EMBO/soda-nel huggingface dataset:
Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease. (2007)
Figure 3.

Autophagic degradation is impeded in Tsg101- and Vps24-depleted cells. HeLa cells stably expressing GFP-LC3 (green) were transfected with control (A), Vps24 (B), or Tsg101 (C) siRNA for 5 d and processed for immunofluorescence analysis. Cells were labeled with antibodies against p62 (red) and analyzed by confocal microscopy. Colocalization is indicated in yellow. Bar, 10 μm. (D) The total level of p62 and GFP-LC3 (normalized to control cells) and their degree of colocalization (% of total) in control and Tsg101- and Vps24-depleted cells were quantified using the Zeiss LSM 510 Meta software. 30 cells from three independent experiments were used for quantification. Error bars = SEM. (E) Western blot showing accumulation of LC3-II and p62 in HeLa cells depleted of Vps24 and Tsg101. p62 also accumulates in the insoluble fraction (bottom picture), indicating a shift to a more aggregated form upon depleted of Vps24 and Tsg101. (F) Western blot showing that LC3-II levels were similar in control and Vps24-depleted cells treated with Bafilomycin A.
Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease. (2007)
Figure 4.

Formation of autolysosomes is inhibited in cells depleted of Tsg101 or Vps24. (A) The double-tagged LC3 protein (mCherry-GFP-LC3) will emit yellow (green merged with red) fluorescence in non-acidic structures and appear as red only in the autolysosomes due to quenching of GFP in these acidic structures. (B–F) HeLa cells were transfected with control (B), Tsg101 (C), or Vps24 (D) siRNA for 4 d and then with mCherry-GFP-LC3 for another 24 h before confocal microscopy analysis. Bar, 5 μm. (E) The mCherry-LC3 (red) signal was quantified in ∼30 cells with similar expression levels from three independent experiments using the Zeiss LSM 510 Meta software and the data are presented as % of total mCherry-GFP-LC3. Error bar = SEM. (F) Degradation of [14C]-valine- labeled proteins in cells incubated in complete medium (control), serum- and amino acid-deficient medium (starve), or starvation media in the presence of 3-methyl adenine (3-MA) to inhibit autophagic degradation. Average degradation per hour from four experiments done in duplicates ±1 SEM are shown.
Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease. (2007)
Figure 5.

Autophagosomes and amphisomes do form in ESCRT-depleted cells. HeLa cells stably expressing GFP-LC3 were transfected with control (A), Tsg101 (B, D, and E), or Vps24 (C) siRNA and processed for immuno-EM analysis. Cryosections were incubated with antibodies against GFP (15-nm gold) and LBPA (10-nm gold). Amphisomes (Am), characterized by their content of electron-dense LC3-positive areas and intraluminal vesicles, were observed in both control (A) and ESCRT-depleted cells (B and C). (D) In the Tsg101-depleted cells we found large clusters of more typical autophagosomes (Au) and (E) clusters of double-membrane structures, consisting of autophagosomes and tubular structures which might represent phagophores, all labeling strongly for GFP-LC3. We never observed similar clusters in control cells and very rarely in Vps24-depleted cells. Bars, 200 nm. (F) Quantification of amphisomes in control cells and cells depleted for Tsg101 or Vps24. Approximately 20 cells were included per siRNA depletion per experiment (n = 3). Error bars = SEM.
Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease. (2007)
Figure 6.

Immuno-EM of p62-positive structures in Vps24- and Tsg101-depleted cells. Cryosections of HeLa cells transfected with Vps24 (A, C, and E) or Tsg101 (B, D, and F) siRNA were incubated with antibodies against p62 (15-nm protein A gold, 10-nm in D). Membrane-free p62-positive aggregates (C and D) or p62-positive structures contained within dense clusters of vesicular-tubular elements (A and B) were detected. Vesicles with early endosomal morphology (EE) or multivesicular appearance (MVB) associated with these clusters. We also observed p62 labeling in electron-dense structures sequestered within amphisomes in cell depleted of Vps24 (E) or Tsg101 (F). The amphisomes typically consist of an electron-dense p62 positive body (E and F, arrowhead), and a multivesicular endosomal structure (E and F, arrow). Bars, 200 nm.
Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease. (2007)
Figure 7.

