( A ) . Bar graph shows quantification of lysosomes ( LAMP1 + ) containing PC1 expressed as % of total number of lysosomes ( mean + / - SEM ) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes and treated with 100 nM BafA1 for 9h . n = 20 cells per condition ; three independent experiments . One - way ANOVA ( P < 0 . 0001 ) with Dunnett ' s multiple comparisons test performed , * * * P < 0 . 0001 .

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(A). Bar graph shows quantification of lysosomes (LAMP1+) containing PC1 expressed as % of total number of lysosomes (mean +/- SEM) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes and treated with 100 nM BafA1 for 9h. n=20 cells per condition; three independent experiments. One-way ANOVA (P<0.0001) with Dunnett's multiple comparisons test performed, ***P<0.0001.


( B ) MEF cell lines lacking the expression of indicated genes were treated for 12h with 50 nM BafA1 , fixed and immunolabeled for PC1 ( 568 , red ) and LAMP1 ( 488 , green ) . Scale bar = 10 µm . Insets show magnification of the boxed area . Bar graph ( on the left ) shows quantification of LAMP1 vesicles positive for PC1 , expressed as % of total lysosomes ( mean + / - SEM ) , n = 12 , 10 , 12 , 10 , 7 , 10 cells per genotype respectively ; 3 independent experiments . One - way ANOVA with Dunnett ' s multiple comparisons test performed and P value adjusted for multiple comparisons . ns ≥ 0 . 05 , * * * P < 0 . 0001 .

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(B) MEF cell lines lacking the expression of indicated genes were treated for 12h with 50 nM BafA1, fixed and immunolabeled for PC1 (568, red) and LAMP1 (488, green). Scale bar = 10 µm. Insets show magnification of the boxed area. Bar graph (on the left) shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), n=12, 10, 12, 10, 7, 10 cells per genotype respectively; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test performed and P value adjusted for multiple comparisons. ns ≥0.05, *** P<0.0001.


( A ) WT and CRISPR - Cas9 Fam134b knockout MEFs were treated as indicated , lysed and analysed by western blot with the indicated antibodies . Western blots are representative of 4 independent experiments .

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(A) WT and CRISPR-Cas9 Fam134b knockout MEFs were treated as indicated, lysed and analysed by western blot with the indicated antibodies. Western blots are representative of 4 independent experiments.


( B ) WT and CRISPR - Cas9 Fam134b MEFs were immunolabeled for PC1 ( 568 , red ) , nuclei stained with Hoechst ( blue ) and analysed by scanning confocal microscopy . Scale bar = 10 µm .

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(B) WT and CRISPR-Cas9 Fam134b MEFs were immunolabeled for PC1 (568, red), nuclei stained with Hoechst (blue) and analysed by scanning confocal microscopy. Scale bar = 10 µm.


( C ) CRISPR FAM134B MEF mock , wild type FAM134B - HA or FAM134Blir - HA transfected were immunolabeled for PC1 ( 568 , red ) , LAMP1 ( 488 , green ) and HA ( 647 , violet ) and analysed by scanning confocal microscopy . Scale bar = 10 µm . Inset panels show magnification of the boxed area . Bar graph shows quantification of LAMP1 vesicles positive for PC1 , expressed as % of total lysosomes ( mean + / - SEM ) , quantification of n = 10 cells per treatment ; 3 independent experiments . One - way ANOVA with Dunnett ' s multiple comparisons test was performed . ns ≥ 0 . 05 , * * * P < 0 . 0001 .

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(C) CRISPR FAM134B MEF mock, wild type FAM134B-HA or FAM134Blir-HA transfected were immunolabeled for PC1 (568, red), LAMP1 (488, green) and HA (647, violet) and analysed by scanning confocal microscopy. Scale bar = 10 µm. Inset panels show magnification of the boxed area. Bar graph shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), quantification of n=10 cells per treatment; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test was performed. ns ≥0.05, *** P<0.0001.


( A ) Bar graph shows quantification of lysosomes ( LAMP1 + ) containing PC1 expressed as % of lysosomes ( mean + / - SEM ) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes , treated with 100 nM BafA1 for 9h . n = 18 cells / treatment ; 3 independent experiments . One - way ANOVA ( P < 0 . 0001 ) with Dunnett ' s multiple comparisons test performed , * * P < 0 . 005 , * * * P < 0 . 0001 .

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(A) Bar graph shows quantification of lysosomes (LAMP1+) containing PC1 expressed as % of lysosomes (mean +/- SEM) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes, treated with 100 nM BafA1 for 9h. n=18 cells/treatment; 3 independent experiments. One-way ANOVA (P<0.0001) with Dunnett's multiple comparisons test performed, ** P<0.005, *** P<0.0001.


( B ) WT and Canx - / - MEFs were untreated or treated with BafA1 ( 10 μM ) for 6 h , lysed and analysed by western blot with indicated antibodies . Filamin and β - actin were used as loading control . Dashed line indicates that unnecessary lanes were removed . Western blot is representative of 3 independent experiments .

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(B) WT and Canx-/- MEFs were untreated or treated with BafA1 (10 μM) for 6 h, lysed and analysed by western blot with indicated antibodies. Filamin and β-actin were used as loading control. Dashed line indicates that unnecessary lanes were removed. Western blot is representative of 3 independent experiments.


( C ) MEF cell lines lacking the indicated genes were treated for 12h with 50 nM BafA1 fixed and immunolabeled for PC1 ( 568 , red ) and LAMP1 ( 488 , green ) . CST was added where indicated . Scale bar = 10 µm . Inset panels show magnification of the boxed area . Bar graph on the right shows quantification of LAMP1 vesicles positive for PC1 , expressed as % of total lysosomes ( mean + / - SEM ) , n = 8 , 8 , 12 , 8 , 8 , 8 cells respectively ; 3 independent experiments . One - way ANOVA with Dunnett ' s multiple comparisons test performed and P value adjusted for multiple comparisons . * * * P < 0 . 0001 .

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(C) MEF cell lines lacking the indicated genes were treated for 12h with 50 nM BafA1 fixed and immunolabeled for PC1 (568, red) and LAMP1 (488, green). CST was added where indicated. Scale bar = 10 µm. Inset panels show magnification of the boxed area. Bar graph on the right shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), n=8, 8, 12, 8, 8, 8 cells respectively; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test performed and P value adjusted for multiple comparisons. *** P<0.0001.


( A , B ) Volcano plot comparing protein fold changes between WT versus Fam134b - / - ( A ) and WT versus Canx - / - MEFs ( B ) . Significantly regulated proteins are labelled in red ( log2 fold change > 1 , - log10 p > 1 . 3 ) . Red dots with blue ring indicate collagen 1 peptides . Graphs represent statistics from three separate experiments for each genotype .

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(A, B) Volcano plot comparing protein fold changes between WT versus Fam134b-/- (A) and WT versus Canx-/- MEFs (B). Significantly regulated proteins are labelled in red (log2 fold change > 1, -log10 p>1.3). Red dots with blue ring indicate collagen 1 peptides. Graphs represent statistics from three separate experiments for each genotype.


( B ) U2OS cells were transfected with empty vector control ( pcDNA3 ) , FAM134B - HA WT or mutant constructs as indicated . Complexes were immune - isolated with HA - magnetic beads , separated by western blot and visualised with antibodies against CANX , FAM134B 5 % of the input is shown . Western blots are representative of 3 independent experiments . Dashed line indicates that unnecessary lanes were removed .

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(B) U2OS cells were transfected with empty vector control (pcDNA3), FAM134B-HA WT or mutant constructs as indicated. Complexes were immune-isolated with HA-magnetic beads, separated by western blot and visualised with antibodies against CANX, FAM134B 5% of the input is shown. Western blots are representative of 3 independent experiments. Dashed line indicates that unnecessary lanes were removed.


U2OS cells were transfected with HALO - PC2 , FAM134B - HA or FAM134Blir - HA constructs , treated with 100 nM BafA1 for 6h and with CST where indicated . Complexes were immune - isolated with HA - magnetic beads , separated by western blot and visualised with antibodies against HALO , CANX , FAM134B , LC3 5 % of the input is shown . Western blots are representative of 3 independent experiments . Dashed line indicates that unnecessary lanes were removed .

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U2OS cells were transfected with HALO-PC2, FAM134B-HA or FAM134Blir-HA constructs, treated with 100 nM BafA1 for 6h and with CST where indicated. Complexes were immune-isolated with HA-magnetic beads, separated by western blot and visualised with antibodies against HALO, CANX, FAM134B, LC3 5% of the input is shown. Western blots are representative of 3 independent experiments. Dashed line indicates that unnecessary lanes were removed.


( B ) SEC trace and SDS - PAGE of purified NHE9 * ( residues 8 to and 574 out of 644 ) as depicted by schematic .

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(B) SEC trace and SDS-PAGE of purified NHE9* (residues 8 to and 574 out of 644) as depicted by schematic.


( C ) Activity of NHE9 * co - reconstituted with ATPase into liposomes to mimic the in vivo situation ( schematic ) . Representative ACMA fluorescence traces of liposome reconstituted NHE9 * ( blue ) , NHE9 * double - mutant N243A - D244A ( red ) , and rat fructose transporter GLUT5 ( black ) . ATP - driven H + pumping establishes a ΔpH ( 0 - 3 min ) . H + efflux is initiated by the addition of 40 mM NaCl , and subsequent addition of NH4Cl ( 4 min ) collapses the proton gradient .

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(C) Activity of NHE9* co-reconstituted with ATPase into liposomes to mimic the in vivo situation (schematic). Representative ACMA fluorescence traces of liposome reconstituted NHE9* (blue), NHE9* double-mutant N243A-D244A (red), and rat fructose transporter GLUT5 (black). ATP-driven H+ pumping establishes a ΔpH (0-3 min). H+ efflux is initiated by the addition of 40 mM NaCl, and subsequent addition of NH4Cl (4 min) collapses the proton gradient.


