Figure 2 ( A - B ) Median GFP intensity of BFP - positive GFP - RBM3 i - neurons measured by flow cytometry upon the sgRNA / Cas9 - mediated KO of top 30 positive ( A ) or negative ( B ) regulator candidates . Statistical analysis is performed between the specific and non - targeting sgRNA groups for positive regulators ( A ) or between the specific sgRNA and reporter groups for negative regulators ( B ) within the 37 ° C or 32 ° C ( 72h ) population . ( C ) qRT - PCR of RBM3 mRNA level normalised to 18s rRNA in i - neurons at 37 ° C or 32 ° C ( 72h ) .

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Figure 2(A-B) Median GFP intensity of BFP-positive GFP-RBM3 i-neurons measured by flow cytometry upon the sgRNA/Cas9-mediated KO of top 30 positive (A) or negative (B) regulator candidates. Statistical analysis is performed between the specific and non-targeting sgRNA groups for positive regulators (A) or between the specific sgRNA and reporter groups for negative regulators (B) within the 37°C or 32°C (72h) population.(C) qRT-PCR of RBM3 mRNA level normalised to 18s rRNA in i-neurons at 37°C or 32°C (72h).


Figure 3RT - PCR of RBM3 mRNA ( Exon 2 - 4 ) in i - neurons at 37 ° C or 32 ° C ( 72h ) in the presence or absence of SMG1 inhibitor . PSI values of RBM3 PE are calculated based on the intensity of PE - included ( red arrows ) and PE - skipped ( green arrow ) isoforms . qRT - PCR using a combination of primers targeting Exon 3a , Exon 3 or Exon 4 - 5 quantifies the PSI values of RBM3 PE ( Exon 3a , including both 3a - L and 3a - S ) relative to Exon 3 or Exon 4 - 5 at 37 ° C or 32 ° C ( 72h ) in the presence or absence of SMG1 inhibitor . RT - PCR of RBM3 mRNA ( Exon 2 - 4 ) in HeLa cells at 37 ° C and 32 ° C ( 48h ) in the presence or absence of SMG1 inhibitor . PSI values of RBM3 PE are depicted in the graph on the right . Schematics and RT - PCR of RBM3 minigene ( Exon 1 - 4 ) , flanked by unique sequences ( thick black bars ) to distinguish it from endogenous transcripts during PCR amplification , expressed in HeLa cells at 37 ° C or 32 ° C ( 48h ) , in the presence or absence of SMG1 inhibitor . PSI values of RBM3 PE are depicted in the graph on the right .

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Figure 3RT-PCR of RBM3 mRNA (Exon 2-4) in i-neurons at 37°C or 32°C (72h) in the presence or absence of SMG1 inhibitor. PSI values of RBM3 PE are calculated based on the intensity of PE-included (red arrows) and PE-skipped (green arrow) isoforms.qRT-PCR using a combination of primers targeting Exon 3a, Exon 3 or Exon 4-5 quantifies the PSI values of RBM3 PE (Exon 3a, including both 3a-L and 3a-S) relative to Exon 3 or Exon 4-5 at 37°C or 32°C (72h) in the presence or absence of SMG1 inhibitor.RT-PCR of RBM3 mRNA (Exon 2-4) in HeLa cells at 37°C and 32°C (48h) in the presence or absence of SMG1 inhibitor. PSI values of RBM3 PE are depicted in the graph on the right.Schematics and RT-PCR of RBM3 minigene (Exon 1-4), flanked by unique sequences (thick black bars) to distinguish it from endogenous transcripts during PCR amplification, expressed in HeLa cells at 37°C or 32°C (48h), in the presence or absence of SMG1 inhibitor. PSI values of RBM3 PE are depicted in the graph on the right.


Figure 4 ( A - B ) qRT - PCR quantifying the PSI values of RBM3 PE relative to RBM3 mRNA in WT i - neurons at 37 ° C ( A ) or 32 ° C ( 72h ) ( B ) , when NMD is blocked by SMG1 inhibitor . Statistical analysis is performed between the specific and non - targeting sgRNA groups . PSI values of RBM3 Exon 3a - L in control and HNRNPH1 - knocked down K562 and HepG2 cells . RNA - Seq data from ENCODE Project , 2 isogenic replicates are included . ( D - E ) RT - PCR of endogenous RBM3 ( D ) and expressed RBM3 minigene ( E ) in control ( scramble siRNA ) or HNRNPH1 - knocked down HeLa cells at 37 ° C or 32 ° C ( 48h ) , in the presence or absence of SMG1 inhibitor . The ratio of PSI values between HNRNPH1 KD and control is shown for only the SMG1 i - treated conditions . ( F - G ) RT - PCR of endogenous RBM3 ( F ) and expressed RBM3 minigene ( G ) in SMG1 inhibitor - treated control ( untransfected ) or FLAG - HNRNPH1 - overexpressing ( OX ) HEK293T cells at 37 ° C . PSI values of RBM3 PE are shown in the graphs on the right respectively . WT i - neurons are transduced with lentiviral constructs expressing BFP ( Control ) or HNRNPH1 - T2A - BFP ( HNRNPH1 overexpression , OX ) at 37 ° C and 32 ° C , followed by RT - PCR of endogenous RBM3 with 24h SMG1 inhibitor treatment . PSI values of RBM3 PE are shown in the graph on the right .

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Figure 4(A-B) qRT-PCR quantifying the PSI values of RBM3 PE relative to RBM3 mRNA in WT i-neurons at 37°C (A) or 32°C (72h) (B), when NMD is blocked by SMG1 inhibitor. Statistical analysis is performed between the specific and non-targeting sgRNA groups. PSI values of RBM3 Exon 3a-L in control and HNRNPH1-knocked down K562 and HepG2 cells. RNA-Seq data from ENCODE Project, 2 isogenic replicates are included.(D-E) RT-PCR of endogenous RBM3 (D) and expressed RBM3 minigene (E) in control (scramble siRNA) or HNRNPH1-knocked down HeLa cells at 37°C or 32°C (48h), in the presence or absence of SMG1 inhibitor. The ratio of PSI values between HNRNPH1 KD and control is shown for only the SMG1 i-treated conditions.(F-G) RT-PCR of endogenous RBM3 (F) and expressed RBM3 minigene (G) in SMG1 inhibitor-treated control (untransfected) or FLAG-HNRNPH1-overexpressing (OX) HEK293T cells at 37°C. PSI values of RBM3 PE are shown in the graphs on the right respectively. WT i-neurons are transduced with lentiviral constructs expressing BFP (Control) or HNRNPH1-T2A-BFP (HNRNPH1 overexpression, OX) at 37°C and 32°C, followed by RT-PCR of endogenous RBM3 with 24h SMG1 inhibitor treatment. PSI values of RBM3 PE are shown in the graph on the right.


Figure 5Western blot and quantification of HNRNPH1 normalised to GAPDH in WT i - neurons at 37 ° C and 32 ° C ( 72h ) . RBM3 blots of the same samples are shown for comparison . RT - PCR of WT and delGGGG RBM3 minigenes in HeLa cells at 37 ° C or 32 ° C ( 48h ) treated with SMG1 inhibitor . PSI values of RBM3 PE are shown in the graphs on the right .

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Figure 5Western blot and quantification of HNRNPH1 normalised to GAPDH in WT i-neurons at 37°C and 32°C (72h). RBM3 blots of the same samples are shown for comparison.RT-PCR of WT and delGGGG RBM3 minigenes in HeLa cells at 37°C or 32°C (48h) treated with SMG1 inhibitor. PSI values of RBM3 PE are shown in the graphs on the right.


Figure 1Snapshots of ChIP - seq signals of Sen1 ( this study ) , RNAP3 ( this study ) and RNAP2 ( data from ( Larochelle et al , 2018 ) ) across ( A ) a 25 kb region of chromosome 3 containing several tRNA and 5S rRNA genesSnapshots of ChIP - seq signals of Sen1 ( this study ) , RNAP3 ( this study ) and RNAP2 ( data from ( Larochelle et al , 2018 ) ) across the U6 snRNA snu6Snapshots of ChIP - seq signals of Sen1 ( this study ) , RNAP3 ( this study ) and RNAP2 ( data from ( Larochelle et al , 2018 ) ) across the srp7 loci . Genome - wide pairwise Pearson correlation coefficient matrix at a resolution of 10 bp . ChIP - qPCR analysis of Flag - tagged Sen1 at the indicated loci in a population of cycling cells ( mean ± standard deviation from 4 biological replicates ) . ChIP - qPCR analysis of the Flag - tagged RNAP3 subunit Rpc37 and Flag - tagged Sen1 across the SPCTRNAARG . 10 tRNA locus , whose TATA box was either mutated ( TATA - ) or not ( TATA + ) . The mutations introduced to disrupt the putative TATA boxes are indicated in red above the graphs . The enrichment values were normalized to SPCTRNATHR . 10 ( mean ± standard deviation from 4 biological replicates ; p - value obtained using the Wilcoxon - Mann Whitney statistical test ) .

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Figure 1Snapshots of ChIP-seq signals of Sen1 (this study), RNAP3 (this study) and RNAP2 (data from (Larochelle et al, 2018)) across (A) a 25 kb region of chromosome 3 containing several tRNA and 5S rRNA genesSnapshots of ChIP-seq signals of Sen1 (this study), RNAP3 (this study) and RNAP2 (data from (Larochelle et al, 2018)) across the U6 snRNA snu6Snapshots of ChIP-seq signals of Sen1 (this study), RNAP3 (this study) and RNAP2 (data from (Larochelle et al, 2018)) across the srp7 loci.Genome-wide pairwise Pearson correlation coefficient matrix at a resolution of 10 bp.ChIP-qPCR analysis of Flag-tagged Sen1 at the indicated loci in a population of cycling cells (mean ± standard deviation from 4 biological replicates).ChIP-qPCR analysis of the Flag-tagged RNAP3 subunit Rpc37 and Flag-tagged Sen1 across the SPCTRNAARG.10 tRNA locus, whose TATA box was either mutated (TATA-) or not (TATA+). The mutations introduced to disrupt the putative TATA boxes are indicated in red above the graphs. The enrichment values were normalized to SPCTRNATHR.10 (mean ± standard deviation from 4 biological replicates; p-value obtained using the Wilcoxon-Mann Whitney statistical test).


