Yoon MJ , Choi B , Kim EJ , Ohk J , Yang C , Choi YG , Lee J , Kang C , Song HK , Kim YK , Woo JS , Cho Y , Choi EJ , Jung H , Kim C .

             autophagy
             chaperone binding

	Fig. 1: A p62 binding protein, UXT colocalizes with protein aggregates. a Domain structure of p62. b p62 knockout HeLa cells (HeLa/p62KO) were transiently transfected with FLAG-tagged p62 (wild type or ΔZZ–LB). Cells were treated with 5 µM MG132 for 6 h, and stained with anti-ubiquitin and anti-p62 antibodies followed by anti-rabbit IgG-TRITC and anti-mouse IgG-Alexa-350 antibodies. DIC differential interference contrast images. Scale bar, 10 µm. c Clustering indexes (Supplementary Fig. 2) of p62 wild type or ΔZZ–LB mutant-transfected cells are calculated and represented as scatter plots (n = 82 and 101 cells examined over three independent experiments, mean ± SEM). ***p < 0.001 (two-sided Mann–Whitney test). d Yeast two-hybrid assays showing the interaction between the ZZ–LB domain of p62 and UXT using LacZ, URA3, and ADE2 reporters. P and N, positive and negative control, respectively. e HEK293T cells were transfected with HA-p62 and GFP-UXT, and their interaction was demonstrated by detection of GFP-UXT in precipitates of HA-p62. f Proximity ligation assay (PLA) dots (red) representing close proximity of antibodies against p62 and UXT (right) were increased compared to isotype control mouse and rabbit IgG antibodies (left). Nuclei were stained with DAPI (blue). Scale bar, 20 µm. g Number of PLA dots per nucleus is shown as bar graphs (n = 6 and 7 random fields for each condition over three independent experiments, mean ± SEM). **p < 0.01 (two-sided Mann–Whitney test). h Interaction between UXT and p62 deletion mutants was analyzed as in e. i Diagram of p62 LB domain-deletion mutants. j Interactions between UXT and p62 mutants were analyzed as in e. k HeLa cells stably expressing GFP-CFTR(ΔF508) were treated with 2.5 µM MG132 for 12 h, and localization of the CFTR mutant and endogenous UXT (stained with anti-UXT antibody followed by anti-mouse IgG-Alexa-350) and p62 (stained with anti-p62 antibody followed by anti-rabbit IgG-TRITC) were analyzed and compared to the untreated control. Scale bar, 25 µm. l Under the same conditions used for k, the levels of UXT and p62 protein in the RIPA-insoluble fraction were analyzed. White and black arrowheads indicate LC3-I and -II, respectively.
	Fig. 2: UXT overexpression protects neuronal cells from proteasome inhibition-induced cell death. a Immunocytochemistry of dorsal root ganglion (DRG) neurons. DRG neurons were treated with 2.5 µM MG132 for 12 h and compared to untreated controls. Endogenous p62 and ubiquitin-positive protein aggregates (upper panels) or p62 and UXT (lower panels) were stained under both conditions with their specific primary antibodies, and subsequently with anti-rabbit IgG-TRITC and anti-mouse IgG-Alexa-350 as secondary antibodies. Scale bar, 10 µm. b On day 0, DRG neurons (white arrows) were isolated from day 13.5 mouse embryos and cultured for 48 h. On day 2, mCherry or mCherry-UXT was introduced into DRG neurons by lentiviral transduction and cells were cultured for 2 days. On day 4, the DRG neurons were treated with 2.5 µM MG132 and observed under a live imaging microscope with temperature and humidity control. DIC (differential interference contrast) images were collected for 28 h after MG132 treatment at 20 min intervals (Supplementary Movie 1). To identify the transduced cells, fluorescence images were also taken at the end. c DIC images collected at 0, 8, 18, and 24 h and mCherry fluorescence images of DRG neurons are shown. Transduced cells are indicated with red asterisks. Membrane blebs are indicated with arrowheads. Scale bar, 20 µm. d Survival of DRG neurons transduced with mCherry without MG132 treatment (blue), mCherry with MG132 treatment (black), and mCherry-UXT with MG132 treatment (red) was analyzed and is shown with the number of DRG neurons analyzed. This analysis was repeated twice with the essentially same results.