( D ) Michaelis - Menten kinetics for NHE9 * ( red ) , NHE9 ΔCTD ( green ) , N243A - D244A ( blue ) and rat GLUT5 ( black ) as detected by ACMA dequenching following substrate addition . In all experiments errors bars , s . e . m . ; n = 3 technical repeats . The apparent KM values are an average from n = 3 separate protein reconstitutions .

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(D) Michaelis-Menten kinetics for NHE9* (red), NHE9 ΔCTD (green), N243A-D244A (blue) and rat GLUT5 (black) as detected by ACMA dequenching following substrate addition. In all experiments errors bars, s.e.m.; n = 3 technical repeats. The apparent KM values are an average from n = 3 separate protein reconstitutions.


A ) Native mass spectrum of NHE9 * purified in the presence ( top panel ) or the absence of additional brain lipids ( bottom panel ) . In the presence of brain lipids , NHE9 * is detected as exclusively dimeric protein with poorly resolved peaks , indicating the presence of multiple lipid adducts . Without brain lipid addition , NHE9 * appears as sharp peaks , revealing several adducts of approximately 1 kDa each , as well as a minor monomer population .

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A) Native mass spectrum of NHE9* purified in the presence (top panel) or the absence of additional brain lipids (bottom panel). In the presence of brain lipids, NHE9* is detected as exclusively dimeric protein with poorly resolved peaks, indicating the presence of multiple lipid adducts. Without brain lipid addition, NHE9* appears as sharp peaks, revealing several adducts of approximately 1 kDa each, as well as a minor monomer population.


B ) Thermal stabilization of purified dimeric NHE9 * - GFP by lipids . Normalized mean FSEC peak fluorescence before heating ( open bars ) , and after heating and centrifugation ( grey bars ) in presence of different lipids . Data presented are mean values ± data range of n = 4 experiments ( see Materials and Methods ) .

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B) Thermal stabilization of purified dimeric NHE9*-GFP by lipids. Normalized mean FSEC peak fluorescence before heating (open bars), and after heating and centrifugation (grey bars) in presence of different lipids. Data presented are mean values ± data range of n = 4 experiments (see Materials and Methods).


C ) Thermal shift stabilization of purified dimeric NHE9 * - GFP in the presence of PIP2 ( red ) compared to PIP2 - free ( black ) . Data presented are normalized mean FSEC peak fluorescence as mean values ± data range of n = 2 technical repeats ; the apparent Tm was calculated with a sigmoidal 4 - parameter logistic regression function ; the average ΔTm presented is calculated from n = 2 independent titrations .

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C) Thermal shift stabilization of purified dimeric NHE9*-GFP in the presence of PIP2 (red) compared to PIP2-free (black). Data presented are normalized mean FSEC peak fluorescence as mean values ± data range of n = 2 technical repeats; the apparent Tm was calculated with a sigmoidal 4-parameter logistic regression function; the average ΔTm presented is calculated from n = 2 independent titrations.


E ) Thermal shift stabilization of the dimeric NHE9 * - K85Q - K105Q - K107Q - GFP by PIP2 ( red ) compared with thermal shift in the absence ( black ) of PIP2 ; data shown are mean values ± data range of n = 2 technical repeats ; the apparent Tm was calculated with a sigmoidal 4 - parameter logistic regression function ; the average ΔTm presented is calculated from n = 2 independent titrations . ( F ) Native MS of the triple mutant reveals monomeric NHE9 with no notable lipid adducts ; a peak highlighted by * was determined to be a soluble contaminant as it was also apparent at MS conditions where no NHE9 was retained .

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E) Thermal shift stabilization of the dimeric NHE9*-K85Q-K105Q-K107Q-GFP by PIP2 (red) compared with thermal shift in the absence (black) of PIP2; data shown are mean values ± data range of n = 2 technical repeats; the apparent Tm was calculated with a sigmoidal 4-parameter logistic regression function; the average ΔTm presented is calculated from n = 2 independent titrations. (F) Native MS of the triple mutant reveals monomeric NHE9 with no notable lipid adducts; a peak highlighted by * was determined to be a soluble contaminant as it was also apparent at MS conditions where no NHE9 was retained.


E ) Thermal shift stabilization of the dimeric NHE9 * - K85Q - K105Q - K107Q - GFP by PIP2 ( red ) compared with thermal shift in the absence ( black ) of PIP2 ; data shown are mean values ± data range of n = 2 technical repeats ; the apparent Tm was calculated with a sigmoidal 4 - parameter logistic regression function ; the average ΔTm presented is calculated from n = 2 independent titrations .

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E) Thermal shift stabilization of the dimeric NHE9*-K85Q-K105Q-K107Q-GFP by PIP2 (red) compared with thermal shift in the absence (black) of PIP2; data shown are mean values ± data range of n = 2 technical repeats; the apparent Tm was calculated with a sigmoidal 4-parameter logistic regression function; the average ΔTm presented is calculated from n = 2 independent titrations.


Verification of screen hits by mtKeima - based FACS analysis of mitophagy . Upper : representative FACS analysis of mitophagy in HeLa cells expressing sgNTC and sgFBXL4 . Bottom : quantitative analysis of mitophagy levels in HeLa cells expressing the indicated sgRNAs . The percentage of cells with high mitophagy level is indicated in red . Two independent sgRNAs were used for each gene . NTC : non - targeting control .

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Verification of screen hits by mtKeima-based FACS analysis of mitophagy. Upper: representative FACS analysis of mitophagy in HeLa cells expressing sgNTC and sgFBXL4. Bottom: quantitative analysis of mitophagy levels in HeLa cells expressing the indicated sgRNAs. The percentage of cells with high mitophagy level is indicated in red. Two independent sgRNAs were used for each gene. NTC: non-targeting control.


Imaging analysis of mitophagy levels in the indicated HeLa cells . Left : sequencing result of WT and FBXL4 - KO cells and representative mitophagy images , white arrows point to mitolysosomes ( FBXL4 - KO cells were not labeled because of the large numbers of mitolysosomes ) ; middle : percentage of cells with mitophagy ( mitolysosome - positive ) ; right : quantification of mitolysosome / mitochondria area . n : number of cells analyzed ( middle ) ; number of imaging areas ( 20 - 30 cells / area ) analyzed ( right ) .

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Imaging analysis of mitophagy levels in the indicated HeLa cells. Left: sequencing result of WT and FBXL4-KO cells and representative mitophagy images, white arrows point to mitolysosomes (FBXL4-KO cells were not labeled because of the large numbers of mitolysosomes); middle: percentage of cells with mitophagy (mitolysosome-positive); right: quantification of mitolysosome/mitochondria area. n: number of cells analyzed (middle); number of imaging areas (20-30 cells/area) analyzed (right).


FACS analysis of mitophagy levels in the indicated HeLa cells . Left : representative FACS results ; right : quantitative analysis of mitophagy levels .

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FACS analysis of mitophagy levels in the indicated HeLa cells. Left: representative FACS results; right: quantitative analysis of mitophagy levels.


Immunoblot analysis of the indicated HeLa cells . B : BNIP3 ; N : NIX . Two clones were shown for each double and triple knockout cells .

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Immunoblot analysis of the indicated HeLa cells. B: BNIP3; N: NIX. Two clones were shown for each double and triple knockout cells.


Representative live cell imaging ( E ) of mitophagy levels in the indicated HeLa cells . n : number of cells analyzed ( left ) ; number of imaging areas analyzed ( right ) .

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Representative live cell imaging (E) of mitophagy levels in the indicated HeLa cells. n: number of cells analyzed (left); number of imaging areas analyzed (right).


Determination of the submitochondrial localization of FBXL4 by Protease K and trypsin digestion . Mitochondria from HeLa FBXL4 - FLAG stable line were purified and stored as intact mitochondria or treated with hypotonic swelling buffer or lysed with Triton X - 100 buffer . Different mitochondrial preparations were then digested with Protease K or trypsin .

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Determination of the submitochondrial localization of FBXL4 by Protease K and trypsin digestion. Mitochondria from HeLa FBXL4-FLAG stable line were purified and stored as intact mitochondria or treated with hypotonic swelling buffer or lysed with Triton X-100 buffer. Different mitochondrial preparations were then digested with Protease K or trypsin.


Analysis of the association of FBXL4 with membrane by alkaline carbonate extraction . Purified mitochondria from HeLa FBXL4 - FLAG stable line were treated with alkaline carbonate buffer at the indicated pH and then centrifuged to collect the supernatant and the pellet fractions . S : supernatant ; P : pellet .

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Analysis of the association of FBXL4 with membrane by alkaline carbonate extraction. Purified mitochondria from HeLa FBXL4-FLAG stable line were treated with alkaline carbonate buffer at the indicated pH and then centrifuged to collect the supernatant and the pellet fractions. S: supernatant; P: pellet.


Determination of the submitochondrial localization of FBXL4 ( 1 - 23 ) - RFP by Protease K digestion . Experiments were performed similarly as ( A ) .

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Determination of the submitochondrial localization of FBXL4(1-23)-RFP by Protease K digestion. Experiments were performed similarly as (A).


Immunoprecipitation analysis of the FBXL4 - Skp1 - Cullin1 ( SCF - FBXL4 ) complex . HeLa cells were infected with lentiviruses expressing the indicated genes . ∆ F : the F - box deletion mutant of FBXL4 - FLAG ; 4A : FBXL4 - 4A mutant as shown in ( A ) .

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Immunoprecipitation analysis of the FBXL4-Skp1-Cullin1 (SCF-FBXL4) complex. HeLa cells were infected with lentiviruses expressing the indicated genes. ∆F: the F-box deletion mutant of FBXL4-FLAG; 4A: FBXL4-4A mutant as shown in (A).


