Figure 1B Sedimentation assay to quantify condensation of unmodified TDP - 43 versus in vitro phosphorylated TDP - 43 ( + CK1δ , + ATP ) and controls ( CK1δ or ATP only ) ; TDP - 43 detected by Western blot ( rabbit anti - TDP - 43 N - term ) . Due to incomplete TEV cleavage , some TDP - 43 - MBP - His6 remains present and co - fractionates with cleaved TDP - 43 , due to TDP - 43 self - self interaction . C Quantification of band intensities of cleaved TDP - 43 shown as means of Supernatant / ( Supernatant + Condensate ) [ S / ( S + C ) ] ratio of three independent experimental replicates ( n = 3 ) ± SD . * * * p < 0 . 0002 by one - way ANOVA with Dunnett ´ s multiple comparison test to Wt . Representative bright field microscopic images of TDP - 43 condensates ( in Hepes buffer ) , Bar , 25 µm ( F ) and quantification of condensate number ( G ) Box plots show the comparison of median and inter - quartile range ( upper and lower quartiles ) of all fields of view ( FOV ) from Min to Max ( whiskers ) of two replicates ( ≥ 22 FOV per condition ) . * p < 0 . 0332 , * * p < 0 . 0021 and * * * p < 0 . 0002 by one - way ANOVA with Dunnett ´ s multiple comparison test to Wt , comparing the respective concentration condition ( 5 , 10 , 20 µM ) .

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

Figure 1B Sedimentation assay to quantify condensation of unmodified TDP-43 versus in vitro phosphorylated TDP-43 (+CK1δ, +ATP) and controls (CK1δ or ATP only); TDP-43 detected by Western blot (rabbit anti-TDP-43 N-term). Due to incomplete TEV cleavage, some TDP-43-MBP-His6 remains present and co-fractionates with cleaved TDP-43, due to TDP-43 self-self interaction. C Quantification of band intensities of cleaved TDP-43 shown as means of Supernatant/(Supernatant+Condensate) [S/(S+C)] ratio of three independent experimental replicates (n=3) ± SD. ***p < 0.0002 by one-way ANOVA with Dunnett´s multiple comparison test to Wt.Representative bright field microscopic images of TDP-43 condensates (in Hepes buffer), Bar, 25 µm (F) and quantification of condensate number (G) Box plots show the comparison of median and inter-quartile range (upper and lower quartiles) of all fields of view (FOV) from Min to Max (whiskers) of two replicates (≥ 22 FOV per condition). *p < 0.0332, **p < 0.0021 and ***p < 0.0002 by one-way ANOVA with Dunnett´s multiple comparison test to Wt, comparing the respective concentration condition (5, 10, 20 µM).


Figure 2B Representative images of FRAP experiments at indicated time - points . Boxes indicate bleached area ( half - bleach of condensate ) . Bar , 5 µm . C FRAP curves after half - bleach of freshly formed Alexa488 - labeled TDP - 43 condensates . Values represent means ± SD of three independent experimental replicates ( n = 3 ) of ≥ 9 droplets analyzed per condition . * * * p < 0 . 0002 by one - way ANOVA with Tukey ´ s multiple comparison test for area under the curve ( AUC ) of individual droplets . D Confocal images of Alexa488 - labeled TDP - 43 aggregates formed in an in vitro aggregation assay ( with TEV protease cleavage ) . Bar , 100 µm . Zoom shows magnified view of aggregates at the 24h time point . Bar , 20 µm .

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

Figure 2B Representative images of FRAP experiments at indicated time-points. Boxes indicate bleached area (half-bleach of condensate). Bar, 5 µm. C FRAP curves after half-bleach of freshly formed Alexa488-labeled TDP-43 condensates. Values represent means ± SD of three independent experimental replicates (n=3) of ≥ 9 droplets analyzed per condition. ***p < 0.0002 by one-way ANOVA with Tukey´s multiple comparison test for area under the curve (AUC) of individual droplets.D Confocal images of Alexa488-labeled TDP-43 aggregates formed in an in vitro aggregation assay (with TEV protease cleavage). Bar, 100 µm. Zoom shows magnified view of aggregates at the 24h time point. Bar, 20 µm.


Figure 4A Hormone - inducible nuclear import assay , representative still images of GCR2 - EGFP2 - TDP - 43 Wt , 12D and 12A before and during import triggered by addition of dexamethasone . Images were live recorded by spinning disc confocal microscopy . Bar , 20 µm . B Quantification of the hormone - inducible nuclear import measured during a total time course of 50 min . Values represent the mean fluorescence intensity of GCR2 - EGFP2 - TDP - 43 in the cytoplasm for three independent replicates ± SEM ( ≥ 42 cells per condition ) . C Phosphomimetic 12D TDP - 43 is competent in autoregulating TDP - 43 expression . SDS - PAGE followed by TDP - 43 Western blot showing downregulation of endogenous TDP - 43 through autoregulation ( 60 ) after 48 h expression of Wt , 12D and 12A variants in HeLa cells . TDP - 43 was detected using rabbit anti - TDP - 43 C - term antibody ( Proteintech ) , Histone H3 ( rabbit anti - Histone H3 antibody , Abcam ) was visualized as a loading control . * denotes an unspecific band . E Splicing analysis by RT - PCR of known TDP - 43 splice targets ( SKAR exon 3 and Bim exon 3 ) in HeLa cells . Silencing of endogenous TDP - 43 by siRNA leads to altered splice isoforms of SKAR and Bim ( second vs . first lane ) . These splicing alterations can be rescued by re - expression of TDP - 43 Wt , but also 12D or 12A variants , demonstrating that phosphomimetic TDP - 43 is fully competent in regulation splicing of these TDP - 43 splice targets .

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

Figure 4A Hormone-inducible nuclear import assay, representative still images of GCR2-EGFP2-TDP-43 Wt, 12D and 12A before and during import triggered by addition of dexamethasone. Images were live recorded by spinning disc confocal microscopy. Bar, 20 µm. B Quantification of the hormone-inducible nuclear import measured during a total time course of 50 min. Values represent the mean fluorescence intensity of GCR2-EGFP2-TDP-43 in the cytoplasm for three independent replicates ± SEM (≥ 42 cells per condition).C Phosphomimetic 12D TDP-43 is competent in autoregulating TDP-43 expression. SDS-PAGE followed by TDP-43 Western blot showing downregulation of endogenous TDP-43 through autoregulation (60) after 48 h expression of Wt, 12D and 12A variants in HeLa cells. TDP-43 was detected using rabbit anti-TDP-43 C-term antibody (Proteintech), Histone H3 (rabbit anti-Histone H3 antibody, Abcam) was visualized as a loading control. * denotes an unspecific band.E Splicing analysis by RT-PCR of known TDP-43 splice targets (SKAR exon 3 and Bim exon 3) in HeLa cells. Silencing of endogenous TDP-43 by siRNA leads to altered splice isoforms of SKAR and Bim (second vs. first lane). These splicing alterations can be rescued by re-expression of TDP-43 Wt, but also 12D or 12A variants, demonstrating that phosphomimetic TDP-43 is fully competent in regulation splicing of these TDP-43 splice targets.


Figure 5B Reduced SG association of TDP - 43 by 12D mutations or in vitro phosphorylation . Bar , 20 µm . F SG recruitment of different TDP - 43 - NLSmut variants in intact HeLa cells in the absence of endogenous TDP - 43 . After TDP - 43 silencing and expression of NLSmut Wt , 12D and 12A variants , SGs were induced by H2O2 treatment and SG recruitment of TDP - 43 was monitored by TDP - 43 and G3BP1 immunostaining . For clarity , signals were converted to grey values in the individual channels ( upper two rows ) . In the merge ( lower row ) , nuclei were stained in DAPI ( turquoise ) , TDP - 43 ( green ) and G3BP1 ( red ) . Bar , 40 µm . H Recruitment of TDP - 43 into arsenite - induced nuclear bodies ( NBs ) in HeLa cells . After TDP - 43 silencing and expression myc - TDP - 43 Wt , 12D and 12A , NBs were induced by sodium arsenite treatment and NB recruitment of TDP - 43 was monitored by TDP - 43 immunostaining . Bar , 20 µm . J Intensity profiles ( right ) of TDP - 43 Wt , 12D and 12A variants ( green ) along white lines ( left ) . Bar , 10 µm .

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

Figure 5B Reduced SG association of TDP-43 by 12D mutations or in vitro phosphorylation. Bar, 20 µm.F SG recruitment of different TDP-43-NLSmut variants in intact HeLa cells in the absence of endogenous TDP-43. After TDP-43 silencing and expression of NLSmut Wt, 12D and 12A variants, SGs were induced by H2O2 treatment and SG recruitment of TDP-43 was monitored by TDP-43 and G3BP1 immunostaining. For clarity, signals were converted to grey values in the individual channels (upper two rows). In the merge (lower row), nuclei were stained in DAPI (turquoise), TDP-43 (green) and G3BP1 (red). Bar, 40 µm.H Recruitment of TDP-43 into arsenite-induced nuclear bodies (NBs) in HeLa cells. After TDP-43 silencing and expression myc-TDP-43 Wt, 12D and 12A, NBs were induced by sodium arsenite treatment and NB recruitment of TDP-43 was monitored by TDP-43 immunostaining. Bar, 20 µm.J Intensity profiles (right) of TDP-43 Wt, 12D and 12A variants (green) along white lines (left). Bar, 10 µm.


