( A ) Upper panel : CHO cells stably transfected with GFP ‐ GATE ‐ 16 were preincubated in the presence or absence of 10 mM NAC or 1000 μ / ml catalase for 10 min before starvation for 2 h in the presence or absence of these drugs , or grown in a control medium containing the drugs for 2 h . The cells were then fixed , permeabilized and stained with anti ‐ GFP monoclonal antibodies . Representative images are shown . Lower panel : HEK 293 cells were transfected with GFP ‐ GATE ‐ 16 . At 24 h post ‐ transfection , the cells were treated with NAC or catalase and starved as explained above , lysed in Ripa buffer and 100 ‐ g of each lysate was separated on 10 % SDS - PAGE and subsequently analyzed with anti ‐ GFP antibodies to detect the transfected GATE ‐ 16 and anti ‐ tubulin antibodies as control . The data were quantified using NIH image program and are depicted as the percentage of lipidated protein from the total GATE ‐ 16 . ( * ) indicates non ‐ lipidated and ( * * ) indicates lipidated GFP ‐ GATE ‐ 16 .

O O O O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O B-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O

(A) Upper panel: CHO cells stably transfected with GFP‐GATE‐16 were preincubated in the presence or absence of 10 mM NAC or 1000 μ/ml catalase for 10 min before starvation for 2 h in the presence or absence of these drugs, or grown in a control medium containing the drugs for 2 h. The cells were then fixed, permeabilized and stained with anti‐GFP monoclonal antibodies. Representative images are shown. Lower panel: HEK 293 cells were transfected with GFP‐GATE‐16. At 24 h post‐transfection, the cells were treated with NAC or catalase and starved as explained above, lysed in Ripa buffer and 100 ‐g of each lysate was separated on 10% SDS-PAGE and subsequently analyzed with anti‐GFP antibodies to detect the transfected GATE‐16 and anti‐tubulin antibodies as control. The data were quantified using NIH image program and are depicted as the percentage of lipidated protein from the total GATE‐16. (*) indicates non‐lipidated and (**) indicates lipidated GFP‐GATE‐16.


( B ) CHO cells treated with NAC or catalase and starved as detailed in ( A ) were incubated with DCFDA and visualized by a confocal microscope or analyzed by a fluorimeter as in Figure 1 .

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(B) CHO cells treated with NAC or catalase and starved as detailed in (A) were incubated with DCFDA and visualized by a confocal microscope or analyzed by a fluorimeter as in Figure 1.


( C ) The rate of degradation of long ‐ lived proteins was measured in CHO cells incubated in either α ‐ MEM medium or EBSS medium , or following pretreatment with 10 mM NAC for 10 min or with 1000 μ / ml catalase overnight . A representative experiment is shown .

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(C) The rate of degradation of long‐lived proteins was measured in CHO cells incubated in either α‐MEM medium or EBSS medium, or following pretreatment with 10 mM NAC for 10 min or with 1000 μ/ml catalase overnight. A representative experiment is shown.


( A ) CHO cells ( for mammalian Atg8s ) or cells of S . cerevisiae ( for ScAtg8 ) were incubated in starvation medium ( EBSS or SD ‐ N , respectively ) for different time periods as indicated before isolation of RNA by the Tri ‐ reagent as explained in Materials and methods . For each gene , data obtained from non ‐ starved cells were set to an arbitrary value of 1 , and results from the corresponding starved cells were normalized accordingly . Results represent the means ± s . d . of three separate experiments .

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(A) CHO cells (for mammalian Atg8s) or cells of S. cerevisiae (for ScAtg8) were incubated in starvation medium (EBSS or SD‐N, respectively) for different time periods as indicated before isolation of RNA by the Tri‐reagent as explained in Materials and methods. For each gene, data obtained from non‐starved cells were set to an arbitrary value of 1, and results from the corresponding starved cells were normalized accordingly. Results represent the means±s.d. of three separate experiments.


( B ) HEK 293 cells were grown in control medium or starved for 2 h in EBSS in the absence or presence of 100 nM bafilomycin A1 . Lysates ( 100 μg ) obtained in Ripa buffer were run on 12 % SDS - PAGE and analyzed by Western blot , using anti ‐ GATE ‐ 16 or anti ‐ tubulin antibodies .

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(B) HEK 293 cells were grown in control medium or starved for 2 h in EBSS in the absence or presence of 100 nM bafilomycin A1. Lysates (100 μg) obtained in Ripa buffer were run on 12% SDS-PAGE and analyzed by Western blot, using anti‐GATE‐16 or anti‐tubulin antibodies.


( C ) CHO cells were grown in a control medium in the presence or absence of 1 mM H2O2 for 1 h , or starved for 3 or 13 h . Lysates ( 10 μg ) obtained in Ripa buffer were incubated with recombinant His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) in 50 KT reaction buffer ( 25 mM Tris , pH 7 . 4 , 50 mM KCl ) at 30 ° C for 45 min , in the presence or absence of 1 mM DTT . The reaction was stopped by addition of sample buffer and boiling , after which the samples were resolved on 15 % SDS - PAGE and subsequently analyzed by Western blot , using anti ‐ His monoclonal antibodies . The experiment was repeated six times ; a representative blot is shown .

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(C) CHO cells were grown in a control medium in the presence or absence of 1 mM H2O2 for 1 h, or starved for 3 or 13 h. Lysates (10 μg) obtained in Ripa buffer were incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer (25 mM Tris, pH 7.4, 50 mM KCl) at 30°C for 45 min, in the presence or absence of 1 mM DTT. The reaction was stopped by addition of sample buffer and boiling, after which the samples were resolved on 15% SDS-PAGE and subsequently analyzed by Western blot, using anti‐His monoclonal antibodies. The experiment was repeated six times; a representative blot is shown.


( D ) Left panel : HeLa cells transfected with Myc ‐ GATE ‐ 16 ‐ HA or with an empty vector as control were labeled with [ 35S ] methionine for 10 min and lysed immediately or chased for 1 h before lysis in Ripa buffer . Lysates were immunoprecipitated using anti ‐ Myc antibodies and the immunoprecipitates were resolved on 15 % SDS - PAGE . Middle panel : HeLa cells transfected with Myc ‐ GATE ‐ 16 ‐ HA were kept in control medium or starved for 30 min or 13 h in EBSS before labeling with [ 35S ] methionine for 10 min , immediate lysis , analysis by SDS - PAGE and quantification ( right panel ) using NIH image program . Values are presented as the average percentage of unprimed form out of the total GATE ‐ 16 . ( * ) in all sections of this figure indicates non ‐ cleaved His6 ‐ GATE ‐ 16 ‐ HA or Myc ‐ GATE ‐ 16 ‐ HA and ( * * ) indicates cleaved His6 ‐ GATE ‐ 16 or Myc ‐ GATE ‐ 16 .

O O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O

(D) Left panel: HeLa cells transfected with Myc‐GATE‐16‐HA or with an empty vector as control were labeled with [35S]methionine for 10 min and lysed immediately or chased for 1 h before lysis in Ripa buffer. Lysates were immunoprecipitated using anti‐Myc antibodies and the immunoprecipitates were resolved on 15% SDS-PAGE. Middle panel: HeLa cells transfected with Myc‐GATE‐16‐HA were kept in control medium or starved for 30 min or 13 h in EBSS before labeling with [35S]methionine for 10 min, immediate lysis, analysis by SDS-PAGE and quantification (right panel) using NIH image program. Values are presented as the average percentage of unprimed form out of the total GATE‐16. (*) in all sections of this figure indicates non‐cleaved His6‐GATE‐16‐HA or Myc‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16 or Myc‐GATE‐16.


( A ) Cleavage activity was tested by incubation of recombinant His6 ‐ HsAtg4A ( 0 . 1 μg ) and His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) in 50 KT reaction buffer at 30 ° C for 45 min in the presence of indicated concentrations of DTT followed by Western blot analysis , using anti ‐ His monoclonal antibodies . The resulting bands from three separate experiments were quantified with a densitometer using the Bio ‐ Rad Multi ‐ Analyst program and are presented as the average percentage of cleaved form out of the total GATE ‐ 16 . ( * ) indicates non ‐ cleaved His6 ‐ GATE ‐ 16 ‐ HA and ( * * ) indicates cleaved His6 ‐ GATE ‐ 16 .

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(A) Cleavage activity was tested by incubation of recombinant His6‐HsAtg4A (0.1 μg) and His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer at 30°C for 45 min in the presence of indicated concentrations of DTT followed by Western blot analysis, using anti‐His monoclonal antibodies. The resulting bands from three separate experiments were quantified with a densitometer using the Bio‐Rad Multi‐Analyst program and are presented as the average percentage of cleaved form out of the total GATE‐16. (*) indicates non‐cleaved His6‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16.


( B ) His6 ‐ HsAtg4A was incubated in the presence of 200 μM DTT at 4 ° C for 10 min . Reduced His6 ‐ HsAtg4A ( 0 . 1 μg ) was then incubated in 50 KT ( to obtain 15 μM DTT ) with the indicated concentrations of H2O2 at 25 ° C for 5 min , after which recombinant His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) was added and incubation proceeded at 30 ° C for 45 min . Reaction mixtures from three separate experiments were analyzed and are presented as explained in ( A ) .

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(B) His6‐HsAtg4A was incubated in the presence of 200 μM DTT at 4°C for 10 min. Reduced His6‐HsAtg4A (0.1 μg) was then incubated in 50 KT (to obtain 15 μM DTT) with the indicated concentrations of H2O2 at 25°C for 5 min, after which recombinant His6‐GATE‐16‐HA (0.3 μg) was added and incubation proceeded at 30°C for 45 min. Reaction mixtures from three separate experiments were analyzed and are presented as explained in (A).


