Figure 5A . Atp5488 ( left panel ) or [ 35S ] Atp5 ( right panel ) were imported into mitochondria for indicated times and in the presence or absence of a membrane potential ( ∆ ψ ) . After proteinase K treatment , samples were separated by SDS - Page and analyzed by fluorescence scanning or digital autoradiography . Prec . , purified precursor protein ; p , precursor ; m , mature protein . B . Import of Atp5488 with increasing concentrations of CCCP to study dependency of import on membrane potential ( ∆ ψ ) . P , precursor ; m , mature protein . D . [ 35S ] Atp5 was imported with increasing CCCP concentrations as described in B . Samples were separated by SDS - PAGE and analyzed by digital autoradiography . E . Quantification of import of [ 35S ] Atp5 with increasing CCCP concentrations . The amount of imported protease - protected protein in the absence of CCCP was set to 100 % ; error bars indicate SEM ( n = 3 ) . G . Atp5488 ( left panel ) or [ 35S ] Atp5 ( right panel ) were imported into wild - type and atp21 ∆ mitochondria for indicated times and in the presence or absence of a membrane potential ( ∆ ψ ) . After proteinase K treatment , samples were solubilized in digitonin buffer and separated by BN - PAGE . Aliquots of the samples was analyzed by SDS - PAGE ( lower panels ) . Subsequently proteins were visualized by fluorescence scanning or digital autoradiography . Complex V dimer , ( CVD ) and monomer ( CVM ) . Prec . , purified precursor ; p , precursor ; m , mature protein .

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Figure 5A. Atp5488 (left panel) or [35S]Atp5 (right panel) were imported into mitochondria for indicated times and in the presence or absence of a membrane potential (∆ψ). After proteinase K treatment, samples were separated by SDS-Page and analyzed by fluorescence scanning or digital autoradiography. Prec., purified precursor protein; p, precursor; m, mature protein.B. Import of Atp5488 with increasing concentrations of CCCP to study dependency of import on membrane potential (∆ψ). P, precursor; m, mature protein.D. [35S]Atp5 was imported with increasing CCCP concentrations as described in B. Samples were separated by SDS-PAGE and analyzed by digital autoradiography. E. Quantification of import of [35S]Atp5 with increasing CCCP concentrations. The amount of imported protease-protected protein in the absence of CCCP was set to 100%; error bars indicate SEM (n=3).G. Atp5488 (left panel) or [35S]Atp5 (right panel) were imported into wild-type and atp21∆ mitochondria for indicated times and in the presence or absence of a membrane potential (∆ψ). After proteinase K treatment, samples were solubilized in digitonin buffer and separated by BN-PAGE. Aliquots of the samples was analyzed by SDS-PAGE (lower panels). Subsequently proteins were visualized by fluorescence scanning or digital autoradiography. Complex V dimer, (CVD) and monomer (CVM). Prec., purified precursor ; p, precursor; m, mature protein.


Figure 1A Co ‐ localization of endogenous TBC1D5 and VPS35 with mCherry ‐ ATG9A , in steady state and autophagy induced by mTOR inhibitor KU0063794 ( 6 h ) . B , C Quantification of co ‐ localization experiments presented in ( A ) . D Co ‐ immunoprecipitation of HA ‐ Flag ‐ TBC1D5 with myc ‐ VPS29 , transiently overexpressed in 293T cells , DMSO or KU0063794 ( 6 h ) treated . E , F Co ‐ immunoprecipitation of endogenous TBC1D5 and ATG9A with myc ‐ VPS29 ( E ) transiently overexpressed in 293T cells or with HA ‐ Flag ‐ VPS35 ( F ) transiently overexpressed in 293T cells , treated with DMSO or KU0063794 ( 6 h ) . G Co ‐ immunoprecipitation of endogenous VPS29 and ATG9A with HA ‐ Flag ‐ TBC1D5stably expressed in 293T cells , treated with DMSO or KU0063794 ( 6 h ) . H Co ‐ immunoprecipitation of endogenous ULK1 and pATG13 ( Ser318 ) with HA ‐ Flag ‐ TBC1D5 , stably expressed in T ‐ REx 293T cells , induced by doxycycline ( 50 ng / ml ) for 20 h prior to DMSO or KU0063794 ( 6 h ) treatment . I Magnified still images extracted from Supplementary Movie S2 . mCherry ‐ TBC1D5 co ‐ localizes with GFP ‐ ATG9 upon autophagy induction by mTOR inhibitor , KU0063794 . H Co - immunoprecipitation of endogenous ULK1 and pATG13 ( Ser318 ) with HA - Flag - TBC1D5 , stably expressed in T - REx 293T cells , induced by doxycycline ( 50 ng / ml ) for 20 h prior to DMSO or KU0063794 ( 6 h ) treatment .

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Figure 1A Co‐localization of endogenous TBC1D5 and VPS35 with mCherry‐ATG9A, in steady state and autophagy induced by mTOR inhibitor KU0063794 (6 h).B, C Quantification of co‐localization experiments presented in (A).D Co‐immunoprecipitation of HA‐Flag‐TBC1D5 with myc‐VPS29, transiently overexpressed in 293T cells, DMSO or KU0063794 (6 h) treated.E, F Co‐immunoprecipitation of endogenous TBC1D5 and ATG9A with myc‐VPS29(E) transiently overexpressed in 293T cells or with HA‐Flag‐VPS35 (F) transiently overexpressed in 293T cells, treated with DMSO or KU0063794 (6 h).G Co‐immunoprecipitation of endogenous VPS29 and ATG9A with HA‐Flag‐TBC1D5stably expressed in 293T cells, treated with DMSO or KU0063794 (6 h).H Co‐immunoprecipitation of endogenous ULK1 and pATG13 (Ser318) with HA‐Flag‐TBC1D5, stably expressed in T‐REx 293T cells, induced by doxycycline (50 ng/ml) for 20 h prior to DMSO or KU0063794 (6 h) treatment.I Magnified still images extracted from Supplementary Movie S2. mCherry‐TBC1D5 co‐localizes with GFP‐ATG9 upon autophagy induction by mTOR inhibitor, KU0063794.H Co-immunoprecipitation of endogenous ULK1 and pATG13 (Ser318) with HA-Flag-TBC1D5, stably expressed in T-REx 293T cells, induced by doxycycline (50 ng/ml) for 20 h prior to DMSO or KU0063794 (6 h) treatment.


Figure 2A Lysates of U2OS cells stably expressing control shRNA or shRNA targeting VPS29 or TBC1D5 . Cells were treated with DMSO or KU0063794 ( 6 h ) , lysed in RIPA buffer ( 1 % SDS ) and lysates were subjected to SDS - PAGE . B U2OS cells stably expressing mCherry ‐ ATG9 were depleted of VPS29 and TBC1D5 using shRNAs , treated with DMSO or KU0063794 ( 6 h ) , fixed with 2 % PFA , and immunostained with anti ‐ LAMP1 antibody . C Quantification of ATG9 vesicular structures in cells from ( B ) under non ‐ stimulated conditions . D Quantification of ATG9 and LAMP1 co ‐ localization in cells presented in ( B ) . E Quantification of ATG9 and LAMP1 co − localization in cells stably depleted for TBC1D5 ( shRNA # 1 ) or VPS29 ( shRNA # 2 ) , transiently transfected with myc − TBC1D5 plasmid resistant to TBC1D5 shRNA # 1 . Cells were treated with KU0063794 ( 6 h ) , 20 h post − transfection , fixed , and immunostained with anti − LAMP1 and anti − myc antibodies . F Control shRNA and U2OS cell lysates depleted for TBC1D5 ( shRNA # 1 ) , transfected with empty plasmid or with shRNA ‐ resistant myc ‐ TBC1D5 . G Immunofluorescence of TBC1D5 ( shRNA # 1 ) cells transiently transfected with shRNA ‐ resistant myc ‐ TBC1D5 , treated with KU0063794 ( 6 h ) . 20 h post ‐ transfection cells were treated , subsequently fixed , and immunostained with anti ‐ myc and anti ‐ LAMP1 antibodies .

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Figure 2A Lysates of U2OS cells stably expressing control shRNA or shRNA targeting VPS29 or TBC1D5. Cells were treated with DMSO or KU0063794 (6 h), lysed in RIPA buffer (1% SDS) and lysates were subjected to SDS-PAGE.B U2OS cells stably expressing mCherry‐ATG9 were depleted of VPS29 and TBC1D5 using shRNAs, treated with DMSO or KU0063794 (6 h), fixed with 2% PFA, and immunostained with anti‐LAMP1 antibody.C Quantification of ATG9 vesicular structures in cells from (B) under non‐stimulated conditions.D Quantification of ATG9 and LAMP1 co‐localization in cells presented in (B).E Quantification of ATG9 and LAMP1 co−localization in cells stably depleted for TBC1D5 (shRNA#1) or VPS29 (shRNA#2), transiently transfected with myc−TBC1D5 plasmid resistant to TBC1D5 shRNA#1. Cells were treated with KU0063794 (6 h), 20 h post−transfection, fixed, and immunostained with anti−LAMP1 and anti−myc antibodies.F Control shRNA and U2OS cell lysates depleted for TBC1D5 (shRNA#1), transfected with empty plasmid or with shRNA‐resistant myc‐TBC1D5.G Immunofluorescence of TBC1D5 (shRNA#1) cells transiently transfected with shRNA‐resistant myc‐TBC1D5, treated with KU0063794 (6 h). 20 h post‐transfection cells were treated, subsequently fixed, and immunostained with anti‐myc and anti‐LAMP1 antibodies.


Figure 3A HA ‐ Flag ‐ TBC1D5 expression was induced in T ‐ REx HeLa cells for 20 h , lysates were subjected to GSTpull ‐ down , − AP2M1 . Co ‐ precipitatedTBC1D5 was detected with anti − Flag antibody . B Control cells and TBC1D5 ( shRNA # 1 ) U2OS cell lysates subjected to SDS - PAGE , subsequently blotted with endogenous antibodies for AP2 subunits AP2A1 and AP2M1 . C Immunofluorescent staining of AP2A1 in U2OS shRNA control , or shRNA # 1 TBC1D5 cells treated with KU0063974 or DMSO ( 6 h ) . D U2OS cells stably expressing HA ‐ Flag ‐ ATG9A were fixed and stained for endogenous AP2A1 and TBC1D5 . Regions of co ‐ localization are indicated with arrows . E U2OS cells stably expressing HA ‐ Flag ‐ ATG9A were starved in EBSS ( 4 h ) or treated with KU0063794 ( 6 h ) , subsequently fixed and stained with anti ‐ AP2A1 antibody . F U2OS cells stably expressing mCherry ‐ ATG9A were treated with KU0063794 ( 6 h ) , lysed in co ‐ immunoprecipitation buffer . Lysates from DMSO ‐ or KU0063794 ‐ treated cells were split and equal volumes were incubated with RFP ‐ Trap beads or GFP ‐ Trap beads , or agarose as a negative control , overnight at 4 ° C . Precipitated proteins were analyzed by SDS - PAGE .

