EMBO/soda-nel huggingface dataset:
Ral GTPase and the Exocyst Regulate Autophagy in a Tissue-Specific Manner (2015)
Figure 6

Figure 6. The exocyst is required for salivary gland degradation

A Control animals (+/w; UAS-sec5IR/+), n= 19, and those with salivary gland-specific knockdown of sec5 (fkh-GAL4/w; UAS-sec5IR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.

B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test.

C Control animals (+/w; UAS-sec15IR/+), n= 20, and those with salivary gland-specific expression sec15 (fkh-GAL4/w; UAS-sec15IR/+), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.

D Quantification of data from (C). Data are represented as means. Statistical significance was determined using a Chi-square test.

E Control animals (+/w; UAS-sec3IR/+), n= 20, and those with salivary gland-specific expression sec3 (fkh-GAL4/w; UAS-sec3IR/+), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.

F Quantification of data from (E). Data are represented as means. Statistical significance was determined using a Chi-square test.

G Control animals (+/w; UAS-sec8IR/+), n= 20, and those with salivary gland-specific knockdown of sec8 (fkh-GAL4/w; UAS-sec8IR/+), n=19, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.

H Quantification of data from (G). Data are represented as means. Statistical significance was determined using a Chi-square test.

I Control animals (+/w; UAS-exo84IR/+), n= 19, and those with salivary gland-specific knockdown of exo84 (fkh-GAL4/w; UAS-exo84IR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.

J Quantification of data from (I). Data are represented as means. Statistical significance was determined using a Chi-square test.
Ral GTPase and the Exocyst Regulate Autophagy in a Tissue-Specific Manner (2015)
Figure 7

Figure 7. Notch activity is regulated by ral, and is required for autophagy in dying salivary gland cells.

A Salivary glands dissected from late 3rd instar larvae (Gbe Su(H)-lac-Z/w-;+;Fkh-GAL4/+) show less notch activity (left) compared to glands from 14 hours after puparium formation (APF) animals (middle). Down-regulation of ral in salivary glands from 14 hours APF animals (Gbe Su(H)-lac-Z/w-;UAS-ralIR/+;Fkh-GAL4/+) reduces the levels of notch activity (right). Scale bar represents 20µm.

B Quantification of data from (A). Data are represented as means ± SEM; n≥ 4. Statistical significance was determined using a Student’s t test.

C Salivary glands dissected from 14 hours after puparium formation animals expressing mCherry-Atg8a in all cells, and notchIR specifically in GFP-marked clone cells (yw, hsflp/w,UAS-notchIR; pmCherry-Atg8a/+; act< FRT, cd2, FRT> GAL4, UAS-GFP/+) analyzed for mCherry-Atg8a puncta. Scale bar represents 20µm.

D Quantification of data from (C). Data are represented as means ± SEM; n> 10. Statistical significance was determined using a Student’s t test.
The UBA domain of conjugating enzyme Ubc1/Ube2K facilitates assembly of K48/K63-branched ubiquitin chains (2021)
Figure 2

Figure 2. Enzymatic activity assays show the preferential targeting of K63-linked ubiquitin chains by Ubc1 resulting in the assembly of K48/K63-branched polyubiquitin.

A. Reaction scheme for single turnover ubiquitination experiments with Ubc1 in presence of fluorescently labeled monoubiquitin as donor and various C-terminally blocked acceptor ubiquitin molecules.

B. At indicated time points, samples were taken from the reaction mix and analyzed by SDS‑PAGE and fluorescence scan. Product fluorescence over total fluorescence per lane was quantified (rel. fluo.).

C. The relative fluorescence was plotted over time for reactions with different ubiquitin acceptor molecules - for example in presence of K63-linked diubiquitin (K63‑Ub2) or K48‑linked diubiquitin. Initial reaction rates were determined as the slope of a linear fir of the fluorescence increase over 20 min.

D. Initial reaction rates were determined for a panel of acceptor ubiquitin molecules in reactions with either full‑length Ubc1 or Ubc1-ΔUBA (aa1‑150). The binding of the UBA domain to individual ubiquitin molecules was disrupted by introducing an R42A amino acid exchange in ubiquitin, while acceptor sites were blocked by introducing the K48R substitution. Reactions were performed in triplicate. Bars show mean and SEM. For representative scans see Appendix Fig S2.

