( E ) Diameter of CJs . CJs were manually measured on TEM recordings . Red line : Average . Error bars : SD . Samples were compared to WT by a One - way ANOVA test . Data information : n : number of mitochondrial

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(E) Diameter of CJs. CJs were manually measured on TEM recordings. Red line: Average. Error bars: SD. Samples were compared to WT by a One-way ANOVA test. Data information: n: number of mitochondrial sections


( F ) Representative ET reconstructions of mitochondria from a WT cell ( left ) , a Mic10 - KO cell ( center ) and a Mic60 - KO cell ( right ) . The OM is displayed in clear grey , the side of the IM that faces the matrix is shown in dark blue . The IM side that faces the IMS is shown in light blue .

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(F) Representative ET reconstructions of mitochondria from a WT cell (left), a Mic10-KO cell (center) and a Mic60-KO cell (right). The OM is displayed in clear grey, the side of the IM that faces the matrix is shown in dark blue. The IM side that faces the IMS is shown in light blue.


( A Nanoscopy of mitochondria of WT and Mic10 - KO cells , as indicated . ( A ) Mitochondria were immunolabeled for Mic60 and ATPB and visualized by 2D STED nanoscopy .

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(A Nanoscopy of mitochondria of WT and Mic10-KO cells, as indicated. (A) Mitochondria were immunolabeled for Mic60 and ATPB and visualized by 2D STED nanoscopy.


( B ) Mitochondria were immunolabeled for TOM20 and ATPB and recorded with 3D STED nanoscopy . Image data were deconvolved . Depicted are cross - sections of a mitochondrion ( side view ) .

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(B) Mitochondria were immunolabeled for TOM20 and ATPB and recorded with 3D STED nanoscopy. Image data were deconvolved. Depicted are cross-sections of a mitochondrion (side view).


( C ) 3D MINFLUX nanoscopy . Mic10 - KO cells were immunolabeled for Mic60 using a directly labeled antibody . Colors encode depth information .

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(C) 3D MINFLUX nanoscopy. Mic10-KO cells were immunolabeled for Mic60 using a directly labeled antibody. Colors encode depth information.


( D ) 3D SIM of living WT and Mic10 - KO cells . Cells were labeled with Mitotracker Green . Images show maximum intensity projections .

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(D) 3D SIM of living WT and Mic10-KO cells. Cells were labeled with Mitotracker Green. Images show maximum intensity projections.


( E ) FIB - SEM of Mic10 - KO cells . Shown is a representative reconstructed mitochondrion . Dashed box : Orthoslice view on the smaller mitochondrion . The OM and IBM are displayed together in clear grey , the CM in is shown in blue .

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(E) FIB-SEM of Mic10-KO cells. Shown is a representative reconstructed mitochondrion. Dashed box: Orthoslice view on the smaller mitochondrion. The OM and IBM are displayed together in clear grey, the CM in is shown in blue.


( F ) 2D STED nanoscopy of mitochondria . Mic19 - KO cells were transfected with a plasmid for Mic10 - FLAG expression under the control of a tetracycline - inducible promoter . Left panel : Mitochondria from noninduced cell . Right : Mitochondria from induced cell expressing Mic10 - FLAG .

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(F) 2D STED nanoscopy of mitochondria. Mic19-KO cells were transfected with a plasmid for Mic10-FLAG expression under the control of a tetracycline-inducible promoter. Left panel: Mitochondria from noninduced cell. Right: Mitochondria from induced cell expressing Mic10-FLAG.


( A ) Cells were immunolabeled for Mic60 and DNA and visualized by confocal fluorescence microscopy before and after induction of Mic60 expression for 48 h .

(A) Cells were immunolabeled for Mic60 and DNA and visualized by confocal fluorescence microscopy before and after induction of Mic60 expression for 48 h.


( B ) Protein levels of MICOS proteins after Mic60 induction . Cell lysates were analyzed by western blotting at the indicated time points .

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(B) Protein levels of MICOS proteins after Mic60 induction. Cell lysates were analyzed by western blotting at the indicated time points.


( C ) Recovery of mitochondrial networks upon Mic60 re - expression . Cells were induced with doxycycline hyclate for 72 h , immunolabeled for TOM20 and visualized by confocal microscopy .

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(C) Recovery of mitochondrial networks upon Mic60 re-expression. Cells were induced with doxycycline hyclate for 72 h, immunolabeled for TOM20 and visualized by confocal microscopy.


( D - G ) TEM of Mic60 - TO cells before and after induction . ( D ) TEM recordings of Mic60 - TO cells at the indicated time points . ( E ) Quantification of the cristae morphology . ( F ) CJ frequency . The number of CJs on TEM recordings was normalized to the length of the OM . At least 90 mitochondrial sections were analyzed for each sample . ( G ) Quantification of the cristae morphology of Mic60 - TO cells . Only mitochondria exhibiting at least one CJ were analyzed . Data information : n : Number of mitochondrial sections . Scale bars : 20 µm ( A ) , 10 µm ( C ) , 500 nm ( D ) .

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(D-G) TEM of Mic60-TO cells before and after induction. (D) TEM recordings of Mic60-TO cells at the indicated time points. (E) Quantification of the cristae morphology. (F) CJ frequency. The number of CJs on TEM recordings was normalized to the length of the OM. At least 90 mitochondrial sections were analyzed for each sample. (G) Quantification of the cristae morphology of Mic60-TO cells. Only mitochondria exhibiting at least one CJ were analyzed. Data information: n: Number of mitochondrial sections. Scale bars: 20 µm (A), 10 µm (C), 500 nm (D).


( A , B ) Live - cell nanoscopy of Mic10 - TO cells . Cells expressing COX8A - SNAP were labeled with SNAP - cell SiR and recorded with STED nanoscopy . ( A ) Cristae architecture in noninduced cells . ( B ) Cristae architecture after Mic10 re - expression ( 24 h induction with doxycycline ) . STED image data were deconvolved .

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(A,B) Live-cell nanoscopy of Mic10-TO cells. Cells expressing COX8A-SNAP were labeled with SNAP-cell SiR and recorded with STED nanoscopy. (A) Cristae architecture in noninduced cells. (B) Cristae architecture after Mic10 re-expression (24 h induction with doxycycline). STED image data were deconvolved.


( C ) STED nanoscopy of fixed Mic10 - TO cells . Cells were induced for 24 h and immunolabeled for Mic60 and Mic10 - FLAG . Upper row : Cell with weak Mic10 expression level . Lower row : Cell with strong Mic10 expression level .

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(C) STED nanoscopy of fixed Mic10-TO cells. Cells were induced for 24 h and immunolabeled for Mic60 and Mic10-FLAG. Upper row: Cell with weak Mic10 expression level. Lower row: Cell with strong Mic10 expression level.


( D - G ) TEM of Mic10 - TO cells . ( D ) Cells induced for the indicated period of time were recorded with TEM . Arrow indicates an intermediately shaped crista . ( E ) Quantification of the cristae morphology . n : Number of mitochondrial sections . ( F ) CJ frequency in Mic10 - TO cells . Number of CJs was normalized to the length of the OM . The same sections were analyzed as in ( E ) . ( G ) Diameter of CJs estimated on TEM recordings . Data information : n : Number of analyzed mitochondrial sections ( E ) or CJs ( G ) . .

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(D-G) TEM of Mic10-TO cells. (D) Cells induced for the indicated period of time were recorded with TEM. Arrow indicates an intermediately shaped crista. (E) Quantification of the cristae morphology. n: Number of mitochondrial sections. (F) CJ frequency in Mic10-TO cells. Number of CJs was normalized to the length of the OM. The same sections were analyzed as in (E). (G) Diameter of CJs estimated on TEM recordings. Data information: n: Number of analyzed mitochondrial sections (E) or CJs (G)..


( A ) FIB - SEM of Mic10 - TO cells . Cells were induced for 0 h , 16 h and 24 h . The representative reconstructions show mitochondria from noninduced , rescued and intermediate cells , as indicated . The OM is shown together with the IBM in clear grey , the CM in blue .

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(A) FIB-SEM of Mic10-TO cells. Cells were induced for 0 h, 16 h and 24 h. The representative reconstructions show mitochondria from noninduced, rescued and intermediate cells, as indicated. The OM is shown together with the IBM in clear grey, the CM in blue.


( B ) ET of Mic10 - TO cells . Mic10 expression was induced for 0 h and 16 h . Shown are reconstructions of representative noninduced and almost completely rescued mitochondria . The OM is displayed in clear grey , the side of the IM that faces the matrix is shown in dark blue . The IM side that faces the IMS is shown in light blue .

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(B) ET of Mic10-TO cells. Mic10 expression was induced for 0 h and 16 h. Shown are reconstructions of representative noninduced and almost completely rescued mitochondria. The OM is displayed in clear grey, the side of the IM that faces the matrix is shown in dark blue. The IM side that faces the IMS is shown in light blue.


Knockdown ( KD ) of OPA1 in HeLa cells . WT and Mic60 - KO cells were transfected with a scrambled control ( Ctrl . ) or siRNA pools against OPA1 ( OPA1 - KD ) for 5 d . ( A ) Cell lysates were analyzed by western blotting .

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Knockdown (KD) of OPA1 in HeLa cells. WT and Mic60-KO cells were transfected with a scrambled control (Ctrl.) or siRNA pools against OPA1 (OPA1-KD) for 5 d. (A) Cell lysates were analyzed by western blotting.


( B ) TEM recordings from OPA1 depleted WT and Mic60 - KO cells . Arrows point to a septum .