Figure 6A Biochemical fractionation into RIPA - soluble ( S ) and RIPA - insoluble ( I ) fractions to analyze solubility of the different myc - TDP - 43 variants ( Wt , 12D and 12A ) expressed in HeLa cells for 48 h . TDP - 43 was detected by TDP - 43 Western blot ( upper blot , rabbit anti - TDP - 43 C - term , Proteintech ) and Myc Western blot ( lower blot , mouse anti - Myc 9E10 ) . B Quantification of myc - TDP - 43 variants ( Wt , 12D and 12A ) in ( S ) versus ( I ) fractions extracted from TDP - 43 Western blots of 4 independent replicates ± SD , plotted as S / ( S + I ) . * p < 0 . 0332 by one way ANOVA with Dunnett ´ s multiple comparison test to Wt . D Primary hippocampal neurons expressing EGFP - TDP - 43 Wt , 12D or 12A with additional NLS mutation . Bar , 80 µm . Right : Zoomed images of white squares ( TDP - 43 signal ) . Bar , 10 µm . E SG recruitment of EGFP - TDP - 43 NLS mutant variants ( Wt , 12D , 12A ) in primary hippocampal neurons . SG formation was induced by 1h heat shock at 42 ° C . SGs and TDP - 43 were monitored by G3BP1 antibody staining and EGFP fluorescence , respectively . For clarity , signals were converted to grey values in the individual channels ( first two columns ) . In the merge ( third column ) , EGFP - TDP - 43 shown in green , G3BP1 in red and nuclei ( DAPI staining ) in turquoise . Bar , 20 µm .

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

Figure 6A Biochemical fractionation into RIPA-soluble (S) and RIPA-insoluble (I) fractions to analyze solubility of the different myc-TDP-43 variants (Wt, 12D and 12A) expressed in HeLa cells for 48 h. TDP-43 was detected by TDP-43 Western blot (upper blot, rabbit anti-TDP-43 C-term, Proteintech) and Myc Western blot (lower blot, mouse anti-Myc 9E10). B Quantification of myc-TDP-43 variants (Wt, 12D and 12A) in (S) versus (I) fractions extracted from TDP-43 Western blots of 4 independent replicates ± SD, plotted as S/(S+I). *p < 0.0332 by one way ANOVA with Dunnett´s multiple comparison test to Wt.D Primary hippocampal neurons expressing EGFP-TDP-43 Wt, 12D or 12A with additional NLS mutation. Bar, 80 µm. Right: Zoomed images of white squares (TDP-43 signal). Bar, 10 µm.E SG recruitment of EGFP-TDP-43 NLS mutant variants (Wt, 12D, 12A) in primary hippocampal neurons. SG formation was induced by 1h heat shock at 42°C. SGs and TDP-43 were monitored by G3BP1 antibody staining and EGFP fluorescence, respectively. For clarity, signals were converted to grey values in the individual channels (first two columns). In the merge (third column), EGFP-TDP-43 shown in green, G3BP1 in red and nuclei (DAPI staining) in turquoise. Bar, 20 µm.


Figure 1Characteristic micrographs for both data sets , respectively ( left and right ) .

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Figure 1Characteristic micrographs for both data sets, respectively (left and right).


Figure 1B . Protein interaction analysis by Nano LC - MS . Unique peptide counts and protein sequence coverage , as well as signal intensities ( PSI ) for Tailor ( bait ) and CG16940 / dmDis3l2 in control IP ( FLAG - GFP ) and FLAG - TailorIP are indicated . A . u . , arbitrary units . D . Co - immunoprecipitation of FLAG - dmDis3l2 ( bait ) and GFP - Tailor full - length and truncations expressed in Drosophila S2cells . GFP - Nibbler ( Nbr ) served as negative control . Domain architecture of GFP - Tailor truncations are indicated . E - G . Recombinant protein interaction analysis using the indicated epitope tags and purification methods . Domain architecture of bait and prey are indicated . E - G . Recombinant protein interaction analysis using the indicated epitope tags and purification methods . Domain architecture of bait and prey are indicated . E - G . Recombinant protein interaction analysis using the indicated epitope tags and purification methods . Domain architecture of bait and prey are indicated . H and I . Immunostaining and imaging of Myc - Tailor and GFP - dmDis3l2 ( H ) or endogenous dmDis3l2 ( I ) in S2 cells . Single color channel images show total inversions . Scale bar = 5 µm .

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

Figure 1B. Protein interaction analysis by Nano LC-MS. Unique peptide counts and protein sequence coverage, as well as signal intensities (PSI) for Tailor (bait) and CG16940/dmDis3l2 in control IP (FLAG-GFP) and FLAG-TailorIP are indicated. A.u., arbitrary units.D. Co-immunoprecipitation of FLAG-dmDis3l2 (bait) and GFP-Tailor full-length and truncations expressed in Drosophila S2cells. GFP-Nibbler (Nbr) served as negative control. Domain architecture of GFP-Tailor truncations are indicated.E- G. Recombinant protein interaction analysis using the indicated epitope tags and purification methods. Domain architecture of bait and prey are indicated.E- G. Recombinant protein interaction analysis using the indicated epitope tags and purification methods. Domain architecture of bait and prey are indicated.E- G. Recombinant protein interaction analysis using the indicated epitope tags and purification methods. Domain architecture of bait and prey are indicated.H and I. Immunostaining and imaging of Myc-Tailor and GFP-dmDis3l2 (H) or endogenous dmDis3l2 (I) in S2 cells. Single color channel images show total inversions. Scale bar = 5 µm.


Figure 2B . In vitro exoribonuclease assay using 5 ′ radiolabeled RNA substrate and immunopurified wild - type ( WT ) or catalytic mutant ( CM ) FLAG - dmDis3l2 ( mutation indicated in Figure 1C ) , incubated for the indicated time and separated on a 15 % polyacrylamide gel followed by phosphorimaging . Quantification of degraded substrate in percent is indicated . C . Change in abundance of 256 different substrate RNAs as determined by high - throughput sequencing of substrates in experiment shown in ( B ) . D . Decay rates of 256 different substrate RNAs . Data shown in ( C ) was normalized to overall decrease in substrate abundance as determined by phosphorimaging ( as shown in B ) and fit to the indicated model for exponential decay . Apparent decay rate of all substrates , as determined by phosphorimaging is indicated in red . E . Selected examples for individual substrates shown in ( D ) . Values report the observed decay rate ( kobs ) in min - 1 . F . Overview of decay rates ( kobs ) of all 256 different substrate RNAs , as determined in ( D ) . Error represents SEM of curve fit . G . Impact of RNA secondary structures , as determined by RNAfold ( Gruber et al , 2008 ) , and reported as effective free energy ( EFE , kcal / mol ) , on decay rates ( kobs ) are shown . Error bars represent SEM of curve fit . Grey boxes represent tukey boxplot of individual EFE groups . White line indicates median . P - Value was determined by Mann - Whitney test comparing individual EFE groups against all substrates . Unstructured substrates used for subsequent analysis are indicated ( EFE groups 4 to 6 ) . H . Nucleotide content of single - stranded RNA substrates , ranked from slow ( top ) to fast ( bottom ) decay by dmDis3l2 . I . Cumulative distribution of decay rates for RNA substrates with the indicated nucleotide at each one of four randomized position at the 3 ′ end of the substrate RNA . P - Value was determined by Mann - Whitney test comparing individual nts against all . J . Functional coupling between two base positions within the randomized 3 ′ end of substrate RNA . Red squares show average increase in decay rate compared to all substrates . Black squares show no or negative effect . K . Boxplot decay rates of substrates containing no , one , two , or three uridine ( s ) within the randomized 3 ′ end of the substrate RNA . Median decay rates SEM of fit are indicated . L . Decay rate of all substrates , or substrates containing no , one , two , or three uridine ( s ) within the randomized 3 ′ end of the substrate RNA . Median and quartile ranges are indicated . P - values were determined by Mann - Whitney test . Substrate containing 4 uridines ( 4U ) is indicated .

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

Figure 2B. In vitro exoribonuclease assay using 5′ radiolabeled RNA substrate and immunopurified wild-type (WT) or catalytic mutant (CM) FLAG-dmDis3l2 (mutation indicated in Figure 1C), incubated for the indicated time and separated on a 15 % polyacrylamide gel followed by phosphorimaging. Quantification of degraded substrate in percent is indicated.C. Change in abundance of 256 different substrate RNAs as determined by high-throughput sequencing of substrates in experiment shown in (B).D. Decay rates of 256 different substrate RNAs. Data shown in (C) was normalized to overall decrease in substrate abundance as determined by phosphorimaging (as shown in B) and fit to the indicated model for exponential decay. Apparent decay rate of all substrates, as determined by phosphorimaging is indicated in red.E. Selected examples for individual substrates shown in (D). Values report the observed decay rate (kobs) in min-1.F. Overview of decay rates (kobs) of all 256 different substrate RNAs, as determined in (D). Error represents SEM of curve fit.G. Impact of RNA secondary structures, as determined by RNAfold (Gruber et al, 2008), and reported as effective free energy (EFE, kcal/mol), on decay rates (kobs) are shown. Error bars represent SEM of curve fit. Grey boxes represent tukey boxplot of individual EFE groups. White line indicates median. P-Value was determined by Mann-Whitney test comparing individual EFE groups against all substrates. Unstructured substrates used for subsequent analysis are indicated (EFE groups 4 to 6).H. Nucleotide content of single-stranded RNA substrates, ranked from slow (top) to fast (bottom) decay by dmDis3l2.I. Cumulative distribution of decay rates for RNA substrates with the indicated nucleotide at each one of four randomized position at the 3′ end of the substrate RNA. P-Value was determined by Mann-Whitney test comparing individual nts against all.J. Functional coupling between two base positions within the randomized 3′ end of substrate RNA. Red squares show average increase in decay rate compared to all substrates. Black squares show no or negative effect.K. Boxplot decay rates of substrates containing no, one, two, or three uridine(s) within the randomized 3′ end of the substrate RNA. Median decay rates SEM of fit are indicated.L. Decay rate of all substrates, or substrates containing no, one, two, or three uridine(s) within the randomized 3′ end of the substrate RNA. Median and quartile ranges are indicated. P-values were determined by Mann-Whitney test. Substrate containing 4 uridines (4U) is indicated.