Figure 2 ( left ) ChIP - qPCR analysis of Rpc37 in the presence or absence of Sen1 at the indicated loci in a population of cycling cells ( mean ± standard deviation from 4 biological replicates ) . ( right ) Western blot analysis of Rpc37 protein levels in the presence or absence of Sen1 . Tubulin was used as a loading control . ( left ) Total RNA from the indicated strains were separated on a 2 , 8 % agarose gel . ( right ) Quantification of overall tRNA levels ( mean ± standard deviation from 3 biological replicates ) . Strand - specific RT - qPCR was used to quantify the indicated RNAP3 transcripts . Transcript levels were normalized to act1 ( mean ± standard deviation from 3 biological replicates ) .

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Figure 2(left) ChIP-qPCR analysis of Rpc37 in the presence or absence of Sen1 at the indicated loci in a population of cycling cells (mean ± standard deviation from 4 biological replicates). (right) Western blot analysis of Rpc37 protein levels in the presence or absence of Sen1. Tubulin was used as a loading control.(left) Total RNA from the indicated strains were separated on a 2,8% agarose gel. (right) Quantification of overall tRNA levels (mean ± standard deviation from 3 biological replicates).Strand-specific RT-qPCR was used to quantify the indicated RNAP3 transcripts. Transcript levels were normalized to act1 (mean ± standard deviation from 3 biological replicates).


Figure 3Cells were grown in minimal medium during 18 hours to induce the strong expression of RnhA . R - ChIP using a catalytically - inactive RNase H1 ( Rnh1 - D129N ) was used to quantify R - loop formation at RNAP3 - transcribed genes ( mean ± standard deviation from 4 biological replicates ) . ( left ) ChIP - qPCR of the 13myc - tagged RNAP3 subunit Rpc25 in the indicated strains at the indicated loci ( mean ± standard deviation of 4 biological replicates ) . ( right ) Western blot analysis of Myc - tagged Rpc25 and Flag - tagged RnhA protein levels in the indicated strains . Tubulin was used as a loading control .

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Figure 3Cells were grown in minimal medium during 18 hours to induce the strong expression of RnhA. R-ChIP using a catalytically-inactive RNase H1 (Rnh1-D129N) was used to quantify R-loop formation at RNAP3-transcribed genes (mean ± standard deviation from 4 biological replicates).(left) ChIP-qPCR of the 13myc-tagged RNAP3 subunit Rpc25 in the indicated strains at the indicated loci (mean ± standard deviation of 4 biological replicates). (right) Western blot analysis of Myc-tagged Rpc25 and Flag-tagged RnhA protein levels in the indicated strains. Tubulin was used as a loading control.


Figure 4Snapshots of ChIP - seq signals of the RNAP3 subunits Rpc1 and Rpc2 in the presence or absence of Sen1 across a representative ( A ) tRNA gene , ( B ) 5S rRNA gene and ( C ) srp7 . Boxed regions highlight the increased density of reads in the downstream region of genes in the absence of Sen1 . Average ChIP - seq profile of Rpc1 and Rpc2 across all isolated tRNA and 5S rRNA genes in the presence and absence of Sen1 . Scanning of Rpc37 - 3flag occupancy at three different tDNA loci in the absence of Sen1 by ChIP - qPCR ( mean ± standard deviation from 6 biological replicates ) .

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Figure 4Snapshots of ChIP-seq signals of the RNAP3 subunits Rpc1 and Rpc2 in the presence or absence of Sen1 across a representative (A) tRNA gene, (B) 5S rRNA gene and (C) srp7. Boxed regions highlight the increased density of reads in the downstream region of genes in the absence of Sen1.Average ChIP-seq profile of Rpc1 and Rpc2 across all isolated tRNA and 5S rRNA genes in the presence and absence of Sen1.Scanning of Rpc37-3flag occupancy at three different tDNA loci in the absence of Sen1 by ChIP-qPCR (mean ± standard deviation from 6 biological replicates).


Figure 5Cells of the indicated genotypes that carried or not the DRT5T dimeric tRNA construct ( schematized on top ) were grown either in the presence of the optimum concentration of adenine ( left ) or in the presence of a limiting concentration of adenine ( right ) . Two independent clones of the same genotype ( # 1 and # 2 ) were used . See text for details . 3F refers to the 3 Flag epitope tag at the C - terminus of Rpc37 . Strand - specific RT - qPCR was used to quantify the levels of read - through transcripts ( see Methods ) . The mean ± standard deviation from 4 biological replicates is represented here . P - values were obtained using the Wilcoxon - Mann Whitney statistical test . Northern blot analysis of the tRNA SPATRNAPRO . 02 using an intron - specific probe ( TCTAAACTCAGCATACAAGTGGGG ) . U5 snRNA was used as a loading control .

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Figure 5Cells of the indicated genotypes that carried or not the DRT5T dimeric tRNA construct (schematized on top) were grown either in the presence of the optimum concentration of adenine (left) or in the presence of a limiting concentration of adenine (right). Two independent clones of the same genotype (#1 and #2) were used. See text for details. 3F refers to the 3 Flag epitope tag at the C-terminus of Rpc37.Strand-specific RT-qPCR was used to quantify the levels of read-through transcripts (see Methods). The mean ± standard deviation from 4 biological replicates is represented here. P-values were obtained using the Wilcoxon-Mann Whitney statistical test.Northern blot analysis of the tRNA SPATRNAPRO.02 using an intron-specific probe (TCTAAACTCAGCATACAAGTGGGG). U5 snRNA was used as a loading control.


Figure 6ChIP - qPCR analysis of Rpc37 around SPCTRNAARG . 10 gene in the strong terminator mutant ( mean ± standard deviation from 4 biological replicates ) . Strand specific RT - qPCR was used to quantify read - through transcripts at SPCTRNAARG . 10 ( arg10 ) and SPCTRNASER . 09 ( ser09 ) in the strong terminator mutant ( mean ± standard deviation from 4 biological replicates ; p - value obtained using the Wilcoxon - Mann Whitney statistical test ) .

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Figure 6ChIP-qPCR analysis of Rpc37 around SPCTRNAARG.10 gene in the strong terminator mutant (mean ± standard deviation from 4 biological replicates).Strand specific RT-qPCR was used to quantify read-through transcripts at SPCTRNAARG.10 (arg10) and SPCTRNASER.09 (ser09) in the strong terminator mutant (mean ± standard deviation from 4 biological replicates; p-value obtained using the Wilcoxon-Mann Whitney statistical test).


Figure 1B . Western blot of oocyte lysate ( left ) and biotinylation - purified surface ( right ) samples showing the expression of PC1 and PC2 in Xenopus oocytes and enhanced surface trafficking of the PC1 / PC2 complex compared to either protein expressed separately . Anti - PC1 C - terminus antibody [ 29 ] recognized both full - length ( asterisk ) and GPS - cleaved CTF ( open circle ) of PC1 . A higher - glycosylated 130 kDa PC2 ( star ) band was only seen when PC1 is coexpressed . C . Co - IP followed by western blot showing the association between PC1 and PC2 that were expressed in Xenopus oocytes . IP was done with an anti - FLAG antibody . Bands of full - length ( asterisk ) and GPS - cleaved CTF ( open circle ) of PC1 are indicated . Both 120 and 130 kDa bands of PC2 were seen in the IPed product . D . Representative current - voltage relationship ( I - V ) curves ( left ) and a scatter plot and bar graph ( right ) showing coexpression of WT PC1 and PC2 produced no current in TEVC recording . The current of the GOF PC2 _ F604P is included as a control . Currents at + 60 mV are shown in the bar graph . Each point represents the recorded current from one oocyte . Oocyte numbers for scatter plot and bar graph are indicated in parentheses . Data are presented as mean ± SD ( * * * P < 0 . 001 , Student ' s t - test ) .

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Figure 1B. Western blot of oocyte lysate (left) and biotinylation-purified surface (right) samples showing the expression of PC1 and PC2 in Xenopus oocytes and enhanced surface trafficking of the PC1/PC2 complex compared to either protein expressed separately. Anti-PC1 C-terminus antibody [29] recognized both full-length (asterisk) and GPS-cleaved CTF (open circle) of PC1. A higher-glycosylated 130 kDa PC2 (star) band was only seen when PC1 is coexpressed.C. Co-IP followed by western blot showing the association between PC1 and PC2 that were expressed in Xenopus oocytes. IP was done with an anti-FLAG antibody. Bands of full-length (asterisk) and GPS-cleaved CTF (open circle) of PC1 are indicated. Both 120 and 130 kDa bands of PC2 were seen in the IPed product.D. Representative current-voltage relationship (I-V) curves (left) and a scatter plot and bar graph (right) showing coexpression of WT PC1 and PC2 produced no current in TEVC recording. The current of the GOF PC2_F604P is included as a control. Currents at +60 mV are shown in the bar graph. Each point represents the recorded current from one oocyte. Oocyte numbers for scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD (***P < 0.001, Student's t-test).