E. Schematic transition state for the reaction of Ubc1 with K63-linked diubiquitin. Binding of the distal moiety in K63-Ub2 to the UBA domain involves R42 in ubiquitin and enhances targeting of K48 in the proximal moiety for discharge of donor ubiquitin by the UBC domain. This results in the assembly of a K48/K63-branched chain.
The UBA domain of conjugating enzyme Ubc1/Ube2K facilitates assembly of K48/K63-branched ubiquitin chains (2021)
Figure 3

Figure 3. Branched chain formation by Ubc1 relies on the association of the UBA domain with the UBC domain and is selective for K63-linked chains.

a. Residues associated with the binding of the UBA domain to the UBC domain were analyzed through sequence alignment of Ubc1 with other E2 enzymes from S. cerevisiae. Y76 and W144 in Ubc1 were predicted to be hot spot residues of the interface, while L143 and L147 were classified as contributing amino acids (Fig 1e).

b. L143E and/or L147E amino acid substitutions were introduced into Ubc1 to disrupt the hydrophobic UBC/UBA domain interface. Single turnover ubiquitination experiments were performed with these Ubc1 variants in presence of K63‑Ub2 with Ub(K48R) at the distal position or monoubiquitin as acceptor. Experiment and initial rate determination as described in Fig 2.

c. Ubc1 variants were generated with deletions in the linker region (aa152‑167), which connects the UBA domain and the UBC domain. Single turnover ubiquitination reaction mixes were incubated for 5 min with K63-Ub2 or for 20 min with monoubiquitin as acceptor. Final product formation was analyzed by SDS-PAGE and fluorescence scan. Product fluorescence over total fluorescence (rel. fluo.) was quantified.

d. C-terminally blocked M1-linked diubiquitin with or without a K48R amino acid substitution at the distal or proximal position were employed in single turnover experiments with full-length Ubc1 or Ubc1-ΔUBA. Reactions with acceptor K63-Ub2 were carried out controls. Initial rate determination as described in Fig 2.

Data information: All reactions were performed in triplicate. Bars show mean and SEM. For representative scans for the experiments shown in panels (B-D) see Appendix Fig S3A-C.
The UBA domain of conjugating enzyme Ubc1/Ube2K facilitates assembly of K48/K63-branched ubiquitin chains (2021)
Figure 5

Figure 5. Branched chain formation by Ubc1 is conserved among species. Ubc1 is slower but more selective than bona fide chain building E2 enzymes.

a. The activity of Ubc1 was compared with the K48-linked chain building enzyme Ubc7/Cue1 in single turnover ubiquitination experiments in presence of either monoubiquitin, K48‑Ub2 or K63-Ub2. Initial rates were determined as described in Fig 2.

b. The K63-linked chain building enzyme Ubc13 with its cofactor Uev1a or the Ubc1 homologue Ube2K (h. sapiens) were employed in single turnover ubiquitination experiments in presence of K63-Ub2 with Ub(K48R) at the proximal or distal position or in presence of K48‑Ub2. Initial rate determination as described in Fig 2.

c. Experiment as in (B) but with the Ubc1 homologue ubc-20 (c. elegans).

Data information: All reactions were performed in triplicate. Bars show mean and SEM. For representative scans of gels from the experiments shown here, see Appendix Fig S3D-E.
The UBA domain of conjugating enzyme Ubc1/Ube2K facilitates assembly of K48/K63-branched ubiquitin chains (2021)
Figure 6

Figure 6. Branched chain formation by Ubc1 outperforms its capacity for de novo chain synthesis.

A. Cartoon representation of reaction conditions used in (B-D), which simultaneously enable de novo chain assembly as well as the formation of K48/K63-branched chains.

B. In vitro ubiquitination reactions with full‑length Ubc1 or Ubc1-ΔUBA were performed. Samples were taken at indicated time points and analyzed by SDS-PAGE and fluorescence scan. Free chain synthesis from Alexa488-labeled monoubiquitin was observed over time. Fluorescence of all reaction products was quantified and normalized to number of fluorophores per product ("de novo"). Double cross (‡) indicates ubiquitinated E2 enzyme.