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(B) TEM recordings from OPA1 depleted WT and Mic60-KO cells. Arrows point to a septum.


( C , D ) Quantification of the cristae morphology of OPA1 deficient WT ( C ) and Mic60 - KO ( D ) cells . The number of CJs was normalized to the length of the OM ( C ) . The number of septated mitochondria in Mic60 - KO cells was estimated ( D ) .

(C,D) Quantification of the cristae morphology of OPA1 deficient WT (C) and Mic60-KO (D) cells. The number of CJs was normalized to the length of the OM (C). The number of septated mitochondria in Mic60-KO cells was estimated (D).


( E , F ) ET of mitochondria from OPA1 - KD cells . WT cells were transfected with a scrambled control ( E ) or siRNA pools against OPA1 ( F ) for 48 h . Reconstructions of representative mitochondria are displayed . The OM is displayed in clear grey , the side of the IM that faces the matrix is shown in dark blue . The IM side that faces the IMS is shown in light blue .

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(E,F) ET of mitochondria from OPA1-KD cells. WT cells were transfected with a scrambled control (E) or siRNA pools against OPA1 (F) for 48 h. Reconstructions of representative mitochondria are displayed. The OM is displayed in clear grey, the side of the IM that faces the matrix is shown in dark blue. The IM side that faces the IMS is shown in light blue.


WT and Mic10 - KO cells were transfected with a scrambled control ( Ctrl . ) or siRNA pools against OPA1 ( OPA1 - KD ) for 72 h . ( A , B ) STED nanoscopy of OPA1 depleted WT ( A ) and Mic10 - KO ( B ) cells . Cells were immunolabeled for Mic60 and ATPB .

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WT and Mic10-KO cells were transfected with a scrambled control (Ctrl.) or siRNA pools against OPA1 (OPA1-KD) for 72 h. (A,B) STED nanoscopy of OPA1 depleted WT (A) and Mic10-KO (B) cells. Cells were immunolabeled for Mic60 and ATPB.


( C ) TEM of OPA1 depleted Mic10 - KO cells . Left : Representative TEM recordings showing CJs marked by arrows in a control cell and a similar view for an OPA1 - KD cell . The CMs are colored in green and the OM and IBM together in magenta . Right : Quantification of mitochondria containing CJs or septa junctions . Sections that contained CJ - like structures were analyzed . Of these , the number of sections exhibiting septa or cristae structures ( other ) was quantified .

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(C) TEM of OPA1 depleted Mic10-KO cells. Left: Representative TEM recordings showing CJs marked by arrows in a control cell and a similar view for an OPA1-KD cell. The CMs are colored in green and the OM and IBM together in magenta. Right: Quantification of mitochondria containing CJs or septa junctions. Sections that contained CJ-like structures were analyzed. Of these, the number of sections exhibiting septa or cristae structures (other) was quantified.


( D Depletion of ATP5ME in HeLa cells . Cells were transfected with a scrambled control or siRNA pools against ATP5ME for 4 d . TEM recording ( left ) and quantification of the number of cristae per mitochondrial section ( right ) .

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(D Depletion of ATP5ME in HeLa cells. Cells were transfected with a scrambled control or siRNA pools against ATP5ME for 4 d. TEM recording (left) and quantification of the number of cristae per mitochondrial section (right).


( E ) Cells depleted of ATP5ME were immunolabeled for Mic60 and recorded with STED . The arrows point to crossing points of the opposite Mic60 distribution bands .

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(E) Cells depleted of ATP5ME were immunolabeled for Mic60 and recorded with STED. The arrows point to crossing points of the opposite Mic60 distribution bands.


( A - D ) Cells expressing Rpl5 - GFP ( A and B ) were grown in rich medium ( before starvation ) and starved in SD - N for the indicated periods ( starvation ) . ( A and C , left ) Degradation of GFP - tagged proteins was analyzed by anti - GFP blot of whole cell extracts . ( right ) The ratio between cleaved GFP and full - length protein was quantified for every time point in four independent experiments . a . u . , arbitrary unit .

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(A-D) Cells expressing Rpl5-GFP (A and B) were grown in rich medium (before starvation) and starved in SD-N for the indicated periods (starvation). (A and C, left) Degradation of GFP-tagged proteins was analyzed by anti-GFP blot of whole cell extracts. (right) The ratio between cleaved GFP and full-length protein was quantified for every time point in four independent experiments. a.u., arbitrary unit.


( A - D ) Cells expressing Rpl5 - GFP ( A and B ) were grown in rich medium ( before starvation ) and starved in SD - N for the indicated periods ( starvation ) . ( B and D ) Cells before starvation or starved for 24 h ( starvation ) were examined both by fluorescence microscopy and differential interferential contrast ( DIC ) . Note that 100 % of wild - type ( WT ) and mutant cells expressed GFP - tagged proteins in the cytoplasm before starvation . C , cytoplasmic localization ; V , vacuolar accumulation ; C + V , localization in both cytoplasm and vacuole .

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(A-D) Cells expressing Rpl5-GFP (A and B) were grown in rich medium (before starvation) and starved in SD-N for the indicated periods (starvation). (B and D) Cells before starvation or starved for 24 h (starvation) were examined both by fluorescence microscopy and differential interferential contrast (DIC). Note that 100% of wild-type (WT) and mutant cells expressed GFP-tagged proteins in the cytoplasm before starvation. C, cytoplasmic localization; V, vacuolar accumulation; C + V, localization in both cytoplasm and vacuole.


( A - D ) Cells expressing Rpl25 - GFP ( C and D ) were grown in rich medium ( before starvation ) and starved in SD - N for the indicated periods ( starvation ) . ( A and C , left ) Degradation of GFP - tagged proteins was analyzed by anti - GFP blot of whole cell extracts . ( right ) The ratio between cleaved GFP and full - length protein was quantified for every time point in four independent experiments . a . u . , arbitrary unit .

(A-D) Cells expressing Rpl25-GFP (C and D) were grown in rich medium (before starvation) and starved in SD-N for the indicated periods (starvation). (A and C, left) Degradation of GFP-tagged proteins was analyzed by anti-GFP blot of whole cell extracts. (right) The ratio between cleaved GFP and full-length protein was quantified for every time point in four independent experiments. a.u., arbitrary unit.


( A - D ) Cells expressing Rpl25 - GFP ( C and D ) were grown in rich medium ( before starvation ) and starved in SD - N for the indicated periods ( starvation ) . ( B and D ) Cells before starvation or starved for 24 h ( starvation ) were examined both by fluorescence microscopy and differential interferential contrast ( DIC ) . Note that 100 % of wild - type ( WT ) and mutant cells expressed GFP - tagged proteins in the cytoplasm before starvation . C , cytoplasmic localization ; V , vacuolar accumulation ; C + V , localization in both cytoplasm and vacuole .

(A-D) Cells expressing Rpl25-GFP (C and D) were grown in rich medium (before starvation) and starved in SD-N for the indicated periods (starvation). (B and D) Cells before starvation or starved for 24 h (starvation) were examined both by fluorescence microscopy and differential interferential contrast (DIC). Note that 100% of wild-type (WT) and mutant cells expressed GFP-tagged proteins in the cytoplasm before starvation. C, cytoplasmic localization; V, vacuolar accumulation; C + V, localization in both cytoplasm and vacuole.


( A ) GFP or Rpl25 - GFP were expressed in cells transformed ( + ) or not transformed ( − ) with a plasmid encoding 6His - ubiquitin . ( top ) Expression levels of GFP and Rpl25 - GFP in whole cell lysates were verified with anti - GFP blotting . ( middle ) Purified 6His - ubiquitin conjugates were examined by anti - GFP blotting . ( bottom ) 6His - ubiquitin expression and efficiency of purification was controlled using an anti - 6His antibody . White lines indicate that intervening lanes have been spliced out .

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(A) GFP or Rpl25-GFP were expressed in cells transformed (+) or not transformed (−) with a plasmid encoding 6His-ubiquitin. (top) Expression levels of GFP and Rpl25-GFP in whole cell lysates were verified with anti-GFP blotting. (middle) Purified 6His-ubiquitin conjugates were examined by anti-GFP blotting. (bottom) 6His-ubiquitin expression and efficiency of purification was controlled using an anti-6His antibody. White lines indicate that intervening lanes have been spliced out.


( B ) Analysis of ubiquitylated forms of Rpl25 - GFP in wild - type and the indicated mutant cells as in A .

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(B) Analysis of ubiquitylated forms of Rpl25-GFP in wild-type and the indicated mutant cells as in A.


( C ) Expression of Rpl25 - GFP and its modified form were analyzed by anti - GFP blot of extracts from wild - type and mutant cells starved for the indicated periods . Ub , ubiquitin ; WT , wild type .

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(C) Expression of Rpl25-GFP and its modified form were analyzed by anti-GFP blot of extracts from wild-type and mutant cells starved for the indicated periods. Ub, ubiquitin; WT, wild type.


( A ) Indicated strains were transformed with plasmids encoding either Rpl25 - GFP ( Rpl25 ) or rpl25 K74 , 75R - GFP ( rpl25KR ) before the deletion of a genomic copy of RPL25 . ( top ) Comparable expression levels of Rpl25 - GFP or rpl25 K74 , 75R - GFP in whole cell lysates were confirmed with anti - GFP blotting . Purified 6His - ubiquitin - conjugated forms of Rpl25 - GFP or rpl25 K74 , 75R - GFP ( middle ) and 6His - ubiquitin expression ( bottom ) were analyzed as in Fig . 2 . Ub , ubiquitin .