( C ) His6 ‐ HsAtg4A ( 0 . 1 μg ) was incubated with recombinant His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) after the following procedures : no treatment ( lane 1 ) ; pretreatment with 200 μM DTT at 25 ° C for 5 min ( lane 2 ) ; treatment with 200 μM DTT followed by treatment with 1 mM H2O2 at 25 ° C for 5 min ( lane 3 ) ; and treatment with 200 μM DTT followed by treatment with 1 mM H2O2 for 5 min and then 2 mM DTT ( lane 4 ) . Reaction mixtures were analyzed by Western blot using anti ‐ His monoclonal antibodies .

O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

(C) His6‐HsAtg4A (0.1 μg) was incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) after the following procedures: no treatment (lane 1); pretreatment with 200 μM DTT at 25°C for 5 min (lane 2); treatment with 200 μM DTT followed by treatment with 1 mM H2O2 at 25°C for 5 min (lane 3); and treatment with 200 μM DTT followed by treatment with 1 mM H2O2 for 5 min and then 2 mM DTT (lane 4). Reaction mixtures were analyzed by Western blot using anti‐His monoclonal antibodies.


( A ) Recombinant His6 ‐ HsAtg4AWT , His6 ‐ HsAtg4AC77A or His6 ‐ HsAtg4AC81S ( 0 . 1 μg ) was incubated with His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) in 50 KT reaction buffer at 30 ° C for 45 min in the presence or absence of 1 mM DTT . Reaction mixtures were analyzed by Western blot using anti ‐ His monoclonal antibodies . ( * ) indicates non ‐ cleaved His6 ‐ GATE ‐ 16 ‐ HA and ( * * ) indicates cleaved His6 ‐ GATE ‐ 16 .

O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O 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-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O

(A) Recombinant His6‐HsAtg4AWT, His6‐HsAtg4AC77A or His6‐HsAtg4AC81S (0.1 μg) was incubated with His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer at 30°C for 45 min in the presence or absence of 1 mM DTT. Reaction mixtures were analyzed by Western blot using anti‐His monoclonal antibodies. (*) indicates non‐cleaved His6‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16.


( B ) His6 ‐ HsAtg4AC81S ( 0 . 1 μg ) was incubated with recombinant His6 ‐ GATE ‐ 16 ‐ HA ( 0 . 3 μg ) following the same procedure as detailed in Figure 5C . Reaction mixtures were analyzed and are presented as explained in ( A ) .

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(B) His6‐HsAtg4AC81S (0.1 μg) was incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) following the same procedure as detailed in Figure 5C. Reaction mixtures were analyzed and are presented as explained in (A).


( A ) CHO cells stably expressing GFP ‐ GATE ‐ 16 were transiently transfected with HsAtg4AWT ‐ Myc ‐ His6 or HsAtg4AC81S ‐ Myc ‐ His6 . At 24 h post ‐ transfection , cells were starved for 2 . 5 h after which they were fixed , permeabilized and incubated with anti ‐ Myc monoclonal antibodies . Upper panel : typical images of cells transfected with the above constructs , as visualized using a confocal microscope ; representative autophagosomes are indicated by arrows . Lower panel : quantification of the average number of autophagosomes per cell in the different transfectants . Images of the fixed cells were visualized using a Nikon eclipse TE300 fluorescent microscope and used for quantification of the number of autophagosomes per cell . The results presented are the means ± s . d . of a total of 100 cells from three separate experiments . ( * ) indicates significance at P0 . 001 .

O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) CHO cells stably expressing GFP‐GATE‐16 were transiently transfected with HsAtg4AWT‐Myc‐His6 or HsAtg4AC81S‐Myc‐His6. At 24 h post‐transfection, cells were starved for 2.5 h after which they were fixed, permeabilized and incubated with anti‐Myc monoclonal antibodies. Upper panel: typical images of cells transfected with the above constructs, as visualized using a confocal microscope; representative autophagosomes are indicated by arrows. Lower panel: quantification of the average number of autophagosomes per cell in the different transfectants. Images of the fixed cells were visualized using a Nikon eclipse TE300 fluorescent microscope and used for quantification of the number of autophagosomes per cell. The results presented are the means±s.d. of a total of 100 cells from three separate experiments. (*) indicates significance at P0.001.


( B ) Left panel : HEK 293 cells were cotransfected with GFP ‐ GATE ‐ 16 and each of the Atg4A constructs mentioned in ( A ) . At 40 h post ‐ transfection , the cells were starved for 2 . 5 h , lysed in Ripa buffer and 100 μg of each lysate was loaded to 10 % SDS - PAGE and subsequently analyzed with anti ‐ GFP antibodies to detect the transfected GATE ‐ 16 , anti ‐ Myc antibodies to detect the transfected HsAtg4A and anti ‐ tubulin antibodies as control . ( * ) indicates non ‐ lipidated GFP ‐ GATE ‐ 16 and ( * * ) indicates lipidated GFP ‐ GATE ‐ 16 . Right panel : results from three separate experiments , as detailed in the left panel , were analyzed using NIH image program and quantified as follows : the amount of lipidated GFP ‐ GATE ‐ 16 out of the total of GFP ‐ GATE ‐ 16 was calculated for each mutant in each experiment . The value obtained for HsAtg4AWT in each experiment was set to 100 % and the relative lipidation in cells transfected with the mutant was calculated accordingly .

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(B) Left panel: HEK 293 cells were cotransfected with GFP‐GATE‐16 and each of the Atg4A constructs mentioned in (A). At 40 h post‐transfection, the cells were starved for 2.5 h, lysed in Ripa buffer and 100 μg of each lysate was loaded to 10% SDS-PAGE and subsequently analyzed with anti‐GFP antibodies to detect the transfected GATE‐16, anti‐Myc antibodies to detect the transfected HsAtg4A and anti‐tubulin antibodies as control. (*) indicates non‐lipidated GFP‐GATE‐16 and (**) indicates lipidated GFP‐GATE‐16. Right panel: results from three separate experiments, as detailed in the left panel, were analyzed using NIH image program and quantified as follows: the amount of lipidated GFP‐GATE‐16 out of the total of GFP‐GATE‐16 was calculated for each mutant in each experiment. The value obtained for HsAtg4AWT in each experiment was set to 100% and the relative lipidation in cells transfected with the mutant was calculated accordingly.


( A ) HeLa cells were transiently transfected with GFP - HsAtg4BWT or GFP ‐ HsAtg4BC78S . At 24 h post ‐ transfection , cells were starved for 2 . 5 h in the presence of 4 mM H2O2 after which they were fixed , permeabilized and incubated with anti ‐ LC3 polyclonal antibodies . Cells were visualized ( upper panel ) and quantified ( lower panel ) as explained in Figure 8 . Representative autophagosomes are indicated in the upper panel by arrows . The results presented in the lower panel are the means ± s . d . of a total of 100 cells from three separate experiments . ( * ) indicates significance at P0 . 001 .

O O O B-CELL_LINE O O O O O B-GENEPROD O O O B-GENEPROD 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-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) HeLa cells were transiently transfected with GFP-HsAtg4BWT or GFP‐HsAtg4BC78S. At 24 h post‐transfection, cells were starved for 2.5 h in the presence of 4 mM H2O2 after which they were fixed, permeabilized and incubated with anti‐LC3 polyclonal antibodies. Cells were visualized (upper panel) and quantified (lower panel) as explained in Figure 8. Representative autophagosomes are indicated in the upper panel by arrows. The results presented in the lower panel are the means±s.d. of a total of 100 cells from three separate experiments. (*) indicates significance at P0.001.


( B ) Left panel : HeLa cells transfected and treated as in ( A ) were lysed in Ripa buffer and 40 μg of each lysate was loaded to 12 % SDS - PAGE and subsequently analyzed with anti ‐ LC3 antibodies and anti ‐ GFP antibodies to detect the transfected HsAtg4B and anti ‐ tubulin antibodies as control . ( * ) indicates non ‐ lipidated LC3 and ( * * ) indicates lipidated LC3 . Right panel : results from three separate experiments , as detailed in the left panel , were analyzed as explained in Figure 8B .

O O O O O O B-CELL_LINE O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O 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) Left panel: HeLa cells transfected and treated as in (A) were lysed in Ripa buffer and 40 μg of each lysate was loaded to 12% SDS-PAGE and subsequently analyzed with anti‐LC3 antibodies and anti‐GFP antibodies to detect the transfected HsAtg4B and anti‐tubulin antibodies as control. (*) indicates non‐lipidated LC3 and (**) indicates lipidated LC3. Right panel: results from three separate experiments, as detailed in the left panel, were analyzed as explained in Figure 8B.


( A - E ) GFP - pho8Δ60 - expressing wild - type ( WT ) , atg5Δ , atg7Δ , and atg5Δpep4Δ cells were starved ( A ) or treated with AmphoB ( B - E ) and subjected to western blotting . Cells were treated with 2 . 5 µg / ml AmphoB for the indicated time ( B ) or with the indicated doses for 24 hr ( C - E ) . Generation of free GFP was a marker of proteolysis .

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(A-E) GFP-pho8Δ60-expressing wild-type (WT), atg5Δ, atg7Δ, and atg5Δpep4Δ cells were starved (A) or treated with AmphoB (B-E) and subjected to western blotting. Cells were treated with 2.5 µg/ml AmphoB for the indicated time (B) or with the indicated doses for 24 hr (C-E). Generation of free GFP was a marker of proteolysis.