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Figure 3A HA‐Flag‐TBC1D5 expression was induced in T‐REx HeLa cells for 20 h, lysates were subjected to GSTpull‐down, −AP2M1. Co‐precipitatedTBC1D5 was detected with anti−Flag antibody.B Control cells and TBC1D5 (shRNA#1) U2OS cell lysates subjected to SDS-PAGE, subsequently blotted with endogenous antibodies for AP2 subunits AP2A1 and AP2M1.C Immunofluorescent staining of AP2A1 in U2OS shRNA control, or shRNA#1 TBC1D5 cells treated with KU0063974 or DMSO (6 h).D U2OS cells stably expressing HA‐Flag‐ATG9A were fixed and stained for endogenous AP2A1 and TBC1D5. Regions of co‐localization are indicated with arrows.E U2OS cells stably expressing HA‐Flag‐ATG9A were starved in EBSS (4 h) or treated with KU0063794 (6 h), subsequently fixed and stained with anti‐AP2A1 antibody.F U2OS cells stably expressing mCherry‐ATG9A were treated with KU0063794 (6 h), lysed in co‐immunoprecipitation buffer. Lysates from DMSO‐ or KU0063794‐treated cells were split and equal volumes were incubated with RFP‐Trap beads or GFP‐Trap beads, or agarose as a negative control, overnight at 4°C. Precipitated proteins were analyzed by SDS-PAGE.


Figure 4A U2OS cells stably expressing HA ‐ Flag ‐ ATG9A transfected with siRNA targeting AP2A1 ( 40 nM ) , or siRNA AllStar control ( 40 nM ) were treated with KU0063794 or DMSO ( 6 h ) 4 days post ‐ transfection , fixed , and stained with anti ‐ HA antibody . B Co ‐ localization of HA ‐ Flag ‐ ATG9A with TGN46 . C Lysates of U2OS cells transfected with siRNA targeting AP2A1 , or siRNA control AllStars analyzed by SDS - PAGE . D U2OS cells stably expressing mCherry − ATG9 were transiently transfected with GFP − Dynamin 2 or GFP − Dynamin 2 ( K44A ) , 20 h post − transfection cells were fixed and with anti − TBC1D5 antibody . ATG9A and Dynamin 2co − localization is indicated by arrows .

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Figure 4A U2OS cells stably expressing HA‐Flag‐ATG9A transfected with siRNA targeting AP2A1 (40 nM), or siRNA AllStar control (40 nM) were treated with KU0063794 or DMSO (6 h) 4 days post‐transfection, fixed, and stained with anti‐HA antibody.B Co‐localization of HA‐Flag‐ATG9A with TGN46.C Lysates of U2OS cells transfected with siRNA targeting AP2A1, or siRNA control AllStars analyzed by SDS-PAGE.D U2OS cells stably expressing mCherry−ATG9 were transiently transfected with GFP−Dynamin 2 or GFP−Dynamin 2 (K44A), 20 h post−transfection cells were fixed and with anti−TBC1D5 antibody. ATG9A and Dynamin 2co−localization is indicated by arrows.


Figure 5A U2OS cells stably expressing HA − Flag − ATG9A treated with DMSO or Dynasore ( 100 μM ) 15 min , fixed and immunostained with anti − HA and anti − TBC1D5 antibodies . B Quantification of ATG9Aand AP2A1co ‐ localization from experiment in ( A ) , pictures from 3 independent experiments were analyzed and co ‐ localization quantified , statistics is calculated as described in Supplementary Materials and Methods . C RGB ‐ line profile across the vesicle indicated with arrow in ( B ) ; red - AP2A1 ; green - ATG9A ; blue - TBC1D5 . D Quantification of ATG9A and AP2 co ‐ localization . Cells were starved in EBSS media ( 45 min ) or pretreated with Dynasore ( 15 min ) and subsequently starved for 45 min in combination with Dynasore . Control cells were treated with DMSO . E Quantification of LC3 puncta in cells stably expressing HA ‐ Flag ‐ ATG9A , treated and presented as in ( D ) . 100 cells were quantified in 3 independent experiments . F T ‐ REx HeLa cells were transfected with siRNA Control oligo or siRNA oligo # 1 targeting ( 40 nM ) . 72 h post − transfection , expression of HA ‐ Flag − TBC1D5 was induced with doxycycline ( 50 ng / ml ) , and 96 h post − transfection cells were treated with Dynasore for 15 min , or pretreated with Dynasore and subsequently starved in combination with Dynasore for additional 45 min . Cells were lysed in co ‐ immunoprecipitation buffer , and lysates were incubated with Myc antibody or M2 antibody coupled with agarose overnight at 4 ° C . Beads were washed 3 times with incubation buffer and subjected to SDS ‐ PAGE .

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Figure 5A U2OS cells stably expressing HA−Flag−ATG9A treated with DMSO or Dynasore (100 μM) 15 min, fixed and immunostained with anti−HA and anti−TBC1D5 antibodies.B Quantification of ATG9Aand AP2A1co‐localization from experiment in (A), pictures from 3 independent experiments were analyzed and co‐localization quantified, statistics is calculated as described in Supplementary Materials and Methods.C RGB‐line profile across the vesicle indicated with arrow in (B); red-AP2A1; green-ATG9A; blue-TBC1D5.D Quantification of ATG9A and AP2 co‐localization. Cells were starved in EBSS media (45 min) or pretreated with Dynasore (15 min) and subsequently starved for 45 min in combination with Dynasore. Control cells were treated with DMSO.E Quantification of LC3 puncta in cells stably expressing HA‐Flag‐ATG9A, treated and presented as in (D). 100 cells were quantified in 3 independent experiments.F T‐REx HeLa cells were transfected with siRNA Control oligo or siRNA oligo#1 targeting (40 nM). 72 h post−transfection, expression of HA‐Flag−TBC1D5 was induced with doxycycline (50 ng/ml), and 96 h post−transfection cells were treated with Dynasore for 15 min, or pretreated with Dynasore and subsequently starved in combination with Dynasore for additional 45 min. Cells were lysed in co‐immunoprecipitation buffer, and lysates were incubated with Myc antibody or M2 antibody coupled with agarose overnight at 4°C. Beads were washed 3 times with incubation buffer and subjected to SDS‐PAGE.


Figure 1 ( A ) Clone P15 induces HA - LC3 lipidation . 293T cells were transfected with P15 plus plasmids expressing HA - LC3A and / or the apoptotic inhibitor p35 ( as shown ) , and lysed for western blotting against the indicated molecules ( anti ‐ HA for HA - LC3A ) . The figure shows that expression of P15 induces HA - LC3A conversion to a lower molecular weight form indicative of protein lipidation . This activity remains unchanged by p35 . ( B ) P15 induces GFP - LC3 translocation to a vesiculated pattern . 293T cells were transfected with the indicated plasmids mixed with vectors expressing GFP - LC3A and p35 . The known autophagic inducer bNIP3L constituted a positive control . Representative confocal microscopy pictures are shown . ( C ) TMEM59 induces HA - LC3 lipidation in different cell lines lacking the SV40 large T − antigen plasmid amplification system . Cells were transfected with the shown plasmids , vectors expressing HA - LC3A ( left ) or HA - LC3B ( right ) and GST ( as transfection control ) , and lysed for western blotting against the indicated molecules . ( D ) TMEM59 induces GFP - LC3 activation . 293 cells were transfected with the indicated plasmids and GFP - LC3A ( top ) or GFP - LC3B ( bottom ) . Representative confocal microscopy pictures are shown . ( E ) Quantification of the phenotype in D . The number of GFP - LC3 ‐ positive vesicles per transfected cell was scored for at least fifty cells . Data are expressed as means ± s . d . of one representative experiment of three repetitions . Source data for this figure is available on the online supplementary information page .

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Figure 1(A) Clone P15 induces HA-LC3 lipidation. 293T cells were transfected with P15 plus plasmids expressing HA-LC3A and/or the apoptotic inhibitor p35 (as shown), and lysed for western blotting against the indicated molecules (anti‐HA for HA-LC3A). The figure shows that expression of P15 induces HA-LC3A conversion to a lower molecular weight form indicative of protein lipidation. This activity remains unchanged by p35.(B) P15 induces GFP-LC3 translocation to a vesiculated pattern. 293T cells were transfected with the indicated plasmids mixed with vectors expressing GFP-LC3A and p35. The known autophagic inducer bNIP3L constituted a positive control. Representative confocal microscopy pictures are shown.(C) TMEM59 induces HA-LC3 lipidation in different cell lines lacking the SV40 large T−antigen plasmid amplification system. Cells were transfected with the shown plasmids, vectors expressing HA-LC3A (left) or HA-LC3B (right) and GST (as transfection control), and lysed for western blotting against the indicated molecules.(D) TMEM59 induces GFP-LC3 activation. 293 cells were transfected with the indicated plasmids and GFP-LC3A (top) or GFP-LC3B (bottom). Representative confocal microscopy pictures are shown.(E) Quantification of the phenotype in D. The number of GFP-LC3‐positive vesicles per transfected cell was scored for at least fifty cells. Data are expressed as means ±s.d. of one representative experiment of three repetitions.Source data for this figure is available on the online supplementary information page.


Figure 2 ( A ) TMEM59 ID is required for HA - LC3 lipidation . 293 cells were transfected with full ‐ length TMEM59 ( FL ) or a deleted version lacking the ID ( ΔID , Δ263 - 323 ) , HA - LC3A ( left ) or HA - LC3B ( right ) and GST . Cells were lysed for western blotting against the shown molecules . ( B ) The ID is necessary for GFP - LC3 activation . 293 cells were transfected with the indicated TMEM59 constructs and GFP - LC3A or GFP - LC3B ( as indicated ) . Representative confocal pictures are shown . ( C ) Quantification of the phenotype in B . Scoring and data expression were as in Figure 1E . ( D ) TMEM59 ID ( amino acids 263 - 323 ) suffices for HA - LC3A lipidation . 293 cells were transfected with the indicated CD16 : 7 chimera ( Control : empty chimera ) and vectors encoding HA - LC3A and GST , subjected to aggregation with the shown amounts of anti ‐ CD16 antibody and lysed for western blotting . The right panel shows control WBs demonstrating equal loading ( ACTIN ) , transfection ( GST ) and chimera expression ( CD16 ) in unaggregated samples , as all experimental points per chimera derive from a single transfection . ( E ) Comparable surface levels of CD16 : 7 constructs . Transfected 293 cells were processed for anti ‐ CD16 flow cytometry . The graph displays percentages of positive cells ( left axis ) and means of fluorescence of positive cells ( MF , right axis ) obtained from triplicates . Data are expressed as means ± s . d . of the triplicates . ( F ) The ID suffices for HA - LC3B lipidation . ( G ) The ID suffices for GFP - LC3 activation . 293 cells were transfected with the indicated chimeras and GFP - LC3A or GFP - LC3B , aggregated and mounted . Representative confocal microscopy pictures are shown . Activated GFP - LC3 appears as a collapsed mass of indiscernible vacuoles . ( H ) Quantification of the phenotype in G . As individual vesicles could not be counted , quantification was done as the percentage of transfected cells showing redistributed GFP - LC3 . At least 10 different fields were counted ( about 400 cells ) . The experiment was repeated three times . Data are expressed as means ± s . d . of the triplicates . Source data for this figure is available on the online supplementary information page .