Figure 3A . RNA decay assay using synthetic , 5 ′ radiolabeled pre - miR - 1003 , immunopurified dmDis3l2 and wild - type ( WT ) or catalytic mutant ( CM ) Tailor . Reactions were incubated for the indicated time and separated by 15 % denaturing polyacrylamide gel electrophoresis and subjected to phosphorimaging . B . Quantification of at least three independent replicates of experiment shown in A . Data represent mean SD . Half - life was determined by fitting data to single exponential ( dmDis3l2 and dmDis3l2 / TailorCM ) or sigmoidal ( dmDis3l2 / TailorWT ) reaction kinetics . C . RNA decay assay using synthetic , 5 ′ radiolabeled pre - miR - 1003 containing the indicated number or uridine residues at the 3 ′ end and immunopurified dmDis3l2 . D . Quantification of three independent replicates of experiment shown in C . Data represent mean SD . Data were fit to single exponential reaction kinetics . E . Reaction kinetics ( kobs ) determined in D plotted against number of 3 ′ terminal uridine residues . Error of fit is shown as SEM .

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Figure 3A. RNA decay assay using synthetic, 5′ radiolabeled pre-miR-1003, immunopurified dmDis3l2 and wild-type (WT) or catalytic mutant (CM) Tailor. Reactions were incubated for the indicated time and separated by 15% denaturing polyacrylamide gel electrophoresis and subjected to phosphorimaging.B. Quantification of at least three independent replicates of experiment shown in A. Data represent mean SD. Half-life was determined by fitting data to single exponential (dmDis3l2 and dmDis3l2/TailorCM) or sigmoidal (dmDis3l2/TailorWT) reaction kinetics.C. RNA decay assay using synthetic, 5′ radiolabeled pre-miR-1003 containing the indicated number or uridine residues at the 3′ end and immunopurified dmDis3l2.D. Quantification of three independent replicates of experiment shown in C. Data represent mean SD. Data were fit to single exponential reaction kinetics.E. Reaction kinetics (kobs) determined in D plotted against number of 3′ terminal uridine residues. Error of fit is shown as SEM.


Figure 4A and E . Western blot analysis of genetically modified Drosophila S2 cells . Unmodified S2 cells ( wild - type , wt ) , or S2 cells depleted of Tailor ( tailorko ) by CRISPR / Cas9 or expressing FLAG - tagged Tailor ( TailorOE ) were analyzed . Antibodies are indicated . Note , that panels were assembled from two technical replicates of identical lysates ( see source data ) . B , C , F and G . Lysate of S2 cells ( as characterized in A and E ) were incubated with - 32P - UTP ( B and F ) or - 32P - ATP ( C and G ) for the indicated time followed by RNA extraction , denaturing polyacrylamide gel electrophoresis and phosphorimaging . D and H . Quantification of at least five ( D ) or seven ( H ) independent replicates of experiment shown in B / C and F / G , respectively . Uridylation signal intensity for substrate S1 ( indicated in B and F ) was corrected for adenylation activity and normalized to wild - type 2 min time point . Data represent mean SEM . P - values were determined using Student ' s t - test . * p < 0 . 05 , * * p < 10 - 2 , * * * p < 10 - 3 , * * * * p < 10 - 4 . A and E . Western blot analysis of genetically modified Drosophila S2 cells . Unmodified S2 cells ( wild - type , wt ) , or S2 cells depleted of dmDis3l2 ( dmDis3l2ko ) by CRISPR / Cas9 or expressing FLAG - tagged catalytic mutant dmDis3l2 ( dmDis3l2CM OE ) were analyzed . Antibodies are indicated . Note , that panels were assembled from two technical replicates of identical lysates ( see source data ) . B , C , F and G . Lysate of S2 cells ( as characterized in A and E ) were incubated with - 32P - UTP ( B and F ) or - 32P - ATP ( C and G ) for the indicated time followed by RNA extraction , denaturing polyacrylamide gel electrophoresis and phosphorimaging . D and H . Quantification of at least five ( D ) or seven ( H ) independent replicates of experiment shown in B / C and F / G , respectively . Uridylation signal intensity for substrate S1 ( indicated in B and F ) was corrected for adenylation activity and normalized to wild - type 2 min time point . Data represent mean SEM . P - values were determined using Student ' s t - test . * p < 0 . 05 , * * p < 10 - 2 , * * * p < 10 - 3 , * * * * p < 10 - 4 .

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

Figure 4A and E. Western blot analysis of genetically modified Drosophila S2 cells. Unmodified S2 cells (wild-type, wt), or S2 cells depleted of Tailor (tailorko) by CRISPR/Cas9 or expressing FLAG-tagged Tailor (TailorOE) were analyzed. Antibodies are indicated. Note, that panels were assembled from two technical replicates of identical lysates (see source data).B, C, F and G. Lysate of S2 cells (as characterized in A and E) were incubated with -32P-UTP (B and F) or -32P-ATP (C and G) for the indicated time followed by RNA extraction, denaturing polyacrylamide gel electrophoresis and phosphorimaging.D and H. Quantification of at least five (D) or seven (H) independent replicates of experiment shown in B/C and F/G, respectively. Uridylation signal intensity for substrate S1 (indicated in B and F) was corrected for adenylation activity and normalized to wild-type 2 min time point. Data represent mean SEM. P-values were determined using Student's t-test. * p<0.05, ** p<10-2, *** p<10-3, **** p<10-4.A and E. Western blot analysis of genetically modified Drosophila S2 cells. Unmodified S2 cells (wild-type, wt), or S2 cells depleted of dmDis3l2 (dmDis3l2ko) by CRISPR/Cas9 or expressing FLAG-tagged catalytic mutant dmDis3l2 (dmDis3l2CM OE) were analyzed. Antibodies are indicated. Note, that panels were assembled from two technical replicates of identical lysates (see source data).B, C, F and G. Lysate of S2 cells (as characterized in A and E) were incubated with -32P-UTP (B and F) or -32P-ATP (C and G) for the indicated time followed by RNA extraction, denaturing polyacrylamide gel electrophoresis and phosphorimaging.D and H. Quantification of at least five (D) or seven (H) independent replicates of experiment shown in B/C and F/G, respectively. Uridylation signal intensity for substrate S1 (indicated in B and F) was corrected for adenylation activity and normalized to wild-type 2 min time point. Data represent mean SEM. P-values were determined using Student's t-test. * p<0.05, ** p<10-2, *** p<10-3, **** p<10-4.


Figure 5A . Protein / RNA co - immunopurification experiments . FLAG - tagged versions of indicated proteins were expressed in S2 cells , followed by immunopurification and RNA isolation . RNA was visualized by CIP / PNK treatment using - 32P - ATP followed by denaturing polyacrylamide gel electrophoresis and phosphorimaging . Star indicates non - specific contaminant .

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Figure 5A. Protein/RNA co-immunopurification experiments. FLAG-tagged versions of indicated proteins were expressed in S2 cells, followed by immunopurification and RNA isolation. RNA was visualized by CIP/PNK treatment using -32P-ATP followed by denaturing polyacrylamide gel electrophoresis and phosphorimaging. Star indicates non-specific contaminant.


Figure 6B . Meta - analysis of 3 ′ end counts in dmDis3l2CM - IP mapping to the indicated region around the annotated 3 ′ end of 68 tRNA loci in the Drosophila melanogaster genome . Cumulative relative distribution of 3 ′ end signal , relative to the annotated 3 ′ end , is reported ( bottom ) . The average genome thymine ( T ) - content for the same regions is displayed ( top ) . C . Northern hybridization experiment using total RNA from adult whole male flies using probes against 3 ′ trailer or mature tRNAAla ( TGC ) . Wild - type flies ( w1118 ) , or flies bearing a homozygous frame - shift mutation in the first coding exon ( dmDis3l2 - / - ) or a homozygous amino acid - exchange mutation in the catalytic site ( dmDis3l2CM ) in the endogenous dmdis3l2 locus in the same genetic background were used . Probes against 2S rRNA served as loading control . D . Quantification of four independent biological replicates of experiment shown in C . P - Value determined by Student ' s t - test . E . Exoribonuclease assay using immunopurified dmDis3l2 and in vitro transcribed , 5 ′ radiolabeled mature tRNA , containing a CCA modification or 3 ′ trailer - containing tRNA . Reactions were incubated for the indicated time and subjected to polyacrylamide gel electrophoresis and phosphorimaging . F . Quantification of three independent replicates of experiment shown in E . Data represent mean SD . Decay rates ( kobs ) SEM of fit were determined upon fitting to single exponential decay kinetics .

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

Figure 6B. Meta-analysis of 3′ end counts in dmDis3l2CM-IP mapping to the indicated region around the annotated 3′ end of 68 tRNA loci in the Drosophila melanogaster genome. Cumulative relative distribution of 3′ end signal, relative to the annotated 3′ end, is reported (bottom). The average genome thymine (T)-content for the same regions is displayed (top).C. Northern hybridization experiment using total RNA from adult whole male flies using probes against 3′ trailer or mature tRNAAla(TGC). Wild-type flies (w1118), or flies bearing a homozygous frame-shift mutation in the first coding exon (dmDis3l2-/-) or a homozygous amino acid-exchange mutation in the catalytic site (dmDis3l2CM) in the endogenous dmdis3l2 locus in the same genetic background were used. Probes against 2S rRNA served as loading control.D. Quantification of four independent biological replicates of experiment shown in C. P-Value determined by Student's t-test.E. Exoribonuclease assay using immunopurified dmDis3l2 and in vitro transcribed, 5′ radiolabeled mature tRNA, containing a CCA modification or 3′ trailer-containing tRNA. Reactions were incubated for the indicated time and subjected to polyacrylamide gel electrophoresis and phosphorimaging.F. Quantification of three independent replicates of experiment shown in E. Data represent mean SD. Decay rates (kobs) SEM of fit were determined upon fitting to single exponential decay kinetics.