( F ) GFP - pho8Δ60 - and Pep4 - mRFP - expressing atg5Δ cells were cultured with ( AmphoB ) or without ( NT ) AmphoB ( 2 . 5 µg / ml , 24 hr ) , and localization of GFP - pho8Δ60 and Pep4 - mRFP was observed by confocal microscopy . Arrowheads indicate the cells in which GFP - pho8Δ60 was delivered to the vacuoles . Scale bars = 2 µm .

O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O

(F) GFP-pho8Δ60- and Pep4-mRFP-expressing atg5Δ cells were cultured with (AmphoB) or without (NT) AmphoB (2.5 µg/ml, 24 hr), and localization of GFP-pho8Δ60 and Pep4-mRFP was observed by confocal microscopy. Arrowheads indicate the cells in which GFP-pho8Δ60 was delivered to the vacuoles. Scale bars = 2 µm.


( G , H ) Accumulation of autophagic body ( AB ) - like structures by AmphoB in atg5Δpep4Δ cells . pep4Δ and atg5Δpep4Δ cells were starved for 3 hr or treated with AmphoB ( 2 . 5 µg / ml , 24 hr ) . Scale bars = 2 µm . Cells containing AB - like structures were counted under phase - contrast microscopy . Representative images ( G ) and the percentage of cells with AB - like structures ( H ) are shown . * p < 0 . 01 vs the value of pep4Δ cells . ( I - K ) Induction of AB - like structures in atg5Δpep4Δ cells treated with AmphoB ( 2 . 5 µg / ml , 24 hr ) . Representative images of quick - frozen replicas ( I ) and thin section of the frozen - substituted material ( J ) are shown .

O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G, H) Accumulation of autophagic body (AB)-like structures by AmphoB in atg5Δpep4Δ cells. pep4Δ and atg5Δpep4Δ cells were starved for 3 hr or treated with AmphoB (2.5 µg/ml, 24 hr). Scale bars = 2 µm. Cells containing AB-like structures were counted under phase-contrast microscopy. Representative images (G) and the percentage of cells with AB-like structures (H) are shown. *p < 0.01 vs the value of pep4Δ cells. (I-K) Induction of AB-like structures in atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml, 24 hr). Representative images of quick-frozen replicas (I) and thin section of the frozen-substituted material (J) are shown.


( I ) AB - like structures accumulated in the vacuole . Scale bar = 0 . 5 µm . ( J ) AB - like structures containing lipid particles and mitochondria accumulated in the cross - section of the vacuole . An autophagosome ( AP ) - like double - membrane compartment containing a ribosome and Golgigranule is present in the cytosol . Scale bar = 0 . 5 µm . A magnified photo is available in Appendix Fig . S4B . ( K ) The cells containing AB - like structures were counted under electron microscopy [ no treatment ( NT ) : n = 20 , AmphoB : n = 192 ] . * p < 0 . 01 vs the value of NT .

O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-SUBCELLULAR O O 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-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

(I) AB-like structures accumulated in the vacuole. Scale bar = 0.5 µm. (J) AB-like structures containing lipid particles and mitochondria accumulated in the cross-section of the vacuole. An autophagosome (AP)-like double-membrane compartment containing a ribosome and Golgigranule is present in the cytosol. Scale bar = 0.5 µm. A magnified photo is available in Appendix Fig. S4B. (K) The cells containing AB-like structures were counted under electron microscopy [no treatment (NT): n = 20, AmphoB: n = 192]. * p < 0.01 vs the value of NT.


( A - D ) Generation of Golgi stacks by AmphoB . Stacked Golgicisternae are observed in quick - frozen replicas ( A - C ) and substituted thin section ( D ) of atg5Δpep4Δ cells treated with AmphoB ( 2 . 5 µg / ml ) for the indicated time . ( A ) Scale bar = 1 µm . ( B ) The magnified image shows the area indicated by the square in ( A ) . Scale bar = 0 . 5 µm . ( C , D ) A representative Golgi stack with four cisternae is shown . Scale bar = 0 . 5 µm .

O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O

(A-D) Generation of Golgi stacks by AmphoB. Stacked Golgicisternae are observed in quick-frozen replicas (A-C) and substituted thin section (D) of atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml) for the indicated time. (A) Scale bar = 1 µm. (B) The magnified image shows the area indicated by the square in (A). Scale bar = 0.5 µm. (C, D) A representative Golgi stack with four cisternae is shown. Scale bar = 0.5 µm.


( E - G ) Generation of AP - like double - membrane compartment from the stacked Golgi . A quick - frozen replica ( E ) and a substituted thin section ( F , G ) are shown . Scale bars = 0 . 5 µm . The distal stacks are extended , curved , and form AP - like structure ( E , F ) . Mitochondria are encircled by the Golgi membrane ( F ) . ( G ) The AP - like structure was separated away from Golgi stacks .

O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O

(E-G) Generation of AP-like double-membrane compartment from the stacked Golgi. A quick-frozen replica (E) and a substituted thin section (F, G) are shown. Scale bars = 0.5 µm. The distal stacks are extended, curved, and form AP-like structure (E, F). Mitochondria are encircled by the Golgi membrane (F). (G) The AP-like structure was separated away from Golgi stacks.


( H , I ) The scene that AP - like structure was fused with vacuole . A magnified image of ( H ) is shown in ( I ) . In atg5Δpep4Δ cells treated with AmphoB ( 2 . 5 µg / ml , 24 hr ) , outer membrane of AP - like structure ( red arrows ) was fused with vacuolar membrane ( blue arrows ) . Inner membranes of AP - like structure ( yellow arrowheads ) were entered into the vacuoles to become AB - like structure . Green arrows indicate the membrane fusion site . Scale bars = 0 . 5 µm ( H ) and 0 . 2 µm ( I ) .

O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O

(H, I) The scene that AP-like structure was fused with vacuole. A magnified image of (H) is shown in (I). In atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml, 24 hr), outer membrane of AP-like structure (red arrows) was fused with vacuolar membrane (blue arrows). Inner membranes of AP-like structure (yellow arrowheads) were entered into the vacuoles to become AB-like structure. Green arrows indicate the membrane fusion site. Scale bars = 0.5 µm (H) and 0.2 µm (I).


( J ) The number of elongated single cisternae ( green bars ) , Golgi stacks ( red bars ) , and AB - like structures ( blue bars ) was counted over time using electron microscopy images . Error bars indicate s . e . m . ( 3 hr : n = 21 , 6 hr : n = 20 , 12 hr : n = 193 , 24 hr : n = 192 ) .

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(J) The number of elongated single cisternae (green bars), Golgi stacks (red bars), and AB-like structures (blue bars) was counted over time using electron microscopy images. Error bars indicate s.e.m. (3 hr: n = 21, 6 hr: n = 20, 12 hr: n = 193, 24 hr: n = 192).


( K ) Translocation of the trans - Golgi protein to the vacuolar membrane induced by AmphoB treatment . GFP - Sft2 - expressing atg5Δ cells ( trans - Golgi marker protein ) were incubated with or without AmphoB ( 2 . 5 µg / ml , 24 hr ) , and localization of GFP - Sft2 was assessed by confocal microscopy . Arrowhead indicates the GFP signal on the vacuolar membrane . Scale bars = 2 µm .

O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O

(K) Translocation of the trans-Golgi protein to the vacuolar membrane induced by AmphoB treatment. GFP-Sft2-expressing atg5Δ cells (trans-Golgi marker protein) were incubated with or without AmphoB (2.5 µg/ml, 24 hr), and localization of GFP-Sft2 was assessed by confocal microscopy. Arrowhead indicates the GFP signal on the vacuolar membrane. Scale bars = 2 µm.


( L - O ) Grh1 and Gvp36 involvement in AmphoB - induced autophagy - like proteolysis . ( L , M ) Representative electron microscopy images of grh1Δatg5Δpep4Δ ( L ) and gvp36Δatg5Δpep4Δ cells ( M ) treated with AmphoB ( 2 . 5 µg / ml , 24 hr ) . AP - like and AB - like structures were absent . ( L ) A unilamellar Golgi cisterna was observed in the cytoplasm ( dotted square ) . Scale bar = 1 µm . ( M ) Formation of a Golgi stack , but not Golgi curvature , was observed ( dotted square ) . Bar = 0 . 5 µm .

O O O O O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O

(L-O) Grh1 and Gvp36 involvement in AmphoB-induced autophagy-like proteolysis. (L, M) Representative electron microscopy images of grh1Δatg5Δpep4Δ (L) and gvp36Δatg5Δpep4Δ cells (M) treated with AmphoB (2.5 µg/ml, 24 hr). AP-like and AB-like structures were absent. (L) A unilamellar Golgi cisterna was observed in the cytoplasm (dotted square). Scale bar = 1 µm. (M) Formation of a Golgi stack, but not Golgi curvature, was observed (dotted square). Bar = 0.5 µm.


( L - O ) Grh1 and Gvp36 involvement in AmphoB - induced autophagy - like proteolysis . ( N ) Indicated cells were treated with AmphoB for 24 hr and the number of cells containing AB - like structures was counted by phase - contrast microscopy . * p < 0 . 01 vs the value of atg5Δpep4Δ cells .

