, tor1-1972A, and taf12-5A tor1-1972A. Each value represents the mean percentage and SE of differentiating cells to the total number of cells, averaged from 6 independent experiments. At least 200 cells from the indicated genotypes were counted in each experiment. Statistical significance was determined by 2-way ANOVA followed by Tukey's multiple comparison tests (n = 6).


( B ) Chlamydomonas reinhardtii ( Cr ) UBA5 and UFC1 are active E1 and E2 enzymes . SDS - PAGE analysis showing transfer of UFM1 to CrUBA5 and CrUFC1 . The gels are run in non - reducing conditions except where otherwise specified . The presented gel is representative of two independent experiments . BME : β - mercaptoethanol ; ATP : Adenosine triphosphate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O

(B) Chlamydomonas reinhardtii (Cr) UBA5 and UFC1 are active E1 and E2 enzymes. SDS-PAGE analysis showing transfer of UFM1 to CrUBA5 and CrUFC1. The gels are run in non-reducing conditions except where otherwise specified. The presented gel is representative of two independent experiments. BME: β-mercaptoethanol; ATP: Adenosine triphosphate.


( C ) RPL26 mono - and di - UFMylation is lost in Chlamydomonas reinhardtii ( Cr ) uba5 and ufl1 mutants . Liquid TAP cultures were either left untreated ( control ) or treated for 24 hours with 200 ng / mL tunicamycin . Protein extracts were analyzed by immunoblotting with anti - UFM1 antibodies . Total proteins were analyzed by Ponceau S staining . 12 hours and 24 hours treatment replicates are shown in Fig . S3C . Right Panel , Quantification of UFMylated RPL26 . RPL26 mono - UFMylated ; RPL26 - ( UFM1 ) 2 : RPL26 di - UFMylated .

O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) RPL26 mono- and di-UFMylation is lost in Chlamydomonas reinhardtii (Cr) uba5 and ufl1 mutants. Liquid TAP cultures were either left untreated (control) or treated for 24 hours with 200 ng/mL tunicamycin. Protein extracts were analyzed by immunoblotting with anti-UFM1 antibodies. Total proteins were analyzed by Ponceau S staining. 12 hours and 24 hours treatment replicates are shown in Fig. S3C. Right Panel, Quantification of UFMylated RPL26. RPL26 mono-UFMylated; RPL26-(UFM1)2: RPL26 di-UFMylated.


( D ) Chlamydomonas reinhardtii ( Cr ) UFMylation pathway mutants are sensitive to ER stress triggered by tunicamycin . Liquid TAP cultures of wild type ( wt ) , uba5 , ufl1 and ire1 mutants were either left untreated ( control ) or treated for 3 days with 200 ng / mL of tunicamycin . Left panel , representative images of control and treated liquid cultures taken 3 - days after incubation . Middle Panel , optical density ( OD ) 600 ( OD600 ) quantification of each genetic background under control conditions . Bars represent the mean ( ± SD ) of 5 biological replicates . Two - tailed unpaired t - tests were performed to analyze the differences between wild type and mutants . Right Panel , normalized OD600 quantification of each genetic background under tunicamycin treatment conditions . Bars represent the mean ( ± SD ) of 5 biological replicates . Two - tailed unpaired t - tests were performed to analyze the differences between wild type and mutants . ns , p - value > 0 . 05 ; * * * , p - value < 0 . 001 . BR : Biological Replicate .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Chlamydomonas reinhardtii (Cr) UFMylation pathway mutants are sensitive to ER stress triggered by tunicamycin. Liquid TAP cultures of wild type (wt), uba5, ufl1 and ire1 mutants were either left untreated (control) or treated for 3 days with 200 ng/mL of tunicamycin. Left panel, representative images of control and treated liquid cultures taken 3-days after incubation. Middle Panel, optical density (OD) 600 (OD600) quantification of each genetic background under control conditions. Bars represent the mean (± SD) of 5 biological replicates. Two-tailed unpaired t-tests were performed to analyze the differences between wild type and mutants. Right Panel, normalized OD600 quantification of each genetic background under tunicamycin treatment conditions. Bars represent the mean (± SD) of 5 biological replicates. Two-tailed unpaired t-tests were performed to analyze the differences between wild type and mutants. ns, p-value > 0.05; ***, p-value < 0.001. BR: Biological Replicate.