C. Reactions as in (B) were performed in presence of C‑terminally capped and Alexa647-labeled K63‑linked Ub3. Monoubiquitin and K63-Ub3 were used in a 3:1 molar ratio. Analysis as in (B). Modification of K63-Ub3 was identified through correlation of fluorescence channels and their fluorescence quantified in the 488 channel ("branched"). The image from the 647 channel was cropped below the point indicated by "647" (see Fig EV2).

D. Alexa488-labeled monoubiquitin and C-terminally capped K63-Ub4 were used in a 4:1 molar ratio for in vitro ubiquitination reactions with full‑length Ubc1 or Ubc1-ΔUBA. Where indicated, Alexa488-labeled monoubiquitin was methylated prior to the reaction (met-Ub) to block de novo chain assembly. Reactions were incubated overnight, quenched and half the reaction mix from each condition was treated with the K63 specific deubiquitinating enzyme AMSH (lanes 4-6). Samples were analyzed by SDS-PAGE and fluorescence scan.
The UBA domain of conjugating enzyme Ubc1/Ube2K facilitates assembly of K48/K63-branched ubiquitin chains (2021)
Figure 7

Figure 7. Assembly of K48/K63-branched ubiquitin chains is conserved in S. cerevisiae. Yeast cells harboring ubiquitin-binding-deficient Ubc1 display specific proteomic alterations and are sensitized to stress conditions.

a. A flow-chart showing the sample preparation for the SILAC-based relative quantification of K48/K63-branched ubiquitin chains from yeast by mass spectrometry. 10xHis-Ub(R54A) was overexpressed in wildtype cells (wt) and either in cells deleted for UBC1 (Δubc1) or cells expressing ubiquitin-binding-deficient Ubc1-EA. The R54A substitution in ubiquitin enables the production of a marker peptide for K48/K63-branched ubiquitin by trypsin digest, which can be detected by targeted proteomics (PRM).

B. Log2 transformed fold changes of peptides between the Δubc1 strain and wildtype were determined. The forward and reverse SILAC experiments are shown on the x or y axis, respectively. The different ubiquitin marker peptides are indicated in the figure. For a list of fold-changes see Fig EV4F.

C. Experiment as in (B) but for the UBC1-EA strain and wildtype.

D. A flow-chart depicting the sample preparation for a SILAC-based proteomic shotgun experiment aimed to explore changes in protein abundance in Ubc1-EA expressing yeast cells.

E. Log2 transformed fold changes of proteins between the UBC1-EA strain and wildtype were determined. The forward and reverse SILAC experiments are shown on the x or y axis, respectively. Genes, which were determined to be significantly different in their protein abundance, are marked in red and labeled. Asterisks (*) indicate genes, which are upregulated at a transcriptional and/or translational level upon heat stress (Mühlhofer et al, 2019). A double cross (‡) marks genes, for which the protein product changes in abundance and/or localization in response to DNA replication stress (Tkach et al, 2012).

F. Ubc1-EA was genomically integrated into various yeast strains. Exponentially growing cells of the indicated genotype were spotted onto YPD plates in serial 10‑fold dilutions. Where noted cells were incubated at elevated temperature (37 °C) or in presence of hydroxyurea (HU) to induce DNA replication stress.

Expanded View Figure legends
Liquid-Liquid Phase Separation Underpins the Formation of Replication Factories in Rotaviruses (2021)
Figure 4