O O O O O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O

(L-O) Grh1 and Gvp36 involvement in AmphoB-induced autophagy-like proteolysis. (N) Indicated cells were treated with AmphoB for 24 hr and the number of cells containing AB-like structures was counted by phase-contrast microscopy. *p < 0.01 vs the value of atg5Δpep4Δ cells.


( L - O ) Grh1 and Gvp36 involvement in AmphoB - induced autophagy - like proteolysis . ( O ) GFP - pho8Δ60 - expressing atg5Δ , grh1Δatg5Δ and gvp36Δatg5Δ cells were cultured with or without AmphoB ( 2 . 5 µg / ml , 24 hr ) and subjected to western blotting for GFP . An unidentified band at 32kD ( asterisk ) is non - specific band because it was present in Pep4 - lacking grh1Δatg5Δ cells .

O O O O O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O

(L-O) Grh1 and Gvp36 involvement in AmphoB-induced autophagy-like proteolysis. (O) GFP-pho8Δ60-expressing atg5Δ, grh1Δatg5Δ and gvp36Δatg5Δ cells were cultured with or without AmphoB (2.5 µg/ml, 24 hr) and subjected to western blotting for GFP. An unidentified band at 32kD (asterisk) is non-specific band because it was present in Pep4-lacking grh1Δatg5Δ cells.


( A ) Localization of cellular PI ( 4 ) P . GFP - 2xPHOsh2 - expressing atg5Δ , sac1 - 23 / atg5Δ , and pik1 - 83 / atg5Δ cells were incubated with or without AmphoB ( 2 . 5 µg / ml ) for 6 hr at 37 ° C , and their localization was observed by confocal microscopy . Scale bars = 2 µm .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

(A) Localization of cellular PI(4)P. GFP-2xPHOsh2-expressing atg5Δ, sac1-23/atg5Δ, and pik1-83/atg5Δ cells were incubated with or without AmphoB (2.5 µg/ml) for 6 hr at 37 °C, and their localization was observed by confocal microscopy. Scale bars = 2 µm.


( B ) The same experiments were performed using GFP - GOLPH3 ( a GolgiPI ( 4 ) P - monitoring protein ) instead of GFP - 2xPHOsh2 . Scale bars = 2 µm .

O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O

(B) The same experiments were performed using GFP-GOLPH3 (a GolgiPI(4)P-monitoring protein) instead of GFP-2xPHOsh2. Scale bars = 2 µm.


( C , D ) Reduction of AmphoB - induced GOMED by the deletion of Golgi PI ( 4 ) phosphatases . ( C ) Indicated cells expressing GFP - pho8Δ60 were cultured with or without AmphoB ( 2 . 5 µg / ml , 24 hr ) and subjected to western blotting for GFP .

O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O

(C, D) Reduction of AmphoB-induced GOMED by the deletion of Golgi PI(4) phosphatases. (C) Indicated cells expressing GFP-pho8Δ60 were cultured with or without AmphoB (2.5 µg/ml, 24 hr) and subjected to western blotting for GFP.


( C , D ) Reduction of AmphoB - induced GOMED by the deletion of Golgi PI ( 4 ) phosphatases . ( D ) The number of AP - like and AB - like structures was counted using EM images ( Fig . EV2B , C ) . ( E − H ) Induction of GOMED by the deletion of Golgi PI ( 4 ) kinase .

O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) Reduction of AmphoB-induced GOMED by the deletion of Golgi PI(4) phosphatases. (D) The number of AP-like and AB-like structures was counted using EM images (Fig. EV2B, C). (E−H) Induction of GOMED by the deletion of Golgi PI(4) kinase.


( E − H ) Induction of GOMED by the deletion of Golgi PI ( 4 ) kinase . ( E ) Indicated cells were incubated at 37 ° C ( temperature shift ) for 6 hr , and cells containing AB - like structures were counted by phase - contrast microscopy . * p < 0 . 01 vs the value of atg5Δpep4Δ cells .

O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O

(E−H) Induction of GOMED by the deletion of Golgi PI(4) kinase. (E) Indicated cells were incubated at 37 °C (temperature shift) for 6 hr, and cells containing AB-like structures were counted by phase-contrast microscopy. *p < 0.01 vs the value of atg5Δpep4Δ cells.


( E − H ) Induction of GOMED by the deletion of Golgi PI ( 4 ) kinase . ( F ) Representative electron microscopy image of pik1 - 83 / atg5Δpep4Δ cells at 3 hr after temperature shift . AP - like structure ( arrow ) and AB - like structures were generated . Scale bar = 0 . 2 µm . ( G ) The number of AP - like and AB - like structures was counted using EM images . Error bars indicate s . e . m . ( atg5Δpep4Δ cells at 37 ° C : n = 54 and pik1 - 83 / atg5Δpep4Δ cells at 37 ° C : n = 63 ) . * p < 0 . 01 vs the value of atg5Δpep4Δ cells .

O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O

(E−H) Induction of GOMED by the deletion of Golgi PI(4) kinase. (F) Representative electron microscopy image of pik1-83/atg5Δpep4Δ cells at 3 hr after temperature shift. AP-like structure (arrow) and AB-like structures were generated. Scale bar = 0.2 µm. (G) The number of AP-like and AB-like structures was counted using EM images. Error bars indicate s.e.m. (atg5Δpep4Δ cells at 37 °C: n = 54 and pik1-83/atg5Δpep4Δ cells at 37 °C: n = 63). *p < 0.01 vs the value of atg5Δpep4Δ cells.


( E − H ) Induction of GOMED by the deletion of Golgi PI ( 4 ) kinase . ( H ) Sec7 - HA - expressing pik1 - 83 / atg5Δ cells were incubated at 37 ° C ( temperature shift ) for 3 hr , and freeze replica immunolabelling was performed as described in Methods . Sec7 - HA - positive signals ( labelled with 15 nm gold particle ) were observed on the surface membrane of AP - like structure and Golgi membrane . Scale bar = 0 . 1 µm .

O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O 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-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O

(E−H) Induction of GOMED by the deletion of Golgi PI(4) kinase. (H) Sec7-HA-expressing pik1-83/atg5Δ cells were incubated at 37 °C (temperature shift) for 3 hr, and freeze replica immunolabelling was performed as described in Methods. Sec7-HA-positive signals (labelled with 15 nm gold particle) were observed on the surface membrane of AP-like structure and Golgi membrane. Scale bar = 0.1 µm.


( I − L ) Genetic ( I , J ) and pharmacological ( K , L ) alterations in anterograde trafficking from the Golgi are required for GOMED . ( I ) Indicated cells were incubated at 37 ° C ( temperature switch ) for 3 hr , and the cells containing AB - like structures were counted by phase - contrast microscopy . * p < 0 . 01 vs the value of atg5Δpep4Δ cells .

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-SUBCELLULAR O O O O 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-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O

(I−L) Genetic (I, J) and pharmacological (K, L) alterations in anterograde trafficking from the Golgi are required for GOMED. (I) Indicated cells were incubated at 37 °C (temperature switch) for 3 hr, and the cells containing AB-like structures were counted by phase-contrast microscopy. *p < 0.01 vs the value of atg5Δpep4Δ cells.


( I − L ) Genetic ( I , J ) and pharmacological ( K , L ) alterations in anterograde trafficking from the Golgi are required for GOMED . ( J ) Indicated cells were also observed using EM . Arrow indicates AP - like structure . Scale bar = 0 . 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O O O O

(I−L) Genetic (I, J) and pharmacological (K, L) alterations in anterograde trafficking from the Golgi are required for GOMED. (J) Indicated cells were also observed using EM. Arrow indicates AP-like structure. Scale bar = 0.5 µm.


Genetic ( I , J ) and pharmacological ( K , L ) alterations in anterograde trafficking from the Golgi are required for GOMED . ( K , L ) atg5Δpep4Δ cells were treated with ( CBM ) or without ( NT ) CBM ( 1 mM , 3 hr ) , and cells containing AB - like structures were counted by phase - contrast microscopy ( K ) . * p < 0 . 01 vs the value of NT .

O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O

Genetic (I, J) and pharmacological (K, L) alterations in anterograde trafficking from the Golgi are required for GOMED. (K, L) atg5Δpep4Δ cells were treated with (CBM) or without (NT) CBM (1 mM, 3 hr), and cells containing AB-like structures were counted by phase-contrast microscopy (K). * p < 0.01 vs the value of NT.


Genetic ( I , J ) and pharmacological ( K , L ) alterations in anterograde trafficking from the Golgi are required for GOMED . ( K , L ) atg5Δpep4Δ cells were treated with ( CBM ) or without ( NT ) CBM ( 1 mM , 3 hr ) . Cells were also observed using EM ( L ) . AB - like structures were observed . Scale bar = 0 . 5 µm .

O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O O O O O O

Genetic (I, J) and pharmacological (K, L) alterations in anterograde trafficking from the Golgi are required for GOMED. (K, L) atg5Δpep4Δ cells were treated with (CBM) or without (NT) CBM (1 mM, 3 hr). Cells were also observed using EM (L). AB-like structures were observed. Scale bar = 0.5 µm.


( M − O ) Disturbance of anterograde trafficking from the Golgi to the PM in the cells with GOMED . Indicated cells expressing Hsp150 - HA ( 3 × ) were treated as follows : NT , no treatment ; starve , starvation for 6 hr ; AmphoB , 2 . 5 µg / ml for 24 hr ( M ) ; CBM , 1 mM for 24 hr ( N ) ; temperature shift , from 25 ° C to 37 ° C for 3 hr ( O ) . The cells were then lysed and examined for Hsp150 - HA ( 3 × ) expression by western blotting . Accumulation of O - glycosylated Hsp150 - HA ( 3 × ) indicated the trafficking failure from the Golgi to the PM .