( F , G ) C53 IDR is essential for UFM1 interaction . HsC53 ( B ) and AtC53 ( C ) IDRs are necessary to mediate the interaction with AtUFM1 and HsUFM1 respectively . MBP - AtC53ΔIDR : MBP - AtC53 ( 1 - 239 , ( KGSGSTSGSG ) 2 , 373 - 549 ) ; MBP - HsC53ΔIDR : HsC53 ( 1 - 262 , ( KGSGSTSGSG ) , 317 - 506 ) . F , G ) Bacterial lysates containing recombinant protein were mixed and pulled down with glutathione magnetic agarose beads . Input and bound proteins were visualized by immunoblotting with anti - GST and anti - MBP antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F, G) C53 IDR is essential for UFM1 interaction. HsC53 (B) and AtC53 (C) IDRs are necessary to mediate the interaction with AtUFM1 and HsUFM1 respectively. MBP-AtC53ΔIDR: MBP-AtC53(1-239, (KGSGSTSGSG)2, 373-549); MBP-HsC53ΔIDR: HsC53(1-262, (KGSGSTSGSG), 317-506). F, G) Bacterial lysates containing recombinant protein were mixed and pulled down with glutathione magnetic agarose beads. Input and bound proteins were visualized by immunoblotting with anti-GST and anti-MBP antibodies.


( H ) AtC53sAIM cannot interact with AtUFM1 . Individual or combinatorial mutations in sAIM1 ( 1A : W276A ) , sAIM2 ( 2A : W287A ) and sAIM3 ( 3A : W335A ) suggest sAIM1 is crucial for UFM1 interaction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) AtC53sAIM cannot interact with AtUFM1. Individual or combinatorial mutations in sAIM1 (1A: W276A), sAIM2 (2A: W287A) and sAIM3 (3A: W335A) suggest sAIM1 is crucial for UFM1 interaction.


( B ) The cAIM peptide cannot outcompete HsUFM1 - HsC53 interaction . Bacterial lysates containing recombinant protein were mixed and pulled down with glutathione magnetic agarose beads . Input and bound proteins were visualized by immunoblotting with anti - GST and anti - MBP antibodies . cAIM peptide was used to a final concentration of 200 µM . HsC53sAIM : HsC53W269A , W294A , W312A .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The cAIM peptide cannot outcompete HsUFM1-HsC53 interaction. Bacterial lysates containing recombinant protein were mixed and pulled down with glutathione magnetic agarose beads. Input and bound proteins were visualized by immunoblotting with anti-GST and anti-MBP antibodies. cAIM peptide was used to a final concentration of 200 µM. HsC53sAIM: HsC53W269A, W294A, W312A.


( C , D ) Microscopy - based protein - protein interaction assays showing unlike GABARAP - C53 interaction , UFM1 - C53 interaction is insensitive to cAIM peptide competition . Glutathione - sepharose beads were prepared by incubating them with GST - GABARAP ( C ) or GST - HsUFM1 ( D ) . The pre - assembled beads were then washed and mixed with 1 µM of HsC53 containing increasing concentrations of cAIM peptide ( 0 - 100 µM ) . The beads were then imaged using a confocal microscope . Left Panel , representative confocal images ( inverted grayscale ) for each condition are shown . Right panel , normalized fluorescence is shown for each condition with the mean ( ± SD ) of 4 replicates . Unpaired two - samples Wilcoxon test with continuity correction was performed to analyze the differences between wild type and wild type with 100 µM AIM peptide . ns , not significant , p - value > 0 . 05 , * * * , p - value < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) Microscopy-based protein-protein interaction assays showing unlike GABARAP-C53 interaction, UFM1-C53 interaction is insensitive to cAIM peptide competition. Glutathione-sepharose beads were prepared by incubating them with GST-GABARAP (C) or GST-HsUFM1 (D). The pre-assembled beads were then washed and mixed with 1 µM of HsC53 containing increasing concentrations of cAIM peptide (0-100 µM). The beads were then imaged using a confocal microscope. Left Panel, representative confocal images (inverted grayscale) for each condition are shown. Right panel, normalized fluorescence is shown for each condition with the mean (± SD) of 4 replicates. Unpaired two-samples Wilcoxon test with continuity correction was performed to analyze the differences between wild type and wild type with 100 µM AIM peptide. ns, not significant, p-value > 0.05, ***, p-value < 0.001.