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

Figure 2(A) TMEM59 ID is required for HA-LC3 lipidation. 293 cells were transfected with full‐length TMEM59 (FL) or a deleted version lacking the ID (ΔID, Δ263-323), HA-LC3A (left) or HA-LC3B (right) and GST. Cells were lysed for western blotting against the shown molecules.(B) The ID is necessary for GFP-LC3 activation. 293 cells were transfected with the indicated TMEM59 constructs and GFP-LC3A or GFP-LC3B (as indicated). Representative confocal pictures are shown.(C) Quantification of the phenotype in B. Scoring and data expression were as in Figure 1E.(D) TMEM59 ID (amino acids 263-323) suffices for HA-LC3A lipidation. 293 cells were transfected with the indicated CD16:7 chimera (Control: empty chimera) and vectors encoding HA-LC3A and GST, subjected to aggregation with the shown amounts of anti‐CD16 antibody and lysed for western blotting. The right panel shows control WBs demonstrating equal loading (ACTIN), transfection (GST) and chimera expression (CD16) in unaggregated samples, as all experimental points per chimera derive from a single transfection.(E) Comparable surface levels of CD16:7 constructs. Transfected 293 cells were processed for anti‐CD16 flow cytometry. The graph displays percentages of positive cells (left axis) and means of fluorescence of positive cells (MF, right axis) obtained from triplicates. Data are expressed as means ±s.d. of the triplicates.(F) The ID suffices for HA-LC3B lipidation.(G) The ID suffices for GFP-LC3 activation. 293 cells were transfected with the indicated chimeras and GFP-LC3A or GFP-LC3B, aggregated and mounted. Representative confocal microscopy pictures are shown. Activated GFP-LC3 appears as a collapsed mass of indiscernible vacuoles.(H) Quantification of the phenotype in G. As individual vesicles could not be counted, quantification was done as the percentage of transfected cells showing redistributed GFP-LC3. At least 10 different fields were counted (about 400 cells). The experiment was repeated three times. Data are expressed as means ±s.d. of the triplicates.Source data for this figure is available on the online supplementary information page.


Figure 3 ( A , B ) Ability of serial C ‐ terminal deletions of TMEM59 to activate LC3 . 293 cells were transfected with the indicated TMEM59 C ‐ terminal deletions , HA - LC3A ( A ) or HA - LC3B ( B ) and GST , and lysed for western blotting against the indicated molecules . ( C ) Amino acids 263 - 281 retain the full potential of TMEM59 ID to promote HA - LC3 conversion . 293 cells were transfected with the shown CD16 : 7 chimeras , HA - LC3A or HA - LC3B ( as indicated ) and GST , and subjected to anti ‐ CD16 aggregation before lysing them for western blotting . The lower panels show control WBs ( unaggregated samples ) . D Amino acids 263 - 281 suffice for GFP - LC3 activation and retain the full potential of TMEM59 ID to promote GFP - LC3 activation . 293 cells were transfected with the indicated CD16 : 7 chimeras and GFP - LC3A or GFP - LC3B ( as indicated ) , and subjected to anti ‐ CD16 aggregation before fixing them for microscopy . The graph shows percentages of transfected cells exhibiting redistributed GFP - LC3 . Quantification and data expression were done as in Figure 2H . ( E ) Amino acids 282 - 323 of TMEM59 lack LC3 activation potential . 293 cells were transfected with the shown chimeras , HA - LC3A or HA - LC3B and GST , and processed for western blotting against the indicated molecules . Control WBs of unaggregated samples are shown in the lower panels . ( F ) Surface expression levels of CD16 : 7 chimeras . Procedures and data expression were as in Figure 2E . The figure shows that the functional differences observed between CD16 : 7 chimeras ( C - E ) are not caused by differential surface expression .

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

Figure 3(A, B) Ability of serial C‐terminal deletions of TMEM59 to activate LC3. 293 cells were transfected with the indicated TMEM59 C‐terminal deletions, HA-LC3A (A) or HA-LC3B (B) and GST, and lysed for western blotting against the indicated molecules.(C) Amino acids 263-281 retain the full potential of TMEM59 ID to promote HA-LC3 conversion. 293 cells were transfected with the shown CD16:7 chimeras, HA-LC3A or HA-LC3B (as indicated) and GST, and subjected to anti‐CD16 aggregation before lysing them for western blotting. The lower panels show control WBs (unaggregated samples).D Amino acids 263-281 suffice for GFP-LC3 activation and retain the full potential of TMEM59 ID to promote GFP-LC3 activation. 293 cells were transfected with the indicated CD16:7 chimeras and GFP-LC3A or GFP-LC3B (as indicated), and subjected to anti‐CD16 aggregation before fixing them for microscopy. The graph shows percentages of transfected cells exhibiting redistributed GFP-LC3. Quantification and data expression were done as in Figure 2H.(E) Amino acids 282-323 of TMEM59 lack LC3 activation potential. 293 cells were transfected with the shown chimeras, HA-LC3A or HA-LC3B and GST, and processed for western blotting against the indicated molecules. Control WBs of unaggregated samples are shown in the lower panels.(F) Surface expression levels of CD16:7 chimeras. Procedures and data expression were as in Figure 2E. The figure shows that the functional differences observed between CD16:7 chimeras (C-E) are not caused by differential surface expression.


Figure 4 ( A ) Endocytic vesicles containing aggregated CD16 : 7 - 263 - 281 become labelled with GFP - LC3 . JAR cells were transfected with vectors expressing the indicated CD16 : 7 chimeras and GFP - LC3A , and subjected to anti ‐ CD16 aggregation for 8 h before staining for the endocytosed chimera ( red ) . Representative confocal pictures are shown . The inset highlights a vesicle where the peripheral GFP - LC3 staining is particularly distinguishable . The right panel displays control WBs showing that chimera aggregation for 8 h activates HA - LC3A conversion in JAR cells . ( B ) Colocalization of endocytosed CD16 : 7 - 263 - 281 with EEA1 and CD63 . JAR cells were transfected with CD16 : 7 - 263 - 281 and subjected to aggregation before staining them for the endocytosed chimera ( red ) and EEA1 or CD63 ( green ; FITC ‐ coupled primary antibodies ) , as indicated . Representative confocal microscopy pictures are shown . ( C ) Immunoelectron microscopy assay showing that endocytosed CD16 : 7 - 263 - 281 localizes to LC3 − labelled , single − membrane vesicles . JAR cells were transfected with the CD16 : 7 - 263 - 281 chimera and a construct expressing human IgG1 fused to LC3A . Cells were subjected to anti − CD16 aggregation for 8 h and processed for immunoelectron microscopy . Thick gold signal ( 18 nm ) : aggregated , endocytosed chimera ; thin gold signal ( 12 nm ) : IgG1 - LC3 . Arrows indicate single membrane ( black ) or IgG1 - LC3A ( white ) . Scale bar : 400 nm . ( D ) Lysosomal inhibition increases HA - LC3II levels promoted by CD16 : 7 - 263 - 281 . 293 cells were transfected with the indicated chimera and HA - LC3A , and subjected to anti − CD16 aggregation in the absence or presence of bafilomycin ( 200 nM , added 4 h post aggregation ) before lysing them for western blotting against the indicated molecules . ( E ) Aggregation of CD16 : 7 - 263 - 281 promotes its own degradation . 293 cells were transfected with the indicated constructs , aggregated and lysed for western blotting . Shown are overexposed anti ‐ CD16 WBs . ( F ) Degradation of CD16 : 7 - 263 - 281 is inhibited by ATG5 depletion . 293 cells were transfected with the indicated siRNAs and subsequently with the shown CD16 : 7 constructs , aggregated and lysed for anti ‐ CD16 western blotting ( left panel ) . The right panel displays control WBs showing ATG5 depletion . Source data for this figure is available on the online supplementary information page .

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

Figure 4(A) Endocytic vesicles containing aggregated CD16:7-263-281 become labelled with GFP-LC3. JAR cells were transfected with vectors expressing the indicated CD16:7 chimeras and GFP-LC3A, and subjected to anti‐CD16 aggregation for 8 h before staining for the endocytosed chimera (red). Representative confocal pictures are shown. The inset highlights a vesicle where the peripheral GFP-LC3 staining is particularly distinguishable. The right panel displays control WBs showing that chimera aggregation for 8 h activates HA-LC3A conversion in JAR cells.(B) Colocalization of endocytosed CD16:7-263-281 with EEA1 and CD63. JAR cells were transfected with CD16:7-263-281 and subjected to aggregation before staining them for the endocytosed chimera (red) and EEA1 or CD63 (green; FITC‐coupled primary antibodies), as indicated. Representative confocal microscopy pictures are shown.(C) Immunoelectron microscopy assay showing that endocytosed CD16:7-263-281 localizes to LC3−labelled, single−membrane vesicles. JAR cells were transfected with the CD16:7-263-281 chimera and a construct expressing human IgG1 fused to LC3A. Cells were subjected to anti−CD16 aggregation for 8 h and processed for immunoelectron microscopy. Thick gold signal (18 nm): aggregated, endocytosed chimera; thin gold signal (12 nm): IgG1-LC3. Arrows indicate single membrane (black) or IgG1-LC3A (white). Scale bar: 400 nm.(D) Lysosomal inhibition increases HA-LC3II levels promoted by CD16:7-263-281. 293 cells were transfected with the indicated chimera and HA-LC3A, and subjected to anti−CD16 aggregation in the absence or presence of bafilomycin (200 nM, added 4 h post aggregation) before lysing them for western blotting against the indicated molecules.(E) Aggregation of CD16:7-263-281 promotes its own degradation. 293 cells were transfected with the indicated constructs, aggregated and lysed for western blotting. Shown are overexposed anti‐CD16 WBs.(F) Degradation of CD16:7-263-281 is inhibited by ATG5 depletion. 293 cells were transfected with the indicated siRNAs and subsequently with the shown CD16:7 constructs, aggregated and lysed for anti‐CD16 western blotting (left panel). The right panel displays control WBs showing ATG5 depletion.Source data for this figure is available on the online supplementary information page.