O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O

(M−O) Disturbance of anterograde trafficking from the Golgi to the PM in the cells with GOMED. Indicated cells expressing Hsp150-HA(3×) were treated as follows: NT, no treatment; starve, starvation for 6 hr; AmphoB, 2.5 µg/ml for 24 hr (M); CBM, 1 mM for 24 hr (N); temperature shift, from 25 °C to 37 °C for 3 hr (O). The cells were then lysed and examined for Hsp150-HA(3×) expression by western blotting. Accumulation of O-glycosylated Hsp150-HA(3×) indicated the trafficking failure from the Golgi to the PM.


( A − I ) Induction of GOMED in Atg5 KO MEFs by CBM treatment . ( A , B ) EM analysis revealed the formation of AP - like and AL - like structures . Atg5 KO MEFs were treated with CBM ( 2 mM ) for 3 hr . A representative low - magnification image ( A ) and high - magnification images ( B ) are shown . Scale bar = 1 µm ( A ) and 0 . 2 µm ( B ) . The arrowheads and arrows indicate AL - like and AP - like structures , respectively .

O O O O O O O O O B-GENEPROD O B-CELL_LINE O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O B-GENEPROD O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O

(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (A, B) EM analysis revealed the formation of AP-like and AL-like structures. Atg5 KO MEFs were treated with CBM (2 mM) for 3 hr. A representative low-magnification image (A) and high-magnification images (B) are shown. Scale bar = 1 µm (A) and 0.2 µm (B). The arrowheads and arrows indicate AL-like and AP-like structures, respectively.


( A − I ) Induction of GOMED in Atg5 KO MEFs by CBM treatment . ( C - F ) Involvement of Golgi membranes in the generation of AL - like structures . ( C ) CLEM analysis of mCherry - syntaxin 6 - expressing Atg5 KO MEFs . Cells were treated with CBM ( 2 mM ) for 24 hr and observed using fluorescence and electron microscopy . mCherry - syntaxin 6puncta merged with the AL - like structures . Scale bar = 5 µm . " N " indicates nucleus . A magnified image of the dashed square is shown in the inset . Scale bars = 2 µm .

O O O O O O O O O B-GENEPROD O B-CELL_LINE O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O

(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (C-F) Involvement of Golgi membranes in the generation of AL-like structures. (C) CLEM analysis of mCherry-syntaxin 6-expressing Atg5 KO MEFs. Cells were treated with CBM (2 mM) for 24 hr and observed using fluorescence and electron microscopy. mCherry-syntaxin 6puncta merged with the AL-like structures. Scale bar = 5 µm. "N" indicates nucleus. A magnified image of the dashed square is shown in the inset. Scale bars = 2 µm.


( A − I ) Induction of GOMED in Atg5 KO MEFs by CBM treatment . ( C - F ) Involvement of Golgi membranes in the generation of AL - like structures . ( D , E ) Requirement of Grasp65 in the generation of AL - like structures . Atg5 KO and Grasp65 - silenced Atg5 KO MEFs were treated without ( NT ) or with CBM ( 2 mM ) for the indicated times followed by immunostaining with an anti - Lamp2 antibody . Representative images ( at 24 h ) are shown in ( D ) . Scale bars = 5 µm . A magnified image of the dashed square is shown in the inset . Scale bars = 1 µm . CBM induced large ring - like Lamp2 fluorescence . ( E ) Percentages of cells with large ring - like Lamp2 immunofluorescence ( mean ± s . e . m . , n = 4 ) . * p < 0 . 01 vs the value of Atg5 KO MEF .

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(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (C-F) Involvement of Golgi membranes in the generation of AL-like structures. (D, E) Requirement of Grasp65 in the generation of AL-like structures. Atg5 KO and Grasp65-silenced Atg5 KO MEFs were treated without (NT) or with CBM (2 mM) for the indicated times followed by immunostaining with an anti-Lamp2 antibody. Representative images (at 24 h) are shown in (D). Scale bars = 5 µm. A magnified image of the dashed square is shown in the inset. Scale bars = 1 µm. CBM induced large ring-like Lamp2 fluorescence. (E) Percentages of cells with large ring-like Lamp2 immunofluorescence (mean ± s.e.m., n = 4). *p < 0.01 vs the value of Atg5 KO MEF.


( A − I ) Induction of GOMED in Atg5 KO MEFs by CBM treatment . ( C - F ) Involvement of Golgi membranes in the generation of AL - like structures . ( F ) Colocalization of Lamp2 and GFP - syntaxin 6 in CBM - treated Atg5 KO MEFs . GFP - syntaxin 6 - expressing Atg5 KO MEFs were treated with ( CBM ) or without ( NT ) CBM ( 2 mM ) for 6 hr and immunostained with an anti - Lamp2 antibody . The ring - like Lamp2 fluorescence merged with the signal for syntaxin 6 . Scale bar = 5 µm . A magnified image of the dashed square is shown in the inset . Scale bars = 2 µm .

O O O O O O O O O B-GENEPROD O B-CELL_LINE O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (C-F) Involvement of Golgi membranes in the generation of AL-like structures. (F) Colocalization of Lamp2 and GFP-syntaxin 6 in CBM-treated Atg5 KO MEFs. GFP-syntaxin 6-expressing Atg5 KO MEFs were treated with (CBM) or without (NT) CBM (2 mM) for 6 hr and immunostained with an anti-Lamp2 antibody. The ring-like Lamp2 fluorescence merged with the signal for syntaxin 6. Scale bar = 5 µm. A magnified image of the dashed square is shown in the inset. Scale bars = 2 µm.


( G ) The monomeric red fluorescent protein ( mRFP ) - green fluorescent protein ( GFP ) tandem protein assay revealed the induction of GOMED . Atg5 KO MEFs stably expressing tandem mRFP - GFP were incubated without ( no treatment ) or with CBM ( 2 mM ) for 24 hr . Red signals indicate acidic compartments . Lysosomes were counterstained with an anti - Lamp2 antibody ( cyan ) . Scale bars = 5 µm . Regions of interest ( ROI ) are indicated by dashed squares . Arrowheads indicate GOMED structure . Scale bars = 2 µm .

O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) The monomeric red fluorescent protein (mRFP)-green fluorescent protein (GFP) tandem protein assay revealed the induction of GOMED. Atg5 KO MEFs stably expressing tandem mRFP-GFP were incubated without (no treatment) or with CBM (2 mM) for 24 hr. Red signals indicate acidic compartments. Lysosomes were counterstained with an anti-Lamp2 antibody (cyan). Scale bars = 5 µm. Regions of interest (ROI) are indicated by dashed squares. Arrowheads indicate GOMED structure. Scale bars = 2 µm.


( A − I ) Induction of GOMED in Atg5 KO MEFs by CBM treatment . ( H , I ) Degradation of GFP - fused proteins indicated the induction of GOMED . Atg5 KO MEFs stably expressing VSVG - GFP ( H ) or M6PR - GFP ( I ) were incubated with CBM ( 5 mM ) for the indicated times . Cell lysates were subjected to immunoblotting with the anti - GFP antibody .

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(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (H, I) Degradation of GFP-fused proteins indicated the induction of GOMED. Atg5 KO MEFs stably expressing VSVG-GFP (H) or M6PR-GFP (I) were incubated with CBM (5 mM) for the indicated times. Cell lysates were subjected to immunoblotting with the anti-GFP antibody.


( J − M ) Genetic induction of GOMED in Atg5 KO MEFs . Atg5 KO MEFs were treated with the indicated small interfering ( si ) RNAs for 24 hr and GOMED was assessed by EM ( J - L ) . Representative images are shown in ( J , K ) . Arrows indicate AL - like structures . Scale bars = 2 µm . ( L ) The number of AP - like and AL - like structures was calculated from the EM images of cells treated with the indicated siRNAs . Error bars indicate s . e . m . ( siControl : n = 23 , siPI4k2α + siPI4k3β : n = 20 , siArfaptin1 : n = 20 ) . * p < 0 . 01 vs the value of siControl .

O O O O O O O O O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J−M) Genetic induction of GOMED in Atg5 KO MEFs. Atg5 KO MEFs were treated with the indicated small interfering (si)RNAs for 24 hr and GOMED was assessed by EM (J-L). Representative images are shown in (J, K). Arrows indicate AL-like structures. Scale bars = 2 µm. (L) The number of AP-like and AL-like structures was calculated from the EM images of cells treated with the indicated siRNAs. Error bars indicate s.e.m. (siControl: n = 23, siPI4k2α + siPI4k3β: n = 20, siArfaptin1: n = 20). *p < 0.01 vs the value of siControl.


( J − M ) Genetic induction of GOMED in Atg5 KO MEFs . Atg5 KO MEFs were treated with the indicated small interfering ( si ) RNAs for 24 hr and GOMED was assessed by the mRFP - GFP tandem protein assay ( M ) . ( M ) The same experiments as in ( G ) were performed using cells treated with the indicated siRNAs . Representative images are shown . Arrowheads indicate GOMED structure . Scale bars = 5 µm .

O O O O O O O O O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J−M) Genetic induction of GOMED in Atg5 KO MEFs. Atg5 KO MEFs were treated with the indicated small interfering (si)RNAs for 24 hr and GOMED was assessed by the mRFP-GFP tandem protein assay (M). (M) The same experiments as in (G) were performed using cells treated with the indicated siRNAs. Representative images are shown. Arrowheads indicate GOMED structure. Scale bars = 5 µm.


