EMBO/soda-nel huggingface dataset:
Identification of TAPBPL as a novel negative regulator of T cell function (2021)
Figure 7

Figure 7. An anti-hTAPBPL mAb neutralizes T cell inhibitory activity of hTAPBPL in vitro and inhibits tumor growth in vivo. (A-C) Anti-hTAPBPL mAb (clone 54) neutralized the inhibitory activity of hTAPBPL-Ig on (A) T cell proliferation, and (B, C) CD69 expression by CD4+ and CD8+ T cells in vitro. (D-L) DBA/2J mice were injected s.c. with 1× 105 P388 murine leukemia cells, followed by injection of the anti-hTAPBPL mAb (25, 50, or 100µg) or isotype Ab (25, 50, or 100 µg) 3 times per week. (D) Tumors size was measured over time. The mean tumor diameter (mm3) + S.D. at the indicated time points are shown. (E-J) At the end of the studies, tumors from 100 µg anti-hTAPBPL mAb- or isotype Ab-treated mice were harvested. Single-cell suspension of the tumors was analyzed for the percentage of (E, F) CD4+ T cells, (G, H) CD8+ T cells, and (I and J) CD4+CD25+FoxP3+ Tregs by flow cytometry. (K, L) Tumor infiltrating CD4+ and CD8+ T cells were examined by immunofluorescence. (M) C57BL/6 mice were injected s.c. with 1x105 B16F10 melanoma cells, and intratumorally with anti-hTAPBPL mAb or isotype Ab (50 µg) 3 times per week. Tumors size was measured over time. The data are expressed as mean + SD and representative of 2 independent experiments with similar results (n=6/group/time). Significance in (A, C) was calculated by one‐way ANOVA with Dunnett test and others by two‐tailed Student's t‐test. * P<0.05, anti-hTAPBPL Ab group was compared with isotype Ab group.

Expanded View Figure Legends:
AP-3 vesicle uncoating occurs after HOPS-dependent vacuole tethering (2020)
Figure 1

Figure 1: Function of possible coat proteins in AP-3 vesicle formation.

(A) Co-localization of c-terminally GFP- or mCherry-tagged Apl5, Sec7 and Vps41 in a vam3 temperature sensitive (ts) background strain. Cells were grown over night at 23°C and where indicated shifted to 37°C for 3 h prior to imaging. White arrowheads indicate co-localization events. Scale bar, 5 µm. Scale bar in inset, 2 µm.

(B) Quantification of co-localization events from (A). Single events were counted and the percentage of co-localization normalized to the number of Apl5-punctae was plotted (n ≥ 187 cells). Bars show the mean percentage of co-localization ± standard deviation. Significance was determined with a two-tailed t-test (***, p ≤ 0.001).

(C) Re-localization of Apl5 upon Vps41 re-localization. Vps41 was tagged with a C-terminal FRBGFP tag in a strain, where Apl5 carries a C-terminal mCherry and Pma1 a FKBP-tag (Auffarth et al, 2014). Cells were grown in either the absence (control) or presence of 10 μM rapamycin, and localization of Vps41 and Apl5 was analysed by fluorescence microscopy. Scale bar, 5 μm.

(D) Effect of clathrin deletion on the AP-3 pathway. GFP-tagged Nyv1 with a C-terminal Snc1 transmembrane domain (GNS) was localized relative to FM4-64 stained vacuoles in the indicated strains. Labeling of the cell surface by GFP indicates a defect in the AP-3 pathway (Cabrera et al., 2009) Scale bar, 5 µm.

(E) Quantification of AP-3 defect from (D). Linear intensity plots were laid over the cells, the AP-3 defect was calculated by dividing GFP-intensities on the vacuolar membrane by the sum of the intensity of vacuolar, and plasma membrane signal (n ≥ 30 cells). Bars show the mean vacuolar signal over total signal ± standard deviation.
AP-3 vesicle uncoating occurs after HOPS-dependent vacuole tethering (2020)
Figure 2

Figure 2: Mitochondrially anchored Vps41 can recruit AP-3.

(A) Model of Vps41 re-localization to mitochondria. In wild-type cells, AP-3 vesicles bud off the TGN and reach the vacuole where they are tethered by the HOPS subunit Vps41. Overexpressed Vps41, C-terminally tagged with GFP-Fis1, localizes to the mitochondria where it is able to tether AP-3 vesicles to the surface. A wild-type copy of Vps41 is still present in the background to minimize disturbance in the endogenous system.