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(A) Indicated strains were transformed with plasmids encoding either Rpl25-GFP (Rpl25) or rpl25 K74,75R-GFP (rpl25KR) before the deletion of a genomic copy of RPL25. (top) Comparable expression levels of Rpl25-GFP or rpl25 K74,75R-GFP in whole cell lysates were confirmed with anti-GFP blotting. Purified 6His-ubiquitin-conjugated forms of Rpl25-GFP or rpl25 K74,75R-GFP (middle) and 6His-ubiquitin expression (bottom) were analyzed as in Fig. 2. Ub, ubiquitin.


( B ) Wild - type and ubp3Δ cells expressing Rpl25 - GFP or rpl25 K74 , 75R - GFP were starved for the indicated period of time . ( C ) Degradation of GFP - tagged proteins was analyzed by anti - GFP blot of whole cell extracts , and the ratio between cleaved GFP and full - length protein was quantified for all time points in four independent experiments . a . u . , arbitrary units . ( D ) Localization of GFP - tagged proteins . C , cytoplasmic localization ; V , vacuolar accumulation ; C + V , localization in both cytoplasm and

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(B) Wild-type and ubp3Δ cells expressing Rpl25-GFP or rpl25 K74,75R-GFP were starved for the indicated period of time. (C) Degradation of GFP-tagged proteins was analyzed by anti-GFP blot of whole cell extracts, and the ratio between cleaved GFP and full-length protein was quantified for all time points in four independent experiments. a.u., arbitrary units. (D) Localization of GFP-tagged proteins. C, cytoplasmic localization; V, vacuolar accumulation; C + V, localization in both cytoplasm and vacuole


( E ) Degradation of Rpl3 was analyzed at 24 h upon starvation by Western blotting of whole cell extracts using anti - Rpl3 ( gift from V . Albanese , Institut Jacques Monod , Paris , France ) or anti - Mex67 ( as a control ) antibodies . Rpl3 and Mex67 expression was quantified and normalized to the expression level at time 0 . Significance of the differences observed for the ribophagy efficiency was evaluated using Student ' s t test . * , P = 0 . 01 - 0 . 05 ; * * , P = 0 . 001 - 0 . 01 ; * * * , P < 0 . 001 . White lanes indicate that intervening lanes have been spliced out . The errors bars correspond to standard deviations .

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(E) Degradation of Rpl3 was analyzed at 24 h upon starvation by Western blotting of whole cell extracts using anti-Rpl3 (gift from V. Albanese, Institut Jacques Monod, Paris, France) or anti-Mex67 (as a control) antibodies. Rpl3 and Mex67 expression was quantified and normalized to the expression level at time 0. Significance of the differences observed for the ribophagy efficiency was evaluated using Student's t test. *, P = 0.01-0.05; **, P = 0.001-0.01; ***, P < 0.001. White lanes indicate that intervening lanes have been spliced out. The errors bars correspond to standard deviations.


( A ) Wild - type cells expressing Bre5 - GFP , Ltn1 - Flag , or ltn1 - ΔRing - Flag were starved for the indicated periods of time .

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(A) Wild-type cells expressing Bre5-GFP, Ltn1-Flag, or ltn1-ΔRing-Flag were starved for the indicated periods of time.


( B ) Cells were treated for 30 min with 100 µM cycloheximide or 100 µM MG132 . Expression of tagged proteins was analyzed by anti - GFP or anti - Flag blotting of whole cell extracts . Five times less extracts were analyzed for ltn1 - ΔRing - Flag cells . A nonspecific band served as a loading control . White lanes indicate that intervening lanes have been spliced out . WT , wild type .

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

(B) Cells were treated for 30 min with 100 µM cycloheximide or 100 µM MG132. Expression of tagged proteins was analyzed by anti-GFP or anti-Flag blotting of whole cell extracts. Five times less extracts were analyzed for ltn1-ΔRing-Flag cells. A nonspecific band served as a loading control. White lanes indicate that intervening lanes have been spliced out. WT, wild type.


A APB Northern blot using a tRNAHis probe . m5C38 levels of tRNAAspGUC were measured by 454 bisulfite sequencing at the indicated time points . Both queuosinylation and methylation levels could be restored by the addition of queuine . The slower migration of tRNAHis is eliminated by oxidizing the ribose with periodate , producing a single faster - migrating band ( ox ) . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

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

A APB Northern blot using a tRNAHis probe. m5C38 levels of tRNAAspGUC were measured by 454 bisulfite sequencing at the indicated time points. Both queuosinylation and methylation levels could be restored by the addition of queuine. The slower migration of tRNAHis is eliminated by oxidizing the ribose with periodate, producing a single faster-migrating band (ox). Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


B The addition of queuine to HCT116 cells cultured in serum free medium resulted in an increase of both queuosinylation and m5C38 levels . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

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

B The addition of queuine to HCT116 cells cultured in serum free medium resulted in an increase of both queuosinylation and m5C38 levels. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


C The addition of 20 nM queuine to the SF medium is sufficient to restore tRNAHisQUG in HeLa , whereas 200 nM of q is necessary for queuosinylation of tRNAHisGUG in HCT116 cells . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

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

C The addition of 20 nM queuine to the SF medium is sufficient to restore tRNAHisQUG in HeLa, whereas 200 nM of q is necessary for queuosinylation of tRNAHisGUG in HCT116 cells. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


B Corresponding queuosinylation and methylation levels , as determined by Northern blot using a tRNAHisGUG probe and bisulfite sequencing of tRNAAspGUC . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

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

B Corresponding queuosinylation and methylation levels, as determined by Northern blot using a tRNAHisGUG probe and bisulfite sequencing of tRNAAspGUC. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


C Representative metaplot of SF cells . 25 - 32 mer ribosome footprint reads were summed across all annotated reading frames with the AUG staring codon at position zero . In - frame 27 - 30 mer ribosome footprints showing the correct periodicity .

C Representative metaplot of SF cells. 25-32 mer ribosome footprint reads were summed across all annotated reading frames with the AUG staring codon at position zero. In-frame 27-30 mer ribosome footprints showing the correct periodicity.


D Representative metagene plot of 27 - 30 mer ribosome footprints in S and SF culture conditions for two biological replicates ( rep1 and rep2 ) . Data information : SF ( serum free medium ) , q ( queuine )

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D Representative metagene plot of 27-30 mer ribosome footprints in S and SF culture conditions for two biological replicates (rep1 and rep2). Data information: SF (serum free medium), q (queuine)


A Heatmap showing codon occupancy according to the color scale . Green bars indicate Q - decoded codons . The Asp - GAC codon translated by Q - and m5C38 - tRNAAspGUC is shown in yellow and Lys and Glu codons are in orange . Lack of Q reduces ribosome translation speed at all Q dependent codons and at near - cognate decoded codons . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

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

A Heatmap showing codon occupancy according to the color scale. Green bars indicate Q-decoded codons. The Asp-GAC codon translated by Q- and m5C38-tRNAAspGUC is shown in yellow and Lys and Glu codons are in orange. Lack of Q reduces ribosome translation speed at all Q dependent codons and at near-cognate decoded codons. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


B Translational speed of C ending codons relative to U ending codons . Error bars : ± SE of the permutated ratios of C - ending codon occupancy relative to U for the indicated conditions ; ( n = 4 ) . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , Q ( queuosine ) , G ( guanine ) .

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

B Translational speed of C ending codons relative to U ending codons. Error bars: ±SE of the permutated ratios of C-ending codon occupancy relative to U for the indicated conditions; (n=4). Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).


C Heatmap displaying increase or decrease of codon frequencies ( ‰ ) in SILAC down - regulated proteins relative to the average frequency of that codon in unchanged proteins .

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C Heatmap displaying increase or decrease of codon frequencies (‰) in SILAC down-regulated proteins relative to the average frequency of that codon in unchanged proteins.


D Codons translated by Q - tRNA are depleted in down - regulated proteins and increased in up - regulated proteins in both forward and reverse SILAC .

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D Codons translated by Q-tRNA are depleted in down-regulated proteins and increased in up-regulated proteins in both forward and reverse SILAC.


A The volcano plot shows differentially translated transcripts against adjusted p - values for SF compared to SF + q . Red dots indicate padj < 0 . 1 in multiple tests . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine )

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A The volcano plot shows differentially translated transcripts against adjusted p-values for SF compared to SF+q. Red dots indicate padj< 0.1 in multiple tests. Data information: S (standard medium), SF (serum free medium), q (queuine)


C Representative polysome profiles of HeLa cells grown in S and SF medium in the absence or presence of q . As a measurement for the global translation rate the fraction of polysomal ribosomes was quantified ; * p < 0 . 05 ( t - test ) ; n = 5 . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , RO ( ribosome occupancy ) .

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

C Representative polysome profiles of HeLa cells grown in S and SF medium in the absence or presence of q. As a measurement for the global translation rate the fraction of polysomal ribosomes was quantified; *p <0.05 (t-test); n=5. Data information: S (standard medium), SF (serum free medium), q (queuine), RO (ribosome occupancy).