Figure 2B . Representative I - V curves ( left ) and a scatter plot and bar graph ( right ) showing that the PC2 _ AA is a GOF mutant and gave rise to a larger current than PC2 _ F604P . Scatter plot and bar graph shows the average current sizes at + 60 mV . The cations included in the bath solution , 100 mM Na + and 2 mM Ca2 + in this case , are indicated by the thick - lined boxes here and in all the following figures . Oocyte numbers for bar graph are indicated in parentheses . Data are presented as mean ± SD in bar graph ( * * * P < 0 . 001 , Student ' s t - test ) . C . Representative currents of PC2 _ F604P and PC2 _ AA mutants in the divalent ion - free bath solution , which contains 100 mM Na + . D . Representative currents of indicated WT and mutants of PC2 in a bath solution containing 70 mM Ca2 + . E . Calcium - activated chloride channel ( CaCC ) blocker MONNA ( 10 μM ) or niflumic acid ( NFA ) ( 1 mM ) partially blocked the current recorded from the PC2 _ AA - injected oocytes in the 70 mM Ca2 + bath solution .

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Figure 2B. Representative I-V curves (left) and a scatter plot and bar graph (right) showing that the PC2_AA is a GOF mutant and gave rise to a larger current than PC2_F604P. Scatter plot and bar graph shows the average current sizes at +60 mV. The cations included in the bath solution, 100 mM Na+ and 2 mM Ca2+ in this case, are indicated by the thick-lined boxes here and in all the following figures. Oocyte numbers for bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).C. Representative currents of PC2_F604P and PC2_AA mutants in the divalent ion-free bath solution, which contains 100 mM Na+.D. Representative currents of indicated WT and mutants of PC2 in a bath solution containing 70 mM Ca2+.E. Calcium-activated chloride channel (CaCC) blocker MONNA (10 μM) or niflumic acid (NFA) (1 mM) partially blocked the current recorded from the PC2_AA-injected oocytes in the 70 mM Ca2+ bath solution.


Figure 3A , B . Representative I - V curves ( left ) and scatter plot and bar graphs ( right ) showing the currents from oocytes expressing PC2 _ AA alone , PC1 with PC2 _ AA , and PC1 with WT PC2 , in bath solutions containing 100 mM Na + ( A ) , and 100 mM Na + and 2 mM Ca2 + ( B ) . Scatter plot and bar graphs show currents at + 80 mV and - 80 ( or - 70 ) mV . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD in bar graph ( n . s . : not significant , * * * P < 0 . 001 , Student ' s t - test ) . C . Representative I - V curves of PC1 / PC2 _ AA recorded in a bath solution containing 100 mM Na + and 2 mM Ca2 + solution in the absence or presence of 10 μM MONNA . D . Representative I - V curves ( left ) and scatter plot and bar graphs ( right ) showing the currents from oocytes expressing PC2 _ AA alone , PC1 with PC2 _ AA , and PC1 with WT PC2 , in bath solutions containing 70 mM Ca2 + . Scatter plot and bar graphs show currents at + 80 mV and - 70 mV . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD in bar graph ( * * * P < 0 . 001 , Student ' s t - test ) . E . Representative I - V curves of PC1 / PC2 _ AA recorded in a bath solution containing 70 mM Ca2 + solution in the absence or presence of 10 μM MONNA . F . Representative currents of PC2 _ AA ( left ) and PC1 / PC2 _ AA ( right ) in solutions containing the indicated Ca2 + concentrations . All solutions contain 20 μM of MONNA to block CaCC current . Corresponding concentrations of NMDG + were added to compensate the osmolarity . See Fig EV2 for currents of the full voltage scale and the details of solutions used . G . Scatter plot and bar graph showing the higher radiolabeled 45Ca uptake rate of oocytes expressing the PC1 / PC2 _ AA complex channel compared to that of oocytes expressing PC1 alone or PC2 alone . For oocytes injected with PC2 alone , five times more concentrated PC2 RNA was injected to increase its surface expression . The purple dashed line indicates the background 45Ca uptake set by the measurement with the water - injected oocytes . Data were averaged from three independent experiments . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD in bar graph ( * * * P < 0 . 001 , Student ' s t - test ) . H . Surface biotinylation followed by western blot showing the expression levels of the indicated proteins in lysate and plasma membrane of oocytes in 45Ca uptake experiments .

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

Figure 3A, B. Representative I-V curves (left) and scatter plot and bar graphs (right) showing the currents from oocytes expressing PC2_AA alone, PC1 with PC2_AA, and PC1 with WT PC2, in bath solutions containing 100 mM Na+ (A), and 100 mM Na+ and 2 mM Ca2+ (B). Scatter plot and bar graphs show currents at +80 mV and -80 (or -70) mV. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, ***P < 0.001, Student's t-test).C. Representative I-V curves of PC1/PC2_AA recorded in a bath solution containing 100 mM Na+ and 2 mM Ca2+ solution in the absence or presence of 10 μM MONNA.D. Representative I-V curves (left) and scatter plot and bar graphs (right) showing the currents from oocytes expressing PC2_AA alone, PC1 with PC2_AA, and PC1 with WT PC2, in bath solutions containing 70 mM Ca2+. Scatter plot and bar graphs show currents at +80 mV and -70 mV. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).E. Representative I-V curves of PC1/PC2_AA recorded in a bath solution containing 70 mM Ca2+ solution in the absence or presence of 10 μM MONNA.F. Representative currents of PC2_AA (left) and PC1/PC2_AA (right) in solutions containing the indicated Ca2+ concentrations. All solutions contain 20 μM of MONNA to block CaCC current. Corresponding concentrations of NMDG+ were added to compensate the osmolarity. See Fig EV2 for currents of the full voltage scale and the details of solutions used.G. Scatter plot and bar graph showing the higher radiolabeled 45Ca uptake rate of oocytes expressing the PC1/PC2_AA complex channel compared to that of oocytes expressing PC1 alone or PC2 alone. For oocytes injected with PC2 alone, five times more concentrated PC2 RNA was injected to increase its surface expression. The purple dashed line indicates the background 45Ca uptake set by the measurement with the water-injected oocytes. Data were averaged from three independent experiments. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).H. Surface biotinylation followed by western blot showing the expression levels of the indicated proteins in lysate and plasma membrane of oocytes in 45Ca uptake experiments.


Figure 4Representative I - V curves ( left ) and scatter plot and bar graphs ( right ) showing the comparison between the currents of full - length PC1 / PC2 _ AA with that of PC1 - CTF / PC2 _ AA in bath solutions containing 100 mM Na + ( B ) , Currents at both + 80 mV and - 80 ( or - 70 ) mV are displayed in the scatter plot and bar graphs . Oocyte numbers are indicated in parentheses . Representative I - V curves ( left ) and scatter plot and bar graphs ( right ) showing the comparison between the currents of full - length PC1 / PC2 _ AA with that of PC1 - CTF / PC2 _ AA in bath solutions containing 100 mM Na + and 2 mM Ca2 + ( C ) Currents at both + 80 mV and - 80 ( or - 70 ) mV are displayed in the scatter plot and bar graphs . Oocyte numbers are indicated in parentheses . Representative I - V curves ( left ) and scatter plot and bar graphs ( right ) showing the comparison between the currents of full - length PC1 / PC2 _ AA with that of PC1 - CTF / PC2 _ AA in bath solutions containing 70 mM Ca2 + ( D ) . Currents at both + 80 mV and - 80 ( or - 70 ) mV are displayed in the scatter plot and bar graphs . Oocyte numbers are indicated in parentheses . E . Scatter plot and bar graph showing that the current of PC1 - CTF / PC2 _ AA is completely abolished when WT PC2 was used , or after introducing R4090W mutation in the putative pore region of PC1 - CTF . Inserted are western blot images showing the expression of the corresponding proteins . Top : anti - PC1 . Bottom : anti - PC2 . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD in bar graph ( * * * P < 0 . 001 , Student ' s t - test ) . Representative I - V curves of oocytes injected with the indicated RNAs in a bath solution containing 100 mM Na + and 2 mM Ca2 + ( F ) , showing PC1 - TLD / PC2 _ AA gave rise to current with similar properties as full - length PC1 / PC2 _ AA channel . Representative I - V curves of oocytes injected with the indicated RNAs in a bath solution containing 100 mM Na + ( G ) , , showing PC1 - TLD / PC2 _ AA gave rise to current with similar properties as full - length PC1 / PC2 _ AA channel . Representative I - V curves of oocytes injected with the indicated RNAs in a bath solution containing 70 mM Ca2 + ( H ) , showing PC1 - TLD / PC2 _ AA gave rise to current with similar properties as full - length PC1 / PC2 _ AA channel . I . Co - IP followed by western blot showing the association between PC2 _ AA and the indicated full - length or fragments of PC1 . Bands of full - length ( asterisk ) , GPS - cleaved CTF or expressed CTF fragment ( open circle ) , and TLD ( star ) of PC1 are indicated . J . Surface biotinylation followed by western blot showing the expression of the complexes formed between PC2 _ AA and full - length PC1 , PC1 - CTF , or PC1 - TLD at the oocyte surface . Besides the blots shown in the figure , surface samples were also blotted with an anti - PC1 N - terminus antibody , and the cleaved NTF fragment was found associated to the plasma membrane in the full - length sample ( shown in Appendix Fig S4 ) . Bands of full - length ( asterisk ) , GPS - cleaved CTF or expressed CTF fragment ( open circle ) , and TLD ( star ) of PC1 are 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 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Representative I-V curves (left) and scatter plot and bar graphs (right) showing the comparison between the currents of full-length PC1/PC2_AA with that of PC1-CTF/PC2_AA in bath solutions containing 100 mM Na+ (B), Currents at both +80 mV and -80 (or -70) mV are displayed in the scatter plot and bar graphs. Oocyte numbers are indicated in parentheses.Representative I-V curves (left) and scatter plot and bar graphs (right) showing the comparison between the currents of full-length PC1/PC2_AA with that of PC1-CTF/PC2_AA in bath solutions containing 100 mM Na+ and 2 mM Ca2+ (C) Currents at both +80 mV and -80 (or -70) mV are displayed in the scatter plot and bar graphs. Oocyte numbers are indicated in parentheses.Representative I-V curves (left) and scatter plot and bar graphs (right) showing the comparison between the currents of full-length PC1/PC2_AA with that of PC1-CTF/PC2_AA in bath solutions containing 70 mM Ca2+ (D). Currents at both +80 mV and -80 (or -70) mV are displayed in the scatter plot and bar graphs. Oocyte numbers are indicated in parentheses.E. Scatter plot and bar graph showing that the current of PC1-CTF/PC2_AA is completely abolished when WT PC2 was used, or after introducing R4090W mutation in the putative pore region of PC1-CTF. Inserted are western blot images showing the expression of the corresponding proteins. Top: anti-PC1. Bottom: anti-PC2. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).Representative I-V curves of oocytes injected with the indicated RNAs in a bath solution containing 100 mM Na+ and 2 mM Ca2+ (F), showing PC1-TLD/PC2_AA gave rise to current with similar properties as full-length PC1/PC2_AA channel.Representative I-V curves of oocytes injected with the indicated RNAs in a bath solution containing 100 mM Na+ (G), , showing PC1-TLD/PC2_AA gave rise to current with similar properties as full-length PC1/PC2_AA channel.Representative I-V curves of oocytes injected with the indicated RNAs in a bath solution containing 70 mM Ca2+ (H), showing PC1-TLD/PC2_AA gave rise to current with similar properties as full-length PC1/PC2_AA channel.I. Co-IP followed by western blot showing the association between PC2_AA and the indicated full-length or fragments of PC1. Bands of full-length (asterisk), GPS-cleaved CTF or expressed CTF fragment (open circle), and TLD (star) of PC1 are indicated.J. Surface biotinylation followed by western blot showing the expression of the complexes formed between PC2_AA and full-length PC1, PC1-CTF, or PC1-TLD at the oocyte surface. Besides the blots shown in the figure, surface samples were also blotted with an anti-PC1 N-terminus antibody, and the cleaved NTF fragment was found associated to the plasma membrane in the full-length sample (shown in Appendix Fig S4). Bands of full-length (asterisk), GPS-cleaved CTF or expressed CTF fragment (open circle), and TLD (star) of PC1 are indicated.