( B ) Binding of AtUFM1 to AtC53 IDR leads to a general drop in signal intensity . NMR signal intensity of AtC53 IDR ( 100 µM ) in the presence of 300 µM AtUFM1 normalized to signal intensity of AtC53 IDR alone . Bars corresponding to residues in the AIMs are highlighted .

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(B) Binding of AtUFM1 to AtC53 IDR leads to a general drop in signal intensity. NMR signal intensity of AtC53 IDR (100 µM) in the presence of 300 µM AtUFM1 normalized to signal intensity of AtC53 IDR alone. Bars corresponding to residues in the AIMs are highlighted.


( D ) The backbone amide signals of AtC53 IDR shift upon AtUFM1 addition in a concentration - dependent manner . Insets of overlaid 1H - 15N HSQC spectra of isotope - labelled AtC53 IDR ( 100 µM ) showing chemical shift perturbations of individual peaks from backbone amides of AIM residues in their free ( gray ) or bound state to unlabelled AtUFM1 . Chemical shifts are indicated with arrows .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) The backbone amide signals of AtC53 IDR shift upon AtUFM1 addition in a concentration-dependent manner. Insets of overlaid 1H-15N HSQC spectra of isotope-labelled AtC53 IDR (100 µM) showing chemical shift perturbations of individual peaks from backbone amides of AIM residues in their free (gray) or bound state to unlabelled AtUFM1. Chemical shifts are indicated with arrows.


( B , Conversion of sAIM into cAIM leads to reduced UFM1 binding and stronger ATG8 interaction . Bacterial lysates containing recombinant proteins were mixed and pulled down with glutathione magnetic agarose beads . Input and bound proteins were visualized by immunoblotting with anti - GST and anti - MBP antibodies . AtC53sAIM : AtC53 ( W276A , W287A , W335A ) ; AtC53cAIM : AtC53 ( IDWD274WDDI , IDWD285WDDI , IDWD333WDDI ) ; ATG8LDS : ATG8YL50AA .

(B, Conversion of sAIM into cAIM leads to reduced UFM1 binding and stronger ATG8 interaction. Bacterial lysates containing recombinant proteins were mixed and pulled down with glutathione magnetic agarose beads. Input and bound proteins were visualized by immunoblotting with anti-GST and anti-MBP antibodies. AtC53sAIM: AtC53 (W276A, W287A, W335A); AtC53cAIM: AtC53(IDWD274WDDI, IDWD285WDDI, IDWD333WDDI); ATG8LDS: ATG8YL50AA.