Immunoprecipitation analysis of the ubiquitination of BNIP3 . FLAG - BNIP3 ( knockin ) , FBXL4 - KO , HA - ubiquitin ( Ub ) HeLa cells were rescued with WT or ∆ F FBXL4 , and treated with DMSO or MG132 ( 20 μM ) for 8 hours . Immunoprecipitation analysis of the ubiquitination of NIX . FLAG - NIX ( knockin ) , FBXL4 - KO , HA - Ub HeLa cells were treated the same as ( F ) .

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Immunoprecipitation analysis of the ubiquitination of BNIP3. FLAG-BNIP3 (knockin), FBXL4-KO, HA-ubiquitin (Ub) HeLa cells were rescued with WT or ∆F FBXL4, and treated with DMSO or MG132 (20 μM) for 8 hours.   Immunoprecipitation analysis of the ubiquitination of NIX. FLAG-NIX (knockin), FBXL4-KO, HA-Ub HeLa cells were treated the same as (F).


Immunoblot analysis of the indicated HeLa cells . FBXL4 - KO HeLa cells were rescued with vector , FBXL4 - FLAG , FBXL4 ( 4A ) - FLAG or FBXL4 ( ∆ F ) - FLAG .

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Immunoblot analysis of the indicated HeLa cells. FBXL4-KO HeLa cells were rescued with vector, FBXL4-FLAG, FBXL4(4A)-FLAG or FBXL4(∆F)-FLAG.


Immunoblot analysis of the indicated HeLa cells . WT and FBXL4 - KO HeLa cells were rescued with vector , WT or mutant FBXL4 - FLAG .

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Immunoblot analysis of the indicated HeLa cells. WT and FBXL4-KO HeLa cells were rescued with vector, WT or mutant FBXL4-FLAG.


Immunoprecipitation analysis of FBXL4 - substrate interaction . FBXL4 - KO HeLa cells were rescued with vector , FBXL4 ( ∆ F ) - FLAG or mutant FBXL4 ( ∆ F ) - FLAG .

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Immunoprecipitation analysis of FBXL4-substrate interaction. FBXL4-KO HeLa cells were rescued with vector, FBXL4(∆F)-FLAG or mutant FBXL4(∆F)-FLAG.


Immunoprecipitation analysis of the integrity of the SCF - FBXL4 complex . Upper : FBXL4 - KO HeLa cells expressing vector , WT or mutant FBXL4 - FLAG were crosslinked with DSP and subject to anti - FLAG immunoprecipitation ; lower : quantification of the ratio of Cullin1 / FLAG band intensities .

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Immunoprecipitation analysis of the integrity of the SCF-FBXL4 complex. Upper: FBXL4-KO HeLa cells expressing vector, WT or mutant FBXL4-FLAG were crosslinked with DSP and subject to anti-FLAG immunoprecipitation; lower: quantification of the ratio of Cullin1/FLAG band intensities.


Viability of the indicated mice . n : number of mice analyzed . Immunoblot analysis of organs from the indicated mice at P0 of age . Elevated BNIP3 and NIX levels are highlighted by red boxes ; reduced mitochondrial proteins are highlighted by blue boxes . Three mice for each genotype were analyzed . Protein samples from HeLa cells treated with DMSO or Actinomycin D ( ActD ) were used as positive control for PARP cleavage .

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Viability of the indicated mice. n: number of mice analyzed.   Immunoblot analysis of organs from the indicated mice at P0 of age. Elevated BNIP3 and NIX levels are highlighted by red boxes; reduced mitochondrial proteins are highlighted by blue boxes. Three mice for each genotype were analyzed. Protein samples from HeLa cells treated with DMSO or Actinomycin D (ActD) were used as positive control for PARP cleavage.


Live cell confocal imaging of mitophagy level in the indicated mouse liver . Left : representative images . The dashed line indicates cell boundary . Right : quantitative analysis of mitophagy levels in hepatocytes . n : number of cells analyzed . Data are mean ± SD . Statistics : two - tailed unpaired Student ' s t - test ; * * * P < 0 . 001 .

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Live cell confocal imaging of mitophagy level in the indicated mouse liver. Left: representative images. The dashed line indicates cell boundary. Right: quantitative analysis of mitophagy levels in hepatocytes. n: number of cells analyzed. Data are mean ± SD. Statistics: two-tailed unpaired Student's t-test; ***P < 0.001.


Viability of the indicated mice . n : number of mice analyzed .

Viability of the indicated mice. n: number of mice analyzed.


Immunoblot analysis of liver samples from the indicated mice at P0 of age . Left : immunoblot analysis , three mice for each genotype were analyzed ; right : quantification of the ratio of mitochondrial protein / actin band intensities . Data are mean + SD from three biological replicates . Statistics : two - tailed unpaired Student ' s t - test ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns , not significant .

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Immunoblot analysis of liver samples from the indicated mice at P0 of age. Left: immunoblot analysis, three mice for each genotype were analyzed; right: quantification of the ratio of mitochondrial protein/actin band intensities. Data are mean + SD from three biological replicates. Statistics: two-tailed unpaired Student's t-test; *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.


GPS reporter cells expressing peptide libraries ending with a random ( X ) or Gly ( G ) residue were treated with dominant - negative ( DN ) cullins and analyzed . Data are presented as mean ± standard deviation from nine replicates . GPS reporter cells carrying indicated libraries were treated with shRNAs against various BC - box proteins and analyzed . VHL and FEM1C serve as unrelated BC - box protein controls . Data are presented as mean ± standard deviation from nine replicates .

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GPS reporter cells expressing peptide libraries ending with a random (X) or Gly (G) residue were treated with dominant-negative (DN) cullins and analyzed. Data are presented as mean ± standard deviation from nine replicates. GPS reporter cells carrying indicated libraries were treated with shRNAs against various BC-box proteins and analyzed. VHL and FEM1C serve as unrelated BC-box protein controls. Data are presented as mean ± standard deviation from nine replicates.


Comparisons of protein stability among wild - type protein and the mutants indicated on the right .

Comparisons of protein stability among wild-type protein and the mutants indicated on the right.


( A ) The relative frequency of Gly in the final 50 residues of functional human proteins with different subcellular localizations . The number on each graph denotes the left - tailed p - value of the - 1 residue . CT - inside and CT - outside represent membrane proteins with their C - termini facing the cytosol or extracellular space , respectively .

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(A) The relative frequency of Gly in the final 50 residues of functional human proteins with different subcellular localizations. The number on each graph denotes the left-tailed p-value of the -1 residue. CT-inside and CT-outside represent membrane proteins with their C-termini facing the cytosol or extracellular space, respectively.


( G ) Confocal images of U2OS cells expressing MTSGLUL - GFP proteins with or without a C - terminal diGly degron . MitoTracker staining shows mitochondria . Scale bar = 20 µm . ( H ) Confocal images of U2OS cells expressing MTSGLUL - GFP - diGly degron fusion proteins co - stained with MitoTracker , and with or without DNCul2 treatment . Scale bar = 20 µm .

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(G) Confocal images of U2OS cells expressing MTSGLUL-GFP proteins with or without a C-terminal diGly degron. MitoTracker staining shows mitochondria. Scale bar = 20 µm. (H) Confocal images of U2OS cells expressing MTSGLUL-GFP-diGly degron fusion proteins co-stained with MitoTracker, and with or without DNCul2 treatment. Scale bar = 20 µm.


Stability analysis of indicated proteins in cells treated with shRNAs against various BC - box proteins . Since GFP was tagged at the protein N - terminus and signal peptide and MTSs need to be exposed to be functional , all tested proteins were mislocalized . Protein stability analyses of wild - type ( WT ) and mutant proteins lacking their extreme terminal Gly ( ∆ G ) or having it masked by adding Leu ( + L ) in cells with or without BC - box protein knockdown ( C ) or overexpression ( D ) .

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Stability analysis of indicated proteins in cells treated with shRNAs against various BC-box proteins. Since GFP was tagged at the protein N-terminus and signal peptide and MTSs need to be exposed to be functional, all tested proteins were mislocalized. Protein stability analyses of wild-type (WT) and mutant proteins lacking their extreme terminal Gly (∆G) or having it masked by adding Leu (+L) in cells with or without BC-box protein knockdown (C) or overexpression (D).


Fractionation and Western blot analysis of wild - type and Flag - tagged PDP2 ( top ) , and wild - type and MTS - deleted ( ∆ MTS ) MRPL28 ( bottom ) . Flag - PDP2 and ∆ MTS MRPL28 were mislocalized . W , C , and M denote whole cell lysates , cytosol fraction and mitochondrial fraction , respectively . Tubulin and VDAC serve as fractionation quality controls .

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Fractionation and Western blot analysis of wild-type and Flag-tagged PDP2 (top), and wild-type and MTS-deleted (∆MTS) MRPL28 (bottom). Flag-PDP2 and ∆MTS MRPL28 were mislocalized. W, C, and M denote whole cell lysates, cytosol fraction and mitochondrial fraction, respectively. Tubulin and VDAC serve as fractionation quality controls.


Protein abundance analysis of HA - tagged wild - type or mutant secretory proteins with their signal peptide deleted ( ∆ SP ) in cells with or without KLHDC3 or APPBP2 expression . The HA epitope was inserted immediately after the signal peptide ( SP ) , which had no impact on protein secretion .

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Protein abundance analysis of HA-tagged wild-type or mutant secretory proteins with their signal peptide deleted (∆SP) in cells with or without KLHDC3 or APPBP2 expression. The HA epitope was inserted immediately after the signal peptide (SP), which had no impact on protein secretion.