Ubiquitin and p62 positive structures accumulate in cells expressing CHMP2 mutants. HeLa cells transfected with myc-tagged wild-type CHMP2B (A), CHMP2BIntron5 (B), or CHMP2BΔ10 (C) were labeled with antibodies against c-Myc (red), ubiquitin (green), and p62 (blue) and analyzed by confocal microscopy. Single channel images in black and white are shown. Bars,10 μm. (D) Total levels of p62 in these cells were quantified in 20 cells from three independent experiments using the Zeiss LSM 510 Meta software and the average normalized to the average p62 level in control (mock transfected) cells. Error bars = SEM. (E) Western blot analysis showing accumulation of p62 in cells transfected with myc-CHMP2BIntron5 compared with mock-transfected cells (control) and cells transfected with CHMP2Bwt. (F) Immuno-EM showing p62-positive aggregates containing dense clusters of vesicular–tubular structures in cells transfected with myc-CHMP2BIntron5. Bar, 200 nm.
Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease. (2007)
Figure 8.

TDP-43 accumulates in cytoplasmic ubiquitin-positive structures in Tsg101- and Vps24-depleted cells. HeLa cells transfected with control (A), Tsg101 (B), or Vps24 (C) siRNA were fixed, permeabilized, and stained with antibodies against TDP-43 (green), ubiquitin (red), and p62 (blue). Single channel images in black and white are shown. Colocalization of all proteins is indicated in white in the merged picture. Bars, 10 μm.
Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease. (2007)
Figure 9.

Vps24 is required for efficient clearance of Htt inclusions. HeLa cells expressing HttQ65-mCFP (A) or HttQ103-mCFP (B) and N2a cells expressing HttQ103-mCFP (C) were transfected with control or Vps24 siRNA for 2 d and then incubated in the absence or presence of doxycycline for 3 d to turn off expression of HttQ65/Q103-mCFP. Vps24 depletion was determined by Western blot analysis. (A and C) Clearance of SDS-insoluble Htt inclusions was analyzed by filter-trap assays. (B) Alternatively, aggregate clearance was analyzed by confocal quantification of the number of HeLa HttQ103 cells having visible inclusions after 3 d in the absence or presence of dox. 300 cells from three independent experiments were counted for each condition. Error bars = SD.
p62 filaments capture and present ubiquitinated cargos for autophagy (2018)
Figure 2

Figure 2. Endogenously tagged p62 is oligomeric and forms clusters with ubiquitin.
(a) Top: schematic representation of the STG-p62 construct. Bottom: characterization of several STG-p62 clones in comparison to parental Hap1 cells. (b) Wild type or STG-p62 Hap1 cells were mock or puromycin treated for 2 hours and stained for ubiquitin. The endogenous p62 was stained with an anti-p62 antibody in the parental Hap1 cells, while the fluorescence of the GFP tag was recorded in STG-p62 cells. Arrowheads indicate co-localizing puncta. (c) Purified STG-p62 was tested for cluster formation in presence of 20µM GST-4xUb. Averages and SDs of three independent replicates are shown for the +GST-4xUb sample and of two for STG-p62 alone. (d,e) FCS analysis of diffusion (d) and brightness (e) of the GFP1-5 ruler and of STG-p62 in the cytoplasm of Hap1 cells. Dashed lines represent the linear regression of the GFP1-5 ruler data. Regression coefficients are indicated. For all data points averages and SDs (on both axes for STG-p62) are shown (see methods for a list of the number of measurements performed for each construct). MW: molecular weight. D: diffusion coefficient (µm2/s).
p62 filaments capture and present ubiquitinated cargos for autophagy (2018)
Figure 3

Figure 3. p62-dependent cluster formation is triggered by poly-ubiquitinated proteins.