( G ) AtC53cAIM forms more GFP - ATG8A colocalizing puncta upon ER stress . Upper Panel , representative confocal images of transgenic Arabidopsis seedlings co - expressing C53 - mCherry ( magenta ) , C53sAIM - mCherry and C53cAIM - mCherry with GFP - ATG8a in c53 mutant background under normal condition and after tunicamycin stress . 6 - day old seedlings were incubated in liquid 1 / 2 MS medium with 1 % sucrose supplemented with DMSO as control or tunicamycin ( 10 μg / ml ) for 6 hours before imaging . Scale bars , 30 μm . Inset scale bars , 10 μm . Right Panel , Quantification of the C53 - autophagosomes ( C53 - APG ) per normalized Z - stacks . Bars represent the mean ( ± SD ) of at least twenty roots from 3 biological replicates for each genotype and treatment . Unpaired two - samples Wilcoxon test with continuity correction was performed to analyze the differences between wild type and mutants . * * * , p - value < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) AtC53cAIM forms more GFP-ATG8A colocalizing puncta upon ER stress. Upper Panel, representative confocal images of transgenic Arabidopsis seedlings co-expressing C53-mCherry (magenta), C53sAIM-mCherry and C53cAIM-mCherry with GFP-ATG8a in c53 mutant background under normal condition and after tunicamycin stress. 6-day old seedlings were incubated in liquid 1/2 MS medium with 1% sucrose supplemented with DMSO as control or tunicamycin (10 μg/ml) for 6 hours before imaging. Scale bars, 30 μm. Inset scale bars, 10 μm. Right Panel, Quantification of the C53-autophagosomes (C53-APG) per normalized Z-stacks. Bars represent the mean (± SD) of at least twenty roots from 3 biological replicates for each genotype and treatment. Unpaired two-samples Wilcoxon test with continuity correction was performed to analyze the differences between wild type and mutants. ***, p-value < 0.001.


B . Genotype distribution in litters from heterozygous matings ( Fbxl4 + / - x Fbxl4 + / - ) at embryonic stage 13 . 5 ( E13 . 5 ) and of live animals at weaning . Data is presented as a percentage . Embryos n = 79 , weaned mice n = 339 . Dashed lines indicate the expected Mendelian ratios . Chi2 test vs expected Mendelian ratios ; * * * , p < 0 . 001 .

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B. Genotype distribution in litters from heterozygous matings (Fbxl4+/- x Fbxl4+/-) at embryonic stage 13.5 (E13.5) and of live animals at weaning. Data is presented as a percentage. Embryos n=79, weaned mice n=339. Dashed lines indicate the expected Mendelian ratios. Chi2 test vs expected Mendelian ratios; ***, p<0.001.


C . Representative images of wild - type ( + / + ) , heterozygous knockout ( + / - ) and homozygous Fbxl4 knockout embryos ( - / - ) at E13 . 5 .

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C. Representative images of wild-type (+/+), heterozygous knockout (+/-) and homozygous Fbxl4 knockout embryos (-/-) at E13.5.


D . Relative levels of mtDNA of Fbxl4 knockout ( - / - ) embryos and the corresponding wild type ( + / + ) embryos at E13 . 5 . Data represent mean ± SEM , n = 5 . Student ' s t - test ; * , p < 0 . 05 .

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D. Relative levels of mtDNA of Fbxl4 knockout (-/-) embryos and the corresponding wild type (+/+) embryos at E13.5. Data represent mean ± SEM, n=5. Student's t-test; *, p<0.05.


E . Representative images of male Fbxl4 knockout ( - / - ) and matched wild - type ( + / + ) animals at one year of age ( left ) . A hunch - back phenotype was observed both in male and female knockout animals ( right , indicated by arrow ) .

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E. Representative images of male Fbxl4 knockout (-/-) and matched wild-type (+/+) animals at one year of age (left). A hunch-back phenotype was observed both in male and female knockout animals (right, indicated by arrow).


F - I . Whole body ( F ) , heart ( G ) , liver ( H ) and kidney ( I ) weights of 1 - year old animals . Data represent mean ± SEM , n ≥ 5 animals . Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . , not significant ( p ≥ 0 . 05 ) .

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F-I. Whole body (F), heart (G), liver (H) and kidney (I) weights of 1-year old animals. Data represent mean ± SEM, n ≥ 5 animals. Student's t-test; *, p<0.05; **, p<0.01; ***, p<0.001; n.s., not significant (p ≥ 0.05).