Figure 7A . Impact of RNA secondary structures on RNA decay rates ( kobs ) of 256 different RNA substrates subjected to E . coli RNase R ( ecoRNaseR ) - or Drosophila melanogaster Dis3l2 ( dmDis3l2 ) - directed RNA decay assay as described in Fig . 2 . RNA secondary structures were determined by RNAfold ( Gruber et al , 2008 ) and reported as effective free energy ( EFE ) . Error bars represent SEM of curve fit . Grey boxes represent tukey boxplot of individual EFE groups . White line indicates median . P - Value determined by Mann - Whitney test comparing individual EFE groups against all substrates . Unstructured RNA used for subsequent analysis are indicated ( EFE groups 4 to 6 ) . B . Nucleotide content of randomized 3 ′ end sequence among the indicated number of substrate RNAs , ranked according to decay kinetics from slow ( left ) to fast ( right ) . Fold - change in abundance for individual nucleotides is reported ( bottom ) . Only unstructured RNA substrates ( see A ) were considered . C . Decay rate of the indicated number of sequences , grouped according to individual nucleotide content in the randomized 3 ′ end sequence . Data is represented as boxplot , outliers are not shown . P - Value reports significant differences compared to all substrates as determined by Mann - Whitney test . Grey area represents the inner quartile range and white line the median of all substrates .

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Figure 7A. Impact of RNA secondary structures on RNA decay rates (kobs) of 256 different RNA substrates subjected to E.coli RNase R (ecoRNaseR)- or Drosophila melanogaster Dis3l2 (dmDis3l2)-directed RNA decay assay as described in Fig. 2. RNA secondary structures were determined by RNAfold (Gruber et al, 2008) and reported as effective free energy (EFE). Error bars represent SEM of curve fit. Grey boxes represent tukey boxplot of individual EFE groups. White line indicates median. P-Value determined by Mann-Whitney test comparing individual EFE groups against all substrates. Unstructured RNA used for subsequent analysis are indicated (EFE groups 4 to 6).B. Nucleotide content of randomized 3′ end sequence among the indicated number of substrate RNAs, ranked according to decay kinetics from slow (left) to fast (right). Fold-change in abundance for individual nucleotides is reported (bottom). Only unstructured RNA substrates (see A) were considered.C. Decay rate of the indicated number of sequences, grouped according to individual nucleotide content in the randomized 3′ end sequence. Data is represented as boxplot, outliers are not shown. P-Value reports significant differences compared to all substrates as determined by Mann-Whitney test. Grey area represents the inner quartile range and white line the median of all substrates.


Figure 1 ( A ) Isolation of FTCD from rat liver extract . The p47 ( 1 - 170 ) fragment , which does not contain the p97 - binding site , was immobilized on beads and incubated with rat liver extract . Bound proteins were fractionated by SDS - PAGE , and stained with CBB . ( B ) FTCD binds directly to p47 . GST - tagged p47 ( 0 . 54 μg ) or GST - tagged p37 ( 0 . 45 μg ) was incubated with His - tagged FTCD ( 0 . 80 μg ) , and then isolated on glutathione beads . The blots were probed with antibodies to FTCD and GST . ( C ) FTCD and p47 form a complex in cells . HepG2 cells were solubilized and incubated with anti - p47 antibodies . The immunoprecipitates were fractionated by SDS - PAGE , followed by Western blotting with antibodies to p47 and FTCD . ( D ) FTCD , p47 and p97 form a complex in cells . HepG2 cells were solubilized and incubated with anti - FTCD antibodies . The immunoprecipitates were fractionated by SDS - PAGE , followed by Western blotting with antibodies to FTCD , VCIP135 , p97 , p47 and syntaxin5 . ( E ) p47 binds to the CD domain of FTCD . GST - tagged p47 ( 0 . 50 μg ) was incubated with either His - tagged FTCD ( 1 - 326 ) ( FT , 0 . 88 μg ) or His - tagged FTCD ( 327 - 541 ) ( CD , 0 . 63 μg ) . The blots were probed with antibodies to His - tag and GST . ( F ) p47 ( E86K , E88K ) does not bind to FTCD . GST - tagged p47wt / mutant ( 0 . 50 μg ) was incubated with the indicated His - tagged proteins ( p97 , 1 . 0 μg ; FTCD , 0 . 75 μg ) . The blots were probed with antibodies to p97 , FTCD and GST . ( G ) FTCD ( R382A ) , which lacks binding ability to polyglutamates , does not bind to p47 . GST - tagged p47 ( 0 . 35 μg ) was incubated with His - tagged FTCDwt / mutant ( 0 . 53 μg ) . The blots were probed with antibodies to His - tag and GST . ( H ) p47 binds to FTCD in Golgi membranes . Salt - washed Golgi membranes were first incubated with either GST - tagged p47wt , p47 ( E86K , E88K ) or p47 ( F253S ) . The isolated membranes were solubilized , and then GST - tagged p47wt / mutants and their binding proteins were recovered with glutathione - beads . The bound proteins were assayed by Western blotting with antibodies to FTCD and GST .

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

Figure 1(A) Isolation of FTCD from rat liver extract. The p47(1-170) fragment, which does not contain the p97-binding site, was immobilized on beads and incubated with rat liver extract. Bound proteins were fractionated by SDS-PAGE, and stained with CBB.(B) FTCD binds directly to p47. GST-tagged p47 (0.54 μg) or GST-tagged p37 (0.45 μg) was incubated with His-tagged FTCD (0.80 μg), and then isolated on glutathione beads. The blots were probed with antibodies to FTCD and GST.(C) FTCD and p47 form a complex in cells. HepG2 cells were solubilized and incubated with anti-p47 antibodies. The immunoprecipitates were fractionated by SDS-PAGE, followed by Western blotting with antibodies to p47 and FTCD.(D) FTCD, p47 and p97 form a complex in cells. HepG2 cells were solubilized and incubated with anti-FTCD antibodies. The immunoprecipitates were fractionated by SDS-PAGE, followed by Western blotting with antibodies to FTCD, VCIP135, p97, p47 and syntaxin5.(E) p47 binds to the CD domain of FTCD. GST-tagged p47 (0.50 μg) was incubated with either His-tagged FTCD(1-326) (FT, 0.88 μg) or His-tagged FTCD(327-541) (CD, 0.63 μg). The blots were probed with antibodies to His-tag and GST.(F) p47(E86K, E88K) does not bind to FTCD. GST-tagged p47wt/mutant (0.50 μg) was incubated with the indicated His-tagged proteins (p97, 1.0 μg; FTCD, 0.75 μg). The blots were probed with antibodies to p97, FTCD and GST.(G) FTCD(R382A), which lacks binding ability to polyglutamates, does not bind to p47. GST-tagged p47 (0.35 μg) was incubated with His-tagged FTCDwt/mutant (0.53 μg). The blots were probed with antibodies to His-tag and GST.(H) p47 binds to FTCD in Golgi membranes. Salt-washed Golgi membranes were first incubated with either GST-tagged p47wt, p47(E86K, E88K) or p47(F253S). The isolated membranes were solubilized, and then GST-tagged p47wt/mutants and their binding proteins were recovered with glutathione-beads. The bound proteins were assayed by Western blotting with antibodies to FTCD and GST.


Figure 2 ( A ) FTCD directly binds to p97 . GST - tagged p97 ( 2 . 1 μg ) was incubated with His - tagged FTCD ( 0 . 80 μg ) , and then isolated on glutathione beads . The blots were probed with antibodies to FTCD and GST . ( B ) Nucleotide dependency of the binding of FTCD with p97 . FTCD ( 0 . 80 μg ) and GST - tagged p97 ( 2 . 1 μg ) were incubated in the presence of the indicated nucleotide ( 1 mM ) . The blots were probed with antibodies to FTCD and GST . ( C ) FTCD binds to the C - terminal region of p97 . GST - tagged p97 fragments were incubated with either FTCD or p47 . The blots were probed with antibodies to FTCD and p47 . ( D ) p97 also binds to the CD domain of FTCD . GST - tagged p97 ( 2 μg ) was incubated with either His - tagged FTCD ( 1 - 326 ) ( FT , 0 . 88 μg ) or His - tagged FTCD ( 327 - 541 ) ( CD , 0 . 62 μg ) . The blots were probed with antibodies to His - tag and GST . ( E ) p97 ( V722K , E724K ) shows very low binding affinity to FTCD . GST - tagged p97wt / mutant ( 2 . 0 μg ) was incubated with the indicated His - tagged proteins ( FTCD , 2 . 0 μg ; p47 , 0 . 5 μg ) . The blots were probed with antibodies to p47 , FTCD and GST . ( F ) FTCD ( R382A ) , which lacks binding ability to polyglutamates , shows a very low binding affinity to p97 . GST - tagged p97 ( 1 . 4 μg ) was incubated with His - tagged FTCDwt / mutant ( 0 . 53 μg ) . The blots were probed with antibodies to His - tag and GST . ( G ) p97 binds to FTCD in Golgi membranes . Salt - washed Golgi membranes were incubated with either GST - tagged p97wt or p97 ( V722k , E724K ) . The isolated membranes were solubilized , and then GST - tagged p97wt / mutants and their binding proteins were recovered with glutathione - beads . The bound proteins were assayed by Western blotting with antibodies to FTCD and GST .

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Figure 2(A) FTCD directly binds to p97. GST-tagged p97 (2.1 μg) was incubated with His-tagged FTCD (0.80 μg), and then isolated on glutathione beads. The blots were probed with antibodies to FTCD and GST.(B) Nucleotide dependency of the binding of FTCD with p97. FTCD (0.80 μg) and GST-tagged p97 (2.1 μg) were incubated in the presence of the indicated nucleotide (1 mM). The blots were probed with antibodies to FTCD and GST.(C) FTCD binds to the C-terminal region of p97. GST-tagged p97 fragments were incubated with either FTCD or p47. The blots were probed with antibodies to FTCD and p47.(D) p97 also binds to the CD domain of FTCD. GST-tagged p97 (2 μg) was incubated with either His-tagged FTCD(1-326) (FT, 0.88 μg) or His-tagged FTCD(327-541) (CD, 0.62 μg). The blots were probed with antibodies to His-tag and GST.(E) p97(V722K, E724K) shows very low binding affinity to FTCD. GST-tagged p97wt/mutant ( 2.0 μg) was incubated with the indicated His-tagged proteins (FTCD, 2.0 μg; p47, 0.5 μg). The blots were probed with antibodies to p47, FTCD and GST.(F) FTCD(R382A), which lacks binding ability to polyglutamates, shows a very low binding affinity to p97. GST-tagged p97 (1.4 μg) was incubated with His-tagged FTCDwt/mutant (0.53 μg). The blots were probed with antibodies to His-tag and GST.(G) p97 binds to FTCD in Golgi membranes. Salt-washed Golgi membranes were incubated with either GST-tagged p97wt or p97(V722k, E724K). The isolated membranes were solubilized, and then GST-tagged p97wt/mutants and their binding proteins were recovered with glutathione-beads. The bound proteins were assayed by Western blotting with antibodies to FTCD and GST.