Figure 5 ( A ) Identification of critical amino acids for HA - LC3 conversion . 293 cells were transfected with the indicated CD16 : 7 - 263 - 281 mutants and HA - LC3A , subjected to anti ‐ CD16 aggregation and lysed for western blotting against the indicated molecules . Asterisks mark mutations with reduced activity . Shown is one representative experiment of six repetitions . ( B ) Identification of critical amino acids for GFP - LC3 activation and colocalization with the endocytosed chimera . JAR cells were transfected with the indicated CD16 : 7 - 263 - 281 mutants and GFP - LC3A , aggregated and stained for the endocytosed chimera ( red ) . Preparations were scored by blindly counting the number of red vesicles ( chimera ) and green vesicles ( GFP - LC3A ) per cell , as well as the number of red vesicles colocalizing with green ones . The percentage of green vesicles colocalizing with red ones was close to 100 % for all mutants , that is , virtually no GFP - LC3A vesicles were unrelated to endocytosed chimera ( not shown ) . At least 50 cells were scored per experimental point . The experiment was repeated three times . The graph shows the number of GFP - LC3A vesicles per cell expressed as the percentage of the value obtained for the wild − type chimera ( left axis ) , and the percentage of chimera vesicles labelled with GFP - LC3A ( right axis ) . Data are expressed as means ± s . d . of the triplicates . Asterisks indicate significant differences with respect to wild − type values ( paired Student ' s t − test ; P < 0 . 01 ) . ( C ) Representative confocal microscopy pictures of the phenotype produced by the indicated mutations . Procedures were as in B . ( D ) Simultaneous mutation of the four essential amino acids to alanine blocks HA - LC3 conversion induced by CD16 : 7 - 263 - 281 . 293 cells were transfected with the indicated chimeras ( 4M , quadruple mutant ) and HA - LC3A , aggregated and lysed for western blotting . ( E ) Simultaneous mutation of the four essential amino acids blocks GFP - LC3 activation and colocalization with the endocytosed chimera . JAR cells were transfected with the indicated CD16 : 7 - 263 - 281 constructs and GFP - LC3A , aggregated and stained for the endocytosed chimera ( red ) . Representative confocal microscopy images are shown . ( F ) Quantification of the phenotype in E . Data gathering and expression were as in B . Source data for this figure is available on the online supplementary information page .

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

Figure 5(A) Identification of critical amino acids for HA-LC3 conversion. 293 cells were transfected with the indicated CD16:7-263-281 mutants and HA-LC3A, subjected to anti‐CD16 aggregation and lysed for western blotting against the indicated molecules. Asterisks mark mutations with reduced activity. Shown is one representative experiment of six repetitions.(B) Identification of critical amino acids for GFP-LC3 activation and colocalization with the endocytosed chimera. JAR cells were transfected with the indicated CD16:7-263-281 mutants and GFP-LC3A, aggregated and stained for the endocytosed chimera (red). Preparations were scored by blindly counting the number of red vesicles (chimera) and green vesicles (GFP-LC3A) per cell, as well as the number of red vesicles colocalizing with green ones. The percentage of green vesicles colocalizing with red ones was close to 100% for all mutants, that is, virtually no GFP-LC3A vesicles were unrelated to endocytosed chimera (not shown). At least 50 cells were scored per experimental point. The experiment was repeated three times. The graph shows the number of GFP-LC3A vesicles per cell expressed as the percentage of the value obtained for the wild−type chimera (left axis), and the percentage of chimera vesicles labelled with GFP-LC3A (right axis). Data are expressed as means ±s.d. of the triplicates. Asterisks indicate significant differences with respect to wild−type values (paired Student's t−test; P<0.01).(C) Representative confocal microscopy pictures of the phenotype produced by the indicated mutations. Procedures were as in B.(D) Simultaneous mutation of the four essential amino acids to alanine blocks HA-LC3 conversion induced by CD16:7-263-281. 293 cells were transfected with the indicated chimeras (4M, quadruple mutant) and HA-LC3A, aggregated and lysed for western blotting.(E) Simultaneous mutation of the four essential amino acids blocks GFP-LC3 activation and colocalization with the endocytosed chimera. JAR cells were transfected with the indicated CD16:7-263-281 constructs and GFP-LC3A, aggregated and stained for the endocytosed chimera (red). Representative confocal microscopy images are shown.(F) Quantification of the phenotype in E. Data gathering and expression were as in B.Source data for this figure is available on the online supplementary information page.


Figure 6 ( A ) Apposition events between GFP - ATG16L1 and endocytosed CD16 : 7 - 263 - 281 . JAR cells were transfected with CD16 : 7 - 263 - 281 and GFP - ATG16L1 or GFP - BECLIN ( as indicated ) , aggregated for 4 h and stained for the endocytosed chimera ( red ) . Representative confocal microscopy pictures are shown . Two different examples are provided for GFP - ATG16L1 . ( B - D ) 293T cells were transfected with the indicated constructs , lysed and subjected to GST immunoprecipitation using agarose beads coupled to glutathione ( IP , immunoprecipitation ; TL , total lysate ) . Shown are WBs against the indicated molecules . ( B ) AU - ATG16L1 co ‐ precipitates with wild ‐ type ( WT ) TMEM59 - GST but not with a mutated version where the four essential amino acids were mutated to alanine ( TMEM59 - 4M - GST ) . ( C ) Full ‐ length TMEM59 and TMEM59 - Δ282 co ‐ precipitate with GST - ATG16L1 , whereas the respective 4M versions do not . ( D ) AU - ATG16L1 co ‐ precipitates with a fusion protein between GST and the minimal active peptide of TMEM59 ( GST - 263 - 281 ) , but not with a 4M version ( GST - 263 - 281 - 4M ) or a GST fusion protein with the inactive portion of TMEM59 - ID ( GST - 282 - 323 ) . ( E ) HA - ATG16L1 expressed in bacteria co − precipitates with a GST - 263 - 281 recombinant protein purified from bacterial cultures , but not with a 4M version of the same construct or GST - 282 - 323 . The indicated GST partners were expressed in bacteria and purified using agarose beads coupled to glutathione . The loaded beads were then used for HA - ATG16L1 pull − down from crude bacterial lysates . Shown are WBs against the indicated molecules ( PD , pull − down ) . A Coomasie staining of a protein gel with the purified GST fusion proteins is shown . The right panel compares the amount of HA - ATG16L1 pulled down by GST - 263 - 281 with the signal provided by direct anti − HA immunoprecipitation . This result shows that about 20 - 25 % of the available HA - ATG16L1 protein is precipitated by GST - 263 - 281 . Asterisks indicate irrelevant bands in B and D . Source data for this figure is available on the online supplementary information page .

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

Figure 6(A) Apposition events between GFP-ATG16L1 and endocytosed CD16:7-263-281. JAR cells were transfected with CD16:7-263-281 and GFP-ATG16L1 or GFP-BECLIN (as indicated), aggregated for 4 h and stained for the endocytosed chimera (red). Representative confocal microscopy pictures are shown. Two different examples are provided for GFP-ATG16L1.(B-D) 293T cells were transfected with the indicated constructs, lysed and subjected to GST immunoprecipitation using agarose beads coupled to glutathione (IP, immunoprecipitation; TL, total lysate). Shown are WBs against the indicated molecules. (B) AU-ATG16L1 co‐precipitates with wild‐type (WT) TMEM59-GST but not with a mutated version where the four essential amino acids were mutated to alanine (TMEM59-4M-GST). (C) Full‐length TMEM59 and TMEM59-Δ282 co‐precipitate with GST-ATG16L1, whereas the respective 4M versions do not. (D) AU-ATG16L1 co‐precipitates with a fusion protein between GST and the minimal active peptide of TMEM59 (GST-263-281), but not with a 4M version (GST-263-281-4M) or a GST fusion protein with the inactive portion of TMEM59-ID (GST-282-323).(E) HA-ATG16L1 expressed in bacteria co−precipitates with a GST-263-281 recombinant protein purified from bacterial cultures, but not with a 4M version of the same construct or GST-282-323. The indicated GST partners were expressed in bacteria and purified using agarose beads coupled to glutathione. The loaded beads were then used for HA-ATG16L1 pull−down from crude bacterial lysates. Shown are WBs against the indicated molecules (PD, pull−down). A Coomasie staining of a protein gel with the purified GST fusion proteins is shown. The right panel compares the amount of HA-ATG16L1 pulled down by GST-263-281 with the signal provided by direct anti−HA immunoprecipitation. This result shows that about 20-25% of the available HA-ATG16L1 protein is precipitated by GST-263-281. Asterisks indicate irrelevant bands in B and D.Source data for this figure is available on the online supplementary information page.


Figure 7 ( A - F ) 293T cells were transfected with the indicated constructs , lysed and subjected to GST immunoprecipitation with agarose beads coupled to glutathione . Shown are WBs against the indicated molecules . ( A ) A deleted version of ATG16L1 lacking the WD domain ( HA - ATG16L1 - ΔWD ) does not co ‐ precipitate with TMEM59 - GST . ( B ) TMEM59 does not co ‐ precipitate with ATG16L1 - ΔWD fused to GST . ( C ) The WD domain of ATG16L1 ( HA - ATG16L1 - WD ) suffices to co ‐ precipitate with TMEM59 - GST . ( D ) TMEM59 co ‐ precipitates with ATG16L1 - WD fused to GST . ( E ) HA - ATG16L1 - WD does not bind a 4M version of TMEM59 - GST . ( F ) TMEM59 - 4M does not co ‐ precipitate with the ATG16L1 - WD fused to GST . Source data for this figure is available on the online supplementary information page .

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

Figure 7(A-F) 293T cells were transfected with the indicated constructs, lysed and subjected to GST immunoprecipitation with agarose beads coupled to glutathione. Shown are WBs against the indicated molecules. (A) A deleted version of ATG16L1 lacking the WD domain (HA-ATG16L1-ΔWD) does not co‐precipitate with TMEM59-GST. (B) TMEM59 does not co‐precipitate with ATG16L1-ΔWD fused to GST. (C) The WD domain of ATG16L1 (HA-ATG16L1-WD) suffices to co‐precipitate with TMEM59-GST. (D) TMEM59 co‐precipitates with ATG16L1-WD fused to GST. (E) HA-ATG16L1-WD does not bind a 4M version of TMEM59-GST. (F) TMEM59-4M does not co‐precipitate with the ATG16L1-WD fused to GST.Source data for this figure is available on the online supplementary information page.