(B) Scheme of the Vps41 primary sequence with indicated motifs. The Vps41 protein harbours two motifs in its N-terminal region that have been shown to affect Apl5 binding, a PEST motif as well as an amphipathic lipid packaging sequence (ALPS) motif (Cabrera et al, 2010). Furthermore, phosphorylation sites at residues S367, S368, S371 and S372 are shown, which are mutated in a phosphomimetic mutant (SD).

(C) Tethering of AP-3 vesicles to the mitochondrial surface. Cells were grown at 30°C in YPG and kept in logarithmic phase. Just prior to imaging, the mitochondrial DNA was stained with DAPI as described in the methods. Localization of Apl6 and Vps41 was analysed by fluorescence microscopy. First column shows DIC (difference interference contrast) image of cells. Scale bar, 5 µm. The inset shows the indicated magnification. Scale bar in inset, 2 µm.

(D) Quantification of (C). Apl5-dots co-localizing with GFP signal were counted and plotted in percent of the total number of Apl5-dots. Bars show the mean percentage of co-localization ± standard deviation (n ≥ 60 cells).

(E, F) Purification of mitochondria from selected strains. Mitochondria were purified from the indicated strains as described in the methods. The amount corresponding to 1 % of the total volume was loaded of the 'load'' sample, 5% were used for 'crude' and 'pure' mitochondria fractions. Samples were loaded on a 10% SDS PAGE gel and analysed by Western Blot. White asterisks indicate unspecific bands.

(G) Quantification of relative Nyv1 signal intensity from (F). Signal intensity was determined using ImageJ and a ratio of pure over crude signal intensity normalized to Tom40 signal intensity was plotted. Bars show the relative Nvy1 signal intensity ± standard deviation from n = 3 independent experiments.
AP-3 vesicle uncoating occurs after HOPS-dependent vacuole tethering (2020)
Figure 3

Figure 3: Ultrastructural analysis and immuno labeling of cells expressing mitochondrially anchored Vps41.

(A-H) Indicated strains were grown in YPG for expression of Vps41-GFP-Fis constructs. Two representative images are shown for each strain. Mitochondria (M) are highlighted in red and vesicular structures in teal (unlabeled for VPS41-GFP-Fis) or green (unlabeled for VPS41-GFP-Fis). Cell wall (CW), plasma membrane (PM), and vacuoles (V) are indicated. Scale bars, 250 nm.
AP-3 vesicle uncoating occurs after HOPS-dependent vacuole tethering (2020)
Figure 4

Figure 4: Effect of mistargeting Vps41 on the AP-3 pathway.

(A) GNS was localized by fluorescence microscopy in the indicated strains grown in medium containing glucose or galactose (see Figure 1D). Images were inverted to visualize possible defects. First column shows DIC image of cells. Scale bar, 5 µm.

(B) Quantification of the AP-3 defect from (A). Linear intensity plots were laid over cells grown in medium containing glucose (D) or galactose (G), the AP-3 defect was calculated by dividing GFP-intensities on the vacuolar membrane by the sum of the intensity of vacuolar, and plasma membrane signal (n ≥ 15 cells). Bars show the mean vacuolar signal over total signal ± standard deviation.

(C) Effect of different truncations and deletions on AP-3 targeting to mitochondria. Indicated strains were grown at 30°C in YPG and kept in logarithmic phase. Localization of Apl5 or Apl6 and Vps41 was analysed by fluorescence microscopy. First column shows DIC image of cells. Scale bar, 5 µm. Scale bar in inset, 2 µm.

(D) Quantification of (C). (-) Control corresponds to Vps41(ΔPEST)-GFP-Fis1 Apl6-mCherry (CUY12558) and (+) corresponds to Vps41(S-D)-GFP-Fis1 Apl6-mCherry (CUY12560). Apl5- /Apl6-dots co-localizing with GFP signal were counted and plotted in percent of the total number of Apl5- /Apl6-dots (n ≥ 63). Bars show the mean percentage of co-localization ± standard deviation.
AP-3 vesicle uncoating occurs after HOPS-dependent vacuole tethering (2020)
Figure 5

Figure 5: Analysis of AP-3 interactors by proximity labelling.

(A) Scheme of Apl5 with C-terminal TurboID tag.