Figure 5A . Representative I - V curves of the PC2 _ AA and PC1 - CTF / PC2 _ AA channels in divalent ion - free bath solutions containing 100 mM of indicated ions , showing the differences in reversal potential . B . Bar graphs showing the markedly different reversal potentials of PC2 _ AA channel and the PC1 - CTF / PC2 _ AA channel in bath solutions containing 100 mM of the indicated ions . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD ( n . s . : not significant , * * * P < 0 . 001 , Student ' s t - test ) . Bar graphs showing the permeability ratios of the indicated ions to that of Na + ( Px : PNa ) ( C ) These ratios are strikingly different between the two channels . The ratios of the particular ions , which are the values of the bars , are indicted on top of the bars . Bar graphs showing the permeability ratios of the indicated ions to that of NMDG + ( Px : PNMDG ) ( D ) . These ratios are strikingly different between the two channels . The ratios of the particular ions , which are the values of the bars , are indicted on top of the bars .

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 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 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

Figure 5A. Representative I-V curves of the PC2_AA and PC1-CTF/PC2_AA channels in divalent ion-free bath solutions containing 100 mM of indicated ions, showing the differences in reversal potential.B. Bar graphs showing the markedly different reversal potentials of PC2_AA channel and the PC1-CTF/PC2_AA channel in bath solutions containing 100 mM of the indicated ions. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, ***P < 0.001, Student's t-test).Bar graphs showing the permeability ratios of the indicated ions to that of Na+ (Px: PNa) (C) These ratios are strikingly different between the two channels. The ratios of the particular ions, which are the values of the bars, are indicted on top of the bars.Bar graphs showing the permeability ratios of the indicated ions to that of NMDG+ (Px: PNMDG) (D). These ratios are strikingly different between the two channels. The ratios of the particular ions, which are the values of the bars, are indicted on top of the bars.


Figure 6Bar graphs showing the reversal potentials of PC1 - CTF / PC2 _ AA and the indicated mutants tested in bath solutions containing 100 mM of the indicated ions . Two PC1 and two PC2 mutations caused dramatic changes in the reversal potential for almost all tested ions ( B ) , indicating that these amino acids are essential for ion permeability . Statistical significance between reversal potentials of all pore mutants and that of the PC1 - CTF / PC2 _ AA channel are indicated . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD ( n . s . : not significant , * * P < 0 . 01 , * * * P < 0 . 001 , Student ' s t - test ) . Bar graphs showing the reversal potentials of PC1 - CTF / PC2 _ AA and the indicated mutants tested in bath solutions containing 100 mM of the indicated ions . Another two PC1 mutations and one PC2 mutation only led to relatively small changes of reversal potential for some ions but not others ( C ) , indicating a less important role of these amino acids in ion permeability . Statistical significance between reversal potentials of all pore mutants and that of the PC1 - CTF / PC2 _ AA channel are indicated . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD ( n . s . : not significant , * * P < 0 . 01 , * * * P < 0 . 001 , Student ' s t - test ) . Bar graphs showing the permeability ratios of the indicated ions to that of Na + ( Px : PNa ) ( D ) Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases . Bar graphs showing the permeability ratios of the indicated ions to that of NMDG + ( Px : PNMDG ) ( E ) . Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases .

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 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 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

Figure 6Bar graphs showing the reversal potentials of PC1-CTF/PC2_AA and the indicated mutants tested in bath solutions containing 100 mM of the indicated ions. Two PC1 and two PC2 mutations caused dramatic changes in the reversal potential for almost all tested ions (B), indicating that these amino acids are essential for ion permeability. Statistical significance between reversal potentials of all pore mutants and that of the PC1-CTF/PC2_AA channel are indicated. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).Bar graphs showing the reversal potentials of PC1-CTF/PC2_AA and the indicated mutants tested in bath solutions containing 100 mM of the indicated ions. Another two PC1 mutations and one PC2 mutation only led to relatively small changes of reversal potential for some ions but not others (C), indicating a less important role of these amino acids in ion permeability. Statistical significance between reversal potentials of all pore mutants and that of the PC1-CTF/PC2_AA channel are indicated. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).Bar graphs showing the permeability ratios of the indicated ions to that of Na+ (Px: PNa) (D) Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases.Bar graphs showing the permeability ratios of the indicated ions to that of NMDG+ (Px: PNMDG) (E). Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases.


Figure 7Representative currents and a scatter plot and bar graph showing the currents of the full - length WT and mutant PC1s associated with PC2 _ AA in bath solutions containing 100 mM Na + and 2 mM Ca2 + ( B ) Currents were normalized to the average current of PC1 / PC2 _ AA recorded from the same batch of oocytes . Oocyte numbers in scatter plot and bar graph are indicated in parentheses . Data are presented as mean ± SD in bar graph ( n . s . : not significant , * * P < 0 . 01 , * * * P < 0 . 001 , Student ' s t - test ) . Representative currents and a scatter plot and bar graph showing the currents of the full - length WT and mutant PC1s associated with PC2 _ AA in bath solutions containing 100 mM Na + and 70 mM Ca2 + ( C ) . Currents were normalized to the average current of PC1 / PC2 _ AA recorded from the same batch of oocytes . Oocyte numbers in scatter plot and bar graph are indicated in parentheses . Data are presented as mean ± SD in bar graph ( n . s . : not significant , * * P < 0 . 01 , * * * P < 0 . 001 , Student ' s t - test ) . D . Surface biotinylation followed by western blot showing the surface expression of the PC1 _ T3041V / PC2 _ AA complex , but not the PC1 _ L3040H / PC2 _ AA complex , in Xenopus oocytes . PC2 _ AA is completely absent from the surface sample when PC1 _ L3040H was coexpressed , indicating that , in our experimental condition , almost all PC2 _ AA were in the complex with PC1 _ L3040H and trapped in the process of cell surface trafficking . Bands of full - length ( asterisk ) and GPS - cleaved CTF ( open circle ) of PC1 , and 130 kDa PC2 ( star ) are 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 O O O O O O 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 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

Figure 7Representative currents and a scatter plot and bar graph showing the currents of the full-length WT and mutant PC1s associated with PC2_AA in bath solutions containing 100 mM Na+ and 2 mM Ca2+ (B) Currents were normalized to the average current of PC1/PC2_AA recorded from the same batch of oocytes. Oocyte numbers in scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).Representative currents and a scatter plot and bar graph showing the currents of the full-length WT and mutant PC1s associated with PC2_AA in bath solutions containing 100 mM Na+ and 70 mM Ca2+ (C). Currents were normalized to the average current of PC1/PC2_AA recorded from the same batch of oocytes. Oocyte numbers in scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).D. Surface biotinylation followed by western blot showing the surface expression of the PC1_T3041V/PC2_AA complex, but not the PC1_L3040H/PC2_AA complex, in Xenopus oocytes. PC2_AA is completely absent from the surface sample when PC1_L3040H was coexpressed, indicating that, in our experimental condition, almost all PC2_AA were in the complex with PC1_L3040H and trapped in the process of cell surface trafficking. Bands of full-length (asterisk) and GPS-cleaved CTF (open circle) of PC1, and 130 kDa PC2 (star) are indicated.