Figure 8 ( C , D , E , G , H ) 293T cells were transfected with the indicated constructs , lysed and subjected to GST immunoprecipitation with agarose beads coupled to glutathione . Shown are WBs against the indicated molecules . ( C ) The N ‐ terminal CARD of NOD2 ( NOD2 - CARD1 - HA ) , but not the CARD of NOD1 ( NOD1 - CARD - HA ) , co ‐ precipitates with GST - ATG16L1 . ( D ) The ID of TLR2 ( HA - TLR2 - ID ) co ‐ precipitates with GST - ATG16L1 . ( E ) Mutated versions ( MUT ) of NOD2 - CARD1 - HA and HA - TLR2 - ID ( as shown ) do not co ‐ precipitate with GST - ATG16L1 . ( F ) Amino ‐ acid sequences of T3JAM and DEDD2 including the motif . Residues identified by the Prosite algorithm are highlighted . ( G ) Wild ‐ type ( WT ) versions of HA - T3JAM and HA - DEDD2 ( as indicated ) co ‐ precipitate with GST - ATG16L1 . ( H ) Mutated versions ( MUT ) of HA - T3JAM and HA - DEDD2 do not co ‐ precipitate with GST - ATG16L1 . Asterisks indicate irrelevant bands in C , D and G . Source data for this figure is available on the online supplementary information page .

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

Figure 8(C, D, E, G, H) 293T cells were transfected with the indicated constructs, lysed and subjected to GST immunoprecipitation with agarose beads coupled to glutathione. Shown are WBs against the indicated molecules. (C) The N‐terminal CARD of NOD2 (NOD2-CARD1-HA), but not the CARD of NOD1 (NOD1-CARD-HA), co‐precipitates with GST-ATG16L1. (D) The ID of TLR2 (HA-TLR2-ID) co‐precipitates with GST-ATG16L1. (E) Mutated versions (MUT) of NOD2-CARD1-HA and HA-TLR2-ID (as shown) do not co‐precipitate with GST-ATG16L1. (F) Amino‐acid sequences of T3JAM and DEDD2 including the motif. Residues identified by the Prosite algorithm are highlighted. (G) Wild‐type (WT) versions of HA-T3JAM and HA-DEDD2 (as indicated) co‐precipitate with GST-ATG16L1. (H) Mutated versions (MUT) of HA-T3JAM and HA-DEDD2 do not co‐precipitate with GST-ATG16L1. Asterisks indicate irrelevant bands in C, D and G.Source data for this figure is available on the online supplementary information page.


Figure 9 ( A ) The right panel shows successful TMEM59 depletion ( rabbit anti ‐ TMEM59immunoprecipitation plus chicken anti ‐ TMEM59WB ) . ( A ) Depletion of TMEM59 inhibits LC3II generation by SA at early infection times . HeLa cells were transfected with the indicated siRNAs and , 48 h later , infected with the bacteria ( SA ) for the shown times before lysing them for western blotting against the indicated molecules . The right panel shows successful TMEM59 depletion ( rabbit anti ‐ TMEM59 immunoprecipitation plus chicken anti ‐ TMEM59 WB ) . ( B ) Colocalization events between bacteria ( GFP labelled ) , TMEM59 - HA and Cherry - LC3A in infected cells . HeLa cells stably expressing TMEM59 - HA and Cherry - LC3A were infected for the indicated times ( moi = 10 ) , and processed for anti ‐ HA immunofluorescence . Representative confocal images are shown . The GFP , anti ‐ HA ( Cy5 ) and DAPI signals were pseudocolored in blue , green and white , respectively . Asterisks indicate overlay images : ( * ) , Cherry - LC3A / TMEM59 ; ( * * ) , Cherry - LC3A / TMEM59 / bacteria ; ( * * * ) , Cherry - LC3A / TMEM59 / bacteria / DAPI . ( C ) Colocalization between bacteria , TMEM59 - HA and Cherry - ATG16L1 . HeLa cells stably expressing TMEM59 - HA and Cherry - ATG16L1 were infected with SA ( 2 h , moi = 10 ) , and processed for anti ‐ HA immunofluorescence . Shown are confocal pictures displaying two representative examples . Asterisks indicate overlays as in B . ( D ) SA promotes co ‐ precipitation of endogenous ATG16L1 with endogenous TMEM59 . HeLa cells were infected with SA ( 2 h , moi = 10 ) , lysed and subjected to immunoprecipitation with anti ‐ TMEM59 antibodies or irrelevant protein G beads ( as indicated ) . Immunoprecipitates were processed for western blotting . The right panel shows control WBs of the lysates used for immunoprecipitation . ( E ) Depletion of TMEM59 reduces recovery of SA from infected cells . HeLa cells were transfected with the indicated siRNAs , infected with SA 96 h later ( 5 h , moi = 0 . 1 ) and lysed for CFU evaluation . The experiment was carried out three independent times . The right panel shows successful TMEM59 depletion ( anti ‐ TMEM59 immunoprecipitation plus anti ‐ TMEM59 WB ) . Source data for this figure is available on the online supplementary information page . ( E ) Depletion of TMEM59 reduces recovery of SA from infected cells . HeLa cells were transfected with the indicated siRNAs , infected with SA 96 h later ( 5 h , moi = 0 . 1 ) and lysed for CFU evaluation . The experiment was carried out three independent times . The graph displays average CFU counts obtained for the 10 − 1 dilution of the relevant extracts , ± s . d . of the three data sets . Asterisks indicate significant differences ( P < 0 . 01 , paired Student ' s t ‐ test ) .

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

Figure 9(A) The right panel shows successful TMEM59 depletion (rabbit anti‐TMEM59immunoprecipitation plus chicken anti‐TMEM59WB).(A) Depletion of TMEM59 inhibits LC3II generation by SA at early infection times. HeLa cells were transfected with the indicated siRNAs and, 48 h later, infected with the bacteria (SA) for the shown times before lysing them for western blotting against the indicated molecules. The right panel shows successful TMEM59 depletion (rabbit anti‐TMEM59 immunoprecipitation plus chicken anti‐TMEM59 WB).(B) Colocalization events between bacteria (GFP labelled), TMEM59-HA and Cherry-LC3A in infected cells. HeLa cells stably expressing TMEM59-HA and Cherry-LC3A were infected for the indicated times (moi=10), and processed for anti‐HA immunofluorescence. Representative confocal images are shown. The GFP, anti‐HA (Cy5) and DAPI signals were pseudocolored in blue, green and white, respectively. Asterisks indicate overlay images: (*), Cherry-LC3A/TMEM59; (**), Cherry-LC3A/TMEM59/bacteria; (***), Cherry-LC3A/TMEM59/bacteria/DAPI.(C) Colocalization between bacteria, TMEM59-HA and Cherry-ATG16L1. HeLa cells stably expressing TMEM59-HA and Cherry-ATG16L1 were infected with SA (2 h, moi=10), and processed for anti‐HA immunofluorescence. Shown are confocal pictures displaying two representative examples. Asterisks indicate overlays as in B.(D) SA promotes co‐precipitation of endogenous ATG16L1 with endogenous TMEM59. HeLa cells were infected with SA (2 h, moi=10), lysed and subjected to immunoprecipitation with anti‐TMEM59 antibodies or irrelevant protein G beads (as indicated). Immunoprecipitates were processed for western blotting. The right panel shows control WBs of the lysates used for immunoprecipitation.(E) Depletion of TMEM59 reduces recovery of SA from infected cells. HeLa cells were transfected with the indicated siRNAs, infected with SA 96 h later (5 h, moi=0.1) and lysed for CFU evaluation. The experiment was carried out three independent times. The right panel shows successful TMEM59 depletion (anti‐TMEM59 immunoprecipitation plus anti‐TMEM59 WB).Source data for this figure is available on the online supplementary information page.(E) Depletion of TMEM59 reduces recovery of SA from infected cells. HeLa cells were transfected with the indicated siRNAs, infected with SA 96 h later (5 h, moi=0.1) and lysed for CFU evaluation. The experiment was carried out three independent times. The graph displays average CFU counts obtained for the 10−1 dilution of the relevant extracts, ±s.d. of the three data sets. Asterisks indicate significant differences (P<0.01, paired Student's t‐test).


Figure 2Evaluation of expression by RNA - Seq and number of peptides required to detect an annotated protein by MS in M . pneumoniae . The plot represents the relationship between expression levels ( average expression from RNA - Seq data ) and number of possible unique tryptic peptides ( UTPs ) for two sets of studied proteins : detected ( blue dots ) and not detected ( orange dots ) by MS . B ) Evaluation of thresholds and artefactual signals in MS data . The histogram represents the total number of SEP proteins detected in 116 shotgun MS experiments with 1 UTP , 1 UTP and 1NUTP , or ≥ 2 UTPs for three categories . Color code : annotated ( blue bars ) , putative new ( orange bars ) , and decoy set ( gray bars ) . C ) Number of SEPs detected by increasing the number of experiments . Color code is the same as in panels B . Each line represents the accumulated number of different SEPs detected ( Y - axis ) when combining 1 to 116 MS datasets ( X - axis ) from M . pneumoniae . Each line has an associated error that is shaded and represents the standard deviation within combinations of datasets ( e . g . , x = 80 will present the average number of proteins detected taking every combination of datasets in groups of 80 samples ) .

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Figure 2Evaluation of expression by RNA-Seq and number of peptides required to detect an annotated protein by MS in M. pneumoniae. The plot represents the relationship between expression levels (average expression from RNA-Seq data) and number of possible unique tryptic peptides (UTPs) for two sets of studied proteins: detected (blue dots) and not detected (orange dots) by MS. B) Evaluation of thresholds and artefactual signals in MS data. The histogram represents the total number of SEP proteins detected in 116 shotgun MS experiments with 1 UTP, 1 UTP and 1NUTP, or ≥2 UTPs for three categories. Color code: annotated (blue bars), putative new (orange bars), and decoy set (gray bars). C) Number of SEPs detected by increasing the number of experiments. Color code is the same as in panels B. Each line represents the accumulated number of different SEPs detected (Y-axis) when combining 1 to 116 MS datasets (X-axis) from M. pneumoniae. Each line has an associated error that is shaded and represents the standard deviation within combinations of datasets (e.g., x=80 will present the average number of proteins detected taking every combination of datasets in groups of 80 samples).