D Proliferation analysis of HeLa cells grown under the indicated culture conditions . Population doubling levels of three biological replicates were calculated for each time point . Error bars : ± SD ; n = 3 . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine )

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D Proliferation analysis of HeLa cells grown under the indicated culture conditions. Population doubling levels of three biological replicates were calculated for each time point. Error bars: ±SD; n = 3. Data information: S (standard medium), SF (serum free medium), q (queuine)


A Representative fluorescence confocal images of live HeLa cells expressing Fluc - EGFP and FlucR188Q - EGFP maintained in SF compared to SF + q . Scale bars , 10 μm . Data information : SF ( serum free medium ) , q ( queuine )

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A Representative fluorescence confocal images of live HeLa cells expressing Fluc-EGFP and FlucR188Q-EGFP maintained in SF compared to SF+q. Scale bars, 10 μm. Data information: SF (serum free medium), q (queuine)


B Fluorescence intensity of aggregates was quantified as a fraction of fluorescence of the entire cell . The number of cells showing at least one aggregate is indicated . 10 cells were imaged for Fluc - EGFP and 20 cells for FlucR188Q - EGFP from 3 independent transfections ; * p < 0 . 05 ( t - test ) . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) , AU ( arbitrary units ) .

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

B Fluorescence intensity of aggregates was quantified as a fraction of fluorescence of the entire cell. The number of cells showing at least one aggregate is indicated. 10 cells were imaged for Fluc-EGFP and 20 cells for FlucR188Q-EGFP from 3 independent transfections; *p < 0.05 (t-test). Data information: S (standard medium), SF (serum free medium), q (queuine), AU (arbitrary units).


C Specific luminescence activity of EGFP - tagged sensor proteins maintained in SF compared to SF + q . Luci ferase activity was normalized to the luciferase protein content in the sample . Error bars : ± SD ; * p < 0 . 05 ( t - test ) ; n = 8 . Data information : SF ( serum free medium ) , q ( queuine )

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

C Specific luminescence activity of EGFP-tagged sensor proteins maintained in SF compared to SF+q. Luciferase activity was normalized to the luciferase protein content in the sample. Error bars: ±SD; *p < 0.05 (t-test); n = 8. Data information: SF (serum free medium), q (queuine)


A Transmission electron microscopy images showing the increase of cystic ER vacuolae upon q depletion . Arrowheads indicate rough endoplasmic reticulum while expansions are indicated by asterisks . Scale bar 250 nm . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) .

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A Transmission electron microscopy images showing the increase of cystic ER vacuolae upon q depletion. Arrowheads indicate rough endoplasmic reticulum while expansions are indicated by asterisks. Scale bar 250 nm. Data information: S (standard medium), SF (serum free medium), q (queuine).


B Representative images showing immunofluorescence of HeLa cells grown in S and SF medium in the absence or presence of Q . Anti - KDEL ( ER stress marker ) ; anti - eIF2 - pS51 ( ER stress - induced inhibitor of translation ) ; anti - ATF4 ( transcription factor that activates ER stress - responsive genes ) ; anti - Hps70 ( chaperone ) . Nuclei were stained with Hoechst . Scale bar 10 µm . Data information : S ( standard medium ) , SF ( serum free medium ) , q ( queuine ) .

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

B Representative images showing immunofluorescence of HeLa cells grown in S and SF medium in the absence or presence of Q. Anti-KDEL (ER stress marker); anti-eIF2-pS51 (ER stress-induced inhibitor of translation); anti-ATF4 (transcription factor that activates ER stress-responsive genes); anti-Hps70 (chaperone). Nuclei were stained with Hoechst. Scale bar 10 µm. Data information: S (standard medium), SF (serum free medium), q (queuine).


A APB Northern blot using a tRNAHis probe . m5C38 levels were determined by 454 bisulfite sequencing of RNA from the same liver tissue . Both queuosinylation and methylation levels could be restored by the addition of q to the feed . The results are shown for three biological replicates . Data information : con ( n = 3 ) mice were fed with conventional sterilized food , - q ( n = 3 ) mice were fed with a q - free synthetic diet for 60 days , and + q ( n = 3 ) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days .

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

A APB Northern blot using a tRNAHis probe. m5C38 levels were determined by 454 bisulfite sequencing of RNA from the same liver tissue. Both queuosinylation and methylation levels could be restored by the addition of q to the feed. The results are shown for three biological replicates. Data information: con (n=3) mice were fed with conventional sterilized food, -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.


B Representative polysome profiles showing a reduction of the protein translation rate in the liver of axenic mice fed with a q - free synthetic diet . Addition of q to the q - free diet significantly rescued the translation rate . n = 3 . Asterisks indicate statistically significant ( p < 0 . 05 , t - test ) differences . Data information : con ( n = 3 ) mice were fed with conventional sterilized food , - q ( n = 3 ) mice were fed with a q - free synthetic diet for 60 days , and + q ( n = 3 ) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days .

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

B Representative polysome profiles showing a reduction of the protein translation rate in the liver of axenic mice fed with a q-free synthetic diet. Addition of q to the q-free diet significantly rescued the translation rate. n=3. Asterisks indicate statistically significant (p<0.05, t-test) differences. Data information: con (n=3) mice were fed with conventional sterilized food, -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.


C Box plot showing the body weight of mice fed a q - free synthetic diet for 60 days , center lines show the medians ; box limits indicate the 25th and 75th percentiles , whiskers extend 1 . 5 times the interquartile range ; n = 3 . Addition of q to the q - free diet significantly rescued body weights . Data information : con ( n = 3 ) mice were fed with conventional sterilized food , - q ( n = 3 ) mice were fed with a q - free synthetic diet for 60 days , and + q ( n = 3 ) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days .

C Box plot showing the body weight of mice fed a q-free synthetic diet for 60 days, center lines show the medians; box limits indicate the 25th and 75th percentiles, whiskers extend 1.5 times the interquartile range; n=3. Addition of q to the q-free diet significantly rescued body weights. Data information: con (n=3) mice were fed with conventional sterilized food, -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.


D Dimethyl - labeling analysis of - q versus + q liver tissue . Top 10 % of deregulated proteins are indicated in red and blue . UPR effectors : HSPA5 / BiP , HSP90b1 and CALR are in orange . Data information : - q ( n = 3 ) mice were fed with a q - free synthetic diet for 60 days , and + q ( n = 3 ) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days .

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

D Dimethyl-labeling analysis of -q versus +q liver tissue. Top 10% of deregulated proteins are indicated in red and blue. UPR effectors: HSPA5/BiP, HSP90b1 and CALR are in orange. Data information: -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.


F Liver sections from indicated mice were stained with anti - KDEL antibody . Arrowheads indicate ER stressed hepatocytes . Scale bar 25 µm . Data information : con ( n = 3 ) mice were fed with conventional sterilized food , - q ( n = 3 ) mice were fed with a q - free synthetic diet for 60 days , and + q ( n = 3 ) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days .

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

F Liver sections from indicated mice were stained with anti-KDEL antibody. Arrowheads indicate ER stressed hepatocytes. Scale bar 25 µm. Data information: con (n=3) mice were fed with conventional sterilized food, -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.


B . UTP6 in U - 2 OS cells exemplify proteins localized to whole nucleoli ( HPA025936 ) .

B. UTP6 in U-2 OS cells exemplify proteins localized to whole nucleoli (HPA025936).


C . Fibrillar center localization shown by a UBTF IF staining in U - 2 OS cells ( HPA006385 ) .

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C. Fibrillar center localization shown by a UBTF IF staining in U-2 OS cells (HPA006385).


E . Dual localization of LEO1 to both the fibrillar center and nucleoplasm in GFP - tagged HeLa cells ( magenta ) , also supported by IF antibody staining using HPA040741 ( green ) .

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E. Dual localization of LEO1 to both the fibrillar center and nucleoplasm in GFP-tagged HeLa cells (magenta), also supported by IF antibody staining using HPA040741 (green).


F . The multilocalizing ribosomal protein RPL13 detected in the nucleoli , cytosol and ER in MCF - 7 cells ( HPA051702 ) .

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F. The multilocalizing ribosomal protein RPL13 detected in the nucleoli, cytosol and ER in MCF-7 cells (HPA051702).


A . KRI1 localized to the nucleoli in MCF - 7 cells ( HPA043574 ) . Data information : Protein of interest is shown in green , nuclear marker DAPI in blue and the reference marker of microtubules in red . Scale bar 10 µm .

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A. KRI1 localized to the nucleoli in MCF-7 cells (HPA043574). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.


B . GPATCH4 localized to the nucleoli in U - 2 OS cells ( HPA054319 ) . Data information : Protein of interest is shown in green , nuclear marker DAPI in blue and the reference marker of microtubules in red . Scale bar 10 µm .

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B. GPATCH4 localized to the nucleoli in U-2 OS cells (HPA054319). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.


C . FOXL2NB localized to the fibrillar center in SiHa cells ( HPA061017 ) . Data information : Protein of interest is shown in green , nuclear marker DAPI in blue and the reference marker of microtubules in red . Scale bar 10 µm .

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C. FOXL2NB localized to the fibrillar center in SiHa cells (HPA061017). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.


D . FAM71E1 localized to the fibrillar center in U - 2 OS cells ( HPA048111 ) . Data information : Protein of interest is shown in green , nuclear marker DAPI in blue and the reference marker of microtubules in red . Scale bar 10 µm .

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D. FAM71E1 localized to the fibrillar center in U-2 OS cells (HPA048111). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.


E . LRRC23 localized to the nucleoli in U - 2 OS cells ( HPA057533 ) . Data information : Protein of interest is shown in green , nuclear marker DAPI in blue and the reference marker of microtubules in red . Scale bar 10 µm .

E. LRRC23 localized to the nucleoli in U-2 OS cells (HPA057533). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.


F . ZNF853 localized to the nucleoli in U - 2 OS cells ( HPA067690 ) . Data information : Protein of interest is shown in green , nuclear marker DAPI in blue and the reference marker of microtubules in red . Scale bar 10 µm .