( A ) Suppression of insulin secretion in Atg5 knockout ( KO ) MIN6 cells after glucose deprivation . Atg5 KO MIN6 cells were incubated with normal culture medium ( NT ) or glucose - depleted medium for 1 hr . The amount of secreted insulin is expressed as the percentage of that of the untreated control ( NT ) ( mean ± s . e . m , n = 3 ) . * p < 0 . 01 vs the value of NT .

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(A) Suppression of insulin secretion in Atg5 knockout (KO) MIN6 cells after glucose deprivation. Atg5 KO MIN6 cells were incubated with normal culture medium (NT) or glucose-depleted medium for 1 hr. The amount of secreted insulin is expressed as the percentage of that of the untreated control (NT) (mean ± s.e.m, n = 3). *p < 0.01 vs the value of NT.


( B , C ) EM analysis revealed the induction of GOMED structures . Atg5 KO MIN6 cells were subjected to glucose deprivation for 1 hr . Representative images are shown . The white arrow and black arrows indicate the AP - like structure and insulinvesicles , respectively . Scale bars = 0 . 2 µm . ( D ) The number of AP - like and AL - like structures was calculated from the EM images of cells left untreated ( NT ) or subjected to glucose deprivation . Error bars indicate s . e . m . ( NT : n = 17 , glucose deprivation : n = 84 ) . * p < 0 . 01 and # p < 0 . 05 vs the value of NT .

O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O

(B, C) EM analysis revealed the induction of GOMED structures. Atg5 KO MIN6 cells were subjected to glucose deprivation for 1 hr. Representative images are shown. The white arrow and black arrows indicate the AP-like structure and insulinvesicles, respectively. Scale bars = 0.2 µm. (D) The number of AP-like and AL-like structures was calculated from the EM images of cells left untreated (NT) or subjected to glucose deprivation. Error bars indicate s.e.m. (NT: n = 17, glucose deprivation: n = 84). *p < 0.01 and #p < 0.05 vs the value of NT.


( E ) Involvement of Golgi membrane in the generation of GOMED . Lamp1 - GFP / mCherry - Syntaxin 6 - expressing Atg5 KO MIN6 cells were subjected to glucose deprivation , and analysed by TEM and confocal microscopy . CLEM analysis revealed that AL - like structures ( arrows ) merged with Lamp1 - GFP fluorescence and Syntaxin 6 fluorescence . Scale bars = 5 µm . A magnified image of the dashed square is shown in the inset . Scale bars = 2 µm .

O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Involvement of Golgi membrane in the generation of GOMED. Lamp1-GFP/mCherry-Syntaxin 6-expressing Atg5 KO MIN6 cells were subjected to glucose deprivation, and analysed by TEM and confocal microscopy. CLEM analysis revealed that AL-like structures (arrows) merged with Lamp1-GFP fluorescence and Syntaxin 6 fluorescence. Scale bars = 5 µm. A magnified image of the dashed square is shown in the inset. Scale bars = 2 µm.


( F ) GOMED - mediated degradation of ( pro ) insulin by glucose deprivation of Atg5 KO MIN6 cells . Atg5 KO MIN6 cells were incubated in glucose - depleted medium in the absence or presence of bafilomycin A1 ( 10 nM ) for the indicated times . The cell lysates were then subjected to immunoblot analysis using an anti - ( pro ) insulin antibody .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O B-CELL_LINE O O B-GENEPROD O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O

(F) GOMED-mediated degradation of (pro)insulin by glucose deprivation of Atg5 KO MIN6 cells. Atg5 KO MIN6 cells were incubated in glucose-depleted medium in the absence or presence of bafilomycin A1 (10 nM) for the indicated times. The cell lysates were then subjected to immunoblot analysis using an anti-(pro)insulin antibody.


( G ) Immunofluorescence analysis of ( pro ) insulin and Lamp2 . Atg5 KO MIN6 cells were left untreated ( NT ) or subjected to glucose deprivation for 1 hr in the presence of E64d ( 25 µM ) or bafilomycin A1 ( 10 nM ) , and were stained with anti - ( pro ) insulin and anti - Lamp2 ( lysosomal marker ) antibodies and observed by fluorescence microscopy . Scale bar = 1 µm . Arrowheads indicate the colocalization of ( pro ) insulin with GOMED structures .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O

(G) Immunofluorescence analysis of (pro)insulin and Lamp2. Atg5 KO MIN6 cells were left untreated (NT) or subjected to glucose deprivation for 1 hr in the presence of E64d (25 µM) or bafilomycin A1 (10 nM), and were stained with anti-(pro)insulin and anti-Lamp2 (lysosomal marker) antibodies and observed by fluorescence microscopy. Scale bar = 1 µm. Arrowheads indicate the colocalization of (pro)insulin with GOMED structures.


( A − D ) EM analysis demonstrated the induction of GOMED . Primary Atg7 KO β - cells were subjected to glucose deprivation for 1 hr . A representative AP - like structure containing insulingranule is shown in ( A ) . An AL - like structure containing insulingranules is shown in ( B ) . Scale bars = 0 . 2 µm .

O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

(A−D) EM analysis demonstrated the induction of GOMED. Primary Atg7 KO β-cells were subjected to glucose deprivation for 1 hr. A representative AP-like structure containing insulingranule is shown in (A). An AL-like structure containing insulingranules is shown in (B). Scale bars = 0.2 µm.


( A − D ) EM analysis demonstrated the induction of GOMED . Primary Atg7 KO β - cells were subjected to glucose deprivation for 1 hr . ( C , D ) GOMED structures containing Golgi vesicles . An AP - like structure containing multiple Golgi vesicles ( arrows ) ( C ) and an AL - like structure fusing with a lysosome ( arrowhead ) ( D ) are shown . Scale bar = 0 . 5 µm . A magnified image of the dashed rectangle is shown in the inset . Scale bar = 0 . 2 µm . AP - like structure contained double - membrane .

O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O

(A−D) EM analysis demonstrated the induction of GOMED. Primary Atg7 KO β-cells were subjected to glucose deprivation for 1 hr. (C, D) GOMED structures containing Golgi vesicles. An AP-like structure containing multiple Golgi vesicles (arrows) (C) and an AL-like structure fusing with a lysosome (arrowhead) (D) are shown. Scale bar = 0.5 µm. A magnified image of the dashed rectangle is shown in the inset. Scale bar = 0.2 µm. AP-like structure contained double-membrane.


( E ) The total number of AL - like structure was calculated from the EM images of WT and Atg7 KO β - cells after glucose deprivation . Error bars indicate s . e . m . ( n = 46 − 163 ) . * p < 0 . 01 and # p < 0 . 05 vs the value of 0 min .

O O O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) The total number of AL-like structure was calculated from the EM images of WT and Atg7 KO β-cells after glucose deprivation. Error bars indicate s.e.m. (n = 46−163). *p < 0.01 and #p < 0.05 vs the value of 0 min.


( F ) The total number of GOMED structure was calculated from the EM images of Atg7 KO β - cells after glucose deprivation in the absence or presence of bafilomycin A1 ( 10 nM ) . Error bars indicate s . e . m . ( n = 109 − 116 ) . * p < 0 . 01 vs the value of 60 min .

O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) The total number of GOMED structure was calculated from the EM images of Atg7 KO β-cells after glucose deprivation in the absence or presence of bafilomycin A1 (10 nM). Error bars indicate s.e.m. (n = 109−116). *p < 0.01 vs the value of 60 min.


( G − J ) EM analysis showed the induction of mitophagy . WT and Atg7 KO β - cells were subjected to glucose deprivation for 1 hr . Representative images showing mitophagy in Atg7 KO β - cells are shown in ( G ) . Arrowhead ( G ) indicates mitophagy . Scale bars = 0 . 5 µm . ( H , J ) The number of structures representative of mitophagy ( H ) per cell was calculated from the EM images of β - cells after glucose depletion . Error bars indicate s . e . m . ( n = 46 − 163 ) . * p < 0 . 01 and # p < 0 . 05 vs the value of 0 min .

O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G−J) EM analysis showed the induction of mitophagy. WT and Atg7 KO β-cells were subjected to glucose deprivation for 1 hr. Representative images showing mitophagy in Atg7 KO β-cells are shown in (G). Arrowhead (G) indicates mitophagy. Scale bars = 0.5 µm. (H, J) The number of structures representative of mitophagy (H) per cell was calculated from the EM images of β-cells after glucose depletion. Error bars indicate s.e.m. (n = 46−163). *p < 0.01 and #p < 0.05 vs the value of 0 min.


( G − J ) EM analysis showed the induction of crinophagy / SINGD . WT and Atg7 KO β - cells were subjected to glucose deprivation for 1 hr . Representative images showing crinophagy / SINGD in WT β - cells are shown in ( I ) . White arrow ( I ) indicates crinophagy / SINGD . Arrows indicate insulingranules ( I ) . Scale bars = 0 . 5 µm . ( H , J ) The number of structures representative of crinophagy / SINGD ( J ) per cell was calculated from the EM images of β - cells after glucose depletion . Error bars indicate s . e . m . ( n = 46 − 163 ) . * p < 0 . 01 and # p < 0 . 05 vs the value of 0 min .