	Fig. 3: UXT and p62 cooperate and are involved in autophagy. a HeLa cells were treated with 2.5 μM MG132 and/or 50 nM bafilomycin A1 (Baf-A1) for 12 h, and the whole-cell lysates were subjected to a western blot analysis. b Band intensities of UXT in each condition were normalized to that of GAPDH, and their relative intensities against non-treated condition are shown as bar graphs (n = 3, mean ± SEM). *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA using Bonferroni’s multiple comparison test). c HeLa cells were transfected with p62-siRNA and then whole-cell lysates were subjected to a western blot analysis. d HeLa cells stably expressing GFP-LC3 were starved of amino acids using EBSS media, and this was followed by inhibition of the autophagic flux by treatment with 50 nM Baf-A1 for 4 h. The localization of GFP-LC3 (green), p62 (blue), and UXT (red) were analyzed by immunocytochemistry, using anti-p62 and anti-UXT as primary antibodies, and anti-rabbit IgG-Alexa-350 and anti-mouse IgG-TRITC as secondary antibodies. Scale bar, 10 μm. e Pearson’s correlation coefficients between each protein were shown as scatter plots (n = 12 and 34 cells for control and EBSS + Baf-A1, respectively, mean ± SEM). ***p < 0.001 (two-sided Mann–Whitney test). f UXT-eYFP-10×His construct was transfected into HEK293E cells, and the protein purified using a Ni-NTA column was treated with His-tagged HRV3C protease to cleave the region between UXT and eYFP. Resulting reaction was passed through the second Ni-NTA column and further separated by size-exclusion chromatography (SEC). g Coomassie blue-stained SDS–PAGE gel profile of UXT-containing SEC fractions in f is shown. Arrowhead indicates UXT. h The SEC fractions were further analyzed with western blot using anti-UXT, anti-p62, and anti-LC3 antibodies.
	Fig. 4: UXT mediates the clearance of SOD1(A4V) aggregates by autophagy. a HEK293T cells were transiently transfected with SOD1 (wild type or A4V)-GFP and mCherry-UXT or mCherry-only control. Representative images of transfected cells, merged composites and differential interference contrast (DIC) images are shown. Scale bar, 10 μm. b HEK293T cells were transiently transfected with SOD1(A4V)-GFP and HA-UXT or vector, and were treated with 50 nM bafilomycin A1 (Baf-A1) for 12 h, as shown. SOD1 and UXT were detected by western blotting in the detergent-insoluble fraction. LC3 in whole-cell lysates were analyzed to confirm inhibition of autophagic flux by Baf-A1, and GAPDH was used as an internal loading control. c Band intensities of SOD1(A4V)-GFP in detergent-insoluble fractions were normalized against those of HA-UXT-transfected cells without Baf-A1 treatment, and are shown as bar graphs (n = 5, mean ± SEM). *p < 0.05; **p < 0.01; ns not significant (one-way ANOVA using Bonferroni’s multiple comparison test). d Immediately after HEK293T cells were transiently co-transfected with SOD1(A4V)-GFP and mCherry-UXT or mCherry-only control, the cells were either treated with 50 nM bafilomycin or DMSO as a control, and fluorescence images of SOD1(A4V)-GFP in mCherry-positive cells were captured at 15 min intervals for 24 h. Representative images at 11, 15, 18, and 22 h are shown. Black and white images indicating pixels automatically recognized as aggregate regions by an in-house MATLAB code (Supplementary Code 1) are also shown. Scale bar, 10 µm. e Averages of the aggregation indexes of cells in d that were vector control transfected (gray, n = 10), mCherry-UXT transfected (blue, n = 11), and mCherry-UXT transfected along with bafilomycin A1 (Baf-A1) treatment (yellow, n = 17) are shown. Mean ± SEM is indicated.
	Fig. 5: UXT induces p62 clustering. a HeLa/p62KO cells were co-transfected with SOD1(A4V)-GFP and either FLAG-tagged p62 wild type or a p62(ΔLB) mutant defective for UXT binding. After transfection, cells were treated with 5 μM MG132 for 6 h to induce p62 body formation and were stained with anti-p62 and anti-ubiquitin antibodies followed by anti-mouse IgG-Alexa-350 and anti-rabbit IgG-TRITC. Representative images are shown. Scale bar, 10 μm. b Clustering indexes in cells expressing wild-type p62 or p62(ΔLB) mutant were analyzed and are shown as scatter plots (n = 42 and 65 cells examined over three independent experiments, mean ± SEM). ***p < 0.001 (two-sided Mann–Whitney test). c HEK293T cells were transiently co-transfected with FLAG-tagged p62 (wild type or ΔLB mutant) and HA-UXT or vector control. The detergent-insoluble fraction of cell lysate was analyzed for the presence of these proteins by western blotting. d The degree of p62 wild type and p62(ΔLB) mutant detected in the detergent-insoluble fraction was normalized against the degree of their expression (whole-cell lysates) in each condition, and are shown as bar graphs (n = 3, mean ± SEM). *p < 0.05; **p < 0.01; ns not significant (two-sided unpaired t test). e To detect an oligomeric form of UXT, detergent-free cell lysates of HEK293T cells expressing GFP-UXT were prepared and their components were separated using a size-exclusion column (SEC) with a fluorometer. f SEC profile of GFP-UXT represented by GFP fluorescence of eluant. Molecular weights of the marker are indicated. g HeLa/p62KO cells were transiently co-transfected with FLAG-p62(ΔPB1) mutant, the oligomerization-defective mutant, and mCherry-UXT or mCherry-only control. The cells were stained with anti-p62 followed by anti-mouse IgG-Alexa-350, and representative images are shown. p62(ΔPB1) clusters induced by UXT are indicated with white arrows. Scale bar, 10 μm.