Figure 1 ( A ) PolyP accumulation in vivo . The polyP content of wild - type cells was set to 100 % . Knockout of Vtc1 , Vtc2 , Vtc3 or Vtc4 impacts cellular polyP levels . Data show the mean ± s . d ( biological replicates n = 3 ) . PolyP synthesis curves of purified endogenous Vtc4 / Vtc3 / Vtc1 and Vtc4 / Vtc2 / Vtc1 complexes at different ATP concentrations in the absence or presence of 10 mM InsP6 in vitro . PolyP synthesis by isolated vacuoles carrying VTC complexes in the absence or presence of 1 µM 5 - InsP7 in vitro .

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

Figure 1(A) PolyP accumulation in vivo. The polyP content of wild-type cells was set to 100%. Knockout of Vtc1, Vtc2, Vtc3 or Vtc4 impacts cellular polyP levels. Data show the mean±s.d (biological replicates n=3).PolyP synthesis curves of purified endogenous Vtc4/Vtc3/Vtc1 and Vtc4/Vtc2/Vtc1 complexes at different ATP concentrations in the absence or presence of 10 mM InsP6 in vitro.PolyP synthesis by isolated vacuoles carrying VTC complexes in the absence or presence of 1 µM 5-InsP7 in vitro.


Figure 3Profile of the pore radius of the Vtc4 / Vtc3 / Vtc1 complex .

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Figure 3Profile of the pore radius of the Vtc4/Vtc3/Vtc1 complex.


Figure 4Cellular polyP content of Vtc4p point mutants expressed under the control of their native promoters in the vtc4Δ background . Data show the mean ± s . d ( biological replicates n = 3 ) . Cellular polyP content of VTC4 and VTC3 point mutants expressed under the control of their native promoters in the vtc4Δ and vtc3Δ ( vtc2Δ ) backgrounds , respectively . Data show the mean ± s . d ( biological replicates n = 3 ) . Cellular polyP content of VTC4 point mutants expressed under the control of their native promoters in the vtc4Δ backgrounds . Data show the mean ± s . d ( biological replicates n = 3 ) .

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

Figure 4Cellular polyP content of Vtc4p point mutants expressed under the control of their native promoters in the vtc4Δ background. Data show the mean±s.d (biological replicates n=3).Cellular polyP content of VTC4 and VTC3 point mutants expressed under the control of their native promoters in the vtc4Δ and vtc3Δ(vtc2Δ) backgrounds, respectively. Data show the mean±s.d (biological replicates n=3).Cellular polyP content of VTC4 point mutants expressed under the control of their native promoters in the vtc4Δ backgrounds. Data show the mean±s.d (biological replicates n=3).


Figure 5PolyP synthesis by isolated vacuoles carrying Vtc4 ( ΔSPX ) / Vtc3 / Vtc1 complex , Vtc4 / Vtc3 ( ΔSPX ) / Vtc1 complex or Vtc4 / Vtc3 / Vtc1 complex in the absence or presence of 1 μM 5 - InsP7 or 1 , 5 - InsP8 in vitro . The reaction system is detailed in Methods . Data show the mean ± s . d ( biological replicates n = 3 ) . PolyP synthesis by isolated vacuoles carrying Vtc4 / Vtc3 ( R223E ) / Vtc1 complex , Vtc4 / Vtc3 ( R226E ) / Vtc1 complex or Vtc4 / Vtc3 / Vtc1 complex in the absence or presence of 1 μM 5 - InsP7 or 1 , 5 - InsP8 in vitro . The reaction system is detailed in Methods . Data show the mean ± s . d ( biological replicates n = 3 ) .

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

Figure 5PolyP synthesis by isolated vacuoles carrying Vtc4(ΔSPX)/Vtc3/Vtc1 complex, Vtc4/Vtc3(ΔSPX)/Vtc1 complex or Vtc4/Vtc3/Vtc1 complex in the absence or presence of 1 μM 5-InsP7 or 1,5-InsP8 in vitro. The reaction system is detailed in Methods. Data show the mean±s.d (biological replicates n=3).PolyP synthesis by isolated vacuoles carrying Vtc4/Vtc3(R223E)/Vtc1 complex, Vtc4/Vtc3(R226E)/Vtc1 complex or Vtc4/Vtc3/Vtc1 complex in the absence or presence of 1 μM 5-InsP7 or 1,5-InsP8 in vitro. The reaction system is detailed in Methods. Data show the mean±s.d (biological replicates n=3).


Figure 6PolyP synthesis curves of purified endogenous Vtc4 / Vtc3 / Vtc1 and truncated Vtc4 / Vtc3 ( Δlp ) / Vtc1 complexes at different ATP concentrations in the absence or presence of 10 mM InsP6 in vitro . The reaction system is detailed in Methods . Data show the mean ± s . d ( biological replicates n = 3 ) . ( D ) PolyP synthesis by isolated vacuoles carrying Vtc4 / Vtc3 ( CL2 - Ala6 ) / Vtc1 complex , Vtc4 / Vtc3 ( CL2 - Asp6 ) / Vtc1 complex or Vtc4 / Vtc3 / Vtc1 complex in the absence or presence of 1 μM 5 - InsP7 or 1 , 5 - InsP8 in vitro . The reaction system is detailed in Methods . Data show the mean ± s . d ( biological replicates n = 3 ) .

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

Figure 6PolyP synthesis curves of purified endogenous Vtc4/Vtc3/Vtc1 and truncated Vtc4/Vtc3(Δlp)/Vtc1 complexes at different ATP concentrations in the absence or presence of 10 mM InsP6 in vitro. The reaction system is detailed in Methods. Data show the mean±s.d (biological replicates n=3). (D) PolyP synthesis by isolated vacuoles carrying Vtc4/Vtc3(CL2-Ala6)/Vtc1 complex, Vtc4/Vtc3(CL2-Asp6)/Vtc1 complex or Vtc4/Vtc3/Vtc1 complex in the absence or presence of 1 μM 5-InsP7 or 1,5-InsP8 in vitro. The reaction system is detailed in Methods. Data show the mean±s.d (biological replicates n=3).


Figure 7Cellular polyP content of VTC4 , VTC3 and VTC1 point mutants expressed under the control of their native promoters in the vtc4Δ , vtc3Δ ( vtc2Δ ) and vtc1Δ backgrounds , respectively . Δ indicates that the entire subunit was knocked out . FL indicates full length , indicating that the subunit has not been modified in any way . Data show the mean ± s . d ( biological replicates n = 3 ) .

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Figure 7Cellular polyP content of VTC4, VTC3 and VTC1 point mutants expressed under the control of their native promoters in the vtc4Δ, vtc3Δ(vtc2Δ) and vtc1Δ backgrounds, respectively. Δ indicates that the entire subunit was knocked out. FL indicates full length, indicating that the subunit has not been modified in any way. Data show the mean±s.d (biological replicates n=3).


Figure 1 ( C ) Representative images of HeLa cells transiently transfected with GST - GFP - CENP - E2605 - 2701 and immunostained with PRC1 and CENP - C , scale bar 10μm ( n = 4 ) . ( E ) Top , Representative images of live HeLa cells in cytokinesis transiently transfected with GFP - CENP - E2605 - 2701 and mutants ( green ) , incubated with SiR - tubulin ( red ) . Scale bar , 10μm . Bottom , linescans showing the fluorescence intensity average and standard error of the mean ( SEM ) for the GFP - CENP - E2605 - 2701 and mutants and tubulin across the cell midbody . For GFP - CENP - E2605 - 2701 , n is the number of cells . n = 11 and for the GFP - CENP - E2605 - 2701 mutants FDN2661LTT , YF2660AA , FF2644AA , n = 15 , 16 and 25 respectively . Biological independent replicates were respectively 2 , 3 , 2 and 1 .

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

Figure 1(C) Representative images of HeLa cells transiently transfected with GST-GFP-CENP-E2605-2701 and immunostained with PRC1 and CENP-C, scale bar 10μm (n=4).(E) Top, Representative images of live HeLa cells in cytokinesis transiently transfected with GFP-CENP-E2605-2701 and mutants (green), incubated with SiR-tubulin (red). Scale bar, 10μm. Bottom, linescans showing the fluorescence intensity average and standard error of the mean (SEM) for the GFP-CENP-E2605-2701 and mutants and tubulin across the cell midbody. For GFP-CENP-E2605-2701, n is the number of cells. n=11 and for the GFP-CENP-E2605-2701 mutants FDN2661LTT, YF2660AA, FF2644AA, n= 15, 16 and 25 respectively. Biological independent replicates were respectively 2, 3, 2 and 1.


Figure 2 ( A ) Top . Size - exclusion chromatography elution profile of MBP - CENP - E2605 - 2701 ( green ) , PRC11 - 168 ( pink ) and MBP - CENP - E2605 - 2701 / PRC11 - 168 ( orange ) . Bottom , Coomassie - stained gel showing the size - exclusion chromatography profile of PRC11 - 168 ( pink ) , MBP - CENP - E2605 - 2701 ( green ) and MBP - CENP - E2605 - 2701 / PRC11 - 168 ( orange ) . A shift in the elution volume was only seen in the presence of both CENP - E and PRC1 . ( B ) Top . Size - exclusion chromatography elution profile of MBP - CENP - E2605 - 2701FDN ( green ) and MBP - CENP - E2605 - 2701 FDN / PRC11 - 168 ( orange ) . Bottom , Coomassie - stained gel showing the size - exclusion chromatography profile of MBP - CENP - E2605 - 2701FDN ( green ) and MBP - CENP - E2605 - 2701 FDN / PRC11 - 168 ( orange ) . No shift in the elution profile was observed .

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Figure 2(A) Top. Size-exclusion chromatography elution profile of MBP-CENP-E2605-2701 (green), PRC11-168 (pink) and MBP-CENP-E2605-2701/PRC11-168 (orange). Bottom, Coomassie-stained gel showing the size-exclusion chromatography profile of PRC11-168 (pink), MBP-CENP-E2605-2701 (green) and MBP-CENP-E2605-2701/PRC11-168 (orange). A shift in the elution volume was only seen in the presence of both CENP-E and PRC1. (B) Top. Size-exclusion chromatography elution profile of MBP-CENP-E2605-2701FDN (green) and MBP-CENP-E2605-2701 FDN/PRC11-168 (orange). Bottom, Coomassie-stained gel showing the size-exclusion chromatography profile of MBP-CENP-E2605-2701FDN (green) and MBP-CENP-E2605-2701 FDN/PRC11-168 (orange). No shift in the elution profile was observed.