Figure 1API delivery is blocked at low temperatures . SEY6210 cells were pulse labeled for 5 min and chased for the times indicated . Identical experiments were performed at 10 ° , 14 ° , and 30 ° C . API was recovered by immunoprecipitation followed by SDS - PAGE and detected by a STORM phosphorimager ( Molecular Dynamics , Sunnyvale , CA ) . The positions of prAPI and mAPI are 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

Figure 1API delivery is blocked at low temperatures. SEY6210 cells were pulse labeled for 5 min and chased for the times indicated. Identical experiments were performed at 10°, 14°, and 30°C. API was recovered by immunoprecipitation followed by SDS-PAGE and detected by a STORM phosphorimager (Molecular Dynamics, Sunnyvale, CA). The positions of prAPI and mAPI are indicated.


Figure 2Precursor API is trapped in a prevacuolar compartment in vps18 cells . ( A ) Yeast strain JSR18Δ1 with plasmid pJSR9 ( vps18 ts ) was grown at the permissive temperature of 26 ° C . Before labeling , the cells were incubated at either 26 ° or 38 ° C for 10 min . Pulse labeling was for 5 min , followed by the indicated chase times . ( B ) vps18 spheroplasts were shifted to 38 ° C for 5 min , pulse labeled for 5 min , and then chased for either 0 or 60 min . At each time point the spheroplasts were collected by centrifugation and resuspended in PS0 buffer . The Total ( T ) fraction either received no treatment , was treated with 50 μg / ml proteinase K , or received proteinase K in the presence of 0 . 2 % Triton X - 100 . The supernatant ( S0 ) and pellet ( P0 ) fractions were collected after centrifugation at 10 , 000 g for 5 min .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Precursor API is trapped in a prevacuolar compartment in vps18 cells. (A) Yeast strain JSR18Δ1 with plasmid pJSR9 (vps18 ts) was grown at the permissive temperature of 26°C. Before labeling, the cells were incubated at either 26° or 38°C for 10 min. Pulse labeling was for 5 min, followed by the indicated chase times.(B) vps18 spheroplasts were shifted to 38°C for 5 min, pulse labeled for 5 min, and then chased for either 0 or 60 min. At each time point the spheroplasts were collected by centrifugation and resuspended in PS0 buffer. The Total (T) fraction either received no treatment, was treated with 50 μg/ml proteinase K, or received proteinase K in the presence of 0.2% Triton X-100. The supernatant (S0) and pellet (P0) fractions were collected after centrifugation at 10,000 g for 5 min.


Figure 3A mutant in the API propeptide causes accumulation of prAPI on a nonvacuolar compartment . ( A ) Spheroplasts from an ape1Δ strain containing a plasmid bearing P22L API were subjected to differential lysis in PS200 buffer . The Total ( T ) fraction ( after differential lysis ) either received no treatment , was treated with 50 μg / ml proteinase K , or received proteinase K in the presence of 0 . 2 % Triton X - 100 . ( B ) The supernatant ( S200 ) and pellet ( P200 ) fractions were collected after centrifugation at 5 , 000 g for 5 min . The P200 fraction was resuspended in 10 % Ficoll , and vacuoles were isolated by flotation through 4 % Ficoll . V , vacuole ; I , 4 / 10 % Ficoll interface ; P , gradient pellet . Proteins were detected by Western blotting . The positions of prAPI , mAPI , and mPrA are indicated . ( C ) ape1Δ cells containing P22L API on a plasmid were pulse labeled for 5 min , harvested by centrifugation , and subjected to a nonradioactive chase in either nitrogen - containing ( SMD ) or nitrogen starvation ( SD - N ) medium . Samples were collected at the times indicated and immunoprecipitated with API antiserum . The resulting SDS - PAGE gels were quantified using a Molecular Dynamics STORM phosphorimager .

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Figure 3A mutant in the API propeptide causes accumulation of prAPI on a nonvacuolar compartment. (A) Spheroplasts from an ape1Δ strain containing a plasmid bearing P22L API were subjected to differential lysis in PS200 buffer. The Total (T) fraction (after differential lysis) either received no treatment, was treated with 50 μg/ ml proteinase K, or received proteinase K in the presence of 0.2% Triton X-100.(B) The supernatant (S200) and pellet (P200) fractions were collected after centrifugation at 5,000 g for 5 min. The P200 fraction was resuspended in 10% Ficoll, and vacuoles were isolated by flotation through 4% Ficoll. V, vacuole; I, 4/10% Ficoll interface; P, gradient pellet. Proteins were detected by Western blotting. The positions of prAPI, mAPI, and mPrA are indicated.(C) ape1Δ cells containing P22L API on a plasmid were pulse labeled for 5 min, harvested by centrifugation, and subjected to a nonradioactive chase in either nitrogen-containing (SMD) or nitrogen starvation (SD-N) medium. Samples were collected at the times indicated and immunoprecipitated with API antiserum. The resulting SDS-PAGE gels were quantified using a Molecular Dynamics STORM phosphorimager.


Figure 4Precursor API is trapped within a vesicle in cvt17 mutants . ( A ) Precursor API is in a protease - protected compartment in cvt17 mutants . THY32 ( cvt17 ) spheroplasts were subjected to differential lysis and protease treatment in PS200 buffer as in Fig 3B ) Precursor API is not free in the vacuolar lumen in cvt17 mutants . Differential fractionation was continued by resuspending the P200 fraction in PS0 buffer and separating the supernatant ( S0 ) and pellet ( P0 ) fractions by centrifugation at 10 , 000 g . The S0 and P0 fractions were treated with proteinase K in the presence and absence of Triton X - 100 as indicated . All fractions were precipitated with 10 % TCA and resolved by SDS PAGE ; proteins of interest were detected by Western blotting . The positions of prAPI , mAPI , and mPrA are 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 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

Figure 4Precursor API is trapped within a vesicle in cvt17 mutants. (A) Precursor API is in a protease-protected compartment in cvt17 mutants. THY32 (cvt17) spheroplasts were subjected to differential lysis and protease treatment in PS200 buffer as in Fig 3B) Precursor API is not free in the vacuolar lumen in cvt17 mutants. Differential fractionation was continued by resuspending the P200 fraction in PS0 buffer and separating the supernatant (S0) and pellet (P0) fractions by centrifugation at 10,000 g. The S0 and P0 fractions were treated with proteinase K in the presence and absence of Triton X-100 as indicated. All fractions were precipitated with 10% TCA and resolved by SDS PAGE; proteins of interest were detected by Western blotting. The positions of prAPI, mAPI, and mPrA are indicated.


Figure 5Precursor API is contained within subvacuolar vesicles in cvt17 mutants . ( A ) Isolated vacuoles ( V ) from cvt17 cells were lysed in PS0 buffer , loaded on top of an Optiprep step gradient , and centrifuged at 170 , 000 g for 60 min as described in Materials and Methods . Fractions VV , 0 / 1 . 06 interface ; LD , 1 . 06 region ; SV , 1 . 06 / 1 . 12 interface ; HD , 1 . 12 region . ( B ) An aliquot of fractions VV and SV was treated with proteinase K either in the presence or absence of Triton X - 100 as indicated . Because of the presence of protease inhibitors in the vesicle isolation procedure , an intermediate - sized API degradation product results from proteinase K treatment . The V fraction represents one - tenth of the load of the Optiprep gradient . Proteins were detected by Western blotting . The positions of prAPI and mALP are indicated .

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Figure 5Precursor API is contained within subvacuolar vesicles in cvt17 mutants. (A) Isolated vacuoles (V) from cvt17 cells were lysed in PS0 buffer, loaded on top of an Optiprep step gradient, and centrifuged at 170,000 g for 60 min as described in Materials and Methods. Fractions VV, 0/1.06 interface; LD, 1.06 region; SV, 1.06/1.12 interface; HD, 1.12 region.(B) An aliquot of fractions VV and SV was treated with proteinase K either in the presence or absence of Triton X-100 as indicated. Because of the presence of protease inhibitors in the vesicle isolation procedure, an intermediate-sized API degradation product results from proteinase K treatment. The V fraction represents one-tenth of the load of the Optiprep gradient. Proteins were detected by Western blotting. The positions of prAPI and mALP are indicated.


Figure 6Immuno - EM of cvt17 cells . Yeast strains SEY6210 ( parental wild type ) and THY32 ( cvt17 ) were grown in YPD and prepared for microscopy as described in Materials and Methods . ( A ) Wild - type cells probed with antibody against mature API . ( B ) A cvt17 section showing a cytosolic vesicle containing prAPI probed with antibody against the API proregion . ( C ) A cvt17 section showing subvacuolar vesicles containing API probed with antibody against mature API . N , nucleus ; V , vacuole ; ( arrows ) API containing vesicles . Bars , 0 . 5 μm .

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Figure 6Immuno-EM of cvt17 cells. Yeast strains SEY6210 (parental wild type) and THY32 (cvt17) were grown in YPD and prepared for microscopy as described in Materials and Methods. (A) Wild-type cells probed with antibody against mature API. (B) A cvt17 section showing a cytosolic vesicle containing prAPI probed with antibody against the API proregion. (C) A cvt17 section showing subvacuolar vesicles containing API probed with antibody against mature API. N, nucleus; V, vacuole; (arrows) API containing vesicles. Bars, 0.5 μm.


Figure 7Characterization of API - containing vesicles . ( A ) Fractionations were performed as in Fig . 5 . The total ( T ) , vacuole ( V ) , vacuolar vesicle ( VV ) , and subvacuolar vesicle ( SV ) fractions are shown . Proteins were detected by immunoblotting as indicated in the figure . ( B ) SYPRO orange staining detected by fluorescent scanning mode on a Molecular Dynamics STORM phosphorimager . The relative mobility of the molecular mass standards are indicated on the left . Polypeptides enriched in the subvacuolar vesicles are indicated on the right .