Fractionation and Western blot analysis of endogenous MIC19 in U2OS cells with or without DNKLHDC2 , DNCul2 or IMP - 1088 treatments . IMP - 1088 inhibits NMTs . C and M denote cytosol and mitochondrial fractions , respectively . Tubulin and VDAC serve as fractionation quality controls . The relative abundance of cytosolic MIC19 / tubulin was normalized to that of lane 1 and is indicated under each lane in red . Another replicate of this experiment is shown in Fig . EV3B .

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Fractionation and Western blot analysis of endogenous MIC19 in U2OS cells with or without DNKLHDC2, DNCul2 or IMP-1088 treatments. IMP-1088 inhibits NMTs. C and M denote cytosol and mitochondrial fractions, respectively. Tubulin and VDAC serve as fractionation quality controls. The relative abundance of cytosolic MIC19/tubulin was normalized to that of lane 1 and is indicated under each lane in red. Another replicate of this experiment is shown in Fig. EV3B.


Abundance comparison of GFP N - or C - terminal tagged with the wild - type or mutant N - terminal ( aa . 1 - 24 ) or C - terminal ( a . a . 218 - 227 ) motif of MIC19 in cells with indicated treatments . Data from sh # 1 is shown here .

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Abundance comparison of GFP N- or C-terminal tagged with the wild-type or mutant N-terminal (aa. 1-24) or C-terminal (a.a. 218-227) motif of MIC19 in cells with indicated treatments. Data from sh#1 is shown here.


Fractionation analysis of wild - type HA - MIC19 in U2OS cells treated with IMP - 1088 and with or without inhibition of ZYG11B or KLHDC2 . The normalized relative abundance of cytosolic HA - MIC19 / tubulin is indicated under each lane in red .

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Fractionation analysis of wild-type HA-MIC19 in U2OS cells treated with IMP-1088 and with or without inhibition of ZYG11B or KLHDC2. The normalized relative abundance of cytosolic HA-MIC19/tubulin is indicated under each lane in red.


GST pull - down assay using cells expressing GST or GST - tagged wild - type or mutant KLHDC2 and HA - tagged SDE2 , FAU or TUG .

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GST pull-down assay using cells expressing GST or GST-tagged wild-type or mutant KLHDC2 and HA-tagged SDE2, FAU or TUG.


Relative stability analysis of GAPDH C - terminally tagged with the last 12 residues of indicated proteins with or without DNKLHDC2 treatment by GPS assays . The - 3 , - 4 or - 5 residue of SARS - CoV NSP proteins was mutated to Ala ( A ) as indicated . The experiment was done in triplicate using three independently prepared viruses to express DNKLHDC2 . Data are presented as the mean value of GFP / RFP + / - standard deviation from triplicate experiments .

Relative stability analysis of GAPDH C-terminally tagged with the last 12 residues of indicated proteins with or without DNKLHDC2 treatment by GPS assays. The -3, -4 or -5 residue of SARS-CoV NSP proteins was mutated to Ala (A) as indicated. The experiment was done in triplicate using three independently prepared viruses to express DNKLHDC2. Data are presented as the mean value of GFP/RFP +/- standard deviation from triplicate experiments.


D , E Immunostaining of MCT2 and SMCT1 in tumors of KIC mice treated with βOHB ( 100mg / kg / day , i . p . ) or 0 . 9 % NaCl ( i . p . ) ( n = 5 mice / group ) . Areas of MCT2 ( upper panel ) and SMCT1 ( lower panel ) stainings ( D ) are expressed as mean of percentage of total tissue area ± SEM . Significance was defined by Mann - Whitney test . * : p < 0 . 05 , * * p < 0 . 01 . Representative images of MCT2 and SMCT1 stainings ( E ) in tumors from KIC mice treated with βOHB or NaCl . Scale bar : 100 µm .

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D, E Immunostaining of MCT2 and SMCT1 in tumors of KIC mice treated with βOHB (100mg/kg/day, i.p.) or 0.9% NaCl (i.p.) (n=5 mice/group). Areas of MCT2 (upper panel) and SMCT1 (lower panel) stainings (D) are expressed as mean of percentage of total tissue area ± SEM. Significance was defined by Mann-Whitney test. *: p<0.05, **p<0.01. Representative images of MCT2 and SMCT1 stainings (E) in tumors from KIC mice treated with βOHB or NaCl. Scale bar: 100 µm.


B - D [ U - 13C ] Glucose ( n = 3 mice ) ( B ) , [ U - 13C ] Glutamine ( n = 5 mice ) ( C ) and [ U - 13C ] Acetate ( n = 6 mice ) ( D ) tracing into βOHB in poorly differentiated PDA explants from KIC mice . Data are expressed as mean ± SEM .

B-D [U-13C]Glucose (n=3 mice) (B), [U-13C]Glutamine (n=5 mice) (C) and [U-13C]Acetate (n=6 mice) (D) tracing into βOHB in poorly differentiated PDA explants from KIC mice. Data are expressed as mean ± SEM.


G Volcano plot illustrating proteins significantly under - or over - represented in sg - CTRL and sg - HMGCL PANC - 1 spheroids ( n = 3 independent experiments ) . Significance was defined by one - tailed Student ' s t - test . Protein levels with a q - value < 0 . 05 ( horizontal axis ) and a fold change < - 1 . 5 or > + 1 . 5 ( vertical axis ) are considered as significantly down or up - regulated in sg - HMGCL PANC - 1 spheroids .

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G Volcano plot illustrating proteins significantly under- or over-represented in sg-CTRL and sg-HMGCL PANC-1 spheroids (n=3 independent experiments). Significance was defined by one-tailed Student's t-test. Protein levels with a q-value<0.05 (horizontal axis) and a fold change <-1.5 or >+1.5 (vertical axis) are considered as significantly down or up-regulated in sg-HMGCL PANC-1 spheroids.


A , B Quantification of volume with representative images ( A ) and weight ( B ) of sg - CTRL ( n = 15 for A and n = 9 for B ) , sg HMGCL # 2 ( n = 19 for A and n = 8 for B ) , # 3 ( n = 7 for A and B ) pancreatic tumors . Data are expressed as mean of tumor volume or weight ± SEM . Significance was defined by Mann - Whitney test . * : p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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A, B Quantification of volume with representative images (A) and weight (B) of sg-CTRL (n=15 for A and n=9 for B), sg HMGCL #2 (n=19 for A and n=8 for B), #3 (n=7 for A and B) pancreatic tumors. Data are expressed as mean of tumor volume or weight ± SEM. Significance was defined by Mann-Whitney test. *: p<0.05, **p<0.01, ***p<0.001.


C Extracellular matrix quantification following trichrome staining in sg - CTRL and sg - HMGCL # 2 pancreatic tumors sections ( n = 8 and 9 mice / group respectively , left panel ) . Data are expressed as mean of percentage of total tissue area ± SEM . Significance was defined by Mann - Whitney test . * * : p < 0 . 01 . Representative images of trichrome staining in sg - CTRL and sg - HMGCL pancreatic tumors . Scale bar : 100 µm ( right panel ) .

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C Extracellular matrix quantification following trichrome staining in sg-CTRL and sg-HMGCL #2 pancreatic tumors sections (n=8 and 9 mice/group respectively, left panel). Data are expressed as mean of percentage of total tissue area ± SEM. Significance was defined by Mann-Whitney test. **: p<0.01. Representative images of trichrome staining in sg-CTRL and sg-HMGCL pancreatic tumors. Scale bar: 100 µm (right panel).


D , E Quantification of volume ( D ) and weight ( E ) of sg - CTRL pancreatic tumors treated with 0 . 9 % NaCl ( i . p . ) ( n = 4 ) , and pancreatic tumors from two different clones of sg - HMGCL treated with 0 . 9 % NaCl ( i . p . ) or βOHB ( 100mg / kg / bi - weekly , i . p . ) ( n = 10 / group ) . Data are expressed as mean of tumor volume or weight ± SEM . Significance was defined by Mann - Whitney test . ns : not significant , * * p < 0 . 01 .

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D, E Quantification of volume (D) and weight (E) of sg-CTRL pancreatic tumors treated with 0.9% NaCl (i.p.) (n=4), and pancreatic tumors from two different clones of sg-HMGCL treated with 0.9% NaCl (i.p.) or βOHB (100mg/kg/bi-weekly, i.p.) (n=10/group). Data are expressed as mean of tumor volume or weight ± SEM. Significance was defined by Mann-Whitney test. ns: not significant, **p<0.01.


H [ U - 13C ] βOHB tracing into TCA intermediate : citrate in sg - CTRL and sg - HMGCL # 2 and # 3 PANC - 1 cells cultured in indicated glucose concentrations . Data are expressed as mean ± SEM . ( n = 2 independent experiments ) . Significance was defined by one - way ANOVA followed by a Bonferroni ' s multiple comparisons test , only significances between sg - HMGCL # 2 , # 3 PANC - 1 cells and sg - CTRL PANC - 1 cells under the same culture condition are mentioned . * * * p < 0 . 001 .

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H [U-13C]βOHB tracing into TCA intermediate: citrate in sg-CTRL and sg-HMGCL #2 and #3 PANC-1 cells cultured in indicated glucose concentrations. Data are expressed as mean ± SEM. (n=2 independent experiments). Significance was defined by one-way ANOVA followed by a Bonferroni's multiple comparisons test, only significances between sg-HMGCL #2, #3 PANC-1 cells and sg-CTRL PANC-1 cells under the same culture condition are mentioned. ***p<0.001.


( B ) Box - and - whisker plots of ketone bodies ( KB ) metagene score defined as first component of PCA of genes in primary tumors ( n = 728 ) , in all metastases ( n = 76 ) , and in liver metastases specifically ( n = 35 ) . Box - and - whisker plot were defined with default parameters by median value ( central band at the 50th percentile ) , interquartile ranges ( IQR , box limited by 25th and 75th percentile ) and whisker boundaries defined at 1 . 5x IQR . Significance was defined by Student ' s t - test .