	Fig. 6: L50A/L59A mutations in UXT specifically disrupt interaction between p62 and UXT. a Interactions between p62 and UXT(L50P/L59P), represented as L/P (upper panel) and p62 and UXT(L50A/L59A), represented as L/A (lower panel) were analyzed. b HeLa/p62KO cells were co-transfected with oligomerization-defective p62(ΔPB1) and either UXT wild type, UXT(L50A/L59A), or UXT(L50P/L59P) mutants, and were stained with anti-p62 antibody followed by anti-mouse IgG-Alexa-350. Representative images are shown. p62(ΔPB1) clusters induced by UXT are indicated with white arrows. Scale bar, 10 μm. c Clustering indexes of p62 (left) and UXT (right) were analyzed under each condition, and are shown as scatter plots (n = 98, 82, and 22 cells examined over three independent experiment, mean ± SEM). ***p < 0.001; ns not significant (two-sided Mann–Whitney test). d The effects of L50P/L59P and L50A/L59A UXT mutations on the amount of detergent-insoluble SOD1(A4V) aggregates. e The degree of HA-UXT mutants detected in the detergent-insoluble faction was normalized against that of HA-UXT wild type and shown as bar graphs (n = 3, mean ± SEM). ***p < 0.001; ns not significant (one-way ANOVA using Bonferroni’s multiple comparison test). f SEC profiles of GFP-UXT(L50A/L59A) and GFP-UXT(L50P/L59P) as detected by GFP fluorescence in the eluant.
	Fig. 7: UXT prevents SOD1(A4V)-induced progressive loss of motor ability in Xenopus. a SOD1 and/or UXT in vitro-transcribed capped RNA were expressed in the central nervous system by targeted microinjection. Motor function was assessed by touch-induced swimming responses at stages 28 and 4. b Each tadpole was touched three times on its tail. Each trace in a circle indicates swimming trajectory of the same tadpole within 3 s (time after the touch is color-coded). The best response of three trials was used to classify the swimming ability. c Injecting SOD1 mutant genes did not result in distinguishable morphological changes. Scale bar, 2 mm. d Swimming responses of tadpoles injected with SOD1 mutant and/or UXT (wild type or L50A/L59A mutant) in vitro-transcribed capped RNA are shown. The behavior of tadpoles was recorded in three independent experiments. The Y-axis indicates the percentage of tadpoles showing a normal swimming response, and the X-axis indicates developmental stages. e Quantification of d at stage 43 with the number of tadpoles in each category is shown. Cyan, yellow, and orange bars represent normal, reduced, and no swimming responses, respectively. The Y-axis indicates the percentage of tadpoles. Ctl control; ***p < 0.001 (two-sided Fisher’s exact test). f, g Effect of UXT expression on SOD1(G93A)-GFP-induced motor deficit was tested as in d and e. +p = 0.0968; **p < 0.01 (two-sided Fisher’s exact test). h, i Effect of a p62 translation-blocking antisense morpholino (MO) on swimming responses was analyzed as in d and e. j Quantification of i at stage 43 as in e. ***p < 0.001 (two-sided Fisher’s exact tests). ns not significant. k SOD1(A4V)-GFP was visualized by immunohistochemistry in cell bodies of lower motor neurons localized in the ventral horn of the spinal cord. Red and green arrows indicate SOD1(A4V)-GFP aggregation and SOD1(A4V)-GFP, respectively. Co-injecting UXT completely prevents SOD1(A4V)-GFP aggregation indicated by white arrows. In cells co-injected with UXT(L50A/L59A (L/A)), the SOD1(A4V)-GFP aggregation is often observed as indicated by yellow arrows. Numbers in parenthesis indicate the number of cells with aggregation/the number of transfected cells analyzed. Scale bar, 10 µm.
	Fig. 8: UXT is required in coping with proteotoxicity in Xenopus. a Reverse transcription-PCR of UXT mRNA in whole tadpole and spinal cord tissue. The entire coding sequence of UXT mRNA was amplified with a band of 460 bp. b In situ hybridization of UXT mRNA (left) and immunohistochemistry of acetylated tubulin (right). The green arrow indicates a lower motor neuronal cell body and the yellow arrows indicate the axons of these neurons. Scale bar, 10 µm. c UXT morpholino (MO) was injected to four blastomeres at the eight cells stage. The injected tadpoles showed a dose-dependent defect in gross morphology in contrast to the control group. The numbers indicate the number of embryos with the same morphology as the representative image out of all examined embryos. Scale bar, 1 mm. d Tadpoles injected with 0.5 ng of control or UXT MO together with wild-type SOD1, but showing normal morphology were analyzed for their motor ability as in Fig. 7d. In three independent experiments, each with the full set of groups, the behaviors of 19, 20, 19, and 28 tadpoles were recorded for control, SOD1 WT, UXT MO, and UXT MO + SOD1 WT group, respectively. St stage. e Quantification of d at stage 43. Cyan, yellow, and orange bars indicate normal, reduced, and no swimming responses, respectively. Ctl control. ***p < 0.001 (two-sided Fisher’s exact tests).

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