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Figure 7Characterization of API-containing vesicles. (A) Fractionations were performed as in Fig. 5. The total (T), vacuole (V), vacuolar vesicle (VV), and subvacuolar vesicle (SV) fractions are shown. Proteins were detected by immunoblotting as indicated in the figure.(B) SYPRO orange staining detected by fluorescent scanning mode on a Molecular Dynamics STORM phosphorimager. The relative mobility of the molecular mass standards are indicated on the left. Polypeptides enriched in the subvacuolar vesicles are indicated on the right.


Figure 1C . Proteins from whole cell extracts and culture supernatants of PAKΔretS , the derivative tssA1 mutant ( ΔA1 ) ( both carrying the pBBR plasmid ) and the complemented ΔA1 mutant carrying pBBR - tssA1 ( ΔA1 + A1 ) were analysed by Western blot . Polyclonal antibodies directed against TssA1 ( A1 ) , Hcp1 , VgrG1a and Tse3 were used . The anti - VgrG1a antibody detects VgrG1a , VgrG1b and VgrG1c , as indicated on the left . Cytoplasmic RNA polymerase ( RNAP ) was monitored using monoclonal antibody directed against its β - subunit .

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Figure 1C. Proteins from whole cell extracts and culture supernatants of PAKΔretS, the derivative tssA1 mutant (ΔA1) (both carrying the pBBR plasmid) and the complemented ΔA1 mutant carrying pBBR-tssA1 (ΔA1+A1) were analysed by Western blot. Polyclonal antibodies directed against TssA1 (A1), Hcp1, VgrG1a and Tse3 were used. The anti-VgrG1a antibody detects VgrG1a, VgrG1b and VgrG1c, as indicated on the left. Cytoplasmic RNA polymerase (RNAP) was monitored using monoclonal antibody directed against its β-subunit.


Figure 2A . BTH experiments showing that TssA1 is able to self - interact in vivo . A graphical representation of β - galactosidase activity from co - transformants of E . coli DHM1 cells producing TssA1 ( A1 ) fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits is shown . The values shown on the y axis correspond to the activity in Miller units . In each case , average activity from two independent experiments is shown and error bars indicate the standard deviation ( S . D . ) . Experiments were carried out in quadruplicate . B . In vitro cross - linking experiments of purified His6 - TssA1 . About 30 μg of purified His6 - TssA1 was cross - linked ( 30 min , 1h and 2h ) at room temperature using increasing amount of ethylene glycol - bis ( succinimidylsuccinate ) EGS ( 2 mM and 5 mM ) where indicated . Western blot analysis of cross - linked products using an anti - His6 monoclonal antibody is shown . The cross - linked species are highlighted with stars with the corresponding oligomeric state indicated on the right ( 1x = monomer ; 2x = dimer ; 6x = hexamer ) . Molecular weight markers ( kDa ) are indicated on the left . C . Analysis of the oligomeric state of TssA1 by AUC . Sedimentation data of His6 - TssA1 ( 0 . 56 mg / ml ) recorded at a rotor speed of 20 , 000 rpm is shown . The left panel shows the sedimentation boundary fits and for clarity , only every third scan is shown in the fitted data plots . The experimental absorbance data are shown as black circles , whereas the boundary fits are shown as grey lines . The right panel shows the size - distribution analysis c ( s ) , obtained from fitting the scan boundaries using SEDFIT , revealing a dodecameric peak ( Mr ~ 479 kDa ) at S20 , w value of 16 . 6 S with a minor peak ( Mr ~ 1 MDa ) at S20 , w value of 29 . 01 S .

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Figure 2A. BTH experiments showing that TssA1 is able to self-interact in vivo. A graphical representation of β-galactosidase activity from co-transformants of E. coli DHM1 cells producing TssA1 (A1) fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits is shown. The values shown on the y axis correspond to the activity in Miller units. In each case, average activity from two independent experiments is shown and error bars indicate the standard deviation (S.D.). Experiments were carried out in quadruplicate.B. In vitro cross-linking experiments of purified His6-TssA1. About 30 μg of purified His6-TssA1 was cross-linked (30 min, 1h and 2h) at room temperature using increasing amount of ethylene glycol-bis(succinimidylsuccinate) EGS (2 mM and 5 mM) where indicated. Western blot analysis of cross-linked products using an anti-His6 monoclonal antibody is shown. The cross-linked species are highlighted with stars with the corresponding oligomeric state indicated on the right (1x = monomer; 2x = dimer; 6x = hexamer). Molecular weight markers (kDa) are indicated on the left.C. Analysis of the oligomeric state of TssA1 by AUC. Sedimentation data of His6-TssA1 (0.56 mg/ml) recorded at a rotor speed of 20,000 rpm is shown. The left panel shows the sedimentation boundary fits and for clarity, only every third scan is shown in the fitted data plots. The experimental absorbance data are shown as black circles, whereas the boundary fits are shown as grey lines. The right panel shows the size-distribution analysis c(s), obtained from fitting the scan boundaries using SEDFIT, revealing a dodecameric peak (Mr ~ 479 kDa) at S20,w value of 16.6 S with a minor peak (Mr ~1 MDa) at S20,w value of 29.01 S.


Figure 3A . A representative micrograph of the TssA1 complex ( left panel ) showing discrete ring - shaped particles with a diameter of ~ 260 Å . The rotationally symmetrised average of the data set ( inset panel ) presents two distinct rings ( numbered ) , disclosing the layered nature of the TssA1 complex . Representative class averages of the TssA1 complex ( right panel ) demonstrating the wide range of conformations taken by the ring - shaped structures . Scale bars are 500 Å in the left panel , 130 Å in the inset and 100 Å in the right panels , respectively . B . Double MSA classification : ( i ) representative orientation class averages show TssA1 rings arranged into four - fold ( 4x ) , five - fold ( 5x ) and six - fold ( 6x ) symmetry - like complexes ( indicated in red , blue and green , respectively ) ; ( ii ) sub - class averages obtained from sub - classification of the corresponding orientation class ; and ( iii ) calculated difference images showing some white or black peaks ( coloured circles ) at the outer ring region . Scale bar is 100 Å . D . SEC - MALS analysis of the purified His6 - TssA11 - 245 . The TssA11 - 245 elution peak is shown and the corresponding mass is indicated . The molar mass and the differential refractive index ( RI ) are plotted versus the elution volume . Data were analysed using the ASTRA method ( Slotboom , Duurkens et al . , 2008 ) .

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Figure 3A. A representative micrograph of the TssA1 complex (left panel) showing discrete ring-shaped particles with a diameter of ~260 Å. The rotationally symmetrised average of the data set (inset panel) presents two distinct rings (numbered), disclosing the layered nature of the TssA1 complex. Representative class averages of the TssA1 complex (right panel) demonstrating the wide range of conformations taken by the ring-shaped structures. Scale bars are 500 Å in the left panel, 130 Å in the inset and 100 Å in the right panels, respectively.B. Double MSA classification: (i) representative orientation class averages show TssA1 rings arranged into four-fold (4x), five-fold (5x) and six-fold (6x) symmetry-like complexes (indicated in red, blue and green, respectively); (ii) sub-class averages obtained from sub-classification of the corresponding orientation class; and (iii) calculated difference images showing some white or black peaks (coloured circles) at the outer ring region. Scale bar is 100 Å.D. SEC-MALS analysis of the purified His6-TssA11-245. The TssA11-245 elution peak is shown and the corresponding mass is indicated. The molar mass and the differential refractive index (RI) are plotted versus the elution volume. Data were analysed using the ASTRA method (Slotboom, Duurkens et al., 2008).


Figure 4A . Co - purification experiments using His6 - TssA1 and untagged TssB1C1 . Western blot analysis of the Ni - NTA outputs using antibodies directed against the His6 - tag ( upper panel ) and TssB1 ( lower panel ) . I , Input ; FT , Flow through ; W , Wash and E , Elution . B . SEC elution fraction of the co - purified His6 - TssA1 and TssB1C1 . SDS - PAGE showing the presence of the three proteins His6 - TssA1 ( A1 ) , TssB1 ( B1 ) and TssC1 ( C1 ) . Molecular weight markers ( kDa ) are indicated on the left . C . Representative micrographs of the immunogold - labelled complex His6 - TssA1 / TssB1C1 . The presence of gold particles is indicated with white arrows . Two close - up views of TssB1C1 sheath displaying gold particles at one extremity are shown ( inset panels ) . Scale bars are 2 , 000 Å and 500 Å for images 1 and 2 - 6 , respectively . D . BTH experiment showing interaction between TssA1 ( A1 ) and Hcp1 . A graphical representation of β - galactosidase activity from E . coli DHM1 cells producing the indicated proteins fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits is shown . The values shown on the y axis correspond to the activity in Miller units . In each case , average activity from two independent experiments is shown and error bars indicate the standard deviation ( S . D . ) . Experiments were carried out in triplicate .

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Figure 4A. Co-purification experiments using His6-TssA1 and untagged TssB1C1. Western blot analysis of the Ni-NTA outputs using antibodies directed against the His6-tag (upper panel) and TssB1 (lower panel). I, Input; FT, Flow through; W, Wash and E, Elution.B. SEC elution fraction of the co-purified His6-TssA1 and TssB1C1. SDS-PAGE showing the presence of the three proteins His6-TssA1 (A1), TssB1 (B1) and TssC1 (C1). Molecular weight markers (kDa) are indicated on the left.C. Representative micrographs of the immunogold-labelled complex His6-TssA1/TssB1C1. The presence of gold particles is indicated with white arrows. Two close-up views of TssB1C1 sheath displaying gold particles at one extremity are shown (inset panels). Scale bars are 2,000 Å and 500 Å for images 1 and 2-6, respectively.D. BTH experiment showing interaction between TssA1 (A1) and Hcp1. A graphical representation of β-galactosidase activity from E. coli DHM1 cells producing the indicated proteins fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits is shown. The values shown on the y axis correspond to the activity in Miller units. In each case, average activity from two independent experiments is shown and error bars indicate the standard deviation (S.D.). Experiments were carried out in triplicate.