Figure 3 ( B ) Representative images of live HeLa cells in mitosis transiently transfected with either GFP - Kif4A1133 - 1165 or GFP - Kif4A1133 - 1232 incubated with SiR - Tubulin . Scale bar , 10μm . Quantification of cells with GFP - Kif4A1133 - 1165 ( n = 12 ) or GFP - Kif4A1133 - 1232 ( n = 13 ) localization to the central spindle . Data represented from 2 independent experiments . ( C ) A shift in the elution volume was only seen in the presence of both Kif4A and PRC1 . Size - exclusion chromatography elution profile of GST - Kif4A1133 - 1232 ( green ) and PRC11 - 168 ( pink ) , or together ( orange ) . Bottom , Coomassie - stained gel showing the size - exclusion chromatography profile of GST - Kif4A1133 - 1232 ( green ) , PRC11 - 168 ( pink ) and GST - Kif4A1133 - 1232 / PRC11 - 168 ( orange ) .

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

Figure 3(B) Representative images of live HeLa cells in mitosis transiently transfected with either GFP-Kif4A1133-1165 or GFP-Kif4A1133-1232 incubated with SiR-Tubulin. Scale bar, 10μm. Quantification of cells with GFP-Kif4A1133-1165 (n=12) or GFP-Kif4A1133-1232 (n=13) localization to the central spindle. Data represented from 2 independent experiments.(C) A shift in the elution volume was only seen in the presence of both Kif4A and PRC1. Size-exclusion chromatography elution profile of GST-Kif4A1133-1232 (green) and PRC11-168 (pink), or together (orange). Bottom, Coomassie-stained gel showing the size-exclusion chromatography profile of GST-Kif4A1133-1232 (green), PRC11-168(pink) and GST-Kif4A1133-1232/PRC11-168 (orange).


Figure 4 ( A ) Representative images of GFP - PRC1 ( magenta ) mixed with rhodamine - microtubules . Fire blue - green intensity LUT used to show tubulin intensity and microtubule overlaps . ( B ) Representative images of 647GST - CENP - E2639 - 2671 ( yellow ) mixed with rhodamine - microtubules . ( D ) Representative immunofluorescence images of HeLa cells stained for DNA , microtubules , PRC1 and with Alexa647 - labelled GST - CENP - E2639 - 2671 showing it also recognizes PRC1 in cells . Experiments were repeated > 3 times . ( F ) Representative kymograph showing microtubule - microtubule sliding in the presence of 2 . 5 nM PRC1 and 50 nM full - length CENP - E . ( G ) Example kymograph showing a free microtubule sliding until reaching the end of the immobilized microtubule where it slows down to a stall .

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

Figure 4(A) Representative images of GFP-PRC1 (magenta) mixed with rhodamine-microtubules. Fire blue-green intensity LUT used to show tubulin intensity and microtubule overlaps. (B) Representative images of 647GST-CENP-E2639-2671 (yellow) mixed with rhodamine-microtubules.(D) Representative immunofluorescence images of HeLa cells stained for DNA, microtubules, PRC1 and with Alexa647-labelled GST-CENP-E2639-2671 showing it also recognizes PRC1 in cells. Experiments were repeated >3 times.(F) Representative kymograph showing microtubule-microtubule sliding in the presence of 2.5 nM PRC1 and 50 nM full-length CENP-E. (G) Example kymograph showing a free microtubule sliding until reaching the end of the immobilized microtubule where it slows down to a stall.


Figure 5 ( D ) Live - cell imaging of metaphase spindles in HeLa cells transiently transfected wild - type , phosphomimetic and non - phosphorylatable mutants of GFP - CENP - E2605 - 2701 ( monomeric ) and GFP - GST - CENP - E2605 - 2701 ( dimeric ) and stained for tubulin using SiR - Tubulin . The fraction of cells localizing to the overlapping microtubules is represented as a percentage . Scalebar , 10 μm . ( G ) Bar graph showing mean and standard error for GFP fluorescence intensity at peak fluorescence for GFP - GST - CENP - E2605 - 2701 and mutants at 9 . 7μm , quantified in F . Asterisks indicate ordinary One - way Anova test significance value . * * * * P < 0 . 0001 .

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Figure 5(D) Live-cell imaging of metaphase spindles in HeLa cells transiently transfected wild-type, phosphomimetic and non-phosphorylatable mutants of GFP-CENP-E2605-2701 (monomeric) and GFP-GST-CENP-E2605-2701 (dimeric) and stained for tubulin using SiR-Tubulin. The fraction of cells localizing to the overlapping microtubules is represented as a percentage. Scalebar, 10 μm.(G) Bar graph showing mean and standard error for GFP fluorescence intensity at peak fluorescence for GFP-GST-CENP-E2605-2701 and mutants at 9.7μm, quantified in F. Asterisks indicate ordinary One-way Anova test significance value. ****P<0.0001.


Figure 6 ( B , C ) Elution profile from a size - exclusion chromatography ( SEC , black line , left y - axis ) run with subsequent multi - angle light scattering ( MALS , right y - axis ) analysis for PRC11 - 168 and PRC11 - 168 MEE . Outcome of the MALS analysis for the peak is presented in pink ( molecular weight , right y - axis ) . ( D ) Fire purple - yellow intensity LUT used to show tubulin intensity and microtubule overlaps . Microtubules are shown alone or incubated with 2 . 5 nM full - length PRC1 WT or PRC1 MEE . Scalebar , 10 μm . Experiment was replicated 2 times .

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

Figure 6(B, C) Elution profile from a size-exclusion chromatography (SEC, black line, left y-axis) run with subsequent multi-angle light scattering (MALS, right y-axis) analysis for PRC11-168 and PRC11-168 MEE. Outcome of the MALS analysis for the peak is presented in pink (molecular weight, right y-axis).(D) Fire purple-yellow intensity LUT used to show tubulin intensity and microtubule overlaps. Microtubules are shown alone or incubated with 2.5 nM full-length PRC1 WT or PRC1 MEE. Scalebar, 10 μm. Experiment was replicated 2 times.


Figure 7 ( A ) Representative immunofluorescence images of HeLa cells in late anaphase and telophase , expressing GFP - PRC1 - WT or - MEE and depleted for endogenous PRC1 using a PRC1 siRNA and a PRC1 sgRNA after doxycycline - induced Cas 9 expression . Microtubules , GFP and CENP - E are in white , green and red respectively . DNA is in blue . ( B ) Graph showing the percentage of cells with a PRC1 - localized at the site of abscission for cells treated in ( A ) . Mean and standard deviation are presented , n = 156 and 141 for cells expressing PRC1 - WT and - MEE respectively . Data from 4 biological replicates and 1 technical replicate . Asterisks indicate a T - test significance value . * * * * P < 0 . 0001 . ( C ) Graph showing the percentage of cells with CENP - E localized at the abscission site for cells treated in ( A ) , mean and standard deviation are presented , n = 58 and 66 for cells expressing PRC1 - WT and - MEE respectively . Data from 2 biological replicates . p - value ( p = 0 . 11 ) calculated for an unpaired T - test . ( G ) Representative live - cell images of HeLa cells expressing GFP - PRC1 - WT or - MEE and depleted for endogenous PRC1 using a PRC1 siRNA and a PRC1 sgRNA after doxycycline - induced Cas 9 expression . Microtubules ( SiR - Tubulin ) and DNA ( SPY650 ) are shown in red . The cell membrane ( CellMask ) is highlighted in white and GFP - PRC1 is in green . Scalebar , 10 μm . ( J ) Representative immunofluorescence images of cells expressing GFP - PRC1 - WT or - MEE after 72 hours siRNA depletion and doxycycline - induced knockout of endogenous PRC1 . DNA , microtubules and GFP - PRC1 / GFP - PRC1 - MEE are in blue , red and green respectively . Scalebar , 10μm .

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

Figure 7(A) Representative immunofluorescence images of HeLa cells in late anaphase and telophase, expressing GFP-PRC1-WT or -MEE and depleted for endogenous PRC1 using a PRC1 siRNA and a PRC1 sgRNA after doxycycline-induced Cas 9 expression. Microtubules, GFP and CENP-E are in white, green and red respectively. DNA is in blue. (B) Graph showing the percentage of cells with a PRC1-localized at the site of abscission for cells treated in (A). Mean and standard deviation are presented, n= 156 and 141 for cells expressing PRC1-WT and -MEE respectively. Data from 4 biological replicates and 1 technical replicate. Asterisks indicate a T-test significance value. ****P<0.0001. (C) Graph showing the percentage of cells with CENP-E localized at the abscission site for cells treated in (A), mean and standard deviation are presented, n= 58 and 66 for cells expressing PRC1-WT and -MEE respectively. Data from 2 biological replicates. p-value (p=0.11) calculated for an unpaired T-test.(G) Representative live-cell images of HeLa cells expressing GFP-PRC1-WT or -MEE and depleted for endogenous PRC1 using a PRC1 siRNA and a PRC1 sgRNA after doxycycline-induced Cas 9 expression. Microtubules (SiR-Tubulin) and DNA (SPY650) are shown in red. The cell membrane (CellMask) is highlighted in white and GFP-PRC1 is in green. Scalebar, 10 μm.(J) Representative immunofluorescence images of cells expressing GFP-PRC1-WT or -MEE after 72 hours siRNA depletion and doxycycline-induced knockout of endogenous PRC1. DNA, microtubules and GFP-PRC1/GFP-PRC1-MEE are in blue, red and green respectively. Scalebar, 10μm.


Figure 1G . Endogenous MITOL enhances the K48 - polyubiquitin chains of Parkin . WT or MITOL KO HeLa cells stably expressing HA - Parkin were treated with CCCP ( 10 µM ) for indicated time . MG132 ( 30 μM ) was added 3 h after CCCP treatment . Lysates of cells were fractionated into cytosolic , mitochondrial and ER fractions , and then subjected to an IP - IB assay with the indicated antibodies . K48 - polyubiquitin chains were immunoblotted with K48 - specific ubiquitin antibody .

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Figure 1G. Endogenous MITOL enhances the K48-polyubiquitin chains of Parkin. WT or MITOL KO HeLa cells stably expressing HA-Parkin were treated with CCCP (10 µM) for indicated time. MG132 (30 μM) was added 3 h after CCCP treatment. Lysates of cells were fractionated into cytosolic, mitochondrial and ER fractions, and then subjected to an IP-IB assay with the indicated antibodies. K48-polyubiquitin chains were immunoblotted with K48-specific ubiquitin antibody.


Figure 2E . MITOL regulates mitophagy . MITOL WT HeLa cells , MITOL KO HeLa cells and KO HeLa cells transfected with MITOL WT plasmids 0 . 5µg ( + ) and 5µg ( + + ) stably expressing HA ‐ Parkin and mt ‐ Keima were treated with CCCP ( 10µM ) for 8h . Then , mKeima was measured at 488 ( pH 7 ) and 561 ( pH 4 ) nm lasers using Flow Cytometer . Percentages of mitophagy were calculated from 30 , 000 cells in each independent experiment . Data represent the mean ± SD of three independent experiments . For statistical analysis , a one - way ANOVA with Tukey post - test was performed , * * P < 0 . 01 .