A . Relative levels of mtDNA in tissues of 1 - year - old Fbxl4 knockout mice determined by quantitative real - time PCR ( normalized to controls ) . Data are presented as mean ± SEM , n = 6 for controls and n = 8 for knockout animals . Student ' s t - test ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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A. Relative levels of mtDNA in tissues of 1-year-old Fbxl4 knockout mice determined by quantitative real-time PCR (normalized to controls). Data are presented as mean ± SEM, n=6 for controls and n=8 for knockout animals. Student's t-test; **, p<0.01; ***, p<0.001.


B . Transcript levels in indicated tissues of 1 - year - old Fbxl4 knockout mice determined by quantitative real - time PCR . Relative mRNA levels in control animals were set as 1 ( dashed line ) . Data are presented as mean ± SEM , n = 6 for controls and n = 8 for knockout animals . Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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B. Transcript levels in indicated tissues of 1-year-old Fbxl4 knockout mice determined by quantitative real-time PCR. Relative mRNA levels in control animals were set as 1 (dashed line). Data are presented as mean ± SEM, n=6 for controls and n=8 for knockout animals. Student's t-test; *, p<0.05; **, p<0.01; ***, p<0.001.


C . Western blot analysis of protein steady - state levels in liver protein extracts from wild type ( Fbxl4 + / + ) and Fbxl4 knock - out ( Fbxl4 - / - ) animals . D . Quantification of the western blot in ( C ) . Signal normalized to the average of controls and presented as mean ± SEM , n = 4 for both controls and knockout animals . Student ' s t - test ; * * , p < 0 . 01 ; * * * , p < 0 , 001 .

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C. Western blot analysis of protein steady-state levels in liver protein extracts from wild type (Fbxl4+/+) and Fbxl4 knock-out (Fbxl4-/-) animals. D. Quantification of the western blot in (C). Signal normalized to the average of controls and presented as mean ± SEM, n=4 for both controls and knockout animals. Student's t-test; **, p<0.01; ***, p<0,001.


E . Quantitative TMT - based proteomic analysis of wild - type and Fbxl4 knock - out liver tissue protein extracts . Proteins from three control and three Fbxl4 knock - out liver protein extracts were TMT - labelled , quantified and analyzed separately as described in Materials and Methods ; the data from control and knock - out animals were averaged after the analysis and presented as - log10 of the adjusted p - value vs log2 - fold change ( logFC ) . Mitochondrial proteins were selected according to mouse MitoCarta 2 . 0 ( Calvo et al , 2016 ) and highlighted in red ; lysosomal proteins were selected according to hLGDB database ( Brozzi et al , 2013 ) and highlighted in yellow .

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E. Quantitative TMT-based proteomic analysis of wild-type and Fbxl4 knock-out liver tissue protein extracts. Proteins from three control and three Fbxl4 knock-out liver protein extracts were TMT-labelled, quantified and analyzed separately as described in Materials and Methods; the data from control and knock-out animals were averaged after the analysis and presented as -log10 of the adjusted p-value vs log2-fold change (logFC). Mitochondrial proteins were selected according to mouse MitoCarta 2.0(Calvo et al, 2016) and highlighted in red; lysosomal proteins were selected according to hLGDB database(Brozzi et al, 2013) and highlighted in yellow.


F . Enrichment analysis of lysosomal and mitochondrial proteins according to the logFC median of each group of proteins . Data is presented as 25 to 75 percentile box with the indicated median and whiskers representing the ± 1 . 5x inter - quartile range . Proteins included in each group : total = 1523 , mitochondrial = 324 , lysosomal = 50 .

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F. Enrichment analysis of lysosomal and mitochondrial proteins according to the logFC median of each group of proteins. Data is presented as 25 to 75 percentile box with the indicated median and whiskers representing the ±1.5x inter-quartile range. Proteins included in each group: total=1523, mitochondrial=324, lysosomal=50.