Figure 3 ( A ) Immunofluorescence staining of FTCD and GM130 , a Golgi marker . HepG2 cells were fixed , permeabilized , stained with a monoclonal antibody to FTCD , a polyclonal antibody to GM130 and DAPI , and observed by confocal microscopy . Scale bar = 10 μm . ( B ) Depletion of FTCD by FTCD siRNA duplexes . HepG2 cells were either mock transfected with water or transfected with two distinct siRNA duplexes specific to FTCD . After incubation for 48 hrs , the cells were analyzed by Western blotting with antibodies to FTCD and α - tubulin . ( C ) The Golgi in cells which were depleted of either FTCD or p47 . HepG2 cells , which were treated with either FTCD siRNAs , p47 siRNA or mock for 48 hrs , were fixed and stained with a monoclonal antibody to GM130 . Scale bar = 10 μm . ( D ) The results of quantification of ( C ) . The values are from five sets of independent experiments . Results are expressed as the mean ± SD ( n = 5 ) , with 100 cells counted in each group of each set . Asterisks indicate a significant difference at P < 0 . 01 compared with the mock - treated cells ( Bonferroni method ) . ( E ) Representative EM images of the Golgi in HepG2 cells treated with either mock or FTCD siRNAs for 42 hrs . Scale bar = 0 . 5 μm . ( F ) The results of quantification of ( E ) . Golgi membranes in HepG2 cells were classified into cisternae , vesicles and tubules , and counted as previously described ( Shorter & Warren , 1999 ) . The results are shown as the mean ± SD of five sets of independent experiments , with 15 - 20 cells counted in each group in each independent experiment . An asterisk indicates a significant difference ( P < 0 . 05 ) compared with the mock - treated cells in each category ( Bonferroni method ) .

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Figure 3(A) Immunofluorescence staining of FTCD and GM130, a Golgi marker. HepG2 cells were fixed, permeabilized, stained with a monoclonal antibody to FTCD, a polyclonal antibody to GM130 and DAPI, and observed by confocal microscopy. Scale bar = 10 μm.(B) Depletion of FTCD by FTCD siRNA duplexes. HepG2 cells were either mock transfected with water or transfected with two distinct siRNA duplexes specific to FTCD. After incubation for 48 hrs, the cells were analyzed by Western blotting with antibodies to FTCD and α-tubulin.(C) The Golgi in cells which were depleted of either FTCD or p47. HepG2 cells, which were treated with either FTCD siRNAs, p47 siRNA or mock for 48 hrs, were fixed and stained with a monoclonal antibody to GM130. Scale bar = 10 μm. (D) The results of quantification of (C). The values are from five sets of independent experiments. Results are expressed as the mean±SD (n=5), with 100 cells counted in each group of each set. Asterisks indicate a significant difference at P<0.01 compared with the mock-treated cells (Bonferroni method). (E) Representative EM images of the Golgi in HepG2 cells treated with either mock or FTCD siRNAs for 42 hrs. Scale bar = 0.5 μm. (F) The results of quantification of (E). Golgi membranes in HepG2 cells were classified into cisternae, vesicles and tubules, and counted as previously described (Shorter & Warren, 1999). The results are shown as the mean±SD of five sets of independent experiments, with 15-20 cells counted in each group in each independent experiment. An asterisk indicates a significant difference (P<0.05) compared with the mock-treated cells in each category (Bonferroni method).


Figure 4 ( A ) The localization of FTCD in mitotic cells . Mitotic HepG2 cells were collected by flushing and cultured on coated coverslips for 10 - 40 mins . The cells were fixed , stained with a monoclonal antibody to FTCD and polyclonal antibodies to GM130 and α - tubulin , and observed by confocal microscopy . Panels d , h , and l indicate the images of α - tubulin and the other panels show the distribution of FTCD or / and GM130 . Scale bar = 10 μm . Arrowheads indicate the colocalization of FTCD and GM130 . ( B ) The effects of FTCD siRNA treatment on Golgi reassembly at the end of mitosis . HepG2 cells were treated with either FTCD siRNA ( FTCD siRNA 2 duplex ) or mock for 48 hrs before the collection of mitotic cells . The collected mitotic cells were cultured for 35 mins and then fixed . The cells were stained with a polyclonal antibody to GM130 and a monoclonal antibody to α - tubulin , and observed by confocal microscopy . Panels a - p display representative images . Scale bar = 10 μm . ( C ) Rescue experiments in FTCD siRNA - treated HepG2 cells . The indicated HA - tagged FTCDs , which were insensitive to FTCD siRNA , were expressed 25 hrs after the treatment of cells with FTCD siRNA2 . The cells were further cultured for 23 hrs and then used for the collection of mitotic cells . The collected mitotic cells were cultured for 35 mins and fixed . The cells were stained with a monoclonal antibody to α - tubulin ( panels g and h ) and polyclonal antibodies to GM130 and HA ( panels a - f ) . Scale bar = 10 μm . ( D ) The cells at cytokinesis in ( B ) and ( C ) were analyzed . The results are shown as the mean ± SD of five sets of independent experiments , with 100 cells counted in each group in each independent experiment . Asterisks indicate a significant difference ( P < 0 . 01 ) compared with the mock - treated cells ( Bonferroni method ) .

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

Figure 4(A) The localization of FTCD in mitotic cells. Mitotic HepG2 cells were collected by flushing and cultured on coated coverslips for 10-40 mins. The cells were fixed, stained with a monoclonal antibody to FTCD and polyclonal antibodies to GM130 and α-tubulin, and observed by confocal microscopy. Panels d, h, and l indicate the images of α-tubulin and the other panels show the distribution of FTCD or/and GM130. Scale bar = 10 μm. Arrowheads indicate the colocalization of FTCD and GM130.(B) The effects of FTCD siRNA treatment on Golgi reassembly at the end of mitosis. HepG2 cells were treated with either FTCD siRNA (FTCD siRNA 2 duplex) or mock for 48 hrs before the collection of mitotic cells. The collected mitotic cells were cultured for 35 mins and then fixed. The cells were stained with a polyclonal antibody to GM130 and a monoclonal antibody to α-tubulin, and observed by confocal microscopy. Panels a-p display representative images. Scale bar = 10 μm.(C) Rescue experiments in FTCD siRNA-treated HepG2 cells. The indicated HA-tagged FTCDs, which were insensitive to FTCD siRNA, were expressed 25 hrs after the treatment of cells with FTCD siRNA2. The cells were further cultured for 23 hrs and then used for the collection of mitotic cells. The collected mitotic cells were cultured for 35 mins and fixed. The cells were stained with a monoclonal antibody to α-tubulin (panels g and h) and polyclonal antibodies to GM130 and HA (panels a-f). Scale bar = 10 μm.(D) The cells at cytokinesis in (B) and (C) were analyzed. The results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiment. Asterisks indicate a significant difference (P<0.01) compared with the mock-treated cells (Bonferroni method).


Figure 5 ( A ) p47 requires its association with FTCD for its localization to the Golgi at the end of mitosis . HepG2 cells were treated with FTCD siRNA and then the indicated HA - tagged FTCDs were expressed as described in Figure 4C . The mitotic cells were collected , cultured for 35 mins , and then fixed . The cells were stained with polyclonal antibodies to p47 and HA and a monoclonal antibody to GM130 , and observed by confocal microscopy . Panels a - l show the distribution of p47 or / and GM130 and panels m - o show HA - FTCD expression . Scale bar = 10 μm . ( B ) Insets in ( A ) . p47 ( red ) and the GM130 ( a Golgi marker , green ) were visualized . Scale bar = 5 μm . ( C ) The results of quantification of ( A ) . The results are shown as the mean ± SD of five sets of independent experiments , with 100 cells counted in each group in each independent experiments . Asterisks indicate a significant difference at P < 0 . 01 compared with the mock - treated cells ( Bonferroni method ) . ( D ) p97 requires its association with FTCD for its localization to the Golgi at the end of mitosis . The cells were treated in the same way as ( A ) and stained with polyclonal antibodies to p97 and HA and a monoclonal antibody to GM130 . Panels a - l show the distribution of p97 or / and GM130 and panels m - o show HA - FTCD expression . Scale bar = 10 μm . ( E ) Insets in ( D ) . p97 ( red ) and the GM130 ( a Golgi marker , green ) were visualized . Scale bar = 5 μm . ( F ) The results of quantification of ( D ) . The results are shown as the mean ± SD of five sets of independent experiments , with 100 cells counted in each group in each independent experiments . Asterisks indicate a significant difference at P < 0 . 01 compared with the mock - treated cells ( Bonferroni method ) .