(a) Representative micrographs of cluster formation assays conducted with the indicated proteins. The total amount of ubiquitin moieties was kept identical in each condition (b) Quantification of the data shown in (a). (c) Quantification of cluster formation assays conducted with the indicated proteins. mCherry-p62 was pre-incubated with the indicated 4xUb variants, followed by the addition of streptavidin immediately before imaging. (d) Representative micrographs of the indicated samples taken 60 minutes after addition of streptavidin. (e) Quantification of cluster formation assays conducted with the indicated proteins. (f) Quantification of cluster formation assays conducted with 2µM mCherry-p62 incubated with the indicated concentration of GST-GFP-4xUb. Box: the highest particle number per field detected in each sample was plotted against the relative GST-GFP-4xUb concentration. Data points were fitted to a sigmoidal curve. The regression coefficient is indicated. (g) Quantification of cluster formation assays conducted with GFP-p62 and the indicated GST-tagged ubiquitin chains. (h-i) Quantification of cluster formation assays performed with mCherry-p62 and GST-4xUb in presence of the indicated free ubiquitin chains. Ubiquitin chains were either pre-mixed with p62 before the addition of GST-4xUb (h), or added 3 minutes after the addition of GST-4xUb to p62 (i). (j) Quantification of cluster formation assays conducted with mCherry-2xFKBP-p62 in presence of AP20187, GST-4xUB and the indicated Ub chains. (k) Representative micrographs of glutathione beads coated with the indicated GST-tagged proteins and incubated with the indicated mCherry-p62 variants. In all pictures the brightness was adjusted to highlight the profile of the beads. For all graphs averages and SDs from at least three independent replicates are shown.
p62 filaments capture and present ubiquitinated cargos for autophagy (2018)
Figure 4

Figure 4. p62 and ubiquitin show different motilities within the clusters

(a) mCherry-p62/GST-GFP-4xUb clusters were photobleached and fluorescence recovery in the bleached area was monitored. The mCherry-tagged low complexity domain (LCD) of hnRNPA1 (hnRNPA1LCD) was included as a reference for a liquid droplet-forming protein. Left: representative micrographs of bleached particles. White arrows indicate the position and orientation of the kymographs shown in Figure EV4a. Right: quantification of fluorescence recovery. Individual traces of all particles analyzed from three independent replicates are displayed. (b) p62 puncta in STG-p62 cells were photobleached and the fluorescence recovery was monitored. Left: representative micrographs of bleached cells. White squares indicate the bleached puncta. Insert: magnification of the bleached puncta. Right: quantification of fluorescence recovery. Averages and SDs of 28 (unbleached) and 29 (bleached) puncta from three independent experiments are shown. (c) Representative electron micrographs of negatively stained p62 filaments incubated in presence or absence of GST-4xUb for the indicated times.
p62 filaments capture and present ubiquitinated cargos for autophagy (2018)
Figure 5

Figure 5. NBR1 cooperates with p62.

(a) Top: schematic representation of the domain architecture of the GFP-NBR1 construct. PB1: Phox and Bem1p domain, ZZ: Zinc finger, CC: coiled-coil, LIR: LC3-interacting region, UBA: ubiquitin-associated domain. Bottom: GFP-NBR1 was recruited to glutathione beads coated with the indicated GST fusion proteins and imaged by spinning disc microscopy. Representative micrographs of at least 70 beads imaged per sample from two independent experiments. (b-c) mCherry-p62 variants were recruited to GFP- or GFP-NBR1-coated beads and imaged as in (a). (b) Representative micrographs of at least 70 beads per sample from three independent replicates. (c) Quantification of the data shown in (b). Data were normalized to p62 wild type binding to GFP-NBR1 within each replicate. Averages and SDs of three independent experiments are shown. (d-e) 2µM mCherry-p62 and/or GFP-NBR1 were incubated in presence of 5µM GST-4xUb. Representative micrographs (d) and quantification of particle number for each channel (e) are shown. Averages and SDs from at least three independent experiments are shown.
p62 filaments capture and present ubiquitinated cargos for autophagy (2018)
Figure 6

Figure 6. LIR-mediated cross talk between cluster formation and the autophagy machinery.