B . Relative mtDNA content in fibroblasts from patients and controls as determined by qRT - PCR using an ND6 probe and normalized to nuclear genomic DNA content using an 18S probe . Individual values are presented for each sample , as well as their mean ± SEM values .

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B. Relative mtDNA content in fibroblasts from patients and controls as determined by qRT-PCR using an ND6 probe and normalized to nuclear genomic DNA content using an 18S probe. Individual values are presented for each sample, as well as their mean ± SEM values.


C . Steady - state protein levels in three control and three FBXL4 patient - derived fibroblasts analyzed by western blotting .

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C. Steady-state protein levels in three control and three FBXL4 patient-derived fibroblasts analyzed by western blotting.


D . Quantification of the western blot in ( C ) . Signals from the control and FBXL4 - mutated cells were considered as biological replicates , normalized to the average of controls and displayed as mean ± SEM ( n = 3 for each ) . Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

D. Quantification of the western blot in (C). Signals from the control and FBXL4-mutated cells were considered as biological replicates, normalized to the average of controls and displayed as mean ± SEM (n=3 for each). Student's t-test; *, p<0.05; **, p<0.01; ***, p<0.001.


E . Quantification of nuclear and mitochondrial transcript levels in control and patient fibroblasts . The results from control and patient cells were averaged , normalized to controls and displayed as mean ± SEM ( n = 3 biological replicates for each condition ) .

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E. Quantification of nuclear and mitochondrial transcript levels in control and patient fibroblasts. The results from control and patient cells were averaged, normalized to controls and displayed as mean ± SEM (n=3 biological replicates for each condition).


F . Quantitative TMT - based proteomic analysis of control and patient fibroblasts . Proteins from three control and three patient fibroblast cell lines were TMT - labelled , quantified and analyzed separately as described in Materials and Methods ; the data from control and patients were averaged after the analysis and presented as - log10 of the adjusted p - value vs log2 - fold change ( logFC ) . Mitochondrial proteins were selected according to human MitoCarta 2 . 0 ( Calvo et al , 2016 ) and highlighted in blue ; lysosomal proteins were selected according to hLGDB database ( Brozzi et al , 2013 ) and highlighted in yellow .

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F. Quantitative TMT-based proteomic analysis of control and patient fibroblasts. Proteins from three control and three patient fibroblast cell lines were TMT-labelled, quantified and analyzed separately as described in Materials and Methods; the data from control and patients were averaged after the analysis and presented as -log10 of the adjusted p-value vs log2-fold change (logFC). Mitochondrial proteins were selected according to human MitoCarta 2.0 (Calvo et al, 2016) and highlighted in blue; lysosomal proteins were selected according to hLGDB database (Brozzi et al, 2013) and highlighted in yellow.


G . Log2FC correlation analysis of Mouse Liver proteomic dataset and Fibroblast Proteomic dataset . Proteins with a log2FC > 0 . 5 are highlighted in black and lysosomal proteins are highlighted in yellow .

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G. Log2FC correlation analysis of Mouse Liver proteomic dataset and Fibroblast Proteomic dataset. Proteins with a log2FC > 0.5 are highlighted in black and lysosomal proteins are highlighted in yellow.


B . Steady - state protein levels in wild - type ( WT ) and FBXL4 knockout ( KO ) RKO cells .

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B. Steady-state protein levels in wild-type (WT) and FBXL4 knockout (KO) RKO cells.


C . ( Upper panel ) Pulse - chase labeling of mitochondrial translation products in wild type ( WT ) and FBXL4 knockout ( KO ) cells . The cells were incubated for 1 hour with a 35S methionine - cysteine labeling mix in the presence of anisomycin to inhibit cytosolic translation ( pulse ) . After removal of the medium , the cells were incubated in complete growth medium without inhibitors and labeled amino acids for 16 or 24 hours ( chase ) . A representative experiment of 4 biological replicates is shown . A Coomassie - stained part of the gel is shown as loading control . ( Lower panel ) Quantification of the MTCO1 - ND4 signal presented as mean ± SEM ; n = 4 biological replicates ; Student ' s t - test ; * , p < 0 . 05 .