Figure 4 | Rotavirus NSP5 and NSP2 undergo phase-separation. (a) Recombinantly expressed, Atto647-dye labelled NSP5, Atto488-dye labelled NSP2 and unlabeled NSP5 protein samples (see Materials and Methods) were used for investigating their phase-separation properties. All labelled protein samples were mixed with the unlabeled NSP5 (1:10 molar ratio) to minimise the effect of labelling on LLPS. Top panel, left to right: NSP5-647/NSP2-488 droplets formed upon mixing of both proteins (10 μM each). Both channels are shown, along with an image of both channels overlaid. 4% (v/v) 1,6-hexanediol (1,6HD) dissolves these condensates. Scale bar, 10 µm. Middle panel, left to right: NSP5-647 (50 μM) + poly-arginine (polyArg, 5 μΜ); NSP5-polyArg condensate + 1,6-HD; NSP5-polyArg condensates + 0.5 M NaCl; NSP5 + R9 peptide (Arg-9, 5 μΜ). Bottom row, left to right: NSP5-647 sample (35 μM) alone; after addition of 10% v/v PEG-20K; NSP2-488 (25 μM) alone; after addition of 10% v/v PEG-20K. (b) Phase diagrams generated through droplet microfluidics for the coacervation of NSP2 and NSP5, in the presence of 0% v/v (left), 1.5% v/v (middle), and 4% v/v (right) 1,6HD. Phase diagrams were generated from N = 2206, 2035 and 1470 data points for each 1,6-hexanediol concentrations, respectively, and the data were used to construct the LLPS probability plots. (c) The number of viroplasms (black y axis on the left) and estimated cytoplasmic NSP5 concentration ([NSP5] µm, blue y axis on the right) at 2, 4, and 6 hours post infection. Viroplasms were counted in N = 595 ± 85 cells for each time point, and intracellular NSP5 concentration was determined by quantitative Western blotting, as described in Materials and Methods.
Liquid-Liquid Phase Separation Underpins the Formation of Replication Factories in Rotaviruses (2021)
Figure 5

Figure 5 | NSP5 drives of liquid-liquid phase separation required for viroplasm formation.

(a) DeePhase analysis of the phase-separating properties of NSP5 (regions with a score of > 0.5, i.e., above the dotted line, indicate residues with high LLPS potential). The CTR region (AA 179-197) is highlighted in red. Note that the amino acid numbering represents an average score with a 30-residue sliding window, and not individual residues. Green boxes denote NSP5 residues of previously shown to be essential for viroplasm-like structure formation when co-expressed with NSP2(Eichwald et al, 2004). (b) EMBOSS protein charge plot shown as an alternating blue (negatively charged) and red (positively charged) regions with a 30-residue sliding window. (c) flDPnn Disorder Propensity Plot of NSP5. Regions with a score above the dotted line are predicted to be disordered. (d) NSP5 model predicted using Alphafold2. Region 179-197 of the CTR within the predicted C-terminal alpha helix is highlighted in red. (c) Top panel, left to right: NSP5-∆CTR labelled with Atto-647 (25 μM) incubated with unlabeled NSP2 (10 μΜ); NSP5-∆CTR in the presence of 10% v/v PEG-20K; and with 5 μΜ of poly-arginine (polyArg). Bottom panel: Atto-647-labelled NSP5-∆CTR (5 μΜ) incubated with unlabeled NSP5 (25 μΜ) and Atto-488 labelled NSP2 (10 μΜ). NSP5/NSP2 droplets containing labelled NSP2 (green) also contain NSP5-∆CTR-Atto647 (magenta), shown along with an image of both 488/647 channels overlaid. Scale bar, 10 µm.
Monitoring mitochondrial translation in living cells (2021)
Figure 1

Figure 1. Visualizing mitochondrial translation in the cellular context.

A Schematic presentation of the approach. Cytosolic translation is inhibited with Harringtonine (Harr) while mitochondrial ribosomes are allowed to incorporate the alkyne-containing methionine homolog (HPG, orange) into newly-synthesized proteins. In fixed cells, HPG moieties are clicked to an azide-conjugated fluorophore (star, magenta), through copper-catalyzed Huisgen cycloaddition (click), and can be visualized microscopically.

B WT HeLa cells were incubated with HPG for 30 minutes in the presence or absence of Harr and/or Chloramphenicol (Chl). After fixation, AF647-azide (magenta) was used for click reaction (see A), and cells were immunostained for TOM20 as mitochondrial marker (green). Representative epifluorescent microscopy images are shown. Magenta images shown in the presence of only Chl and in the absence of both inhibitors (none) are presented at lower brightness level than the other panels, duo to their very strong intensities. An inset of Harr panel is zoomed in the panel below for better visualization. Met, methionine. Scale bars, 50 µm and 10 µm for the zoom panel.

C Mitochondrial translation products in osteosarcoma 143B control and Rho0 cell lines as described in A. Scale bars, 25 µm.