Figure 5B . Cartoon representations of the dodecameric gp6 _ 334C structure ( PDB code 3H3W ) . Chains A and B of gp6 dimers are shown in magenta and green respectively , and the conserved blocks mapped onto the assembled dodecameric gp6 structure are shown in orange . A close up view showing the conserved blocks present in regions of gp6 involved in ring assembly , is shown on the right .

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Figure 5B. Cartoon representations of the dodecameric gp6_334C structure (PDB code 3H3W). Chains A and B of gp6 dimers are shown in magenta and green respectively, and the conserved blocks mapped onto the assembled dodecameric gp6 structure are shown in orange. A close up view showing the conserved blocks present in regions of gp6 involved in ring assembly, is shown on the right.


Figure 6A Fluorescence microscopy images showing the presence / absence of TssBC sheaths ( seen as fluorescent foci ) in the ∆ B1 : : B1 - sfGFP ( top panel ) , ∆ B1 ∆ A1 : : B1 - sfGFP , ∆ B1 ∆ E1 : : B1 - sfGFP cells ( middle panels ) and in the complemented strains ∆ B1 ∆ A1 : : B1 - sfGFP + A1 and ∆ B1 ∆ E1 : : B1 - sfGFP + E1 ( bottom panels ) . Scale bars are 2 μm . B . Quantification of TssB1 - sfGFP foci and of diffuse fluorescence in various P . aeruginosa backgrounds imaged in panel A . Values are normalized to the total number of bacteria ( % ) . Data were obtained from three independent experiments , with images and values from representative replicates being shown .

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Figure 6A Fluorescence microscopy images showing the presence/absence of TssBC sheaths (seen as fluorescent foci) in the ∆B1::B1-sfGFP (top panel), ∆B1∆A1::B1-sfGFP, ∆B1∆E1::B1-sfGFP cells (middle panels) and in the complemented strains ∆B1∆A1::B1-sfGFP + A1 and ∆B1∆E1::B1-sfGFP + E1 (bottom panels). Scale bars are 2 μm. B. Quantification of TssB1-sfGFP foci and of diffuse fluorescence in various P. aeruginosa backgrounds imaged in panel A. Values are normalized to the total number of bacteria (%). Data were obtained from three independent experiments, with images and values from representative replicates being shown.


Figure 7BTH screen for interactions between TssA1 and T6SS components of interest . A graphical representation ( bottom ) of β - galactosidase activity from E . coli DHM1 cells producing the indicated proteins fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits and images of corresponding spots on X - gal LB agar plates ( top ) are shown . The combination of T25 / T18 fusion proteins are indicated as 25 or 18 followed by the name of the fused T6SS protein . T6SS proteins are abbreviated as follows : A1 = TssA1 , E1 = TssE1 , F1 = TssF1 , G1 = TssG1 and K1 = TssK1 . The values shown on the y axis correspond to the activity in Miller units . In each case , average activity from two independent experiments is shown and error bars indicate the standard deviation ( S . D . ) . Experiments were carried out in triplicate .

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Figure 7BTH screen for interactions between TssA1 and T6SS components of interest. A graphical representation (bottom) of β-galactosidase activity from E. coli DHM1 cells producing the indicated proteins fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits and images of corresponding spots on X-gal LB agar plates (top) are shown. The combination of T25/T18 fusion proteins are indicated as 25 or 18 followed by the name of the fused T6SS protein. T6SS proteins are abbreviated as follows: A1=TssA1, E1=TssE1, F1=TssF1, G1=TssG1 and K1=TssK1. The values shown on the y axis correspond to the activity in Miller units. In each case, average activity from two independent experiments is shown and error bars indicate the standard deviation (S.D.). Experiments were carried out in triplicate.


Figure 8B . Cartoon representation of the gp6 / gp8 baseplate subcomplex of the T4 phage . The gp6 ring structure ( PDB code 3H3W ) is shown using the same colour code described in figure 5 . The chains A and B of the gp8 dimers ( PDB code 1PDM ) are shown in blue and yellow , respectively . The conserved blocks mapped onto the dimeric structure of gp8 are shown in orange . A close up view of the gp8 structure ( bottom ) shows that the conserved blocks are involved in dimerization and interaction with gp6 .

Figure 8B. Cartoon representation of the gp6/gp8 baseplate subcomplex of the T4 phage. The gp6 ring structure (PDB code 3H3W) is shown using the same colour code described in figure 5. The chains A and B of the gp8 dimers (PDB code 1PDM) are shown in blue and yellow, respectively. The conserved blocks mapped onto the dimeric structure of gp8 are shown in orange. A close up view of the gp8 structure (bottom) shows that the conserved blocks are involved in dimerization and interaction with gp6.


Figure 9A . Structure of the sheath / baseplate complex of the bacteriophage T4 , adapted from Kostyuchenko et al . ( Kostyuchenko , Leiman et al . , 2003 ) . A colour code with corresponding gp protein is provided . The proposed corresponding homolog in the T6SS is indicated with parentheses .

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Figure 9A. Structure of the sheath/baseplate complex of the bacteriophage T4, adapted from Kostyuchenko et al. (Kostyuchenko, Leiman et al., 2003). A colour code with corresponding gp protein is provided. The proposed corresponding homolog in the T6SS is indicated with parentheses.


Figure 1The Arabidopsis pi4kβ1 pi4kβ2 double mutant displays a growth defect and cytokinetic defects . The growth phenotype of the Arabidopsis pi4kβ1 pi4kβ2 double mutant can be complemented by ectopic expression of PI4Kβ1 or PI4Kβ2 , or by mCherry - tagged PI4Kβ1 expressed from a genomic fragment . Plants shown are one month old . Scale bar , 10 cm . The irregularities in the pattern of root epidermal cell division orientation of the pi4kβ1 pi4kβ2 double mutant is also complemented by ectopic expression of PI4Kβ variants , as shown by propidium iodide ( PI ) staining of five - day - old roots . Arrowhead , oblique cell wall . Insets , magnifications of areas showing cell wall stubs . Scale bars , 50 µm . Quantifications of cell wall stubs , oblique cell walls and root meristem size ( wild type , n = 15 roots ; pi4kβ1 , n = 15 roots ; pi4kβ2 , n = 14 roots ; pi4kβ1 pi4kβ2 , n = 18 roots ; pPI4Kβ1 : PI4Kβ1 , n = 14 roots , pPI4Kβ2 : PI4Kβ2 , n = 15 roots ) . n . d . , not detected . Data are mean ± SD . Lowercase letters indicate a significant increase of oblique cell walls ( Welch ANOVA with Games - Howell post - hoc test ; p < 0 . 0001 ) or a significant reduction of root meristem size in the pi4kβ1 pi4kβ2 double mutant ( one - way ANOVA with post - hoc Tukey HSD ; p < 0 . 001 ) . Complementation of the pi4kβ1 pi4kβ2 phenotype by functional FLAG - PI4Kβ1 expressed from the intrinsic pPI4Kβ1 or by the pKNOLLE promoters , as indicated , shown for one - month - old plants . Scale bar , 10 cm . Complementation with the pKNOLLE - driven construct indicates functionality of PI4Kβ1 during cell plate formation in G2 / M phase . PI staining of pKNOLLE : FLAG - PI4Kβ1 expressed in the pi4kβ1 pi4kβ2 double mutant background indicates rescue of cytokinetic defects . Arrowhead , cell wall stub in the pi4kβ1 pi4kβ2 double mutant . Scale bars , 50 µm .

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Figure 1The Arabidopsis pi4kβ1 pi4kβ2 double mutant displays a growth defect and cytokinetic defects. The growth phenotype of the Arabidopsis pi4kβ1 pi4kβ2 double mutant can be complemented by ectopic expression of PI4Kβ1 or PI4Kβ2, or by mCherry-tagged PI4Kβ1 expressed from a genomic fragment. Plants shown are one month old. Scale bar, 10 cm.The irregularities in the pattern of root epidermal cell division orientation of the pi4kβ1 pi4kβ2 double mutant is also complemented by ectopic expression of PI4Kβ variants, as shown by propidium iodide (PI) staining of five-day-old roots. Arrowhead, oblique cell wall. Insets, magnifications of areas showing cell wall stubs. Scale bars, 50 µm.Quantifications of cell wall stubs, oblique cell walls and root meristem size (wild type, n = 15 roots; pi4kβ1, n = 15 roots; pi4kβ2, n = 14 roots; pi4kβ1 pi4kβ2, n = 18 roots; pPI4Kβ1:PI4Kβ1, n = 14 roots, pPI4Kβ2:PI4Kβ2, n = 15 roots). n.d., not detected. Data are mean ± SD. Lowercase letters indicate a significant increase of oblique cell walls (Welch ANOVA with Games-Howell post-hoc test; p < 0.0001) or a significant reduction of root meristem size in the pi4kβ1 pi4kβ2 double mutant (one-way ANOVA with post-hoc Tukey HSD; p < 0.001).Complementation of the pi4kβ1 pi4kβ2 phenotype by functional FLAG-PI4Kβ1 expressed from the intrinsic pPI4Kβ1 or by the pKNOLLE promoters, as indicated, shown for one-month-old plants. Scale bar, 10 cm. Complementation with the pKNOLLE-driven construct indicates functionality of PI4Kβ1 during cell plate formation in G2/M phase.PI staining of pKNOLLE:FLAG-PI4Kβ1 expressed in the pi4kβ1 pi4kβ2 double mutant background indicates rescue of cytokinetic defects. Arrowhead, cell wall stub in the pi4kβ1 pi4kβ2 double mutant. Scale bars, 50 µm.