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

Figure 5(A) p47 requires its association with FTCD for its localization to the Golgi at the end of mitosis. HepG2 cells were treated with FTCD siRNA and then the indicated HA-tagged FTCDs were expressed as described in Figure 4C. The mitotic cells were collected, cultured for 35 mins, and then fixed. The cells were stained with polyclonal antibodies to p47 and HA and a monoclonal antibody to GM130, and observed by confocal microscopy. Panels a-l show the distribution of p47 or/and GM130 and panels m-o show HA-FTCD expression. Scale bar = 10 μm. (B) Insets in (A). p47 (red) and the GM130 (a Golgi marker, green) were visualized. Scale bar = 5 μm. (C) The results of quantification of (A). The results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiments. Asterisks indicate a significant difference at P<0.01 compared with the mock-treated cells (Bonferroni method). (D) p97 requires its association with FTCD for its localization to the Golgi at the end of mitosis. The cells were treated in the same way as (A) and stained with polyclonal antibodies to p97 and HA and a monoclonal antibody to GM130. Panels a-l show the distribution of p97 or/and GM130 and panels m-o show HA-FTCD expression. Scale bar = 10 μm. (E) Insets in (D). p97 (red) and the GM130 (a Golgi marker, green) were visualized. Scale bar = 5 μm. (F) The results of quantification of (D). The results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiments. Asterisks indicate a significant difference at P<0.01 compared with the mock-treated cells (Bonferroni method).


Figure 6 ( A ) Anti - FTCD antibodies inhibit p97 / p47 - mediated Golgi membrane fusion . Golgi fragments were mixed with the indicated components ( p97 , 60 μg / ml ; p47 , 30 μg / ml ; p37 , 24 μg / ml ; p115 , 40 μg / ml ) together with either anti - FTCD or anti - p115 antibodies . Results are expressed as mean + SD of five sets of independent experiments ; 0 % and 100 % represent the buffer only ( 25 . 9 % in cisternal membranes ) and p97 / p47 with no antibodies ( ' + none ' , 48 . 8 % ) , respectively . Asterisks indicate a significant difference at P < 0 . 01 compared with the no antibody groups ( ' + none ' ) ( Bonferroni method ) . ( B ) p47 ( E86K , E88K ) , which lacks binding affinity to FTCD , does not function in p97 / p47 - mediated Golgi membrane fusion . Golgi fragments were incubated with p97 ( 60 μg / ml ) and p47wt / mutant ( 30 μg / ml ) . Data are shown as the mean + SD of five sets of independent experiments ; 0 % and 100 % represent the buffer only ( 27 . 3 % in cisternal membranes ) and p97 + p47wt ( ' + p47wt ' , 50 . 9 % ) , respectively . Asterisks indicate a significant difference at P < 0 . 01 compared with the group in which p47wt as well as p97 were added ( ' + p47wt ' ) ( Bonferroni method ) . ( C ) p97 ( V722K , E724K ) , which lacks binding affinity to FTCD , does not function in p97 / p47 - mediated Golgi membrane fusion . Golgi fragments were incubated with the following components : p97wt / mutant , 60 μg / ml ; p47 , 30 μg / ml ; p37 , 24 μg / ml ; p115 , 40 μg / ml . Data are shown as the mean + SD of five sets of independent experiments ; 0 % and 100 % represent the buffer only ( 29 . 5 % in cisternal membranes ) , and p47 + p97wt ( ' + p97wt ' . 57 . 5 % ) , respectively . An asterisk indicates a significant difference at P < 0 . 01 compared with the group in which p97wt as well as p47 were added ( ' + p97wt ' ) ( Bonferroni method ) . ( D ) Panels a - e show representative EM images . Scale bar = 0 . 5 μm .

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

Figure 6(A) Anti-FTCD antibodies inhibit p97/p47-mediated Golgi membrane fusion. Golgi fragments were mixed with the indicated components (p97, 60 μg/ml; p47, 30 μg/ml; p37, 24 μg/ml; p115, 40 μg/ml) together with either anti-FTCD or anti-p115 antibodies. Results are expressed as mean+SD of five sets of independent experiments; 0% and 100% represent the buffer only (25.9% in cisternal membranes) and p97/p47 with no antibodies ('+ none', 48.8%), respectively. Asterisks indicate a significant difference at P<0.01 compared with the no antibody groups ('+none') (Bonferroni method).(B) p47(E86K, E88K), which lacks binding affinity to FTCD, does not function in p97/p47-mediated Golgi membrane fusion. Golgi fragments were incubated with p97 (60 μg/ml) and p47wt/mutant (30 μg/ml). Data are shown as the mean+SD of five sets of independent experiments; 0% and 100% represent the buffer only (27.3% in cisternal membranes) and p97+p47wt ('+p47wt', 50.9%), respectively. Asterisks indicate a significant difference at P<0.01 compared with the group in which p47wt as well as p97 were added ('+p47wt') (Bonferroni method).(C) p97(V722K,E724K), which lacks binding affinity to FTCD, does not function in p97/p47-mediated Golgi membrane fusion. Golgi fragments were incubated with the following components: p97wt/mutant, 60 μg/ml; p47, 30 μg/ml; p37, 24 μg/ml; p115, 40 μg/ml. Data are shown as the mean+SD of five sets of independent experiments; 0% and 100% represent the buffer only (29.5% in cisternal membranes), and p47+p97wt ('+p97wt'. 57.5%), respectively. An asterisk indicates a significant difference at P<0.01 compared with the group in which p97wt as well as p47 were added ('+p97wt') (Bonferroni method).(D) Panels a-e show representative EM images. Scale bar = 0.5 μm.


Figure 7 ( A ) Two sets of FTCD molecules and a p97 / p47 complex form a quaternary complex . His - tagged FTCD was biotinylated and immobilized on streptavidin beads ( His - FTCD - beads ) . The beads ( 1 . 0 μg ) were incubated with Flag - tagged FTCD ( 2 . 0 μg ) in the absence or presence of p47 ( 1 . 0 μg ) and p97 ( 2 . 0 μg ) . The blots were probed with HRP - conjugated avidin and antibodies to Flag - tag , p97 and p47 . ( B ) Formation of the quaternary complex in the presence of nucleotides . His - FTCD - beads ( 1 . 0 μg ) were incubated with Flag - tagged FTCD ( 2 . 0 μg ) , p97 ( 2 . 0 μg ) and p47 ( 1 . 0 μg ) in the presence of nucleotides ( 1 mM ) . The blots were probed as described in ( A ) . ( C ) Mutants that lack binding affinity disrupt the formation of the quaternary complex . His - FTCD - beads ( 1 . 0 μg ) and Flag - tagged FTCD ( 2 . 0 μg ) were incubated in the presence of AMP - PNP together with p97wt / mutant ( 2 . 0 μg ) and p47wt / mutant ( 1 . 0 μg ) . The blots were probed as described in ( A ) . ( D ) Negative stain electron microscopy of the quaternary complex . The panels show class averages of centered and aligned negative stain particles for the different populations present within the dataset , i . e . , the full quaternary complex FTCD - p97 / p47 - FTCD ( left panel ) , and its partial complexes ( two variations , middle two panels ) . Class averages contain approximately 20 images per class . A class average showing a p97 / p47 side view from cryo - EM data and a projection generated from FTCD structure ( PDB code 1TT9 ) have been included for comparisons . Brackets and arrow heads indicate the p97 hexamer and the FTCD octamer , respectively . Scale bar = 15 nm . ( E ) FTCDwt - beads are aggregated in the presence of both p97 and p47 . Biotinylated FTCDwt and FTCD ( R382A ) were immobilized on streptavidin magnetic beads to form FTCDwt - beads and FTCD ( R382A ) - beads , respectively . The beads were incubated with the indicated proteins ( p97 , 0 . 1 μg / μl ; p47 , 0 . 1 μg / μl ; p37 , 0 . 1 μg / μl ) in the presence of AMP - PNP ( 1 mM ) , and observed under a bright - field microscope . Scale bar = 50 μm . ( F ) The results of quantification of ( E ) . The percentages of beads in the aggregates were calculated . Results are expressed as the mean + SD of ten sets of independent experiments . An asterisk indicates a significant difference at P < 0 . 01 compared with the others ( Bonferroni method ) . ( G ) p97 / p47 - mediated aggregation of FTCDwt - beads is nucleotide - dependent . FTCDwt - beads were incubated together with p97 ( 0 . 1 μg / μl ) and p47 ( 0 . 1 μg / μl ) in the presence of the indicated nucleotides ( 1mM ) . The representative images are presented in Figure EV1E . Data are shown as the mean + SD of nine sets of independent experiments . An asterisk and hash tags indicate significant differences at P < 0 . 01 compared with the others and the negative control ( ' none ' ) , respectively ( Bonferroni method ) . ( H ) p47 and p97 mutants that lack binding affinity inhibit p97 / p47 - mediated aggregation of FTCDwt - beads . FTCDwt - beads were incubated with p97wt / mutant ( 0 . 1 μg / μl ) and p47wt / mutants ( 0 . 1 μg / μl ) in the presence of AMP - PNP . The representative images are presented in Figure EV1F . Data are shown as the mean + SD of nine sets of independent experiments . Asterisks indicate a significant difference at P < 0 . 01 compared with the positive control ( ' p97wt + p47wt ' ) ( Bonferroni method ) .