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(B) Box-and-whisker plots of ketone bodies (KB) metagene score defined as first component of PCA of genes in primary tumors (n=728), in all metastases (n=76), and in liver metastases specifically (n=35). Box-and-whisker plot were defined with default parameters by median value (central band at the 50th percentile), interquartile ranges (IQR, box limited by 25th and 75th percentile) and whisker boundaries defined at 1.5x IQR. Significance was defined by Student's t-test.


D - F Effect of βOHB treatment on liver metastatic incidence , size and status . Representative HPS staining of liver lobes from metastatic mice treated with βOHB ( 100mg / kg / day , i . p . ) or 0 . 9 % NaCl ( i . p . ) ( n = 8 mice / group ) . Metastatic areas are separated from liver by yellow dotted lines . Scale bar : 1mm and 500 µm for insets ( D ) . Number and size of metastasis per lobe and classified in small , medium and large size ( E ) . Pathological status of metastasis from mice treated with βOHB or NaCl ( n = 13 or 5 lobes / group , respectively ) . Data are expressed as percentage of total metastasis in all liver lobes presenting metastasis ( F ) .

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D-F Effect of βOHB treatment on liver metastatic incidence, size and status. Representative HPS staining of liver lobes from metastatic mice treated with βOHB (100mg/kg/day, i.p.) or 0.9% NaCl (i.p.) (n=8 mice/group). Metastatic areas are separated from liver by yellow dotted lines. Scale bar: 1mm and 500 µm for insets (D). Number and size of metastasis per lobe and classified in small, medium and large size (E). Pathological status of metastasis from mice treated with βOHB or NaCl (n=13 or 5 lobes/group, respectively). Data are expressed as percentage of total metastasis in all liver lobes presenting metastasis (F).


( a , b ) Isolated acini from wild - type ( WT ) and Trpml1 - / - mice ( ML1 - / - ) were used to determine the frequency of Ca2 + oscillations in response to stimulation with 3 and 10 pM CCK ( a ) and the response to high concentrations of CCK ( b ) . The average frequency from multiple experiments is given in the columns as mean ± s . e . m in ( a ) . The traces and columns in ( b ) show the mean ± s . e . m of 3 experiments each includes 4 - 6 acini composed of 6 - 10 cells .

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(a, b) Isolated acini from wild-type (WT) and Trpml1-/- mice (ML1-/-) were used to determine the frequency of Ca2+ oscillations in response to stimulation with 3 and 10 pM CCK (a) and the response to high concentrations of CCK (b). The average frequency from multiple experiments is given in the columns as mean±s.e.m in (a). The traces and columns in (b) show the mean±s.e.m of 3 experiments each includes 4-6 acini composed of 6-10 cells.


( c - e ) pancreaticacini were used to measure exocytosis in response to stimulation with the indicated CCK concentrations . ( c ) shows exocytosis during the first 5 min of stimulation , ( d ) shows the two phases , first rapid and second sustained phase , of exocytosis and ( e ) shows exocytosis at the indicated CCK concentrations measured during 30 min of stimulation . The results are averages of 3 - 4 experiments with each mouse line . denotes p < 0 . 01 or better and # denotes p < 0 . 05 or better .

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(c-e) pancreaticacini were used to measure exocytosis in response to stimulation with the indicated CCK concentrations. (c) shows exocytosis during the first 5 min of stimulation, (d) shows the two phases, first rapid and second sustained phase, of exocytosis and (e) shows exocytosis at the indicated CCK concentrations measured during 30 min of stimulation. The results are averages of 3-4 experiments with each mouse line. denotes p<0.01 or better and # denotes p<0.05 or better.


Panel ( a ) shows example TEM ultrastructure images obtained from male and female mice pancreas and the columns shows the granules size distribution . Granules size was analyzed in at least 10 images from 3 wild - type and 5 Trpml1 - / - mice .

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Panel (a) shows example TEM ultrastructure images obtained from male and female mice pancreas and the columns shows the granules size distribution. Granules size was analyzed in at least 10 images from 3 wild-type and 5 Trpml1-/- mice.


( b ) Fixed acini were co - stained with Amylase ( granules ) and LAMP1 ( lysosomes ) and the fold overlap was calculated from 14 and 11 images obtained from 3 wild - type and 3 Trpml1 - / - mice , respectively .

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(b) Fixed acini were co-stained with Amylase (granules) and LAMP1 (lysosomes) and the fold overlap was calculated from 14 and 11 images obtained from 3 wild-type and 3 Trpml1-/- mice, respectively.


( c ) Live wild - type and Trpml1 - / - acini were labeled with mitotracker ( green ) and lysotracker ( red ) . The panels show the merged green , red and DIC images . The large vesicles are marked by white arrows and shown at higher magnification in the inserts . The overlap between lysosomes and granules was determined by ImageJ ( see methods ) . The results are from 14 wild - type and 30 Trpml1 - / - acini each comprising 3 - 7 cells and obtained from 3 wild - type and 4 Trpml1 - / - mice .

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(c) Live wild-type and Trpml1-/- acini were labeled with mitotracker (green) and lysotracker (red). The panels show the merged green, red and DIC images. The large vesicles are marked by white arrows and shown at higher magnification in the inserts. The overlap between lysosomes and granules was determined by ImageJ (see methods). The results are from 14 wild-type and 30 Trpml1-/- acini each comprising 3-7 cells and obtained from 3 wild-type and 4 Trpml1-/- mice.


( d ) Co - IP of lysosomal LAMP1 and granules VAMP8 and Rab27b . Note the increased lysosomal / granules interaction .

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(d) Co-IP of lysosomal LAMP1 and granules VAMP8 and Rab27b. Note the increased lysosomal/granules interaction.


( e ) Enhanced exocytosis of the lysosomal acid phosphatase by Trpml1 - / - acini stimulated with 100 pM CCK . The results are the mean ± s . e . m of 3 experiments with acini obtained from 3 wild - type and 3 Trpml1 - / - mice .

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(e) Enhanced exocytosis of the lysosomal acid phosphatase by Trpml1-/-acini stimulated with 100 pM CCK. The results are the mean±s.e.m of 3 experiments with acini obtained from 3 wild-type and 3 Trpml1-/-mice.


( a , b ) Plasmaamylase ( a ) and pancreaticMPO ( b ) in 3 mice of each line .

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(a, b) Plasmaamylase(a) and pancreaticMPO(b) in 3 mice of each line.


( c ) Edema was evaluated in pancreas of mice treated with PBS or caerulein by H & amp ; amp ; E staining . Shown are examples of images and the average damage in the columns , which show the mean ± s . e . m of 4 experiments with each mouse line .

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(c) Edema was evaluated in pancreas of mice treated with PBS or caerulein by H&amp;E staining. Shown are examples of images and the average damage in the columns, which show the mean±s.e.m of 4 experiments with each mouse line.


( d ) Intracellular trypsin activity was measured in isolated pancreatic acini before and after stimulation with 10 nM CCK for 30 min . Shown are example images and the averages .

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(d) Intracellular trypsin activity was measured in isolated pancreatic acini before and after stimulation with 10 nM CCK for 30 min. Shown are example images and the averages.


( e ) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained for the lipid ceramide . The figure shows example images before stimulation and the averaged fluorescence intensity .

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(e) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained for the lipid ceramide. The figure shows example images before stimulation and the averaged fluorescence intensity.


( f ) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained with the autophagy marker LC3 . The figure shows example images before stimulation and the columns depict the number of autophagosomes as determined by ImageJ . Results in ( c - f ) are given as the mean ± s . e . m of 4 experiments from 4 mice of each line and at least 10 acini in each experiment .

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(f) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained with the autophagy marker LC3. The figure shows example images before stimulation and the columns depict the number of autophagosomes as determined by ImageJ. Results in (c-f) are given as the mean±s.e.m of 4 experiments from 4 mice of each line and at least 10 acini in each experiment.


( a ) Isolated acini from wild - type ( black ) and Trpml1 - / - mice ( red ) loaded with Fura2 were used to determine [ Ca2 + ] i in response to stimulation with 100 µM carbachol . The traces are the mean ± s . e . m . of 6 acinar clusters . Similar results were obtained in 3 experiments .

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(a) Isolated acini from wild-type (black) and Trpml1-/- mice (red) loaded with Fura2 were used to determine [Ca2+]i in response to stimulation with 100 µM carbachol. The traces are the mean±s.e.m. of 6 acinar clusters. Similar results were obtained in 3 experiments.


( b , c ) Saliva was collected from wild - type ( black ) and Trpml1 - / - mice ( red ) injected I . P . with 10 mg / kg pilocarpine ( b ) or 1 mg / kg pilocarpine and 0 . 6 mg / kg isoproterenol ( c ) over 20 - 40 min to stimulate fluid secretion by pilocarpine and amylase secretion by isoproterenol . Each condition used 4 - 5 mice from each like and the results are given as the mean ± s . e . m .

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(b, c) Saliva was collected from wild-type (black) and Trpml1-/- mice (red) injected I.P. with 10 mg/kg pilocarpine (b) or 1 mg/kg pilocarpine and 0.6 mg/kg isoproterenol (c) over 20-40 min to stimulate fluid secretion by pilocarpine and amylase secretion by isoproterenol. Each condition used 4-5 mice from each like and the results are given as the mean±s.e.m.


( d ) the saliva collected in ( c ) was used to determine the amount of secreted amylase .

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(d) the saliva collected in (c) was used to determine the amount of secreted amylase.


( e , f ) Isolated acini from the parotid glands of wild - type ( black ) or Trpml1 - / - mice ( red ) were used to measure amylase secretion in response to 50 nM isoproterenol for the indicated times ( e ) and the indicated isoproterenol concentrations during 30 min stimulation ( f ) . The results are the mean ± s . e . m obtained from two mice of each line .