F. ZNF853 localized to the nucleoli in U-2 OS cells (HPA067690). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.


G . METTL5 localized to the nucleoli in U - 2 OS cells ( HPA038223 ) . Data information : Protein of interest is shown in green , nuclear marker DAPI in blue and the reference marker of microtubules in red . Scale bar 10 µm .

G. METTL5 localized to the nucleoli in U-2 OS cells (HPA038223). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.


B . IF staining of MKI67 shows localization to nucleoli rim in U - 251 cells ( CAB000058 ) . Data information : Protein of interest is shown in green , nuclear marker DAPI / Hoechst in blue and microtubules reference marker in red . Scale bar 10 µm .

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B. IF staining of MKI67 shows localization to nucleoli rim in U-251 cells (CAB000058). Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.


C . IF staining of GNL3 shows localization to nucleoli rim in U - 2 OS cells ( HPA036743 ) . Data information : Protein of interest is shown in green , nuclear marker DAPI / Hoechst in blue and microtubules reference marker in red . Scale bar 10 µm .

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C. IF staining of GNL3 shows localization to nucleoli rim in U-2 OS cells (HPA036743). Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.


D . HEK 293T cells expressing endogenous levels of N - terminus mNG - MKI67 show a faint nucleoli rim localization , although still visible . White arrows indicate cells where the rim could be seen . Data information : Protein of interest is shown in green , nuclear marker DAPI / Hoechst in blue and microtubules reference marker in red . Scale bar 10 µm .

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D. HEK 293T cells expressing endogenous levels of N-terminus mNG-MKI67 show a faint nucleoli rim localization, although still visible. White arrows indicate cells where the rim could be seen. Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.


E . Fixed HEK 293T cells expressing mNG - tagged MKI67 show a clear nucleoli rim localization . Data information : Protein of interest is shown in green , nuclear marker DAPI / Hoechst in blue and microtubules reference marker in red . Scale bar 10 µm .

E. Fixed HEK 293T cells expressing mNG-tagged MKI67 show a clear nucleoli rim localization. Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.


F . HEK 293T cells expressing endogenous levels of C - terminus tagged GNL3 show nucleoli rim localization . White arrows indicate cells where the rim could be seen . Data information : Protein of interest is shown in green , nuclear marker DAPI / Hoechst in blue and microtubules reference marker in red . Scale bar 10 µm .

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F. HEK 293T cells expressing endogenous levels of C-terminus tagged GNL3 show nucleoli rim localization. White arrows indicate cells where the rim could be seen. Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.


A . MKI67 ( HPA000451 ) . B . NOC2L ( HPA044258 ) . C . EMG1 ( HPA039304 ) . D . BMS1 ( HPA043081 ) . E . BRIX1 ( HPA039614 ) . F . RSL1D1 ( HPA043483 ) . Data information : Protein of interest is shown in green , microtubules in red and DAPI in blue . Images of interphase cells were acquired from a different experiment and staining intensities cannot be compared between interphase and mitotic cells . Scale bar 10 μm .

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A. MKI67 (HPA000451). B. NOC2L (HPA044258). C. EMG1 (HPA039304). D. BMS1 (HPA043081). E. BRIX1 (HPA039614). F. RSL1D1 (HPA043483). Data information: Protein of interest is shown in green, microtubules in red and DAPI in blue. Images of interphase cells were acquired from a different experiment and staining intensities cannot be compared between interphase and mitotic cells. Scale bar 10 μm.


C - E . IF stainings of previously uncharacterized mitotic chromosome proteins predicted to be highly disordered . Protein of interest shown in green and DAPI in blue . Scale bar 10 µm . C . CCDC86 ( HPA041117 ) , 100 % predicted disorder level . D . RRP15 ( HPA024639 ) , 87 % predicted disorder level . E . PRR19 ( HPA070350 ) , 67 % predicted disorder level .

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C-E. IF stainings of previously uncharacterized mitotic chromosome proteins predicted to be highly disordered. Protein of interest shown in green and DAPI in blue. Scale bar 10 µm. C. CCDC86 (HPA041117), 100% predicted disorder level. D. RRP15 (HPA024639), 87% predicted disorder level. E. PRR19 (HPA070350), 67% predicted disorder level.


A . MKI67 is detected on the chromosomes throughout all mitotic phases and exemplifies proteins in the early recruitment category . B - F . IF stainings of proteins where localization to mitotic chromosomes is seen after metaphase , showing a typical late recruitment expression pattern . B . NOL12 ( HPA003547 ) . C . TIGD1 ( HPA041717 ) . D . BYSL ( HPA031219 ) . E . ACTL6B ( HPA045126 ) . F . ZNF202 ( HPA059229 ) . Data information : Protein of interest is shown in green , microtubules in red and DAPI in blue . Images of interphase cells were acquired from a different experiment and staining intensities cannot be compared between interphase and mitotic cells . Scale bar 10 μm .

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A. MKI67 is detected on the chromosomes throughout all mitotic phases and exemplifies proteins in the early recruitment category. B-F. IF stainings of proteins where localization to mitotic chromosomes is seen after metaphase, showing a typical late recruitment expression pattern. B. NOL12 (HPA003547). C. TIGD1 (HPA041717). D. BYSL (HPA031219). E. ACTL6B (HPA045126). F. ZNF202 (HPA059229). Data information: Protein of interest is shown in green, microtubules in red and DAPI in blue. Images of interphase cells were acquired from a different experiment and staining intensities cannot be compared between interphase and mitotic cells. Scale bar 10 μm.


A - B . GNL3 and PES1 are localizing to protein aggregates around the chromosomes in the MKI67 KO cells . A . GNL3 ( HPA036743 ) . Median overlapKO = 0 . 539 , median overlapWT = 0 . 795 ( P = 9 . 34x10 - 9 ) and median Pearson correlationKO = - 0 . 335 , median Pearson correlationWT = 0 . 134 ( P = 3 . 58x10 - 6 ) . B . PES1 ( HPA040210 ) . Median overlapKO = 0 . 516 , median overlapWT = 0 . 755 ( P = 1 . 77x10 - 13 ) and median Pearson correlationKO = - 0 . 293 , median Pearson correlationWT = 0 . 129 ( P = 4 . 06x10 - 13 ) . Data information : Protein of interest is shown in green and DAPI in blue . Scale bar 10 μm . For the colocalization analysis data ; biological replicate 1 highlighted in grey and biological replicate 2 in green . Both the overlap and Pearson correlation between DAPI / Protein staining was measured and a two - tailed unpaired Wilcoxon test was used .

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A-B. GNL3 and PES1 are localizing to protein aggregates around the chromosomes in the MKI67 KO cells. A. GNL3 (HPA036743). Median overlapKO = 0.539, median overlapWT = 0.795 (P = 9.34x10-9) and median Pearson correlationKO = -0.335, median Pearson correlationWT = 0.134 (P = 3.58x10-6). B. PES1 (HPA040210). Median overlapKO = 0.516, median overlapWT = 0.755 (P = 1.77x10-13) and median Pearson correlationKO = -0.293, median Pearson correlationWT = 0.129 (P = 4.06x10-13). Data information: Protein of interest is shown in green and DAPI in blue. Scale bar 10 μm. For the colocalization analysis data; biological replicate 1 highlighted in grey and biological replicate 2 in green. Both the overlap and Pearson correlation between DAPI/Protein staining was measured and a two-tailed unpaired Wilcoxon test was used.


C . No protein aggregates were observed in MKI67 KO cells for PWP1 ( HPA038708 ) , despite being localized to mitotic chromosomes in WT cells . Median overlapKO = 0 . 523 , median overlapWT = 0 . 861 ( P = 1 . 05x10 - 13 ) and median Pearson correlationKO = - 0 . 190 , median Pearson correlationWT = 0 . 515 ( P = 8 . 71x10 - 15 ) . Data information : Protein of interest is shown in green and DAPI in blue . Scale bar 10 μm . For the colocalization analysis data ; biological replicate 1 highlighted in grey and biological replicate 2 in green . Both the overlap and Pearson correlation between DAPI / Protein staining was measured and a two - tailed unpaired Wilcoxon test was used . Box limits mark 1st and 3rd quantile , whiskers the 1 . 5x interquartile range and center line the median .

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C. No protein aggregates were observed in MKI67 KO cells for PWP1 (HPA038708), despite being localized to mitotic chromosomes in WT cells. Median overlapKO = 0.523, median overlapWT = 0.861 (P = 1.05x10-13) and median Pearson correlationKO = -0.190, median Pearson correlationWT = 0.515 (P = 8.71x10-15). Data information: Protein of interest is shown in green and DAPI in blue. Scale bar 10 μm. For the colocalization analysis data; biological replicate 1 highlighted in grey and biological replicate 2 in green. Both the overlap and Pearson correlation between DAPI/Protein staining was measured and a two-tailed unpaired Wilcoxon test was used. Box limits mark 1st and 3rd quantile, whiskers the 1.5x interquartile range and center line the median.


D - E . Late recruited proteins do not follow the same recruitment pattern in the MKI67 KO cells compared to WT HeLa cells . White arrows indicate where protein aggregates can be seen . D . DENND4A ( HPA065343 ) . Median overlapKO = 0 . 671 , median overlapWT = 0 . 803 ( P = 1 . 02x10 - 4 ) and median Pearson correlationKO = - 0 . 224 , median Pearson correlationWT = 0 . 236 ( P = 1 . 91x10 - 5 ) . E . NOL12 ( HPA003547 ) . Median overlapKO = 0 . 468 , median overlapWT = 0 . 820 ( P = 1 . 01x10 - 17 ) and median Pearson correlationKO = - 0 . 318 , median Pearson correlationWT = 0 . 381 ( P = 1 . 81x10 - 10 ) . White arrow indicates metaphase cell . Data information : Protein of interest is shown in green and DAPI in blue . Scale bar 10 μm . For the colocalization analysis data ; biological replicate 1 highlighted in grey and biological replicate 2 in green . Both the overlap and Pearson correlation between DAPI / Protein staining was measured and a two - tailed unpaired Wilcoxon test was used . Box limits mark 1st and 3rd quantile , whiskers the 1 . 5x interquartile range and center line the median .