O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O 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-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G−J) EM analysis showed the induction of crinophagy/SINGD. WT and Atg7 KO β-cells were subjected to glucose deprivation for 1 hr. Representative images showing crinophagy/SINGD in WT β-cells are shown in (I). White arrow (I) indicates crinophagy/SINGD. Arrows indicate insulingranules (I). Scale bars = 0.5 µm. (H, J) The number of structures representative of crinophagy/SINGD (J) per cell was calculated from the EM images of β-cells after glucose depletion. Error bars indicate s.e.m. (n = 46−163). *p < 0.01 and #p < 0.05 vs the value of 0 min.


( K ) The number of ( pro ) insulingranule - containing AL - like structure was calculated from the EM images of wild - type ( WT ) and Atg7 KO β - cells after glucose deprivation . Error bars indicate s . e . m . ( n = 46 − 163 ) . * p < 0 . 01 and # p < 0 . 05 vs the value of 0 min .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) The number of (pro)insulingranule-containing AL-like structure was calculated from the EM images of wild-type (WT) and Atg7 KO β-cells after glucose deprivation. Error bars indicate s.e.m. (n = 46−163). *p < 0.01 and #p < 0.05 vs the value of 0 min.


Survival curve of N . furzeri in the FLI facility . Recording of deaths starts at age of 5 wph , which corresponds to sexual maturity , and the colored dashed lines indicate the three age groups analysed in this study ( 5 animals / group ) , namely 5 weeks post hatching ( wph , young , sexual maturity ) , 12 wph ( adult ) and 39 wph ( old , past median lifespan ) of a wild - derived strain that exhibits a median lifespan of 7 - 8 months .

B-EXP_ASSAY O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Survival curve of N. furzeri in the FLI facility. Recording of deaths starts at age of 5 wph, which corresponds to sexual maturity, and the colored dashed lines indicate the three age groups analysed in this study (5 animals/group), namely 5 weeks post hatching (wph, young, sexual maturity), 12 wph (adult) and 39 wph (old, past median lifespan) of a wild-derived strain that exhibits a median lifespan of 7-8 months.


Global protein - transcript correlation for each sample , grouped by age . RPKM and iBAQ values were used to estimate transcript and protein levels from matched RNA - seq and TMT - based proteomics data obtained from the same animal . An ANOVA test was performed to evaluate significance among the age groups ( mean correlation at 5 wph : 0 . 48 ; at 12 wph : 0 . 43 ; and at 39 wph : 0 . 33 ; p = 3 . 05e - 07 , n = 5 per age group ) . In boxplots , the horizontal line represents the median , the bottom and top of the box the 25th and 75th percentile , respectively , and the whiskers extend 1 . 5 folds the interquartile range .

O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Global protein-transcript correlation for each sample, grouped by age. RPKM and iBAQ values were used to estimate transcript and protein levels from matched RNA-seq and TMT-based proteomics data obtained from the same animal. An ANOVA test was performed to evaluate significance among the age groups (mean correlation at 5 wph: 0.48; at 12 wph: 0.43; and at 39 wph: 0.33; p = 3.05e-07, n=5 per age group). In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range.


Scatter plot of log2 fold changes for genes differentially expressed both at transcript and protein levels ( adj . p < 0 . 05 ) . The color gradients indicate gene density in the regions where individual points overlap . Numbers of genes in each quadrant and the value of Pearson ' s coefficient of correlation , r , are reported for each graph . Solid lines represent a spline fit ( r = 0 . 505 for genes significantly affected at both transcript and protein levels , p < 2 . 2 x 10 - 16 , D ) ( r = 0 . 126 , p = 0 . 007 , E ) .

O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Scatter plot of log2 fold changes for genes differentially expressed both at transcript and protein levels (adj. p < 0.05). The color gradients indicate gene density in the regions where individual points overlap. Numbers of genes in each quadrant and the value of Pearson's coefficient of correlation, r, are reported for each graph. Solid lines represent a spline fit (r = 0.505 for genes significantly affected at both transcript and protein levels, p < 2.2 x 10-16, D) (r = 0.126, p = 0.007, E).


Mechanisms affecting protein abundance during aging . Bar plots are based on all the proteins affected in either one of the age comparisons ( adj . p < 0 . 05 ) . Proteins were divided in the following five groups : ( i ) proteins and transcripts with significant and consistent changes ( dark brown ) , ( ii ) proteins with significant changes , and with consistent changes of the transcripts ( light brown ) , ( iii ) proteins with no transcripts detected ( dark gray ) , ( iv ) proteins with transcripts whose translation is potentially regulated by miRNAs ( light gray ) , as assessed by the workflow displayed in ( Figure EV2D ) , ( v ) all the remaining proteins that we classified as regulated by other post - transcriptional mechanisms ( violet ) . pgs = protein groups .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mechanisms affecting protein abundance during aging. Bar plots are based on all the proteins affected in either one of the age comparisons (adj. p < 0.05). Proteins were divided in the following five groups: (i) proteins and transcripts with significant and consistent changes (dark brown), (ii) proteins with significant changes, and with consistent changes of the transcripts (light brown), (iii) proteins with no transcripts detected (dark gray), (iv) proteins with transcripts whose translation is potentially regulated by miRNAs (light gray), as assessed by the workflow displayed in (Figure EV2D), (v) all the remaining proteins that we classified as regulated by other post-transcriptional mechanisms (violet). pgs = protein groups.


Abundance changes of ribosomal proteins and their transcripts during aging . Cytoplasmic ribosomal proteins of large and small subunits are displayed separately and changes are shown for both the age comparisons as box plots . Transcripts are displayed as light blue and proteins as dark blue boxes . Changes of individual proteins are displayed as dots ; orange dots identify significant cases ( adj . p < 0 . 05 , n = 4 per age group for proteome and n = 5 per age group for transcriptome ) . In boxplots , the horizontal line represents the median , the bottom and top of the box the 25th and 75th percentile , respectively , and the whiskers extend 1 . 5 folds the interquartile range .

O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Abundance changes of ribosomal proteins and their transcripts during aging. Cytoplasmic ribosomal proteins of large and small subunits are displayed separately and changes are shown for both the age comparisons as box plots. Transcripts are displayed as light blue and proteins as dark blue boxes. Changes of individual proteins are displayed as dots; orange dots identify significant cases (adj. p < 0.05, n=4 per age group for proteome and n=5 per age group for transcriptome). In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range.


Brains from young ( 5 wph ) and old ( 39 wph ) were homogenized and clarified lysates separated by size - exclusion chromatography ( SEC ) . For each age group , two pools of brains were processed separately . For each experiment ( 4 in total ) , 39 fractions were collected along the chromatogram , digested into peptides and analysed by Data Independent Acquisition ( DIA ) quantitative mass spectrometry .

O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O

Brains from young (5 wph) and old (39 wph) were homogenized and clarified lysates separated by size-exclusion chromatography (SEC). For each age group, two pools of brains were processed separately. For each experiment (4 in total), 39 fractions were collected along the chromatogram, digested into peptides and analysed by Data Independent Acquisition (DIA) quantitative mass spectrometry.


Co - elution profiles for selected protein complexes . For each complex , the median abundance of all the quantified subunits was used to generate the complex profile across fractions ( Dataset EV5 ) . All the complex profiles are scaled to the max value ( set to 1 ) to make profiles comparable across experiments . The estimated molecular weight of the displayed complexes is indicated in brackets .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Co-elution profiles for selected protein complexes. For each complex, the median abundance of all the quantified subunits was used to generate the complex profile across fractions (Dataset EV5). All the complex profiles are scaled to the max value (set to 1) to make profiles comparable across experiments. The estimated molecular weight of the displayed complexes is indicated in brackets.


Statistics of protein complexes undergoing stoichiometry changes with aging . Only protein complexes that had at least 5 members quantified were considered for each comparison . Complexes were considered affected if at least two members showed significant stoichiometry change ( adj . p < 0 . 05 and absolute log2 fold change > 0 . 5 ) . The complete list of stoichiometry changes is available in Dataset EV6 .

O O B-SUBCELLULAR I-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

Statistics of protein complexes undergoing stoichiometry changes with aging. Only protein complexes that had at least 5 members quantified were considered for each comparison. Complexes were considered affected if at least two members showed significant stoichiometry change (adj. p < 0.05 and absolute log2 fold change > 0.5). The complete list of stoichiometry changes is available in Dataset EV6.


Violin plots depicting interquartile ranges ( IQRs ) of individual members of protein complexes during aging . The IQR for each protein complex considered in G was calculated using transcript ( total RNA dataset , light blue ) or protein ( dark blue ) log2 fold changes between two age groups ( n = 4 per age group for proteome and n = 5 per age group for transcriptome ) . * p < 0 . 05 , Wilcoxon Rank Sum test . The central line of the violin plots indicates the median value .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Violin plots depicting interquartile ranges (IQRs) of individual members of protein complexes during aging. The IQR for each protein complex considered in G was calculated using transcript (total RNA dataset, light blue) or protein (dark blue) log2 fold changes between two age groups (n=4 per age group for proteome and n=5 per age group for transcriptome). * p < 0.05, Wilcoxon Rank Sum test. The central line of the violin plots indicates the median value.


Heat map showing relative protein fold changes for members of selected complexes affected by aging . Names of significantly affected members in the 39 wph vs . 12 wph comparison ( adj . p < 0 . 05 and absolute log2 fold change > 0 . 5 ) are highlighted in bold with a star . Related to Figure EV3 and Dataset EV5 and EV6 .

O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Heat map showing relative protein fold changes for members of selected complexes affected by aging. Names of significantly affected members in the 39 wph vs. 12 wph comparison (adj. p < 0.05 and absolute log2 fold change > 0.5) are highlighted in bold with a star. Related to Figure EV3 and Dataset EV5 and EV6.