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

Figure 7(A) Two sets of FTCD molecules and a p97/p47 complex form a quaternary complex. His-tagged FTCD was biotinylated and immobilized on streptavidin beads (His-FTCD-beads). The beads (1.0 μg) were incubated with Flag-tagged FTCD (2.0 μg) in the absence or presence of p47 (1.0 μg) and p97 (2.0 μg). The blots were probed with HRP-conjugated avidin and antibodies to Flag-tag, p97 and p47.(B) Formation of the quaternary complex in the presence of nucleotides. His-FTCD-beads (1.0 μg) were incubated with Flag-tagged FTCD (2.0 μg), p97 (2.0 μg) and p47 (1.0 μg) in the presence of nucleotides (1 mM). The blots were probed as described in (A).(C) Mutants that lack binding affinity disrupt the formation of the quaternary complex. His-FTCD-beads (1.0 μg) and Flag-tagged FTCD (2.0 μg) were incubated in the presence of AMP-PNP together with p97wt/mutant (2.0 μg) and p47wt/mutant (1.0 μg). The blots were probed as described in (A).(D) Negative stain electron microscopy of the quaternary complex. The panels show class averages of centered and aligned negative stain particles for the different populations present within the dataset, i.e., the full quaternary complex FTCD-p97/p47-FTCD (left panel), and its partial complexes (two variations, middle two panels). Class averages contain approximately 20 images per class. A class average showing a p97/p47 side view from cryo-EM data and a projection generated from FTCD structure (PDB code 1TT9) have been included for comparisons. Brackets and arrow heads indicate the p97 hexamer and the FTCD octamer, respectively. Scale bar = 15 nm.(E) FTCDwt-beads are aggregated in the presence of both p97 and p47. Biotinylated FTCDwt and FTCD(R382A) were immobilized on streptavidin magnetic beads to form FTCDwt-beads and FTCD(R382A)-beads, respectively. The beads were incubated with the indicated proteins (p97, 0.1 μg/μl; p47, 0.1 μg/μl; p37, 0.1 μg/μl) in the presence of AMP-PNP (1 mM), and observed under a bright-field microscope. Scale bar = 50 μm. (F) The results of quantification of (E). The percentages of beads in the aggregates were calculated. Results are expressed as the mean+SD of ten sets of independent experiments. An asterisk indicates a significant difference at P<0.01 compared with the others (Bonferroni method). (G) p97/p47-mediated aggregation of FTCDwt-beads is nucleotide-dependent. FTCDwt-beads were incubated together with p97 (0.1 μg/μl) and p47 (0.1 μg/μl) in the presence of the indicated nucleotides (1mM). The representative images are presented in Figure EV1E. Data are shown as the mean+SD of nine sets of independent experiments. An asterisk and hash tags indicate significant differences at P<0.01 compared with the others and the negative control ('none'), respectively (Bonferroni method).(H) p47 and p97 mutants that lack binding affinity inhibit p97/p47-mediated aggregation of FTCDwt-beads. FTCDwt-beads were incubated with p97wt/mutant (0.1 μg/μl) and p47wt/mutants (0.1 μg/μl) in the presence of AMP-PNP. The representative images are presented in Figure EV1F. Data are shown as the mean+SD of nine sets of independent experiments. Asterisks indicate a significant difference at P<0.01 compared with the positive control ('p97wt+p47wt') (Bonferroni method).


Figure 8 ( A ) The expression of FTCDwt - HA - MAO causes the aggregation of mitochondria . HeLa Tet - off cells inducibly expressing either FTCDwt - HA - MAO or FTCD ( R382A ) - HA - MAO were cultured in DOX - free medium for 48 hrs . Cells which were cultured in the DOX - containing medium were used as a control . The cells were fixed and stained with a monoclonal antibody to mitochondria and a polyclonal antibody to HA , followed by observation by confocal microscopy . Panels a - j display representative images . Scale bar = 10 μm . ( B ) The results of quantification of ( A ) . Results are shown as the mean ± SD of five sets of independent experiments , with 100 cells counted in each group in each independent experiment . An asterisk indicates a significant difference at P < 0 . 01 compared with the others ( Bonferroni method ) . ( C ) Representative EM images of a cell expressing FTCDwt - HA - MAO . The HeLa Tet - off cells inducibly expressing FTCDwt - HA - MAO were cultured for 48 hrs in the absence of DOX , and used for EM observation . Panel b displays the inset in panel a . Arrowheads show narrow gaps between mitochondria . Scale bar = 0 . 2 μm . The low magnification image is presented in Figure EV2C . ( D ) Endogenous p47 and p97 are necessary for mitochondria aggregation mediated by FTCDwt - HA - MAO . HeLa Tet - off cells inducibly expressing FTCDwt - HA - MAO were transfected with either mock , p47 siRNA or p97 siRNA duplexes and cultured for 24 hrs . The cells were further cultured in DOX - free medium for 48 hrs for the induction of FTCDwt - HA - MAO , and then analyzed as in ( A ) . Panels a - l display representative images . Scale bar = 10 μm . ( E ) The results of quantification of ( D ) . Results are shown as the mean ± SD of five sets of independent experiments , with 100 cells counted in each group in each independent experiment . An asterisk indicates a significant difference at P < 0 . 01 compared with the others ( Bonferroni method ) . ( F ) Binding between FTCD and p47 and between p47 and p97 is necessary for mitochondria aggregation mediated by FTCDwt - HA - MAO . The HeLa Tet - off cells inducibly expressing FTCDwt - HA - MAO were transfected with mammalian expression constructs of siRNA - insensitive Flag - tagged p47wt / mutants at the same time as the treatment of p47 siRNA , and cultured for 24 hrs . The cells were further cultured in DOX - free medium for 48 hrs for the induction of FTCD - HA - MAO . After fixation , the cells were visualized with a monoclonal antibody to mitochondria and polyclonal antibodies to HA and Flag . Panels a - l display representative images . Scale bar = 10 μm . ( G ) Binding between FTCD and p97 is necessary for mitochondria aggregation mediated by FTCDwt - HA - MAO . The HeLa Tet - off cells inducibly expressing FTCDwt - HA - MAO were transfected with the mammalian expression construct of siRNA - insensitive Flag - tagged p97wt / mutant at the same time as treatment with p97 siRNA . The following procedures were the same as in ( F ) . Panels a - i display representative images . Scale bar = 10 μm . ( H ) The results of quantification of ( F ) and ( G ) . Results are shown as the mean ± SD of five sets of independent experiments , with 100 cells counted in each group in each independent experiment . Asterisks indicate a significant difference at P < 0 . 01 compared with siRNA treatment alone ( ' none ' ) and compared with mutant expression ( Bonferroni method ) .

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

Figure 8(A) The expression of FTCDwt-HA-MAO causes the aggregation of mitochondria. HeLa Tet-off cells inducibly expressing either FTCDwt-HA-MAO or FTCD(R382A)-HA-MAO were cultured in DOX-free medium for 48 hrs. Cells which were cultured in the DOX-containing medium were used as a control. The cells were fixed and stained with a monoclonal antibody to mitochondria and a polyclonal antibody to HA, followed by observation by confocal microscopy. Panels a-j display representative images. Scale bar = 10 μm. (B) The results of quantification of (A). Results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiment. An asterisk indicates a significant difference at P<0.01 compared with the others (Bonferroni method). (C) Representative EM images of a cell expressing FTCDwt-HA-MAO. The HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO were cultured for 48 hrs in the absence of DOX, and used for EM observation. Panel b displays the inset in panel a. Arrowheads show narrow gaps between mitochondria. Scale bar = 0.2 μm. The low magnification image is presented in Figure EV2C.(D) Endogenous p47 and p97 are necessary for mitochondria aggregation mediated by FTCDwt-HA-MAO. HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO were transfected with either mock, p47 siRNA or p97 siRNA duplexes and cultured for 24 hrs. The cells were further cultured in DOX-free medium for 48 hrs for the induction of FTCDwt-HA-MAO, and then analyzed as in (A). Panels a-l display representative images. Scale bar = 10 μm. (E) The results of quantification of (D). Results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiment. An asterisk indicates a significant difference at P<0.01 compared with the others (Bonferroni method). (F) Binding between FTCD and p47 and between p47 and p97 is necessary for mitochondria aggregation mediated by FTCDwt-HA-MAO. The HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO were transfected with mammalian expression constructs of siRNA-insensitive Flag-tagged p47wt/mutants at the same time as the treatment of p47 siRNA, and cultured for 24 hrs. The cells were further cultured in DOX-free medium for 48 hrs for the induction of FTCD-HA-MAO. After fixation, the cells were visualized with a monoclonal antibody to mitochondria and polyclonal antibodies to HA and Flag. Panels a-l display representative images. Scale bar = 10 μm. (G) Binding between FTCD and p97 is necessary for mitochondria aggregation mediated by FTCDwt-HA-MAO. The HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO were transfected with the mammalian expression construct of siRNA-insensitive Flag-tagged p97wt/mutant at the same time as treatment with p97 siRNA. The following procedures were the same as in (F). Panels a-i display representative images. Scale bar = 10 μm. (H) The results of quantification of (F) and (G). Results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiment. Asterisks indicate a significant difference at P<0.01 compared with siRNA treatment alone ('none') and compared with mutant expression (Bonferroni method).


Figure 9 ( A ) FTCD located in mitochondria causes mitochondria aggregation during mitosis . Mitotic cells were collected by flushing from HeLa Tet - off cells in which either FTCDwt - HA - MAO or FTCD ( R382A ) - HA - MAO had been induced for 24 hrs . The mitotic cells were further cultured for 4 hrs to enable complete cytokinesis . Cells in which FTCDwt - HA - MAO was induced for 28 hrs without synchronization were used as the asynchronous control group . Cells were fixed and visualized with a monoclonal antibody to mitochondria and a polyclonal antibody to HA . Panels a - f display representative images . Scale bar = 10 μm . ( B ) The results of quantification of ( A ) . Results are shown as the mean ± SD of seven sets of independent experiments , with 100 cells counted in each group in each independent experiment . An asterisk indicates a significant difference at P < 0 . 01 compared with the others ( Bonferroni method ) . ( C ) A single living cell expressing FTCDwt - HA - MAO was tracked during mitosis . Mitotic cells were collected by flushing from the HeLa Tet - off cells inducibly expressing FTCDwt - HA - MAO , and cultured in the absence or presence of DOX . The cells were stained with MitoTracker at the 22 hr time point and confocal images of 30 - 40 cells were randomly taken at the 23 hr time point . From the 23 hr to 27 hr time point , cells were tracked in the bright field every 30 mins . At the 27 hr time point , confocal images were taken of the 21 - 38 cells that were successfully tracked . Panels a - j display representative images . Asterisks indicate the tracked cells . Scale bar = 10 μm . ( D ) The results of quantification of ( C ) . The experiment was repeated five times and each line shows the average percentage of an independent experiment . The percentages of cells that underwent mitosis are as follows : 41 . 5 % in the DOX ( - ) group and 54 . 7 % in the DOX ( + ) group . ( E ) FTCD located in mitochondria causes mitochondria aggregation at the end of mitosis . Mitotic cells were collected from the HeLa Tet - off cells inducibly expressing FTCDwt - HA - MAO and cultured in the absence or presence of DOX as in ( C ) . Cells were stained with MitoTracker at the 23hr time point and observed by confocal microscopy from the 24 hr to 26 hr time point . Panels a - t display representative images . Scale bar = 10 μm . ( F ) The results of quantification of ( E ) . Cells were classified by their cell cycle phases into the following three categories : prometaphase / metaphase , anaphase / telophase and cytokinesis . Results are shown as the mean ± SD of three sets of independent experiments , with 10 - 14 cells counted in each group in each independent experiment . Asterisks indicate a significant difference at P < 0 . 01 compared with the DOX ( + ) group and with the prometaphase / metaphase category ( Bonferroni method ) .