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(e, f) Isolated acini from the parotid glands of wild-type (black) or Trpml1-/- mice (red) were used to measure amylase secretion in response to 50 nM isoproterenol for the indicated times (e) and the indicated isoproterenol concentrations during 30 min stimulation (f). The results are the mean±s.e.m obtained from two mice of each line.


( a ) shows example TEM images and ( b ) shows the granules size distribution in parotid glands determined from 10 images obtained from 3 wild - type mice and 14 images obtained from 4 Trpml1 - / - mice .

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(a) shows example TEM images and (b) shows the granules size distribution in parotid glands determined from 10 images obtained from 3 wild-type mice and 14 images obtained from 4 Trpml1-/- mice.


( c ) Co - IP of the lysosomal LAMP1 and the secretory granules VAMP8 and Rab27b . Input control is actin .

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(c) Co-IP of the lysosomal LAMP1 and the secretory granules VAMP8 and Rab27b. Input control is actin.


( d ) Acini isolated from the parotid glands of wild - type and Trpml1 - / - mice were incubated with mitotracker ( green ) and lysotracker . The large vesicles in the secretory granules / lysosomal area are marked by white arrows . The average number of enlarged profiles is given in the columns and obtained from 3 acinar preparations of each line .

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(d) Acini isolated from the parotid glands of wild-type and Trpml1-/- mice were incubated with mitotracker (green) and lysotracker. The large vesicles in the secretory granules/lysosomal area are marked by white arrows. The average number of enlarged profiles is given in the columns and obtained from 3 acinar preparations of each line.


( e ) Shown is the time course ( left panel ) and averaged 10 min secretion ( columns ) of the lysosomal marker acid phosphatase in the saliva collected from wild - type ( black ) and Trpml1 - / - mice ( red ) stimulated with 1 mg / kg pilocarpine and 0 . 6 mg / kg isoproterenol . The mean ± s . e . m is from 4 mice of each line .

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(e) Shown is the time course (left panel) and averaged 10 min secretion (columns) of the lysosomal marker acid phosphatase in the saliva collected from wild-type (black) and Trpml1-/- mice (red) stimulated with 1 mg/kg pilocarpine and 0.6 mg/kg isoproterenol. The mean±s.e.m is from 4 mice of each line.


( a , b ) example traces of mEPSCs recorded from wild - type ( a ) and Trpml1 - / - ( b ) micebrain slices as detailed in Methods . The lower traces in each panel show the portion of the traces marked by dotted boxes in an expanded time scale . ( c ) histogram distribution of the events at each mEPSC . The lower columns show the distribution of the higher amplitude mEPSCs . ( d ) the frequency of mEPSCs in each line . The results in ( c , b ) are the mean ± s . e . m of 8 experiments each obtained from 4 mice of each line .

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(a, b) example traces of mEPSCs recorded from wild-type (a) and Trpml1-/-(b) micebrain slices as detailed in Methods. The lower traces in each panel show the portion of the traces marked by dotted boxes in an expanded time scale. (c) histogram distribution of the events at each mEPSC. The lower columns show the distribution of the higher amplitude mEPSCs. (d) the frequency of mEPSCs in each line. The results in (c, b) are the mean±s.e.m of 8 experiments each obtained from 4 mice of each line.


( e ) the mean ± s . e . m of the Ca2 + signal in response to depolarization with 30 mM KCl of 6 and 5 experiments with wild - type and Trpml1 - / - slices , respectively .

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(e) the mean±s.e.m of the Ca2+ signal in response to depolarization with 30 mM KCl of 6 and 5 experiments with wild-type and Trpml1-/- slices, respectively.


( a ) example TEM images of synapses in wild - type and Trpml1 - / - micebraincortex . ( b ) the distribution of synaptic vesicle size determined in 10 and 14 images obtained from 3 wild - type and 3 Trpml1 - / - mice , respectively . ( c ) example TEM images at low magnification of wild - type and Trpml1 - / - micebraincortical slices with the dark PSD profiles marked by white arrows . ( d ) average number of PSD / µm2 analyzed in at least 10 images obtained from 3 mice of each line .

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(a) example TEM images of synapses in wild-type and Trpml1-/-micebraincortex. (b) the distribution of synaptic vesicle size determined in 10 and 14 images obtained from 3 wild-type and 3 Trpml1-/-mice, respectively. (c) example TEM images at low magnification of wild-type and Trpml1-/-micebraincortical slices with the dark PSD profiles marked by white arrows. (d) average number of PSD/µm2 analyzed in at least 10 images obtained from 3 mice of each line.


( e ) Glutamate levels and secretion by cultured cerebral cortical neurons . Total glutamate measured in non - stimulated wild - type ( black ) and Trpml1 - / - ( green ) neurons and neurons stimulated by depolarization with 30 mM KCl ( blue and red ) .

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(e) Glutamate levels and secretion by cultured cerebral cortical neurons. Total glutamate measured in non-stimulated wild-type (black) and Trpml1-/- (green) neurons and neurons stimulated by depolarization with 30 mM KCl (blue and red).


( f ) Rescue of glutamate levels and secretion by expression of TRPML1 in cultured cerebral cortical neurons . Total glutamate measured in non - stimulated Trpml1 - / - neurons ( green ) and Trpml1 - / - neurons transfected with TRPML1 ( black ) and untransfected ( red ) and TRPML1 - transfected ( clue ) Trpml1 - / - neurons stimulated by depolarization with 30 mM KCl .

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(f) Rescue of glutamate levels and secretion by expression of TRPML1 in cultured cerebral cortical neurons. Total glutamate measured in non-stimulated Trpml1-/- neurons (green) and Trpml1-/- neurons transfected with TRPML1 (black) and untransfected (red) and TRPML1-transfected (clue) Trpml1-/- neurons stimulated by depolarization with 30 mM KCl.


( g ) averaged Co - IP of SNAP25 and VAMP2 in three brain regions of wild - type and Trpml1 - / - mice . The images of all blots are given in Extended view Figure E4 .

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(g) averaged Co-IP of SNAP25 and VAMP2 in three brain regions of wild-type and Trpml1-/- mice. The images of all blots are given in Extended view Figure E4.


293T cells were transfected with a plasmid encoding FLAG - Stx17 wild type ( WT ) or the indicated constructs . At 24 h after transfection , cell lysates were immunoprecipitated ( IP ) with anti - FLAG M2 beads , and analyzed by IB using antibodies against PGAM5 and FLAG . Five percent of lysates was analyzed as input .

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293T cells were transfected with a plasmid encoding FLAG-Stx17 wild type (WT) or the indicated constructs. At 24 h after transfection, cell lysates were immunoprecipitated (IP) with anti-FLAG M2 beads, and analyzed by IB using antibodies against PGAM5 and FLAG. Five percent of lysates was analyzed as input.


HeLa cells stably expressing FLAG - Stx17 WT or the K254C mutant were fixed and subjected to PLA using antibodies against FLAG and PGAM5 . Scale bar , 5 μm . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with WT ( paired Student ' s t - test ) .

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HeLa cells stably expressing FLAG-Stx17 WT or the K254C mutant were fixed and subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with WT (paired Student's t-test).


MBP or the MBP - Stx17 constructs attached to amylose resin were mixed with GST - PGAM5 , and the proteins bound to the resin were separated by SDS - PAGE and blotted onto PVDF membranes . The blots were detected by an anti - GST antibody ( upper panels ) or stained with Coomassie Brilliant Blue R - 250 ( lower panels ) . Ten percent of the proteins used for each experiment was analyzed as input . Asterisks and double asterisk may represent MBP dimers and degradation products .

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MBP or the MBP-Stx17 constructs attached to amylose resin were mixed with GST-PGAM5, and the proteins bound to the resin were separated by SDS-PAGE and blotted onto PVDF membranes. The blots were detected by an anti-GST antibody (upper panels) or stained with Coomassie Brilliant Blue R-250 (lower panels). Ten percent of the proteins used for each experiment was analyzed as input. Asterisks and double asterisk may represent MBP dimers and degradation products.


293T cells were cotransfected with plasmids encoding FLAG - Stx17 WT and the indicated PGAM5 - GFP constructs , and

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293T cells were cotransfected with plasmids encoding FLAG-Stx17 WT and the indicated PGAM5-GFP constructs, and analyzed


293T cells were cotransfected with plasmids encoding FLAG - Stx17 or Stx18 and the C - terminally GFP - tagged transmembrane domain of PGAM5 （ amino acids 1 - 35 ） constructs , and

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293T cells were cotransfected with plasmids encoding FLAG-Stx17 or Stx18 and the C-terminally GFP-tagged transmembrane domain of PGAM5（amino acids 1-35）constructs, and analyzed


HeLa cells were treated with DMSO ( Vehicle ) or 0 . 03 mg / ml digitonin ( + Digitonin ) , fixed and then double immunostained for PGAM5 and Tom20 . Scale bar , 5 μm . The bar graph on the right shows the Manders ' coefficients for the colocalization of PGAM5 and Tom20 . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with Vehicle ( paired Student '

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HeLa cells were treated with DMSO (Vehicle) or 0.03 mg/ml digitonin (+Digitonin), fixed and then double immunostained for PGAM5 and Tom20. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of PGAM5 and Tom20. Values are means ± SEM (n = 3). ***P<0.001 as compared with Vehicle (paired Student's


HeLa cells stably expressing FLAG - Stx17 wild type ( WT ) were transfected with a plasmid encoding Su9 - GFP ( mitochondria ) or Sec61β - GFP ( ER ) . At 24 h after transfection , the cells were subjected to PLA using antibodies against FLAG and PGAM5 . Scale bar , 5 μm . The bar graph on the right shows the Manders ' coefficients for the colocalization of PLA dots and Su9 - GFP or Sec61β - GFP . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 ( paired Student ' s t - test ) .