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D-E. Late recruited proteins do not follow the same recruitment pattern in the MKI67 KO cells compared to WT HeLa cells. White arrows indicate where protein aggregates can be seen. D. DENND4A (HPA065343). Median overlapKO = 0.671, median overlapWT = 0.803 (P = 1.02x10-4) and median Pearson correlationKO = -0.224, median Pearson correlationWT = 0.236 (P = 1.91x10-5). E. NOL12 (HPA003547). Median overlapKO = 0.468, median overlapWT = 0.820 (P = 1.01x10-17) and median Pearson correlationKO = -0.318, median Pearson correlationWT = 0.381 (P = 1.81x10-10). White arrow indicates metaphase cell. Data information: Protein of interest is shown in green and DAPI in blue. Scale bar 10 μm. For the colocalization analysis data; biological replicate 1 highlighted in grey and biological replicate 2 in green. Both the overlap and Pearson correlation between DAPI/Protein staining was measured and a two-tailed unpaired Wilcoxon test was used. Box limits mark 1st and 3rd quantile, whiskers the 1.5x interquartile range and center line the median.


A - B . Wild - type and ΔnlpI cells were heated at a range of temperatures and the soluble components were labelled by TMT , combined and quantified by LC - MS , using the published 2D - TPP protocol ( Mateus et al . , 2018 ) . Shown are volcano plots of two replicates depicting changes in : protein abundance ( A ) and thermostability ( B ) . A local FDR ( false discovery rate ) < 0 . 01 was set as a threshold for significance . Highlighted proteins : outer membrane proteins ( OMPs , light green ) , β - barrel assembly machinery ( BAMs , red ) , PG synthases / regulators ( green ) , PG hydrolases and regulators ( blue ) and the Tol - Pal complex ( violet ) . All other proteins were colored grey and not labelled to increase the plot clarity .

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A-B. Wild-type and ΔnlpI cells were heated at a range of temperatures and the soluble components were labelled by TMT, combined and quantified by LC-MS, using the published 2D-TPP protocol (Mateus et al., 2018). Shown are volcano plots of two replicates depicting changes in: protein abundance (A) and thermostability (B). A local FDR (false discovery rate) <0.01 was set as a threshold for significance. Highlighted proteins: outer membrane proteins (OMPs, light green), β-barrel assembly machinery (BAMs, red), PG synthases/regulators (green), PG hydrolases and regulators (blue) and the Tol-Pal complex (violet). All other proteins were colored grey and not labelled to increase the plot clarity.


C - D . Affinity chromatography with immobilised NlpI . Membrane extracts from E . coli were incubated in low and high salt binding conditions ( 50 and 400 mM NaCl , respectively ) , and then eluted with 1 M NaCl or 2 M NaCl to identify possible interaction partners by label - free LC - MS analysis . The plot shows the log2 fold enriched proteins when compared to those eluted from a parallel empty column control , versus the log10 p - value , in low ( 4 replicates ) ( C ) and high ( 2 replicates ) ( D ) salt . Highlighted points are all interactions with PG enzymes and their regulators , as well as members of the divisome . All other proteins were colored grey and not labelled to increase the plot clarity ; many were non - physiological interactions with abundant cytoplasmic proteins .

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C-D. Affinity chromatography with immobilised NlpI. Membrane extracts from E. coli were incubated in low and high salt binding conditions (50 and 400 mM NaCl, respectively), and then eluted with 1 M NaCl or 2 M NaCl to identify possible interaction partners by label-free LC-MS analysis. The plot shows the log2 fold enriched proteins when compared to those eluted from a parallel empty column control, versus the log10 p-value, in low (4 replicates) (C) and high (2 replicates) (D) salt. Highlighted points are all interactions with PG enzymes and their regulators, as well as members of the divisome. All other proteins were colored grey and not labelled to increase the plot clarity; many were non-physiological interactions with abundant cytoplasmic proteins.


A . Dissociation constants for interactions between NlpI with MepM , MepS , PBP4 , PBP7 as determined by Microscale thermophoresis ( MST ) . The values are mean ± standard deviation of three independent experiments . B . MepS - NlpI interaction by MST as an example plot for Fig 2A . MST curve plotted is the mean data + / - SD of three independent experiments . Fl , fluorescently labelled ; FNorm , normalized fluorescence .

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A. Dissociation constants for interactions between NlpI with MepM, MepS, PBP4, PBP7 as determined by Microscale thermophoresis (MST). The values are mean ± standard deviation of three independent experiments. B. MepS-NlpI interaction by MST as an example plot for Fig 2A. MST curve plotted is the mean data +/- SD of three independent experiments. Fl, fluorescently labelled; FNorm, normalized fluorescence.


NlpI has different binding sites for MepS and PBP4 as shown by the ability of labelled MepS to bind preformed NlpI - PBP4 ( C ) complexes by a fixed concentration MST assay . Values are mean ± standard deviation of 3 - 6 independent experiments . To calculate significance , the data were fit using a linear model . Calculated means were compared using Tukey ' s HSD test , resulting in p - values corrected for multiple testing . Relevant p - values are highlighted directly in the figure ( * < 0 . 05 ; * * < 0 . 01 , * * * < 0 . 001 )

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NlpI has different binding sites for MepS and PBP4 as shown by the ability of labelled MepS to bind preformed NlpI-PBP4 (C) complexes by a fixed concentration MST assay. Values are mean ± standard deviation of 3-6 independent experiments. To calculate significance, the data were fit using a linear model. Calculated means were compared using Tukey's HSD test, resulting in p-values corrected for multiple testing. Relevant p-values are highlighted directly in the figure (*<0.05; **<0.01, ***<0.001)


NlpI has different binding sites for MepS and PBP7 as shown by the ability of labelled MepS to bind preformed NlpI - PBP7 ( D ) complexes by a fixed concentration MST assay . Values are mean ± standard deviation of 3 - 6 independent experiments . To calculate significance , the data were fit using a linear model . Calculated means were compared using Tukey ' s HSD test , resulting in p - values corrected for multiple testing . Relevant p - values are highlighted directly in the figure ( * < 0 . 05 ; * * < 0 . 01 , * * * < 0 . 001 )

NlpI has different binding sites for MepS and PBP7 as shown by the ability of labelled MepS to bind preformed NlpI-PBP7 (D) complexes by a fixed concentration MST assay. Values are mean ± standard deviation of 3-6 independent experiments. To calculate significance, the data were fit using a linear model. Calculated means were compared using Tukey's HSD test, resulting in p-values corrected for multiple testing. Relevant p-values are highlighted directly in the figure (*<0.05; **<0.01, ***<0.001)


A . HPLC - based PG digestion assay representing EPase activity . The graph shows the relative percentage of the muropeptide TetraTetra present at the end of the incubation period for each protein as described in materials and methods . MepM and PBP4 were incubated with sacculi , whilst MepS and PBP7 were incubated with soluble muropeptides , both from E . coli MC1061 , respectively . Values are mean ± standard deviation of three independent experiments . To calculate significance , the data were fit using a linear model . Calculated means were compared using Tukey ' s HSD test , resulting in p - values corrected for multiple testing . Relevant p - values are highlighted directly in the figure ( * < 0 . 05 ; * * < 0 . 01 , * * * < 0 . 001 )

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. HPLC-based PG digestion assay representing EPase activity. The graph shows the relative percentage of the muropeptide TetraTetra present at the end of the incubation period for each protein as described in materials and methods. MepM and PBP4 were incubated with sacculi, whilst MepS and PBP7 were incubated with soluble muropeptides, both from E. coli MC1061, respectively. Values are mean ± standard deviation of three independent experiments. To calculate significance, the data were fit using a linear model. Calculated means were compared using Tukey's HSD test, resulting in p-values corrected for multiple testing. Relevant p-values are highlighted directly in the figure (*<0.05; **<0.01, ***<0.001)


B . Genetic interactions of nlpI with EPase genes . Strains were arrayed using a Rotor HDA replicator on Lennox LB agar plates and incubated for 12 h at 37 ° C . Each plate contained 384 colonies , 96 from the wildtype , single mutants and double mutants . An example of a 384 plate is shown . Double mutants were made twice , swapping the resistance markers to the two single mutants . Colony integral opacity was quantified as a fitness readout , using the image analysis software Iris ( Kritikos et al . , 2017 ) . Bar plots shows the averaged values of 2 biological experiments , each having 96 technical replicates ( i = 2 , n = 192 ) . The error bars represent the 95 % confidence interval .

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B. Genetic interactions of nlpI with EPase genes. Strains were arrayed using a Rotor HDA replicator on Lennox LB agar plates and incubated for 12 h at 37°C. Each plate contained 384 colonies, 96 from the wildtype, single mutants and double mutants. An example of a 384 plate is shown. Double mutants were made twice, swapping the resistance markers to the two single mutants. Colony integral opacity was quantified as a fitness readout, using the image analysis software Iris (Kritikos et al., 2017). Bar plots shows the averaged values of 2 biological experiments, each having 96 technical replicates (i=2, n=192). The error bars represent the 95% confidence interval.