D Comparison of mitochondrial translation labeling using radioactive [35S]Met in control HEK cells, or HPG followed by click reactions with AF488-azide, in purified mitochondria. Samples were analyzed by SDS-PAGE and either digital autoradiography or fluorescent gel imaging.

E Similar experiments, performed by click reactions in 143B COX1 mutant cells (m.6930G>A), derived from a patient, or in COA6KO HEK293T cells, in comparison to the respective controls.
Monitoring mitochondrial translation in living cells (2021)
Figure 2

Figure 2. Different levels of gene expression in mitochondria of a single cell.

A Mitochondrial translation products were labeled in WT HeLa cells for indicated time lengths (heat map) and cells immunostained for TOM20 (gray). Representative confocal images are shown for each time point. The same 5-minute image was used in both rows with different brightness as a reference for intensity comparison of the other time points in the row. Scale bars, 10 µm. Mitochondrial protein synthesis was quantified over time. 30 cells from N = 3 independent experiments were analyzed for each time point (Average of the HPG intensity in the mitochondrial area was calculated and the baseline was set to zero by subtracting the value of time point 0 from all the values. Error bars, +/- SEM). A hyperbolic graph was fitted to the data. R-squared, 0.97.

B As in A, Mitochondrial translation products were labeled for increasing time (10 and 20 minutes) in HeLa cells. The number of analyzed cells was increased in the plots from 25 to 300 per time point. 150 or 300 random cells from N = 3 independent experiments were analyzed. Kruskal-Wallis followed by Fisher's test. ****p < 0.0001. Error bars, +/- SEM.

C Mitochondrial translation products were labeled for 30 minutes and clicked with AF647 (magenta) and cells immunostained for TOM20 (green) in fibroblasts, iPSCs-derived cardiomyocytes, and hippocampal neurons. Representative epifluorescent microscopy images are shown. The correlation of HPG and TOM20 channels is represented in fire lookup table, after calculating the Pearson Correlation values over each pixel of the images. Scale bars, 25 µm.
Monitoring mitochondrial translation in living cells (2021)
Figure 3

Figure 3. Utilizing HPG labeling in a disease context and mapping mitochondrial translation with super resolution microscopy.

A Mitochondrial translation products were labeled using a FUNCAT approach and [35S]Met labeling in control and TSFM patient fibroblasts. Representative epifluorescent microscopy and the autoradiography images are shown. Scale bars, 50 µm. Changes in mitochondrial protein synthesis were quantified for both approaches. For microscopy image analysis, >300 cells from N = 3 independent experiments were analyzed for each cell type. Mann-Whitney U test, ****p < 0.0001. For radiolabeling analyses, N =3 independent samples were quantified for each cell type. Student t test, **p < 0.01. Error bars, +/- SEM.

B As in A using control and COA6KO HEK cells. Scale bars, 25 µm. For microscopy image analysis, ****p < 0.0001, and for radiolabeling experiments, *p < 0.05 were calculated. Error bars, +/- SEM.

C Mitochondrial protein synthesis was monitored in hippocampal neurons, using ATTO 590-azide (magenta) labelling and cells immunostained for TOM20 (green). A representative deconvolved STED microscopy image is shown. Scale bars, 1 µm.
Monitoring mitochondrial translation in living cells (2021)
Figure 4

Figure 4. Mitochondrial translation in neurons.

A Mitochondrial translation products were labeled with HPG in hippocampal neurons, immunostained for ANK-G (initial axon segment), and plasma membrane was stained with DiO. Representative epifluorescent images and zoomed insets of neural branches are shown. In both axon (ANK-G positive, arrow) and dendrite (star), HPG incorporation revealing mitochondrial protein synthesis was detected. Scale bars, 20 µm.

B Representative confocal images of a dendrite (top) and an axon (bottom) in which mitochondrial translation products were labeled with HPG (magenta). HOMER1 and VGLUT1 were used as post- and presynapse markers, respectively (green) and DiO stained randomly neurites (gray). In each neurite, an exemplary region was selected and the DiO was outlined to mark the outer edge of the region. As a control, cells were treated with Chloramphenicol (Chl) to inhibit mitochondrial translation. Scale bars, 5 µm.