(B) Model of possible biotinylation events along the AP-3 pathway. AP-3 has to be recruited to the TGN where it concentrates cargo and forms a vesicle. This is transported to the vacuole where it is tethered and subsequently undergoes fusion. During these steps several interactors can come in close proximity to the TurboID Biotin Ligase (grey shade).

(C) Volcano plots of proteins in close proximity to Apl5-TurboID versus control cells, from a label-free proteomics analysis of streptavidin-biotin pulldowns. The logarithmic ratios of protein intensities are plotted against negative logarithmic P values of two tailed Student's t-test, equal variance, performed from n = 3 independent experiments. The red dashed line (significance, 0.05) separates specifically identified proteins (top right portion of plot) from background. Selected top hits are indicated with black dots, and all specific interactors are reported in Dataset EV1. Named hits are marked with colour according to legend in (B). Grey text indicates selected non-significant hits.

(D) Model of possible biotinylation events with AP-3 vesicles tethered to the mitochondrial surface by mitochondrially targeted Vps41.

(E) Volcano plots of proteins in close proximity to Apl5-TurboID versus control cells in a GAL1pr-Vps41-GFP-Fis1 background, from a label-free proteomics analysis of streptavidin-biotin pulldowns with cells grown in YPG. The logarithmic ratios of protein intensities are plotted against negative logarithmic P values of two tailed Student's t-test, equal variance, performed from n = 3 independent experiments. The red dashed line (significance, 0.001) separates specifically identified proteins (top right portion of plot) from background. Selected top hits are indicated with black dots, and all specific interactors are reported in Dataset EV2. Named hits are marked with colour according to legend in (B). Grey text indicates selected non-significant hits.
AP-3 vesicle uncoating occurs after HOPS-dependent vacuole tethering (2020)
Figure 6

Figure 6: Effect of ArfGAP deletions on the AP-3 pathway.

(A) Model of ArfGAP function in vesicle trafficking through the Golgi in yeast. Gcs1 and Glo3 function in trafficking from the cis-Golgi to the ER, whereas Age2 and Gcs1 may function at or beyond the TGN (see text).

(B) AP-3 sorting defects in GAP deletions. The AP-3 reporter GNS (see Figure 1D) was localized relative to FM4-64 stained vacuoles in the indicated strains. First column shows the DIC image of cells. Scale bar, 5 µm.

(C) Table summarizing AP-3 trafficking defects in selected mutants.

(D) Localization of AP-3 in ArfGAP deletion strains. Localization of C-terminally GFP-tagged Apl5 relative to FM4-64 stained vacuoles in the indicated strains. First column shows DIC image of cells. Scale bar, 5 µm.

(E) Quantification of Apl5-dots in (D). Apl5-positve dots were counted and averaged to the number of dots per cell. Box boundaries indicate 25% and 75% of the dataset. The middle line indicates the median and the small square the mean. Whiskers correspond to 5% and 95% values, whereas the small lines show the farthermost outliers (n ≥ 110 cells). Significance was determined with a two-tailed t-test (***, p ≤ 0.001).

(F) Co-localization of C-terminally GFP-tagged Apl5 and C-terminally mCherry-tagged Sec7 in wt and age2Δ background. Cells were grown at 30°C and kept in logarithmic phase. Scale bar, 5 µm. Scale bar in inset, 2 µm.

(G) Quantification of co-localization from (F) normalized per cell. Number of co-localizing dots was determined and normalized to the number of co-localization events per cell. Bars show the mean number of co-localizing dots per cell ± standard deviation (n ≥ 60).
AP-3 vesicle uncoating occurs after HOPS-dependent vacuole tethering (2020)
Figure 7

Figure 7: Requirement of Age2 ArfGAP activity in AP-3 trafficking.

(A, B) AP-3 sorting defects in strains overexpressing Gcs1 (A) or Age2 (B). The AP-3 reporter GNS (see Figure 1D) was localized relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm.

(C) Localization of AP-3 in ArfGAP overexpression strains. Localization of C-terminally GFP-tagged Apl5 relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm.

(D) Quantification of Apl5 dots in (C). Apl5-positve dots were counted and averaged to the number of dots per cell. Box boundaries indicate 25% and 75% of the dataset. The middle line indicates the median and the small square the mean. Whiskers correspond to 5% and 95% values, whereas the small lines show the farthermost outliers (n ≥ 80 cells).