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HeLa cells stably expressing FLAG-Stx17 wild type (WT) were transfected with a plasmid encoding Su9-GFP (mitochondria) or Sec61β-GFP (ER). At 24 h after transfection, the cells were subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of PLA dots and Su9-GFP or Sec61β-GFP. Values are means ± SEM (n = 3). ***P<0.001 (paired Student's t-test).


HeLa cells were treated with DMSO ( Vehicle ) or 20 μM CCCP ( + CCCP ) for 2 h , lysed and subjected to Percoll - based fractionation . Equal amounts of proteins were analyzed by IB using the indicated antibodies . PNS , postnuclear supernatant ; MS , microsomes ; Mt , mitochondria . The amounts of proteins recovered on fractionation were as follows for vehicle and CCCP treatment , respectively : PNS ( 6 . 6 mg and 5 . 5 mg ) , cytosol ( 4 . 8 mg and 4 . 9 mg ) , MS ( 2 . 0 mg and 2 . 2 mg ) , MAM ( 0 . 55 mg and 0 . 46 mg ) and Mt ( 0 . 30 mg and 0 . 28 mg ) .

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HeLa cells were treated with DMSO (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed and subjected to Percoll-based fractionation. Equal amounts of proteins were analyzed by IB using the indicated antibodies. PNS, postnuclear supernatant; MS, microsomes; Mt, mitochondria. The amounts of proteins recovered on fractionation were as follows for vehicle and CCCP treatment, respectively: PNS (6.6 mg and 5.5 mg), cytosol (4.8 mg and 4.9 mg), MS (2.0 mg and 2.2 mg), MAM (0.55 mg and 0.46 mg) and Mt (0.30 mg and 0.28 mg).


Electron microscopic analysis of HeLa cells expressing PGAM5 - GFP and APEX2 - GFP - binding peptide . Arrows indicate the position of 3 , 3 ' - diaminobenzidine reaction at the ER - mitochondria interface . Scale bar , 500 nm .

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Electron microscopic analysis of HeLa cells expressing PGAM5-GFP and APEX2-GFP-binding peptide. Arrows indicate the position of 3,3'-diaminobenzidine reaction at the ER-mitochondria interface. Scale bar, 500 nm.


HeLa cells stably expressing FLAG - Stx17 WT were mock - transfected or transfected with siRNA for Mfn1 , Mfn2 or PACS - 2 . At 72 h after transfection , the cells were subjected to PLA using antibodies against FLAG and PGAM5 . Scale bar , 5 μm . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with Mock ( paired Student ' s t - test ) .

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HeLa cells stably expressing FLAG-Stx17 WT were mock-transfected or transfected with siRNA for Mfn1, Mfn2 or PACS-2. At 72 h after transfection, the cells were subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with Mock (paired Student's t-test).


HeLa cells were mock - transfected or transfected with siRNA for Mfn1 , Stx17 , Mfn2 or PACS - 2 . At 72 h after transfection , the cells were subjected to PLA using antibodies against Drp1 and PGAM5 . Scale bar , 5 μm . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with Mock ( paired Student ' s t - test ) .

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HeLa cells were mock-transfected or transfected with siRNA for Mfn1, Stx17, Mfn2 or PACS-2. At 72 h after transfection, the cells were subjected to PLA using antibodies against Drp1 and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with Mock (paired Student's t-test).


HeLa cells with mock treatment ( Mock ) or depleted of Stx17 ( Stx17 KD ) were fixed and double immunostained for PGAM5 and Tom20 . Scale bar , 5 μm .

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HeLa cells with mock treatment (Mock) or depleted of Stx17 (Stx17 KD) were fixed and double immunostained for PGAM5 and Tom20. Scale bar, 5 μm.


293T cells with mock treatment or depleted of Stx17 were incubated with ethanol ( Vehicle ) or 20 μM CCCP ( + CCCP ) for 2 h , lysed and then analyzed by IB using the indicated antibodies .

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293T cells with mock treatment or depleted of Stx17 were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed and then analyzed by IB using the indicated antibodies.


293T cells transiently expressing FLAG - Stx17 wild type ( WT ) or the K254C mutant were incubated with ethanol ( Vehicle ) or 20 μM CCCP ( + CCCP ) for 2 h , lysed , immunoprecipitated with anti - FLAG M2 beads and then analyzed by IB using the indicated antibodies .

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293T cells transiently expressing FLAG-Stx17 wild type (WT) or the K254C mutant were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed, immunoprecipitated with anti-FLAG M2 beads and then analyzed by IB using the indicated antibodies.


HeLa cells stably expressing FLAG - Stx17 WT were incubated with ethanol ( Vehicle ) or 20 μM CCCP ( + CCCP ) for 2 h , and subjected to PLA using antibodies against FLAG and PGAM5 . Scale bar , 5 μm . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with Vehicle ( paired Student ' s t - test ) .

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HeLa cells stably expressing FLAG-Stx17 WT were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, and subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with Vehicle (paired Student's t-test).


HeLa cells stably expressing GFP - Parkin with mock treatment ( Control ) or depleted ( KD ) of Stx17 , PGAM5 or Drp1 were incubated for 16 h with ethanol ( Vehicle ) or 10 μM CCCP ( + CCCP ) , and analyzed by IB using the indicated antibodies .

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HeLa cells stably expressing GFP-Parkin with mock treatment (Control) or depleted (KD) of Stx17, PGAM5 or Drp1 were incubated for 16 h with ethanol (Vehicle) or 10 μM CCCP (+CCCP), and analyzed by IB using the indicated antibodies.


HeLa cells stably expressing GFP - Parkin with mock treatment ( Mock ) or depleted of Stx17 , PGAM5 or Drp1 were incubated in the absence ( Vehicle ) or presence of 20 μM CCCP ( + CCCP ) for 2 h and analyzed by immunofluorescence microscopy . Scale bar , 5 μm . The bar graph on the right shows the Manders ' coefficients for the colocalization of GFP - Parkin and Tom20 . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with Mock ( paired Student ' s t - test ) .

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HeLa cells stably expressing GFP-Parkin with mock treatment (Mock) or depleted of Stx17, PGAM5 or Drp1 were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h and analyzed by immunofluorescence microscopy. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of GFP-Parkin and Tom20. Values are means ± SEM (n = 3). ***P<0.001 as compared with Mock (paired Student's t-test).


PINK1 - FLAG and GFP - Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence ( ethanol ) or presence of 20 μM CCCP for 2 h and analyzed by IB using the indicated antibodies . F , full - length PINK1 ; C , cleaved PINK1 .

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PINK1-FLAG and GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence (ethanol) or presence of 20 μM CCCP for 2 h and analyzed by IB using the indicated antibodies. F, full-length PINK1; C, cleaved PINK1.


GFP - Parkin stably expressing HeLa cells were mock - transfected ( Mock ) or transfected with siRNA ( KD ) for Stx17 or PGAM5 . At 48 h after siRNA transfection , the cells were transfected with a plasmid encoding FLAG - DFCP1 , incubated for 24 h , treated with 20 μM CCCP for 2 h and then immunostained for FLAG . Scale bars , 5 μm . The bar graph below shows the Manders ' coefficients for the colocalization of GFP - Parkin and FLAG - DFCP1 . Values are means ± SEM ( n = 3 ) . * * P < 0 . 01 as compared with Mock ( paired Student ' s t - test ) .

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GFP-Parkin stably expressing HeLa cells were mock-transfected (Mock) or transfected with siRNA (KD) for Stx17 or PGAM5. At 48 h after siRNA transfection, the cells were transfected with a plasmid encoding FLAG-DFCP1, incubated for 24 h, treated with 20 μM CCCP for 2 h and then immunostained for FLAG. Scale bars, 5 μm. The bar graph below shows the Manders' coefficients for the colocalization of GFP-Parkin and FLAG-DFCP1. Values are means ± SEM (n = 3). **P<0.01 as compared with Mock (paired Student's t-test).


GFP - Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated with 20 μM CCCP for 2 h and immunostained for LC3 . Scale bar , 5 μm . The bar graph below shows the Manders ' coefficients for the colocalization of GFP - Parkin and LC3 . Values are means ± SEM ( n = 3 ) . * * P < 0 . 01 as compared with Mock ( paired Student ' s t - test ) .

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GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated with 20 μM CCCP for 2 h and immunostained for LC3. Scale bar, 5 μm. The bar graph below shows the Manders' coefficients for the colocalization of GFP-Parkin and LC3. Values are means ± SEM (n = 3). **P<0.01 as compared with Mock (paired Student's t-test).


GFP - Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence ( Vehicle ) or presence of 20 μM CCCP ( + CCCP ) for 2 h and analyzed by IB using the indicated antibodies .

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GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h and analyzed by IB using the indicated antibodies.


HeLa cells stably expressing GFP - Parkin with mock treatment ( Control ) or depleted of FUNDC1 ( FUNDC1 KD ) were incubated for 16 h with ethanol ( Vehicle ) or 10 μM CCCP ( + CCCP ) , and analyzed by IB using the indicated antibodies .

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HeLa cells stably expressing GFP-Parkin with mock treatment (Control) or depleted of FUNDC1 (FUNDC1 KD) were incubated for 16 h with ethanol (Vehicle) or 10 μM CCCP (+CCCP), and analyzed by IB using the indicated antibodies.


GFP - Parkin stably expressing HeLa cells with mock treatment ( Mock ) or depleted of FUNDC1 ( FUNDC1 KD ) were incubated with 20 μM CCCP for 2 h and immunostained for Tom20 . Scale bar , 5 μm .

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GFP-Parkin stably expressing HeLa cells with mock treatment (Mock) or depleted of FUNDC1 (FUNDC1 KD) were incubated with 20 μM CCCP for 2 h and immunostained for Tom20. Scale bar, 5 μm.