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Figure 9(A) FTCD located in mitochondria causes mitochondria aggregation during mitosis. Mitotic cells were collected by flushing from HeLa Tet-off cells in which either FTCDwt-HA-MAO or FTCD(R382A)-HA-MAO had been induced for 24 hrs. The mitotic cells were further cultured for 4 hrs to enable complete cytokinesis. Cells in which FTCDwt-HA-MAO was induced for 28 hrs without synchronization were used as the asynchronous control group. Cells were fixed and visualized with a monoclonal antibody to mitochondria and a polyclonal antibody to HA. Panels a-f display representative images. Scale bar = 10 μm. (B) The results of quantification of (A). Results are shown as the mean±SD of seven sets of independent experiments, with 100 cells counted in each group in each independent experiment. An asterisk indicates a significant difference at P<0.01 compared with the others (Bonferroni method). (C) A single living cell expressing FTCDwt-HA-MAO was tracked during mitosis. Mitotic cells were collected by flushing from the HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO, and cultured in the absence or presence of DOX. The cells were stained with MitoTracker at the 22 hr time point and confocal images of 30-40 cells were randomly taken at the 23 hr time point. From the 23 hr to 27 hr time point, cells were tracked in the bright field every 30 mins. At the 27 hr time point, confocal images were taken of the 21-38 cells that were successfully tracked. Panels a-j display representative images. Asterisks indicate the tracked cells. Scale bar = 10 μm. (D) The results of quantification of (C). The experiment was repeated five times and each line shows the average percentage of an independent experiment. The percentages of cells that underwent mitosis are as follows: 41.5 % in the DOX(-) group and 54.7 % in the DOX(+) group. (E) FTCD located in mitochondria causes mitochondria aggregation at the end of mitosis. Mitotic cells were collected from the HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO and cultured in the absence or presence of DOX as in (C). Cells were stained with MitoTracker at the 23hr time point and observed by confocal microscopy from the 24 hr to 26 hr time point. Panels a-t display representative images. Scale bar = 10 μm.(F) The results of quantification of (E). Cells were classified by their cell cycle phases into the following three categories: prometaphase/metaphase, anaphase/telophase and cytokinesis. Results are shown as the mean±SD of three sets of independent experiments, with 10-14 cells counted in each group in each independent experiment. Asterisks indicate a significant difference at P<0.01 compared with the DOX(+) group and with the prometaphase/metaphase category (Bonferroni method).


Figure 2Starvation triggers accumulation of nucleosides , bases and S7P . Wild ‐ type S . cerevisiae cells growing in minimal media were switched to minimal media containing no carbon , no nitrogen or no phosphate . After the indicated duration of starvation , the metabolome was quantified by LC ‐ MS . For media composition , see Supplementary Table S2 . For the list of absolute concentration of metabolties , see Supplementary Table S3 . Data are shown in heat map format , with each line reflecting the dynamics of a particular compound in a particular culture condition . Metabolite levels of biological duplicates were averaged , normalized to cells growing steadily in glucose ( time zero ) , and the resulting fold changes log2 transformed .

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Figure 2Starvation triggers accumulation of nucleosides, bases and S7P. Wild‐type S. cerevisiae cells growing in minimal media were switched to minimal media containing no carbon, no nitrogen or no phosphate. After the indicated duration of starvation, the metabolome was quantified by LC‐MS. For media composition, see Supplementary Table S2. For the list of absolute concentration of metabolties, see Supplementary Table S3. Data are shown in heat map format, with each line reflecting the dynamics of a particular compound in a particular culture condition. Metabolite levels of biological duplicates were averaged, normalized to cells growing steadily in glucose (time zero), and the resulting fold changes log2 transformed.


Figure 3 ( A ) Experimental design for demonstrating metabolite production via macromolecule degradation . Yeast cells were grown on 2 % unlabeled glucose , and then switched to U ‐ 13C ‐ glucose for 70 min , which completely labels free metabolite but only partially labels macromolecules . Thereafter , glucose was removed and the metabolome analyzed by LC ‐ MS . ( B ) Fraction of unlabeled nucleosides , nucleic bases and PPP intermediates as a function of starvation time , in wild ‐ type and autophagy deficient ( atg7 deletion ) yeast . The x axis represents minutes after carbon starvation , and the y axis represents fraction of unlabeled metabolites ( mean ± range of N = 2 biological replicates ) . ( C ) Ratio of metabolite levels in atg7 strain versus wild ‐ type strain in carbon starvation . ( D ) Ratio of metabolite levels in bcy1 strain and snf1 strain versus wild ‐ type strain in carbon starvation . ( E ) Ratio of metabolite levels in rapamycin treatment versus nitrogen starvation for wild ‐ type yeast . In ( C ) to ( E ) , all reported values are log2 transformed ratios ; data are mean of duplicate samples at each time point .

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Figure 3(A) Experimental design for demonstrating metabolite production via macromolecule degradation. Yeast cells were grown on 2% unlabeled glucose, and then switched to U‐13C‐glucose for 70 min, which completely labels free metabolite but only partially labels macromolecules. Thereafter, glucose was removed and the metabolome analyzed by LC‐MS.(B) Fraction of unlabeled nucleosides, nucleic bases and PPP intermediates as a function of starvation time, in wild‐type and autophagy deficient (atg7 deletion) yeast. The x axis represents minutes after carbon starvation, and the y axis represents fraction of unlabeled metabolites (mean±range of N=2 biological replicates).(C) Ratio of metabolite levels in atg7 strain versus wild‐type strain in carbon starvation.(D) Ratio of metabolite levels in bcy1 strain and snf1 strain versus wild‐type strain in carbon starvation.(E) Ratio of metabolite levels in rapamycin treatment versus nitrogen starvation for wild‐type yeast. In (C) to (E), all reported values are log2 transformed ratios; data are mean of duplicate samples at each time point.


Figure 4 ( A ) Ratio of metabolite levels in sdt1 and phm8 strains versus wild ‐ type strain in carbon starvation . All reported values are log2 transformed ratios ; data are mean of duplicate samples at each time point . ( B ) Summary of transcripts data of PHM8 , SDT1 and other enzymes in the pathway ( Gasch et al , 2000 ; Bradley et al , 2009 ; Klosinska et al , 2011 ) . All values are log2 transformed fold changes . ( C ) Screening of Phm8 ' s phosphatase activity against 90 phosphorylated compounds . Phosphatase activity was measured in the presence of 0 . 5 mM substrate and 5 mM Mg2 + ( pH = 7 . 0 , 30 ° C ) . Compounds with specific activity higher than 0 . 1 μmol / mg / min are shown . The x axis represents specific phosphatase activity ( μmol of phosphate produced per minute per mg of enzyme , mean ± range of N = 2 replicates ) . ( D ) Top table : absolute intracellular concentration of nucleotide monophosphates in carbon starvation . Bottom plots : Phosphatase activity of Phm8 and Sdt1 as a function of CMP concentration . The x axis represents CMP concentration and the y axis represents specific phosphatase activity ( μmol of phosphate produced per minute per mg of enzyme , mean ± range of N = 2 replicates ) .

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Figure 4(A) Ratio of metabolite levels in sdt1 and phm8 strains versus wild‐type strain in carbon starvation. All reported values are log2 transformed ratios; data are mean of duplicate samples at each time point.(B) Summary of transcripts data of PHM8, SDT1 and other enzymes in the pathway (Gasch et al, 2000; Bradley et al, 2009; Klosinska et al, 2011). All values are log2 transformed fold changes.(C) Screening of Phm8's phosphatase activity against 90 phosphorylated compounds. Phosphatase activity was measured in the presence of 0.5 mM substrate and 5 mM Mg2+ (pH=7.0, 30°C). Compounds with specific activity higher than 0.1 μmol/mg/min are shown. The x axis represents specific phosphatase activity (μmol of phosphate produced per minute per mg of enzyme, mean±range of N=2 replicates).(D) Top table: absolute intracellular concentration of nucleotide monophosphates in carbon starvation. Bottom plots: Phosphatase activity of Phm8 and Sdt1 as a function of CMP concentration. The x axis represents CMP concentration and the y axis represents specific phosphatase activity (μmol of phosphate produced per minute per mg of enzyme, mean±range of N=2 replicates).


Figure 5Confirmation that Pnp1 is the physiological purine nucleoside phosphorylase , Urh1 is the pyrimidine hydrolase , and Prm15 ( Pgm3 ) is the phosphoribomutase . Ratio of metabolite levels in pnp1 / urh1 , pnp1 , urh1 and prm15 ( pgm3 ) strains versus wild ‐ type strain during carbon starvation . Data are shown in heat map format , with each line reflecting the dynamics of the ratio of the metabolite levels in a particular strain versus wild ‐ type strain . All reported values are log2 transformed ratios ; data are mean of duplicate samples at each time point .

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Figure 5Confirmation that Pnp1 is the physiological purine nucleoside phosphorylase, Urh1 is the pyrimidine hydrolase, and Prm15 (Pgm3) is the phosphoribomutase. Ratio of metabolite levels in pnp1/urh1, pnp1, urh1 and prm15 (pgm3) strains versus wild‐type strain during carbon starvation. Data are shown in heat map format, with each line reflecting the dynamics of the ratio of the metabolite levels in a particular strain versus wild‐type strain. All reported values are log2 transformed ratios; data are mean of duplicate samples at each time point.