(a) Cluster formation assays were conducted in presence or absence of 20µM mCherry-LC3B or mCherry, either pre-mixed with p62 or added 1" after the addition of GST-4xUb. Quantification of particle number (left) and size (right) are shown. (b) Cluster formation assays were conduced with mCherry-p62 wt or LIR mutant in presence of GST-4xUb. Left: representative images of the samples two minutes after the addition of GST-4xUb. Middle: quantification of particle count. The dashed line indicates the time point of images displayed on the left. Right: input gel of the samples after reaction. The relative position of marker bands is indicated. For all graphs, averages and SDs from at least three independent replicates are shown. (c) Top left: Western blots of lysates of CRISPR/Cas9 genome-edited STG-p62 wild type and LIR mutant cells. Clone Lir1B was used for the experiments shown in Figure 6c. Bottom left: Representative pictures of STG-p62 WT or LIR mutant Hap1 cells treated with Puromycin for 90 minutes and stained with anti-Ub conjugates antibody (FK2). Right: quantification of cytoplasmic p62 puncta per cell (top), and of their co-localization upon puromycin treatment (bottom). Averages and SDs from three independent experiments are shown. In total, at least 1440 number of cells were counted per condition.
The DnaJ proteins DJA6 and DJA5 are essential for chloroplast iron-sulfur cluster biogenesis (2021)
Figure 1

Figure 1. dja6 dja5 and RNAi mutants display albino and variegation phenotypes, respectively, and defects in chloroplast development.

A Phenotypes of WT, dja6, dja5, dja6 dja5 and RNAi seedlings on MS medium with 2% sucrose. Scale bars = 0.5 cm.

B,C Phenotypes and chlorophyll fluorescence images of 6-week-old WT, dja6 dja5 and complemented plants (B) on MS medium with 2% sucrose, scale bars = 0.5 cm; WT, dja6, dja5 and RNAi mutants (C) grown on soil,­­­ scale bars = 1 cm. Fluorescences in (B) and (C) were measured with the FluorCam700MF and visualized using a pseudocolor index as indicated on the right.

D Chlorophyll content of total leaves in 6-week-old WT, dja6, dja5, dja6 dja5 and RNAi seedlings. FW, fresh weight. Data are the means ± SEM (n = 7 biological replicates).

E The ultrastructure of plastids from WT, dja6, dja5, dja6 dja5 and RNAi mutants. T, thylakoids. Scale bars = 0.2 μm.
The DnaJ proteins DJA6 and DJA5 are essential for chloroplast iron-sulfur cluster biogenesis (2021)
Figure 2

Figure 2. Loss of DJA6 and DJA5 mainly affects the accumulation of the Fe-S cluster-containing proteins in chloroplasts.

A, B Total protein was extracted from 4-week-old WT, dja6, dja5, dja6 dja5 and RNAi seedlings, respectively. WT and dja6 dja5 used in (A) were grown on MS medium with 2% sucrose, while WT, dja6, dja5 and RNAi mutants used in (B) were grown on soil. 10 μg protein was loaded, except for detection with anti-SUFE1 and anti-SUFA antiserum, for which 25 μg were loaded. Specific bands were identified by immunoblotting and by their molecular weight, and are indicated by asterisks. Designations of photosynthetic protein complexes and their diagnostic components are labeled on the left and right, respectively. The Fe-S types of Fe-S proteins are shown in parentheses. Actin served as controls to normalize protein levels. For each protein, three independent biological replicates were performed and a representative one is shown.
The DnaJ proteins DJA6 and DJA5 are essential for chloroplast iron-sulfur cluster biogenesis (2021)
Figure 3

Figure 3. DJA6 and DJA5 are critical for the maintenance of cellular iron homeostasis.