C . nlpI deletion changes the morphology of EPase - mutant strains . The graph shows the cell width of single and double deletions strains ( 800 < n < 2000 cells ) . The box has a medium between 25 and 75 % . The whiskers with the upper and lower vertical line indicating the 95 and 5 % . The dots are individual points outside the 5 and 95 % range . Above the graph are representative images of cells lacking MepS or PBP4 in combination with a deletion of NlpI . The scale bar equals 2 µm . Gene encoding protein legend : nlpI encodes NlpI , dacB encodes PBP4 , pbpG encode PBP7 , mepM encodes MepM , meps encodes MepS .

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C. nlpI deletion changes the morphology of EPase-mutant strains. The graph shows the cell width of single and double deletions strains (800 < n < 2000 cells). The box has a medium between 25 and 75%. The whiskers with the upper and lower vertical line indicating the 95 and 5%. The dots are individual points outside the 5 and 95% range. Above the graph are representative images of cells lacking MepS or PBP4 in combination with a deletion of NlpI. The scale bar equals 2 µm. Gene encoding protein legend: nlpI encodes NlpI, dacB encodes PBP4, pbpG encode PBP7, mepM encodes MepM, meps encodes MepS.


D . Inducible mepS expression system ( pBAD30 ) strongly overproduces MepS . Strains were grown in LB at 30 ° C and cells were collected at OD600 ≈ 0 . 4 . The level of MepS contained in the membrane fraction was detected using purified anti - MepS antibody .

D. Inducible mepS expression system (pBAD30) strongly overproduces MepS. Strains were grown in LB at 30°C and cells were collected at OD600≈0.4. The level of MepS contained in the membrane fraction was detected using purified anti-MepS antibody.


E . Visualization of the effect of MepS absence or its overexpression on cell width by phase contrast microscopy . Cultures were grown in LB at 30 ° C and aliquots of culture were taken at OD600 ≈ 0 . 1 . The scale bar equals 5 μm .

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E. Visualization of the effect of MepS absence or its overexpression on cell width by phase contrast microscopy. Cultures were grown in LB at 30°C and aliquots of culture were taken at OD600≈0.1. The scale bar equals 5 μm.


F . MepS level modulates cell width . The graph shows the distribution of mean cell width for each cell , with n corresponding to the number of cells measured for each strain and the median width for the population being indicated by a dotted line and referred to as w .

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F. MepS level modulates cell width. The graph shows the distribution of mean cell width for each cell, with n corresponding to the number of cells measured for each strain and the median width for the population being indicated by a dotted line and referred to as w.


G . Relative fitness of ΔnlpI , ΔmepS and ΔnlpIΔmepS mutants . Strains were arrayed using a Rotor HDA replicator on Lennox LB agar plates supplemented 10 % sucrose , or LB agar plates containing 0 mM or 500 mM NaCl . Plates were incubated for 12 h at 37 ° C . Each plate contained 384 colonies , 96 from the wildtype , single mutants and double mutants .

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G. Relative fitness of ΔnlpI, ΔmepS and ΔnlpIΔmepS mutants. Strains were arrayed using a Rotor HDA replicator on Lennox LB agar plates supplemented 10% sucrose, or LB agar plates containing 0 mM or 500 mM NaCl. Plates were incubated for 12 h at 37°C. Each plate contained 384 colonies, 96 from the wildtype, single mutants and double mutants.


H . Cells of wildtype ( WT ) , ΔnlpI , ΔmepS and ΔnlpIΔmepS containing multicopy plasmids with lacZ were grown onto CPRG indicator agar to assay envelope integrity . CPRG ( yellow ) cannot penetrate intact Gram - negative envelopes . Its conversion by intracellular β - galactosidase to CPR ( red ) indicates loss of envelope integrity .

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H. Cells of wildtype (WT), ΔnlpI, ΔmepS and ΔnlpIΔmepS containing multicopy plasmids with lacZ were grown onto CPRG indicator agar to assay envelope integrity. CPRG (yellow) cannot penetrate intact Gram-negative envelopes. Its conversion by intracellular β-galactosidase to CPR (red) indicates loss of envelope integrity.


A . Phase contrast image and corresponding fluorescence SIM image of BW25113 cells that have been grown in LB at 37 ° C and immunolabelled with specific antibodies against NlpI . Scale bar equals 2 µm .

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A. Phase contrast image and corresponding fluorescence SIM image of BW25113 cells that have been grown in LB at 37°C and immunolabelled with specific antibodies against NlpI. Scale bar equals 2 µm.


B . Genetic interactions of NlpI with PG machineries . An example of a 384 plate is shown . Bar plots shows the averaged values of 2 experiments ( i = 2 , n = 192 ) . Error bars denote the 95 % confidence interval of the mean .

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B. Genetic interactions of NlpI with PG machineries. An example of a 384 plate is shown. Bar plots shows the averaged values of 2 experiments (i=2, n=192). Error bars denote the 95% confidence interval of the mean.


C . NlpI deletion exacerbates the morphological defects of the PBP1B / LpoB - mutant strains . The graph shows the cell width of single and double deletions strains ( 800 < n < 2000 cells ) . The box has a medium between 25 and 75 % . The whiskers with the upper and lower vertical line indicating the 95 and 5 % . The dots are individual points outside the 5 and 95 % range . Representative images of the strains are shown above the graph . The scale bar equals 2 µm . Gene encoding protein legend : nlpI encodes NlpI , mrcA encodes PBP1A , lpoA encodes LpoA , mrcB encodes PBP1B , lpoB encodes LpoB .

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C. NlpI deletion exacerbates the morphological defects of the PBP1B/LpoB-mutant strains. The graph shows the cell width of single and double deletions strains (800 < n < 2000 cells). The box has a medium between 25 and 75%. The whiskers with the upper and lower vertical line indicating the 95 and 5%. The dots are individual points outside the 5 and 95% range. Representative images of the strains are shown above the graph. The scale bar equals 2 µm. Gene encoding protein legend: nlpI encodes NlpI, mrcA encodes PBP1A, lpoA encodes LpoA, mrcB encodes PBP1B, lpoB encodes LpoB.


D . Dissociation constants for interactions between PBP1A and LpoA with NlpI , MepM , MepS , PBP4 and PBP7 as determined by MST . The values are mean ± standard deviation of three independent experiments . 1PBP1A was used as fluorescently labelled protein in all assays . 2LpoA was used as unlabelled ligand in all combinations , except with MepM and PBP4 .

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D. Dissociation constants for interactions between PBP1A and LpoA with NlpI, MepM, MepS, PBP4 and PBP7 as determined by MST. The values are mean ± standard deviation of three independent experiments. 1PBP1A was used as fluorescently labelled protein in all assays. 2LpoA was used as unlabelled ligand in all combinations, except with MepM and PBP4.


A - B . PBP4 has different interaction sites for PBP1A / LpoA and NlpI as shown by a single concentration MST assay . Plots show the FNorm or fluorescence values of fluorescently labelled PBP1A or LpoA with or without PBP4 , NlpI or PBP4 - NlpI . Values are mean ± standard deviation of three independent experiments .

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A-B. PBP4 has different interaction sites for PBP1A/LpoA and NlpI as shown by a single concentration MST assay. Plots show the FNorm or fluorescence values of fluorescently labelled PBP1A or LpoA with or without PBP4, NlpI or PBP4-NlpI. Values are mean ± standard deviation of three independent experiments.


A . Estimated number of molecules and molarity of PBP1a / LpoA and EPases . 1Numbers obtained by ribosomal profiling in rich growth medium ( Li et al . , 2014 ) . 2Concentration of monomer . 3Decreases in the presence of NlpI ( Singh et al . , 2015 ) . 4 " no change detected " . The periplasmic concentrations of proteins were estimated for a cell with periplasmic volume of 3 . 33 × 10 - 16 L , where 1 molecule corresponds to 5 nM .

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A. Estimated number of molecules and molarity of PBP1a/LpoA and EPases. 1Numbers obtained by ribosomal profiling in rich growth medium (Li et al., 2014). 2Concentration of monomer. 3Decreases in the presence of NlpI (Singh et al., 2015). 4"no change detected". The periplasmic concentrations of proteins were estimated for a cell with periplasmic volume of 3.33 ×10-16 L, where 1 molecule corresponds to 5 nM.


Immunofluorescence microscopy of HEK293T exogenously expressing ATF6α - V5 tagged . Cells were fixed and stained with anti - calnexin ( green ) or anti - V5 ( red ) . An overlay of the cells is presented in the third panel . Bar = 10 µm .

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Immunofluorescence microscopy of HEK293T exogenously expressing ATF6α-V5 tagged. Cells were fixed and stained with anti-calnexin (green) or anti-V5 (red). An overlay of the cells is presented in the third panel. Bar = 10 µm.


Whole cell lysates from HEK293T cells , with or without stable expression of ATF6α ( as indicated ) were immunoisolated with mouse anti - V5 . RCN1 was transiently transfected into both cell lines in the experiment depicted in panel 3 . Immunoisolated material was separated by SDS - PAGE under reducing conditions followed by western blotting to determine the co - isolation of ER proteins with ATF6α . Ig - HC ( anti - BiP blot ) indicates immunoglobulin heavy chains .

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Whole cell lysates from HEK293T cells, with or without stable expression of ATF6α (as indicated) were immunoisolated with mouse anti-V5. RCN1 was transiently transfected into both cell lines in the experiment depicted in panel 3. Immunoisolated material was separated by SDS-PAGE under reducing conditions followed by western blotting to determine the co-isolation of ER proteins with ATF6α. Ig-HC (anti-BiP blot) indicates immunoglobulin heavy chains.