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C. (Upper panel) Pulse-chase labeling of mitochondrial translation products in wild type (WT) and FBXL4 knockout (KO) cells. The cells were incubated for 1 hour with a 35S methionine-cysteine labeling mix in the presence of anisomycin to inhibit cytosolic translation (pulse). After removal of the medium, the cells were incubated in complete growth medium without inhibitors and labeled amino acids for 16 or 24 hours (chase). A representative experiment of 4 biological replicates is shown. A Coomassie-stained part of the gel is shown as loading control. (Lower panel) Quantification of the MTCO1-ND4 signal presented as mean ± SEM; n=4 biological replicates; Student's t-test; *, p<0.05.


D . ( Upper panel ) Mitochondrial translation products were labeled in wild type ( WT ) and FBXL4 knock - out ( KO ) RKO cells for 60 minutes as in C , followed by 24 h chase in the presence of 5 μM epoxomicin ( epoxo ) or 20 mM ammonium chloride ( NH4Cl ) . A representative experiment of n ≥ 3 biological replicates is shown . A fragment of the Coomassie - stained gel is shown as loading control . ( Lower panel ) Quantification of the ND4 - MTCO1 signal . n ≥ 3 biological replicates , data are presented as mean ± SEM . Student ' s t - test ; * , p < 0 . 05 .

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D. (Upper panel) Mitochondrial translation products were labeled in wild type (WT) and FBXL4 knock-out (KO) RKO cells for 60 minutes as in C, followed by 24 h chase in the presence of 5 μM epoxomicin (epoxo) or 20 mM ammonium chloride (NH4Cl). A representative experiment of n≥3 biological replicates is shown. A fragment of the Coomassie-stained gel is shown as loading control. (Lower panel) Quantification of the ND4-MTCO1 signal. n≥3 biological replicates, data are presented as mean ± SEM. Student's t-test; *, p<0.05.


E - F . Steady - state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with

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E-F. Steady-state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with NH4Cl


E - F . Steady - state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with epoxomicin ( Epoxo , F ) .

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E-F. Steady-state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with epoxomicin (Epoxo, F).


A . Confocal images from the mito - QC - expressing fibroblast lines . Scale bar 25 μm . B . Quantification of the mitolysosomes / cell area ( μm2 ) using the mito - QC counter plugin for ImageJ ( Montava - Garriga et al , 2020 ) . At least 3 technical replicates for each line and n > 15 cells analyzed for each technical replicate . Data is presented as 25 to 75 percentile box with the indicated median and whiskers representing 5 - 95 percentile interval . Non - parametric Mann - Whitney test ; * * * , p < 0 . 001 .

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A. Confocal images from the mito-QC-expressing fibroblast lines. Scale bar 25 μm. B. Quantification of the mitolysosomes/cell area (μm2) using the mito-QC counter plugin for ImageJ (Montava-Garriga et al, 2020). At least 3 technical replicates for each line and n>15 cells analyzed for each technical replicate. Data is presented as 25 to 75 percentile box with the indicated median and whiskers representing 5-95 percentile interval. Non-parametric Mann-Whitney test; ***, p<0.001.


( b ) Temperature - sensitive growth assay . Yeast strains ( wild - type or deletion mutants , indicated on the left ) were transformed with either an empty vector or a plasmid carrying an artificial guide RNA gene targeting U2 pseudouridylation at position 42 and 44 ( indicated on the top ) , and grown at various temperatures ( indicated at the bottom ) .

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(b) Temperature-sensitive growth assay. Yeast strains (wild-type or deletion mutants, indicated on the left) were transformed with either an empty vector or a plasmid carrying an artificial guide RNA gene targeting U2 pseudouridylation at position 42 and 44 (indicated on the top), and grown at various temperatures (indicated at the bottom).