A-C Metal concentrations in the WT, dja6 dja5 and RNAi mutants. Fe concentrations in total leaves (A and B) and total chloroplast (C) from 4-week-old WT, dja6 dja5 and RNAi mutants were quantified by inductively coupled plasma mass spectrometry, and are shown as means ± SEM (n = 3 biological replicates) of μg g-1 dry weight (DW). WT and RNAi mutants used in (A and C) were grown on soil, while WT and dja6 dja5 seedlings used in (B) were grown on MS medium with 2% sucrose. Similar results were obtained in two additional independent biological experiments. Asterisks indicate significant differences from the value of WT (two-sample Student's t-test; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

D Volcano plot of the result from quantitative transcriptome profiling in WT and dja6 dja5 mutant. Statistical analysis was performed using Student's t-test. Data on the X-axis represent the Log2-ratio of FC (fold change) between the absolute abundances of transcripts identified from dja6 dja5 against WT. The Y-axis represents the Log10 of FDR (false discovery rate). All DEGs (differentially expressed genes) with FDR < 0.01 and FC > 2 are marked in grey, while all iron-related DEGs with FDR < 0.01 and FC > 2 are marked in red. The iron-related DEGs labeled with red dots within the black boxes are referred to in the text. All data are based on three biological replicates.
The DnaJ proteins DJA6 and DJA5 are essential for chloroplast iron-sulfur cluster biogenesis (2021)
Figure 4

Figure 4. DJA6 and DJA5 bind iron in vitro and in vivo.

A UV-visible absorption spectra of recombinant DJA6 and DJA5. Recombinant DJA6 after elution from Ni-NTA Sepharose beads followed by concentration is shown on the right.

B Electron paramagnetic resonance spectra of recombinant DJAs. The observed resonance is marked for the g value.

C Analysis of the ferrous iron binding activity of DJA6. Isothermal titration calorimetry measurements of ferrous iron binding to apo DJA6 were carried out on a MicroCal iTC200. Fe2+ was titrated into a solution of 50 μM DJA6 or DJA6 variants. For the background titrations, the ferrous iron was titrated into the corresponding buffer.

D DJA6 can bind Fe2+ in chloroplast. Arabidopsis mesophyll protoplasts were transformed with plasmids that express the indicated gene constructs, respectively. The untransformed protoplasts and protoplasts transformed with non-fused EBFP proteins were used as controls. The protoplasts were further stained by FeRhoNoxTM-1 (red) to visualize the cellular distribution of Fe2+. Green, EBFP fluorescence signals; magenta, chloroplast autofluorescence signals; yellow, regions where the EBFP and FeRhoNoxTM-1 fluorescence signals merged. The speckles which DJA6-EBFP co-localizes with FeRhoNoxTM-1 are indicated by white arrows. Scale bars = 5 μm.

E Diagram of the domain structure of DJA6. Grey, TP (chloroplast transit peptide); blue, J domain; steel blue, GR domain; cyan, CR domain; green, C-terminal domain.

F Ribbon representation of the quarternary structural model of mature dimeric DJA6 prepared with PyMOL (Lam & Siu, 2017). The α subunit and α' subunit of DJA6 dimer are colored blue-to-green and green-to-red, respectively. The relative orientations of the domains differ in the two monomers in the asymmetric unit.

G UV-visible absorption spectra of recombinant DJA6 and DJA6 truncated peptides. Recombinant DJA6 and DJA6 truncated peptides after elution from Ni-NTA Sepharose beads followed by concentration are shown above.