Representative Coomassie - stained SDS - PAGE gel showing the isolation of SDS insoluble aggregates from mouse brain lysates . TH = total homogenate , SUP = supernatant , W1 = SDS - soluble fraction , Pellet = formic - acid soluble fraction ( see Figure EV4A ) . Quantification of the yield of SDS - insoluble aggregates from young and old brain lysates was based on densitometry analysis of Coomassie - stained gel bands obtained from different animals , n = 5 per age group ( Figure EV4B ) ; * * p < 0 . 01 , unpaired t - test . In boxplots , the horizontal line represents the median , the bottom and top of the box the 25th and 75th percentile , respectively , and the whiskers extend 1 . 5 folds the interquartile range .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O B-ORGANISM B-TISSUE O O B-SUBCELLULAR O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative Coomassie-stained SDS-PAGE gel showing the isolation of SDS insoluble aggregates from mouse brain lysates. TH = total homogenate, SUP = supernatant, W1 = SDS-soluble fraction, Pellet = formic-acid soluble fraction (see Figure EV4A). Quantification of the yield of SDS-insoluble aggregates from young and old brain lysates was based on densitometry analysis of Coomassie-stained gel bands obtained from different animals, n=5 per age group (Figure EV4B); ** p < 0.01, unpaired t-test. In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range.


Volcano plot based on protein quantification by label - free mass spectrometry depicting the enrichment of specific proteins in protein aggregates . The x - axis indicates the log2 ratio between protein abundance in aggregates ( Pellet ) and starting total homogenate ( TH ) . The horizontal dashed line indicates a p value cut - off of 0 . 05 and vertical lines a log2 fold change cut off of ± 0 . 5 . Selected proteins are highlighted as colored dots as indicated in the figure legend . Protein quantification was based on samples obtained from 3 independent isolations .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Volcano plot based on protein quantification by label-free mass spectrometry depicting the enrichment of specific proteins in protein aggregates. The x-axis indicates the log2 ratio between protein abundance in aggregates (Pellet) and starting total homogenate (TH). The horizontal dashed line indicates a p value cut-off of 0.05 and vertical lines a log2 fold change cut off of ± 0.5. Selected proteins are highlighted as colored dots as indicated in the figure legend. Protein quantification was based on samples obtained from 3 independent isolations.


Double - labelling of telencephalic sections of N . furzeri with anti - RPS6 ( green ) as ribosomal marker and Proteostat as a marker for aggregated proteins ( red ) . Nuclear counterstaining was performed with DAPI ( blue ) . Scale bar = 10 μm .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-GENEPROD O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O O

Double-labelling of telencephalic sections of N. furzeri with anti-RPS6 (green) as ribosomal marker and Proteostat as a marker for aggregated proteins (red). Nuclear counterstaining was performed with DAPI (blue). Scale bar = 10 μm.


Abundance changes of proteasome proteins and their transcripts during aging . Members of the 19S and 20S complex are displayed separately and changes are shown for both the age comparisons as box plots . Transcripts are displayed as light blue and proteins as dark blue boxes . Changes of individual proteins are displayed as dots , orange dots represent significant cases ( adj . p < 0 . 05 , n = 4 per age group for proteome and n = 5 per age group for transcriptome ) . In boxplots , the horizontal line represents the median , the bottom and top of the box the 25th and 75th percentile , respectively , and the whiskers extend 1 . 5 folds the interquartile range .

O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Abundance changes of proteasome proteins and their transcripts during aging. Members of the 19S and 20S complex are displayed separately and changes are shown for both the age comparisons as box plots. Transcripts are displayed as light blue and proteins as dark blue boxes. Changes of individual proteins are displayed as dots, orange dots represent significant cases (adj. p < 0.05, n=4 per age group for proteome and n=5 per age group for transcriptome). In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range.


Heat map showing transcript and protein fold changes for members of the 26 proteasome ( 19S and 12S complexes ) . Genes are annotated according to significance of their changes at the level of transcript ( adj . p < 0 . 05 : black , adj . p > 0 . 05 : white ) or protein ( adj . p < 0 . 05 : green , adj . p > 0 . 05 : white ) .

O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Heat map showing transcript and protein fold changes for members of the 26 proteasome (19S and 12S complexes). Genes are annotated according to significance of their changes at the level of transcript (adj. p < 0.05: black, adj. p > 0.05: white) or protein (adj. p < 0.05: green, adj. p > 0.05: white).


In - gel proteasome assay following native gel electrophoresis ( top ) and immunoblotting of proteasome complexes ( 30S , 26S and 20S ) ( bottom ) in young ( 5wph ) , adult ( 12 wph ) and old ( 39 wph ) killifish brains . For additional samples and low exposure pictures see Figure EV5B and EV5C .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O

In-gel proteasome assay following native gel electrophoresis (top) and immunoblotting of proteasome complexes (30S, 26S and 20S) (bottom) in young (5wph), adult (12 wph) and old (39 wph) killifish brains. For additional samples and low exposure pictures see Figure EV5B and EV5C.


Bar plots depicting the quantification of chymotrypsin - like ( CT - L ) activity from native gels calculated for doubly capped ( 30S ) or singly capped ( 26S ) proteasomes . n > = 5 per sample group ; error bars indicate standard error of the mean . * p < 0 . 05 , * * p < 0 . 005 , * * * p = 0 . 0001 , * * * * p < 0 . 001 , one - way ANOVA , Holm - Sidak ' s multiple comparison test . For each sample group , the mean value of activity in young samples ( 5 wph ) was set to 100 % .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O

Bar plots depicting the quantification of chymotrypsin-like (CT-L) activity from native gels calculated for doubly capped (30S) or singly capped (26S) proteasomes. n >=5 per sample group; error bars indicate standard error of the mean. * p < 0.05, ** p < 0.005, *** p=0.0001, **** p < 0.001, one-way ANOVA, Holm-Sidak's multiple comparison test. For each sample group, the mean value of activity in young samples (5 wph) was set to 100%.


Percentage ( % ) of chymotrypsin - like ( CT - L ) , trypsin - like ( T - L ) and peptidylglutamyl peptide hydrolysing or caspase - like ( PGPH ) proteasome activities in brain extracts of killifish of different ages . n > = 6 per sample group ; error bars indicate standard error of the mean . * * p < 0 . 005 , * * * p = 0 . 0001 , * * * * p < 0 . 001 , one - way ANOVA , Holm - Sidak ' s multiple comparison test . For each sample group , the mean value of each activity in young samples ( 5 wph ) was set to 100 % .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O B-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O

Percentage (%) of chymotrypsin-like (CT-L), trypsin-like (T-L) and peptidylglutamyl peptide hydrolysing or caspase-like (PGPH) proteasome activities in brain extracts of killifish of different ages. n >=6 per sample group; error bars indicate standard error of the mean. ** p < 0.005, *** p=0.0001, **** p < 0.001, one-way ANOVA, Holm-Sidak's multiple comparison test. For each sample group, the mean value of each activity in young samples (5 wph) was set to 100%.


Age - related changes of proteins involved in the ubiquitin cycle . All the displayed proteins showed significant protein level changes in the 39 wph vs . 12 wph comparison ( adj . p < 0 . 05 ) .

O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

Age-related changes of proteins involved in the ubiquitin cycle. All the displayed proteins showed significant protein level changes in the 39 wph vs. 12 wph comparison (adj. p < 0.05).


Chymotrypsin - like ( CT - L ) proteasome activity in brain extracts of killifish treated with different proteasome inhibitors . The activity was measured at day 4 after the beginning of treatment . n = 8 per sample group ; error bars indicate standard deviation . * * p < 0 . 01 , * * * * p < 0 . 0001 , one - way ANOVA followed by Holm - Sidak ' s multiple comparisons test .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O B-ORGANISM O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Chymotrypsin-like (CT-L) proteasome activity in brain extracts of killifish treated with different proteasome inhibitors. The activity was measured at day 4 after the beginning of treatment. n=8 per sample group; error bars indicate standard deviation. ** p < 0.01, **** p < 0.0001, one-way ANOVA followed by Holm-Sidak's multiple comparisons test.


Proteasome inhibition induces proteasome activators ( PSME4 ) , assembly factors ( POMP ) ubiquitin ligases ( UBE3C ) , mediators of autophagosome formation ( ATG2B ) , heat shock proteins and chaperones in killifish brain . Protein abundances were quantified by DIA mass spectrometry and they are shown relative to the mean value of vehicle control samples ( DMSO ) set to 1 , n = 10 per sample group . Adj . p < 0 . 05 for all the displayed proteins . Error bars indicate mean + / - SD .

B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O B-ORGANISM B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Proteasome inhibition induces proteasome activators (PSME4), assembly factors (POMP) ubiquitin ligases (UBE3C), mediators of autophagosome formation (ATG2B), heat shock proteins and chaperones in killifish brain. Protein abundances were quantified by DIA mass spectrometry and they are shown relative to the mean value of vehicle control samples (DMSO) set to 1, n=10 per sample group. Adj. p < 0.05 for all the displayed proteins. Error bars indicate mean +/-SD.


Bortezomib treatment affects the stoichiometry of a subset of protein complexes . The number of affected proteins ( adj . p < 0 . 25 ) for each protein complex is indicated . Only protein complexes that had at least two members affected are shown .

B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O

Bortezomib treatment affects the stoichiometry of a subset of protein complexes. The number of affected proteins (adj. p < 0.25) for each protein complex is indicated. Only protein complexes that had at least two members affected are shown.