Whole cell lysates from HEK293T cells , with or without stable expression of

Whole cell lysates from HEK293T cells, with or without stable expression of ATF6α


Whole cell lysates from HEK293T cells stably expressing ATF6α were either untreated ( - ) or treated ( + ) with 5 µM thapsigargin ( TG ) for 1 h . THBS4 was transiently transfected into cells in the experiment depicted in panel 2 . ATF6α was either immunoisolated with anti - V5 ( panel 1 and 3 ) or anti - HA ( panel 2 ) . For the experiment in panel 3 the cells were cross - linked prior to immunoisolation . Samples were separated by SDS - PAGE under reducing conditions followed by western blots to determine co - isolation of endogenous PDIR , ERp18 or exogenously expressed THBS4 with ATF6α . HC indicates immunoglobulin heavy chain .

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Whole cell lysates from HEK293T cells stably expressing ATF6α were either untreated (-) or treated (+) with 5 µM thapsigargin (TG) for 1 h. THBS4 was transiently transfected into cells in the experiment depicted in panel 2. ATF6α was either immunoisolated with anti-V5 (panel 1 and 3) or anti-HA (panel 2). For the experiment in panel 3 the cells were cross-linked prior to immunoisolation. Samples were separated by SDS-PAGE under reducing conditions followed by western blots to determine co-isolation of endogenous PDIR, ERp18 or exogenously expressed THBS4 with ATF6α. HC indicates immunoglobulin heavy chain.


HEK293T cells stably expressing ATF6α were either left untransfected ( UT ) or transfected with substrate - trapping mutants of PDI oxidoreductases . ATF6α was immunoisolated from cell lysates with mouse anti - ATF6α , separated by SDS - PAGE under non - reducing conditions and ATF6α detected by western blotting using rabbit anti - HA . Blots confirm mixed - disulfide complexes between ATF6α and ERp18 , ERp57 , and PDIR indicated with arrows ( lanes 2 , 4 , 5 ) . M , D and O refer to ATF6α monomer , dimer and oligomer respectively .

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HEK293T cells stably expressing ATF6α were either left untransfected (UT) or transfected with substrate-trapping mutants of PDI oxidoreductases. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. Blots confirm mixed-disulfide complexes between ATF6α and ERp18, ERp57, and PDIR indicated with arrows (lanes 2, 4, 5). M, D and O refer to ATF6α monomer, dimer and oligomer respectively.


Whole cell lysates of HEK293T cells expressing an ATF6α mutant containing cysteine to alanine mutations in the lumenal domain ( ATF6 DM ) were transfected with PDI substrate - trapping mutants . ATF6α was immunoisolated with mouse anti - ATF6α , separated by SDS - PAGE under non - reducing conditions , and ATF6α detected with rabbit anti - HA .

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Whole cell lysates of HEK293T cells expressing an ATF6α mutant containing cysteine to alanine mutations in the lumenal domain (ATF6 DM) were transfected with PDI substrate-trapping mutants. ATF6α was immunoisolated with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions, and ATF6α detected with rabbit anti-HA.


HEK293T cells were co - transfected with ATF6α S1P and PDI substrate - trapping mutants and either left untreated ( - ) or treated ( + ) for 1 h with 5 µM TG to induce ER stress . ATF6α was immunoisolated from cell lysates with mouse anti - ATF6α , separated by SDS - PAGE under non - reducing conditions and ATF6α detected by western blotting using rabbit anti - HA . Blots indicate that mixed - disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress . M , D and O refer to ATF6α monomer , dimer and oligomer respectively . * indicates positions of the ATF6α - PDI enzymes mixed - disulfide complexes .

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

HEK293T cells were co-transfected with ATF6α S1P and PDI substrate-trapping mutants and either left untreated (-) or treated (+) for 1 h with 5 µM TG to induce ER stress. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. Blots indicate that mixed-disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress. M, D and O refer to ATF6α monomer, dimer and oligomer respectively. *indicates positions of the ATF6α-PDI enzymes mixed-disulfide complexes.


HEK293T cells were co - transfected with ATF6α S1P and PDI substrate - trapping mutants and either left untreated ( - ) or treated ( + ) for 1 h with 5 µg / ml tunicamycin to induce ER stress . ATF6α was immunoisolated from cell lysates with mouse anti - ATF6α , separated by SDS - PAGE under non - reducing conditions and ATF6α detected by western blotting using rabbit anti - HA . Blots indicate that mixed - disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress . M , D and O refer to ATF6α monomer , dimer and oligomer respectively . * indicates positions of the ATF6α - PDI enzymes mixed - disulfide complexes . * * indicates hypoglycosylated ATF6α . Data shown are representative of three independent experiments .

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

HEK293T cells were co-transfected with ATF6α S1P and PDI substrate-trapping mutants and either left untreated (-) or treated (+) for 1 h with 5 µg/ml tunicamycin to induce ER stress. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. Blots indicate that mixed-disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress. M, D and O refer to ATF6α monomer, dimer and oligomer respectively. *indicates positions of the ATF6α-PDI enzymes mixed-disulfide complexes. ** indicates hypoglycosylated ATF6α. Data shown are representative of three independent experiments.


HEK293T cells were co - transfected with ATF6α S1P and PDI substrate - trapping mutants and either left untreated ( - ) or treated ( + ) for 1 h with 20 µM MG132 to induce ER stress . ATF6α was immunoisolated from cell lysates with mouse anti - ATF6α , separated by SDS - PAGE under non - reducing conditions and ATF6α detected by western blotting using rabbit anti - HA . Blots indicate that mixed - disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress . M , D and O refer to ATF6α monomer , dimer and oligomer respectively . * indicates positions of the ATF6α - PDI enzymes mixed - disulfide complexes . * * indicates hypoglycosylated ATF6α . Data shown are representative of three independent experiments .

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

HEK293T cells were co-transfected with ATF6α S1P and PDI substrate-trapping mutants and either left untreated (-) or treated (+) for 1 h with 20 µM MG132 to induce ER stress. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. Blots indicate that mixed-disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress. M, D and O refer to ATF6α monomer, dimer and oligomer respectively. *indicates positions of the ATF6α-PDI enzymes mixed-disulfide complexes. ** indicates hypoglycosylated ATF6α. Data shown are representative of three independent experiments.


HEK293T cells were co - transfected with ATF6α S1P and PDI substrate - trapping mutants and either left untreated ( - ) or treated ( + ) for 1 h with 5 µM TG to induce ER stress . Samples from ( A ) were rerun and probed with anti - sera raised against respective PDI enzymes as indicated ( D ) . * indicates positions of the ATF6α - PDI enzymes mixed - disulfide complexes .

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

HEK293T cells were co-transfected with ATF6α S1P and PDI substrate-trapping mutants and either left untreated (-) or treated (+) for 1 h with 5 µM TG to induce ER stress. Samples from (A) were rerun and probed with anti-sera raised against respective PDI enzymes as indicated (D). *indicates positions of the ATF6α-PDI enzymes mixed-disulfide complexes.


HEK293T cells were co - transfected with the ERp18 substrate - trapping mutant and the indicated ATF6α constructs . Cell were allowed to recover for 24 h post - transfection before being either left untreated ( - ) or treated ( + ) for 1 h with 5 µM TG to induce ER stress . ATF6α was immunoisolated from cell lysates with mouse anti - ATF6α , separated by SDS - PAGE under non - reducing conditions and ATF6α detected by western blotting using rabbit anti - HA . The blot confirms that ATF6α C618A forms a stable complex with ERp18 trapping mutant following ER stress . M , D and O refer to ATF6 monomer , dimer and oligomer respectively . * indicates positions of the ATF6 - ERp18 mixed - disulfide complexes .

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

HEK293T cells were co-transfected with the ERp18 substrate-trapping mutant and the indicated ATF6α constructs. Cell were allowed to recover for 24 h post-transfection before being either left untreated (-) or treated (+) for 1 h with 5 µM TG to induce ER stress. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. The blot confirms that ATF6α C618A forms a stable complex with ERp18 trapping mutant following ER stress. M, D and O refer to ATF6 monomer, dimer and oligomer respectively. *indicates positions of the ATF6-ERp18 mixed-disulfide complexes.


HEK293T cells were co - transfected with GFP - ATF6α and ERp18 trapping mutant constructs as indicated . GFP - ATF6α complexes were immunoisolated using GFP - trap . Immunoisolated material was separated by SDS - PAGE under non - reducing conditions and silver stained to detect ATF6α disulfide - linked complexes . MS analyses confirmed that protein bands indicated by black arrows are a complex comprising ATF6α and ERp18 , whilst bands marked as monomer ( M ) and oligomer ( O ) contain ATF6α only .

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HEK293T cells were co-transfected with GFP-ATF6α and ERp18 trapping mutant constructs as indicated. GFP-ATF6α complexes were immunoisolated using GFP-trap. Immunoisolated material was separated by SDS-PAGE under non-reducing conditions and silver stained to detect ATF6α disulfide-linked complexes. MS analyses confirmed that protein bands indicated by black arrows are a complex comprising ATF6α and ERp18, whilst bands marked as monomer (M) and oligomer (O) contain ATF6α only.


CRISPR / Cas9 - based knockout of ERp18 in HEK293T cells expressing ATF6α . ERp18 levels in control ( WT ) and knockout ( KO ) cells were detected with an antibody to ERp18 .

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CRISPR/Cas9-based knockout of ERp18 in HEK293T cells expressing ATF6α. ERp18 levels in control (WT) and knockout (KO) cells were detected with an antibody to ERp18.