H Size-exclusion profiles for apo and iron-loaded forms of DJA6. 1 mM EDTA, purified DJA6 after incubated with EDTA at a final concentration of 1 mM; 20 μM or 100 μM Ferrous, apo-DJA6 after incubated with Fe(NH4)2(SO4)2 at a final concentration of 20 μM or 100 μM, respectively. Positions of theoretical tetramer and dimer are indicated by dotted lines. Images of the Coomassie Brilliant Blue-stained gel after SEC elution are shown.
The DnaJ proteins DJA6 and DJA5 are essential for chloroplast iron-sulfur cluster biogenesis (2021)
Figure 5

Figure 5. J domain of DJA6 mediates the interactions between DJA6 and chloroplast SUF components (SUFE1 and SUFC).

A BiFC analyses for the interactions between DJA6 and SUFE1 or SUFC in Arabidopsis mesophyll protoplasts. Yellow, YFP fluorescence signals; red, chloroplast autofluorescence signals. Scale bars = 10 μm.

B Co-IP assays for the interactions of DJA6 with SUFE1 and SUFC. Total protein extracts of protoplasts co-transfected with designated constructs were subjected to immunoprecipitation, and then analyzed by immunoblotting with antibodies indicated on the right. Input (left lane) indicates that 100 μg total protein was loaded on the gel.

C Pull-down assays of the interactions between DJA6 and chloroplast SUF components. GST-tagged mature DJA6 was used as the bait, MBP-tagged mature NFS2, SUFE1, SUFB, SUFC, SUFD and MBP were used as the prey. The recombinant proteins were incubated with glutathione-agarose beads, and the bound proteins were eluted and analyzed by staining.

D Pull-down assay of the interactions between SUFC and apo- or iron-bound form of DJA6. GST-tagged mature iron-bound or apo-DJA6 was used as the bait, MBP and MBP-tagged mature SUFC were used as the prey. The bound proteins were eluted and analyzed by staining and immunoblotting.

E Yeast two-hybrid analyses of the interactions between DJA6 and chloroplast SUF components. Positive control, the interaction between murine p53 fused with GAL4 BD and SV40 large T-antigen fused with GAL4 AD; negative control, the coexpression of Human Lamin C fused with GAL4 BD and SV40 large T-antigen fused with GAL4 AD. The dilution ratios are indicated on the left.

F Schematic diagram of DJA6 with five different constructs: grey, TP (chloroplast transit peptide, residues 1-85); blue, J domain (residues 86-147); steel blue, GR domain (residues 148-223); cyan, CR domain (residues 224-287); green, C-terminal domain (residues 288-442). The different truncations of DJA6 are shown in detail below.

G, H BiFC analyses for the interactions between truncated forms of DJA6 and SUFE1 or SUFC in Arabidopsis mesophyll protoplasts. Scale bars = 10 μm.
The DnaJ proteins DJA6 and DJA5 are essential for chloroplast iron-sulfur cluster biogenesis (2021)
Figure 7

Figure 7. The conserved biological role of the DJA6 protein family and evolutionary relationship between DJA6 and the SUF apparatus.

A Evaluation of the evolutionary relationships using the Tol-MirrorTree analyses. Pairwise genetic distances (GD) among designated proteins in representative photosynthetic organisms (Dataset EV2) are indicated by green spots. R, Pearson correlation coefficient; P, significant differences between the Maximum Likelihood trees (one-sided t-test).

B UV-visible absorption spectra of purified AtDJA6 and SynDJA6. Recombinant SynDJA6 after elution from Ni-NTA Sepharose beads followed by concentration is shown on the right.

C Phenotypes and chlorophyll fluorescence images of 6-week-old WT and 35Spro:SynDJA6-FLAG/dja6 dja5 transgenic plants grown on soil. Scale bars = 1 cm.

D Expression of FLAG-tagged SynDJA6 in 35Spro:SynDJA6-FLAG/dja6 dja5 transgenic plants. Total leaf extracts (100 μg proteins) from WT and transgenic seedlings were loaded. FLAG-tagged SynDJA6 is indicated by asterisk.