293T cells with mock treatment ( Mock ) or depleted of Stx17 ( Stx17 KD ) were incubated in the absence ( Vehicle ) or presence of 20 μM CCCP ( + CCCP ) for 2 h , immunoprecipitated with anti - FLAG M2 beads and then analyzed by IB using the indicated antibodies . Three percent of lysates was analyzed as input . During Stx17 knockdown , cells were transfected with a plasmid encoding FLAG - FUNDC1 .

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293T cells with mock treatment (Mock) or depleted of Stx17 (Stx17 KD) were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h, immunoprecipitated with anti-FLAG M2 beads and then analyzed by IB using the indicated antibodies. Three percent of lysates was analyzed as input. During Stx17 knockdown, cells were transfected with a plasmid encoding FLAG-FUNDC1.


GFP - Parkin stably expressing HeLa cells were treated and then subjected to PLA using antibodies against FLAG and PGAM5 . During Stx17 knockdown , cells were transfected with a plasmid encoding FLAG - FUNDC1 . Scale bar , 5 μm . Values are means ± SEM ( n = 3 ) . * P < 0 . 05 and * * P < 0 . 01 as compared with Mock ( + CCCP ) by paired Student ' s t - test .

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GFP-Parkin stably expressing HeLa cells were treated and then subjected to PLA using antibodies against FLAG and PGAM5. During Stx17 knockdown, cells were transfected with a plasmid encoding FLAG-FUNDC1. Scale bar, 5 μm. Values are means ± SEM (n = 3). *P<0.05 and **P<0.01 as compared with Mock (+CCCP) by paired Student's t-test.


A PGAM5 overexpression partially rescues the mitochondrial defects by Stx17 loss . Transmission electron microscope images of the indirect flight muscle in the indicated genotypes ( a , Control ; b , Stx17 - / - ; c , Stx17 - / - and Stx17 overexpression ; d , Stx17 - / - and PGAM5 overexpression ) of 7 - day - old adult flies are shown . The bar graph on the right shows frequency of healthy and abnormal mitochondria presented as percentages ( mean ± SEM ) using the scoring system : Class 0 , normal ; Class 1 , fuzzy or dilated cristae ; Class 2 , fragmented cristae and loss of electron density . * p < 0 . 05 , # p < 0 . 001 vs . the same class of control ( Dunnett ' s test ) . n = 50 - 119 mitochondria from three or four independent samples . Genotypes used were : + / y ; Act5c - GAL4 / + ( a ) , + / y ; Act5c - GAL4 / + ; Stx17LL06330 / Stx17LL06330 ( b ) , + / y ; Act5c - GAL4 / UAS - FLAG - Stx17 ; Stx17LL06330 / Stx17LL06330 ( c ) , + / y ; Act5c - GAL4 / UAS - PGAM5 ; Stx17LL06330 / Stx17LL06330 ( d ) . Scale bars , 200 nm .

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A PGAM5 overexpression partially rescues the mitochondrial defects by Stx17 loss. Transmission electron microscope images of the indirect flight muscle in the indicated genotypes (a, Control; b, Stx17-/-; c, Stx17-/- and Stx17 overexpression; d, Stx17-/- and PGAM5 overexpression) of 7-day-old adult flies are shown. The bar graph on the right shows frequency of healthy and abnormal mitochondria presented as percentages (mean ± SEM) using the scoring system : Class 0, normal; Class 1, fuzzy or dilated cristae; Class 2, fragmented cristae and loss of electron density. *p < 0.05, #p < 0.001 vs. the same class of control (Dunnett's test). n = 50-119 mitochondria from three or four independent samples. Genotypes used were: +/y; Act5c-GAL4/+ (a), +/y; Act5c-GAL4/+; Stx17LL06330/Stx17LL06330 (b), +/y; Act5c-GAL4/UAS-FLAG-Stx17; Stx17LL06330/Stx17LL06330 (c), +/y; Act5c-GAL4/UAS-PGAM5; Stx17LL06330/Stx17LL06330 (d). Scale bars, 200 nm.


B Simultaneous reduction of Stx17 and PGAM5 results in mitochondrial degeneration . a , Stx17 + / - ; b , PGAM5 - / - ; c , Stx17 + / - , PGAM5 - / - . The bar graph on the right represents the mitochondrial phenotypes classified p < 0 . 05 , * * p < 0 . 01 vs . the same class of Stx17 + / - ( Dunnett ' s test ) . n = 62 - 195 mitochondria from three independent samples . Scale bars = 1 µm . Genotypes used were : + / y ; Act5c - GAL4 / + ; Stx17LL06330 / + ( a ) , PGAM51 / y ; Act5c - GAL4 / + ( b ) , PGAM51 / y ; Act5c - GAL4 / UAS - Stx17 - FLAG ; Stx17LL06330 / + ( c ) .

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B Simultaneous reduction of Stx17 and PGAM5 results in mitochondrial degeneration. a, Stx17+/-; b, PGAM5-/-; c, Stx17+/-, PGAM5-/-. The bar graph on the right represents the mitochondrial phenotypes classified p < 0.05, **p < 0.01 vs. the same class of Stx17+/- (Dunnett's test). n = 62-195 mitochondria from three independent samples. Scale bars = 1 µm. Genotypes used were: +/y; Act5c-GAL4/+; Stx17LL06330/+ (a), PGAM51/y; Act5c-GAL4/+ (b), PGAM51/y; Act5c-GAL4/UAS-Stx17-FLAG; Stx17LL06330/+ (c).


( D ) Mean current responses to rapid switching from 140 mM CholGluc to 140 mM KGluc of HEK293T cells expressing WT EAAT1 ( n = 3 ) or WT EAAT2 ( n = 3 ) . Cells were intracellularly dialyzed with a 115mM KGluc - based solution and held at 0 mV .

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(D) Mean current responses to rapid switching from 140 mM CholGluc to 140 mM KGluc of HEK293T cells expressing WT EAAT1 (n=3) or WT EAAT2 (n=3). Cells were intracellularly dialyzed with a 115mM KGluc-based solution and held at 0 mV.


( B ) Representative WT and mutant whole - cell current responses to voltage jumps before and after removal of external K + . Cells were dialyzed with a KNO3 - based pipette solution and the external solution contained either 140 mM CholineNO3 or 140 mM KNO3 .

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(B) Representative WT and mutant whole-cell current responses to voltage jumps before and after removal of external K+. Cells were dialyzed with a KNO3-based pipette solution and the external solution contained either 140 mM CholineNO3 or 140 mM KNO3.


( C ) Relative changes in fluorescence for WT , D312N and D405N GltPh upon a temperature jump in MST experiments as a function of [ K + ] . Solid lines represent non - parametric fits , and shaded area show bootstrapped 99 % confidence intervals for those fits . Non - overlapping confidence intervals indicate significant difference between WT and mutant binding curves . Values are given as mean ± SD ( n ≥ 3 ) .

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(C) Relative changes in fluorescence for WT, D312N and D405N GltPh upon a temperature jump in MST experiments as a function of [K+]. Solid lines represent non-parametric fits, and shaded area show bootstrapped 99% confidence intervals for those fits. Non-overlapping confidence intervals indicate significant difference between WT and mutant binding curves. Values are given as mean ± SD (n≥3).


( D ) Representative whole - cell current recordings from cells internally dialyzed with NaNO3 and glutamate in choline - NO3 - based external solutions or in NaNO3 - based solutions supplemented with 1 mM L - glutamate . Glutamate - induced changes in anion current amplitudes indicate that D399N and D486N EAAT2 ( GltPh D312 and D405 ) are K + - independent , but are functionally expressed in the plasma membrane .

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(D) Representative whole-cell current recordings from cells internally dialyzed with NaNO3 and glutamate in choline-NO3-based external solutions or in NaNO3-based solutions supplemented with 1 mM L-glutamate. Glutamate-induced changes in anion current amplitudes indicate that D399N and D486N EAAT2 (GltPh D312 and D405) are K+-independent, but are functionally expressed in the plasma membrane.


( E ) Current - voltage relationships of steady - state currents from cells dialyzed with a KNO3 - based pipette solution . Currents were measured at external 140 mM KNO3 and normalized to the current at - 170 mV from a consecutive recording of the same cell in K + - free external solutions . Values are given as mean ± SD ( n ≥ 3 )

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(E) Current-voltage relationships of steady-state currents from cells dialyzed with a KNO3-based pipette solution. Currents were measured at external 140 mM KNO3 and normalized to the current at -170 mV from a consecutive recording of the same cell in K+-free external solutions. Values are given as mean ± SD (n≥3)


( E ) Representative transport current recordings from cells expressing L448A or L448T ( GltPh A360 ) or WT EAAT1 upon voltage jumps to ˗ 140 mV before and after superfusion with 1 mM l - glutamate . Cells were intracellularly dialyzed with a K + - free solution without permeating anions to isolate K + - independent transport currents . Transient capacitive currents during the first 5 ms after a voltage jump were blanked .

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(E) Representative transport current recordings from cells expressing L448A or L448T (GltPh A360) or WT EAAT1 upon voltage jumps to ˗140 mV before and after superfusion with 1 mM l-glutamate. Cells were intracellularly dialyzed with a K+-free solution without permeating anions to isolate K+-independent transport currents. Transient capacitive currents during the first 5 ms after a voltage jump were blanked.


( F ) Current - voltage relationship for net transport current amplitudes for WT and L448A / T EAAT1 ( GltPh A360 ) for choline - based pipette solutions . Values are given as mean ± SD with indicated numbers of experiments .

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(F) Current-voltage relationship for net transport current amplitudes for WT and L448A/T EAAT1 (GltPh A360) for choline-based pipette solutions. Values are given as mean ± SD with indicated numbers of experiments.