(E, F) AP-3 sorting defects in GAP-dead mutants of Gcs1 (E) and Age2 (F). The AP-3 reporter GNS (see Figure 1D) was localized relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm.

(G) Localization of AP-3 in GAP-dead mutants of Gcs1 and Age2. Localization of C-terminally GFP-tagged Apl5 relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm.

(H) Quantification of Apl5 dots in (G). Apl5-positve dots were counted and averaged to the number of dots per cell. Box boundaries indicate 25% and 75% of the dataset. The middle line indicates the median and the small square the mean. Whiskers correspond to 5% and 95% values, whereas the small lines show the farthermost outliers (n ≥ 80 cells).
Ubiquitylation by the Ltn1 E3 ligase protects 60S ribosomes from starvation-induced selective autophagy. (2014)
Figure 1.

LTN1 deletion rescues the ribophagy defect of UBP3-null cells. (A–D) Cells expressing Rpl5-GFP (A and B) or Rpl25-GFP (C and D) were grown in rich medium (before starvation) and starved in SD-N for the indicated periods (starvation). (A and C, left) Degradation of GFP-tagged proteins was analyzed by anti-GFP blot of whole cell extracts. (right) The ratio between cleaved GFP and full-length protein was quantified for every time point in four independent experiments. a.u., arbitrary unit. (B and D) Cells before starvation or starved for 24 h (starvation) were examined both by fluorescence microscopy and differential interferential contrast (DIC). Note that 100% of wild-type (WT) and mutant cells expressed GFP-tagged proteins in the cytoplasm before starvation. C, cytoplasmic localization; V, vacuolar accumulation; C + V, localization in both cytoplasm and vacuole. Bars, 5 µm. The significance of the differences observed for the ribophagy efficiency was evaluated using Student’s t test. *, P = 0.01–0.05; **, P = 0.001–0.01. The errors bars correspond to standard deviations.
Ubiquitylation by the Ltn1 E3 ligase protects 60S ribosomes from starvation-induced selective autophagy. (2014)
Figure 2.

The 60S ribosomal protein Rpl25 is a substrate of both Ltn1 and Ubp3. (A) GFP or Rpl25-GFP were expressed in cells transformed (+) or not transformed (−) with a plasmid encoding 6His-ubiquitin. (top) Expression levels of GFP and Rpl25-GFP in whole cell lysates were verified with anti-GFP blotting. (middle) Purified 6His-ubiquitin conjugates were examined by anti-GFP blotting. (bottom) 6His-ubiquitin expression and efficiency of purification was controlled using an anti-6His antibody. White lines indicate that intervening lanes have been spliced out. (B) Analysis of ubiquitylated forms of Rpl25-GFP in wild-type and the indicated mutant cells as in A. (C) Expression of Rpl25-GFP and its modified form were analyzed by anti-GFP blot of extracts from wild-type and mutant cells starved for the indicated periods. Ub, ubiquitin; WT, wild type.
Ubiquitylation by the Ltn1 E3 ligase protects 60S ribosomes from starvation-induced selective autophagy. (2014)
Figure 3.

Ubiquitylation of Rpl25 at K74 regulates 60S ribophagy. (A) Indicated strains were transformed with plasmids encoding either Rpl25-GFP (Rpl25) or rpl25 K74,75R-GFP (rpl25KR) before the deletion of a genomic copy of RPL25. (top) Comparable expression levels of Rpl25-GFP or rpl25 K74,75R-GFP in whole cell lysates were confirmed with anti-GFP blotting. Purified 6His-ubiquitin–conjugated forms of Rpl25-GFP or rpl25 K74,75R-GFP (middle) and 6His-ubiquitin expression (bottom) were analyzed as in Fig. 2. Ub, ubiquitin. (B) Wild-type and ubp3Δ cells expressing Rpl25-GFP or rpl25 K74,75R-GFP were starved for the indicated period of time. (C) Degradation of GFP-tagged proteins was analyzed by anti-GFP blot of whole cell extracts, and the ratio between cleaved GFP and full-length protein was quantified for all time points in four independent experiments. a.u., arbitrary units. (D) Localization of GFP-tagged proteins. C, cytoplasmic localization; V, vacuolar accumulation; C + V, localization in both cytoplasm and vacuole (E) Degradation of Rpl3 was analyzed at 24 h upon starvation by Western blotting of whole cell extracts using anti-Rpl3 (gift from V. Albanese, Institut Jacques Monod, Paris, France) or anti-Mex67 (as a control) antibodies. Rpl3 and Mex67 expression was quantified and normalized to the expression level at time 0. Significance of the differences observed for the ribophagy efficiency was evaluated using Student’s t test. *, P = 0.01–0.05; **, P = 0.001–0.01; ***, P < 0.001. White lanes indicate that intervening lanes have been spliced out. The errors bars correspond to standard deviations.
Ubiquitylation by the Ltn1 E3 ligase protects 60S ribosomes from starvation-induced selective autophagy. (2014)
Figure 4.