Figure 2In vivo localization of mCherry - PI4Kβ1 expressed from the pPI4Kβ1 promoter in root tips of five - day - old complemented pi4kβ1 pi4kβ2 plants . The mCherry - PI4Kβ1 distribution was imaged using a Zeiss LSM880 in Airyscan Virtual Pinhole ( VP ) mode with the pinhole set to 2 . Arrowheads , nascent cell plates decorated by mCherry - PI4Kβ1 . Scale bar , 20 µm . Whole - mount immunostaining of five - day - old seedlings expressing mCherry - PI4kβ1 in the pi4kβ1 pi4kβ2 double mutant background using anti - tubulin ( red ) and anti - mCherry ( green ) antibodies , and DAPI ( blue ) . ( І ) , ( ІІ ) , magnifications of regions marked in b , representing early and late cytokinetic stages . Scale bars , 20 µm . Relative localization of mCherry - PI4Kβ1 and the TGN marker , ARF1 , during cytokinesis . Four - day - old seedlings were immunostainned with anti - mCherry ( green ) and anti - ARF1 ( red ) antibodies . Scale bars are 10 µm .

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Figure 2In vivo localization of mCherry-PI4Kβ1 expressed from the pPI4Kβ1 promoter in root tips of five-day-old complemented pi4kβ1 pi4kβ2 plants. The mCherry-PI4Kβ1 distribution was imaged using a Zeiss LSM880 in Airyscan Virtual Pinhole (VP) mode with the pinhole set to 2. Arrowheads, nascent cell plates decorated by mCherry-PI4Kβ1. Scale bar, 20 µm. Whole-mount immunostaining of five-day-old seedlings expressing mCherry-PI4kβ1 in the pi4kβ1 pi4kβ2 double mutant background using anti-tubulin (red) and anti-mCherry (green) antibodies, and DAPI (blue). (І), (ІІ), magnifications of regions marked in b, representing early and late cytokinetic stages. Scale bars, 20 µm.Relative localization of mCherry-PI4Kβ1 and the TGN marker, ARF1, during cytokinesis. Four-day-old seedlings were immunostainned with anti-mCherry (green) and anti-ARF1 (red) antibodies. Scale bars are 10 µm.


Figure 3The ultrastructure of cytokinetic defects was analyzed by transmission electron microscopy of five - day - old root tip meristems . Images shown are from 3 roots ( wild type ) and 7 roots ( pi4kβ1 pi4kβ2 double mutant ) . In wild type controls ( A ) , vesicles fused at the cell division plane to give rise to tubular network ( TN ) of a nascent cell plate . White arrowheads , fused vesicles at the division plane . n = 8 cells . In the pi4kβ1 pi4kβ2 double mutant ( B ) , vesicles were delivered to the cell division plane at a similar stage as wild type but did not fuse . The nuclei of both wild type and double mutant cells were at telophase or interphase , as judged by the presence of decondensed chromatin and a nuclear envelope . At this stage , failed cell plate formation was never seen in wild type ( A ) . Black arrowheads , unfused vesicles . n = 6 cells . Unfused vesicles clustered around a cell wall stub ( arrow ) . White arrowheads , light vesicles ; black arrowheads , dark vesicles ; black arrow , cell wall stub . n = 8 cells . No cross wall was seen in this cell with two nuclei . n = 6 cells . n , nucleus ; ne , nuclear envelope . Scale bars in magnified figures , 2 μm ; in insets , 1 μm .

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Figure 3The ultrastructure of cytokinetic defects was analyzed by transmission electron microscopy of five-day-old root tip meristems. Images shown are from 3 roots (wild type) and 7 roots (pi4kβ1 pi4kβ2 double mutant). In wild type controls (A), vesicles fused at the cell division plane to give rise to tubular network (TN) of a nascent cell plate. White arrowheads, fused vesicles at the division plane. n = 8 cells. In the pi4kβ1 pi4kβ2 double mutant (B), vesicles were delivered to the cell division plane at a similar stage as wild type but did not fuse. The nuclei of both wild type and double mutant cells were at telophase or interphase, as judged by the presence of decondensed chromatin and a nuclear envelope. At this stage, failed cell plate formation was never seen in wild type (A). Black arrowheads, unfused vesicles. n = 6 cells. Unfused vesicles clustered around a cell wall stub (arrow). White arrowheads, light vesicles; black arrowheads, dark vesicles; black arrow, cell wall stub. n = 8 cells. No cross wall was seen in this cell with two nuclei. n = 6 cells. n, nucleus; ne, nuclear envelope. Scale bars in magnified figures, 2 μm; in insets, 1 μm.


Figure 4The effects of reduced association of PtdIns ( 4 ) P with the cell plate on the recruitment of CLC2 - GFP were analyzed in root meristem cells of five - day - old plants . Top , Cell plate - associated fluorescence of the PtdIns ( 4 ) P reporter , 2 × mCherryFAPP1 - PH , was monitored at the end of cytokinesis in five - day - old wild type controls or pi4kβ1 pi4kβ2 double mutants , as indicated . Scale bars , 10 µm . Bottom , The intensity of 2 × mCherryFAPP1 - PH signals was quantified at the end of cytokinesis at the cell plate ( left plot ) and at the TGN ( right plot ) . Signal intensities at the cell plate were recorded along the dashed lines indicated in the images and normalized to intensities at the apical plasma membrane ( wild type , n = 37 cells , 24 roots ; pi4kβ1 pi4kβ2 , n = 55 cells , 41 roots ) . TGN intensities were recorded using the Airyscan VP mode , and the mean TGN intensity was normalized to mean apical and basal plasma membrane intensity ( wild type , n = 30 cells , 22 roots ; pi4kβ1 pi4kβ2 , n = 37 cells , 31 roots ) . * * * , significant differences ( p < 0 . 0001 ) according to two - tailed Mann - Whitney U - tests . Image series from live cell time lapse analysis of CLC2 - GFP at the cell plate in roots of wild type seedlings costained with FM4 - 64 , as indicated . Arrowheads , appearance of CLC2 - GFP at the cell plate . Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane , defined as t0 . Scale bars , 10 μm . Images are from median focal planes chosen from time lapse series recorded as 3D stacks . Image series from live cell time lapse analysis of CLC2 - GFP at the cell plate in roots of pi4kβ1 pi4kβ2 seedlings costained with FM4 - 64 , as indicated . Arrowheads , appearance of CLC2 - GFP at the cell plate . Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane , defined as t0 . Scale bars , 10 μm . Images are from median focal planes chosen from time lapse series recorded as 3D stacks . Left , quantification of the time of CLC2 - GFP appearance at the cell plate in wild type ( B ) or the pi4kβ1 pi4kβ2 double mutant ( C ) ( wild type , n = 10 cells , 7 roots ; pi4kβ1 pi4kβ2 , n = 24 cells , 19 roots ) . Right , quantification of CLC2 - GFP at the TGN in interphase cells . Five - day - old roots expressing CLC2 - GFP were subjected to immunostaining and the mean TGN intensity was normalized to plasma membrane intensity ( wild type , n = 14 cells , 2 roots ; pi4kβ1 pi4kβ2 , n = 15 cells , 2 roots ) .

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Figure 4The effects of reduced association of PtdIns(4)P with the cell plate on the recruitment of CLC2-GFP were analyzed in root meristem cells of five-day-old plants. Top, Cell plate-associated fluorescence of the PtdIns(4)P reporter, 2×mCherryFAPP1-PH, was monitored at the end of cytokinesis in five-day-old wild type controls or pi4kβ1 pi4kβ2 double mutants, as indicated. Scale bars, 10 µm. Bottom, The intensity of 2×mCherryFAPP1-PH signals was quantified at the end of cytokinesis at the cell plate (left plot) and at the TGN (right plot). Signal intensities at the cell plate were recorded along the dashed lines indicated in the images and normalized to intensities at the apical plasma membrane (wild type, n = 37 cells, 24 roots; pi4kβ1 pi4kβ2, n = 55 cells, 41 roots). TGN intensities were recorded using the Airyscan VP mode, and the mean TGN intensity was normalized to mean apical and basal plasma membrane intensity (wild type, n=30 cells, 22 roots; pi4kβ1 pi4kβ2, n=37 cells, 31 roots). ***, significant differences (p<0.0001) according to two-tailed Mann-Whitney U-tests.Image series from live cell time lapse analysis of CLC2-GFP at the cell plate in roots of wild type seedlings costained with FM4-64, as indicated. Arrowheads, appearance of CLC2-GFP at the cell plate. Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane, defined as t0. Scale bars, 10 μm. Images are from median focal planes chosen from time lapse series recorded as 3D stacks.Image series from live cell time lapse analysis of CLC2-GFP at the cell plate in roots of pi4kβ1 pi4kβ2 seedlings costained with FM4-64, as indicated. Arrowheads, appearance of CLC2-GFP at the cell plate. Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane, defined as t0. Scale bars, 10 μm. Images are from median focal planes chosen from time lapse series recorded as 3D stacks.Left, quantification of the time of CLC2-GFP appearance at the cell plate in wild type (B) or the pi4kβ1 pi4kβ2 double mutant (C) (wild type, n = 10 cells, 7 roots; pi4kβ1 pi4kβ2, n = 24 cells, 19 roots). Right, quantification of CLC2-GFP at the TGN in interphase cells. Five-day-old roots expressing CLC2-GFP were subjected to immunostaining and the mean TGN intensity was normalized to plasma membrane intensity (wild type, n = 14 cells, 2 roots; pi4kβ1 pi4kβ2, n = 15 cells, 2 roots).