( c ) Blockage of pseudouridylation by deletion of specific pseudouridylase genes . RNAs isolated from wild - type and pseudouridylase - deletion strains ( indicated on the top ) were used for U2 pseudouridylation assay ( CMC modification followed by primer - extension ) . Primer - extension pauses / stops correspond to ψ sites ( indicated on the left and right ) . Note : The above - background signal of ψ42 in the snr81Δ lane ( and to some extent , in the snr81Δ pus7Δ lane ) is likely caused by the presence of strong ψ44 band . This is a primer - extension artifact , which often occurs when there is a strong ψ signal in the neighboring position . Alternatively , it is possible that having ψ at position 44 allows for an unidentified enzyme other than snR81 to modify U42 ( albeit inefficiently ) ( Perhaps Pus1 , which is responsible for ψ44 formation , can be a bit " processive " ) . Lanes 1 - 8 and lanes 9 - 16 are from two separate gels .

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(c) Blockage of pseudouridylation by deletion of specific pseudouridylase genes. RNAs isolated from wild-type and pseudouridylase-deletion strains (indicated on the top) were used for U2 pseudouridylation assay (CMC modification followed by primer-extension). Primer-extension pauses/stops correspond to ψ sites (indicated on the left and right). Note: The above-background signal of ψ42 in the snr81Δ lane (and to some extent, in the snr81Δ pus7Δ lane) is likely caused by the presence of strong ψ44 band. This is a primer-extension artifact, which often occurs when there is a strong ψ signal in the neighboring position. Alternatively, it is possible that having ψ at position 44 allows for an unidentified enzyme other than snR81 to modify U42 (albeit inefficiently) (Perhaps Pus1, which is responsible for ψ44 formation, can be a bit "processive"). Lanes 1-8 and lanes 9-16 are from two separate gels.


( d ) Restoration of U2 pseudouridylation by an artificial box H / ACA RNA . RNAs isolated from various yeast strains ( indicated on the top ) , including the snr81Δ pusΔ strain that was transformed with an artificial guide RNA targeting positions 42 and 44 ( lanes 9 and 10 ) , were assayed for pseudouridylation [ see legend to ( c ) ] .

(d) Restoration of U2 pseudouridylation by an artificial box H/ACA RNA. RNAs isolated from various yeast strains (indicated on the top), including the snr81Δ pusΔ strain that was transformed with an artificial guide RNA targeting positions 42 and 44 (lanes 9 and 10), were assayed for pseudouridylation [see legend to (c)].


( a ) Growth assay using the ACT1 - CUP1 reporter system . Yeast cells carrying the ACT1 - CUP1 reporter were deleted of pseudouridylase genes ( indicated on the left ) , and were then assayed for growth at 30 ° C on media containing various concentrations of [ Cu2 + ] ( indicated at the bottom ) .

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(a) Growth assay using the ACT1-CUP1 reporter system. Yeast cells carrying the ACT1-CUP1 reporter were deleted of pseudouridylase genes (indicated on the left), and were then assayed for growth at 30°C on media containing various concentrations of [Cu2+] (indicated at the bottom).


( b ) Growth rescue by restoration of pseudouridylation . The wild - type strain [ from ( a ) ] or the snr81Δ pusΔ strain [ also from ( a ) ] , which exhibited the most severe growth - deficiency phenotype , was transformed with an empty vector or a plasmid carrying a pseudouridylation guide RNA gene targeting either position 42 , position 44 , or both ( indicated ) . The resulting cells were assayed for growth at 30 ° C on media containing various concentrations of [ Cu2 + ] ( indicated at the bottom ) .

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(b) Growth rescue by restoration of pseudouridylation. The wild-type strain [from (a)] or the snr81Δ pusΔ strain [also from (a)], which exhibited the most severe growth-deficiency phenotype, was transformed with an empty vector or a plasmid carrying a pseudouridylation guide RNA gene targeting either position 42, position 44, or both (indicated). The resulting cells were assayed for growth at 30°C on media containing various concentrations of [Cu2+] (indicated at the bottom).