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Figure 2E. MITOL regulates mitophagy. MITOL WT HeLa cells, MITOL KO HeLa cells and KO HeLa cells transfected with MITOL WT plasmids 0.5µg (+) and 5µg (++) stably expressing HA‐Parkin and mt‐Keima were treated with CCCP (10µM) for 8h. Then, mKeima was measured at 488 (pH 7) and 561 (pH 4) nm lasers using Flow Cytometer. Percentages of mitophagy were calculated from 30,000 cells in each independent experiment. Data represent the mean ± SD of three independent experiments. For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01.


Figure 3C . Endogenous MITOL translocates to the ER in later phase of mitophagy . EGFP - MITOL knock in HeLa cells were transfected with HA - Parkin and treated with DMSO or CCCP ( 10 µM ) as indicated times . Cells were fixed , permeabilized and subjected to immunofluorescence analysis with the indicated antibodies . Colocalization was quantified by Manders ' s coefficient . Means ± SEM of more than 10 cells obtained from three independent experiments . For statistical analysis , a one - way ANOVA with Tukey ' s multiple comparisons test were performed , * * * * P < 0 . 0001 . Scale bar represents 1 μm . E . ER - localized MITOL is transported from the mitochondria , and is not newly synthesized protein . HeLa cells stably expressing HA - Parkin were co - transfected with vectors for MITOL - KikGR and FLAG - FKBP38 . After ultraviolet light ( 365 nm ) exposure to the whole plate , cells were incubated with or without CCCP ( 10 µM ) for indicated time , and monitored for red and green KikGR fluorescence . Scale bar , 10 μm . Higher magnification images of the boxed regions are shown in the small panel . Right panel is schematic experimental model for KikGR in this study .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3C. Endogenous MITOL translocates to the ER in later phase of mitophagy. EGFP-MITOL knock in HeLa cells were transfected with HA-Parkin and treated with DMSO or CCCP (10 µM) as indicated times. Cells were fixed, permeabilized and subjected to immunofluorescence analysis with the indicated antibodies. Colocalization was quantified by Manders's coefficient. Means ± SEM of more than 10 cells obtained from three independent experiments. For statistical analysis, a one-way ANOVA with Tukey's multiple comparisons test were performed, ****P < 0.0001. Scale bar represents 1 μm.E. ER-localized MITOL is transported from the mitochondria, and is not newly synthesized protein. HeLa cells stably expressing HA-Parkin were co-transfected with vectors for MITOL-KikGR and FLAG-FKBP38. After ultraviolet light (365 nm) exposure to the whole plate, cells were incubated with or without CCCP (10 µM) for indicated time, and monitored for red and green KikGR fluorescence. Scale bar, 10 μm. Higher magnification images of the boxed regions are shown in the small panel. Right panel is schematic experimental model for KikGR in this study.


Figure 4C . Phosphorylated Parkin accumulates in the ER fraction in the absence of MITOL . WT or MITOL KO HeLa cells stably expressing HA - Parkin were treated with DMSO or CCCP ( 10 µM ) for 48 h . Lysates of cells were fractionated into cytosolic , mitochondrial and ER fractions , and then subjected to an IB assay with the indicated antibodies . Phospho - Ser65 - Parkin is detected by using specific antibody . Right panels are the quantification of IB about Phospho - Ser65 - Parkin protein levels of the ER fraction . The data represent the mean ± SD for three independent experiments . For statistical analysis , a one - way ANOVA with Tukey post - test was performed , * * P < 0 . 01 . D . Parkin is detected at the ER in the absence of MITOL . HeLa cells were transfected with vectors for KikGR - Parkin . After ultraviolet light ( 365 nm ) exposure to the whole plate , cells were incubated with or without CCCP ( 10 µM ) for indicated time , and monitored for red and green KikGR fluorescence . Scale bar , 10 μm . Higher magnification images of the boxed regions are shown in the small panel .

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 O O O O O O O O O O O O O O O O O O O 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 4C. Phosphorylated Parkin accumulates in the ER fraction in the absence of MITOL. WT or MITOL KO HeLa cells stably expressing HA-Parkin were treated with DMSO or CCCP (10 µM) for 48 h. Lysates of cells were fractionated into cytosolic, mitochondrial and ER fractions, and then subjected to an IB assay with the indicated antibodies. Phospho-Ser65-Parkin is detected by using specific antibody. Right panels are the quantification of IB about Phospho-Ser65-Parkin protein levels of the ER fraction. The data represent the mean ± SD for three independent experiments. For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01.D. Parkin is detected at the ER in the absence of MITOL. HeLa cells were transfected with vectors for KikGR-Parkin. After ultraviolet light (365 nm) exposure to the whole plate, cells were incubated with or without CCCP (10 µM) for indicated time, and monitored for red and green KikGR fluorescence. Scale bar, 10 μm. Higher magnification images of the boxed regions are shown in the small panel.


Figure 5D . MITOL KO increases degradation of ER targeting FKBP38 . HeLa cells stably expressing HA - Parkin were transfected with mitochondrial targeting FLAG - FKBP38 ( FKBP38ActA ) and ER targeting FLAG - FKBP38 ( FKBP38IYFFT ) , and treated with DMSO or CCCP ( 10 µM ) for 24 h . Lysates of cells were subjected to an IB assay with FLAG antibodies for organelle targeting FKBP38 or indicated antibodies . Another cell lysates were subsequently separated on a Phos - tag gel , followed by IB with anti - Parkin antibodies ( Phos - tag ) . E - G . MITOL partly prevents CCCP - induced cell death by reducing Parkin - mediated degradation of FKBP38 . WT or MITOL KO HeLa cells stably expressing HA - Parkin and HeLa cells were transfected with the indicated vectors , and treated with DMSO or CCCP ( 10 µM ) for 24 h . Cells were stained with propidium iodide ( PI ) for counting the percentage of dead cells using fluorescence microscopy . Data represent the mean ± SD of five independent experiments ( > 100 individual cells were counted ) . For statistical analysis , a one - way ANOVA with Tukey post - test was performed , * * P < 0 . 01 . KR , FKBP38 K271 / 273R .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5D. MITOL KO increases degradation of ER targeting FKBP38. HeLa cells stably expressing HA-Parkin were transfected with mitochondrial targeting FLAG-FKBP38 (FKBP38ActA) and ER targeting FLAG-FKBP38 (FKBP38IYFFT), and treated with DMSO or CCCP (10 µM) for 24 h. Lysates of cells were subjected to an IB assay with FLAG antibodies for organelle targeting FKBP38 or indicated antibodies. Another cell lysates were subsequently separated on a Phos-tag gel, followed by IB with anti-Parkin antibodies (Phos-tag).E-G. MITOL partly prevents CCCP-induced cell death by reducing Parkin-mediated degradation of FKBP38. WT or MITOL KO HeLa cells stably expressing HA-Parkin and HeLa cells were transfected with the indicated vectors, and treated with DMSO or CCCP (10 µM) for 24 h. Cells were stained with propidium iodide (PI) for counting the percentage of dead cells using fluorescence microscopy. Data represent the mean ± SD of five independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01. KR, FKBP38 K271/273R.


Figure 6A . Mislocalization of Bcl - 2 is observed in MITOL KO cells in mitophagy . WT or MITOL KO HeLa cells stably expressing HA - Parkin were transfected with FLAG - Bcl2 and treated with DMSO or CCCP ( 10 µM ) for the indicated times . Cells were immunostained with indicated antibody . Scale bar , 1 μm . B . MITOL prevents caspase - dependent cell death in mitophagy . WT or MITOL KO HeLa cells stably expressing HA - Parkin and HeLa cells were treated with DMSO or CCCP ( 10 µM ) with or without Z - VAD - FMK ( 10 μM ) for 24 h . Cells were stained with propidium iodide ( PI ) for counting fluorescence microscopy of the percentage of dead cells . Data represent the mean ± SD of three independent experiments ( > 100 individual cells were counted ) . For statistical analysis , a one - way ANOVA with Tukey post - test was performed , * * P < 0 . 01 . C . Cell death in mitophagy caused by MITOL deficiency depends on calcium leak . WT and MITOL KO HeLa cells stably expressing HA - Parkin or HeLa cells were treated with DMSO or CCCP ( 10 µM ) with or without BAPTA - AM ( 10 μM ) for 24 h . Cells were stained with propidium iodide ( PI ) for counting fluorescence microscopy of the percentage of dead cells . Data represent the mean ± SD of three independent experiments ( > 100 individual cells were counted ) . For statistical analysis , a one - way ANOVA with Tukey post - test was performed , * * P < 0 . 01 .

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

Figure 6A. Mislocalization of Bcl-2 is observed in MITOL KO cells in mitophagy. WT or MITOL KO HeLa cells stably expressing HA-Parkin were transfected with FLAG-Bcl2 and treated with DMSO or CCCP (10 µM) for the indicated times. Cells were immunostained with indicated antibody. Scale bar, 1 μm.B. MITOL prevents caspase-dependent cell death in mitophagy. WT or MITOL KO HeLa cells stably expressing HA-Parkin and HeLa cells were treated with DMSO or CCCP (10 µM) with or without Z-VAD-FMK (10 μM) for 24 h. Cells were stained with propidium iodide (PI) for counting fluorescence microscopy of the percentage of dead cells. Data represent the mean ± SD of three independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01. C. Cell death in mitophagy caused by MITOL deficiency depends on calcium leak. WT and MITOL KO HeLa cells stably expressing HA-Parkin or HeLa cells were treated with DMSO or CCCP (10 µM) with or without BAPTA-AM (10 μM) for 24 h. Cells were stained with propidium iodide (PI) for counting fluorescence microscopy of the percentage of dead cells. Data represent the mean ± SD of three independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01.


Figure 1 ( E ) Northern blot analysis of circBtnl1 in different tissues of two - month - old mice , 18S RNA served as a loading control . ( F ， G ) RNA fluorescence in situ hybridization of circBtnl1 in small intestine tissues ( F ) and organoid ( G ) . Green region shows the distribution of circBtnl1 using antisense probe , gray region shows the nuclei staining by DAPI . For ( F ) ， scale bars ， 5um ; for ( G ) ， scale bars ， 20 um .

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Figure 1(E) Northern blot analysis of circBtnl1 in different tissues of two-month-old mice, 18S RNA served as a loading control.(F， G) RNA fluorescence in situ hybridization of circBtnl1 in small intestine tissues (F) and organoid (G). Green region shows the distribution of circBtnl1 using antisense probe, gray region shows the nuclei staining by DAPI. For (F)，scale bars，5um; for (G)，scale bars，20 um.