Ltn1 is rapidly degraded in a RING domain– and proteasome-dependent manner. (A) Wild-type cells expressing Bre5-GFP, Ltn1-Flag, or ltn1-ΔRing-Flag were starved for the indicated periods of time. (B) Cells were treated for 30 min with 100 µM cycloheximide or 100 µM MG132. Expression of tagged proteins was analyzed by anti-GFP or anti-Flag blotting of whole cell extracts. Five times less extracts were analyzed for ltn1-ΔRing-Flag cells. A nonspecific band served as a loading control. White lanes indicate that intervening lanes have been spliced out. WT, wild type.
Centromere Localization and Function of Mis18 Requires 'Yippee-like' Domain- Mediated Oligomerization (2016)
Figure 1

Figure 1. spMis18 possesses a highly conserved N-terminal Yippee-like globular domain and a C-terminal α-helical domain.

A. Schematic representation of domain organization of Mis18 proteins from S. pombe and human (as suggested by the Conserved Domain Database (CDD) and secondary structure predictions).

B. Amino acid conservation of Mis18 proteins among eukaryotes. The alignment includes orthologues from S. pombe (sp), G. gallus (gg), M. musculus (mm), H. sapiens (hs) and B. Taurus (bt). Predicted (light orange; using PsiPred, http://bioinf.cs.ucl.ac.uk/psipred) and observed (bright orange; from the crystal structure shown in Fig 1D) secondary structure elements are shown below the aligned sequences. Amino acid residues mutated in this study are highlighted with circles (dimer interface residues) and asterisks (putative substrate-binding pocket residues).

C. SDS-PAGE showing a representative fraction of the purified spMis18 N-terminal Yippee-like globular domain (amino acid residues 1-120; spMis181-120).

D. Cartoon representation of the crystal structure of spMis181-120 in two different orientations, highlighting key residues within the putative substrate-binding pocket.

E. Substrate-binding pocket mutations Y90A and Y74A/Y90A/T105A/S107K affect the ability of ectopically expressed spMis18fl to rescue the temperature sensitivity of mis18-262 cells to varying degrees. Five-fold serial dilutions of mis18-262 cells transformed with plasmids harbouring the indicated spMis18fl constructs, spotted on PMG – uracil + Phloxine B media supplemented with (repressed) or without (expressed) thiamine, and incubated at the indicated temperatures; dead cells stain dark pink.
Centromere Localization and Function of Mis18 Requires 'Yippee-like' Domain- Mediated Oligomerization (2016)
Figure 2

Figure 2. Yippee-like globular domains of Mis18 proteins possess an intrinsic preference to form homo/hetero-dimers.

A. Cartoon representation of the assembly of spMis18 Yippee-like domains (spMis181-120) as observed in the crystal asymmetric unit. Close up views highlight the amino acid composition and interactions stabilizing the dimeric arrangements. Interface-II is composed of conserved residues and an extensive binding surface as compared to Interface-I (see text for details), suggesting Dimer-II to be a physiologically relevant dimer. Residues mutated in this study are highlighted with circles.

B. Size Exclusion Chromatography (SEC) and SEC-MALS (SEC combined with Multi Angle Light scattering) analyses of the recombinant wild-type (wt) and dimer disrupting mutant (I31A) of spMis181-120. Measured molecular weights from SEC-MALS confirm that spMis181-120 wt is a dimer in solution and spMis181-120 I31A is a monomer.

C. Evaluation of the ability of Yippee-like domains of human Mis18 proteins (hsMis18α77-187 and hsMis18β56-183) to form a heterodimer. SEC and SEC-MALS analyses demonstrate that while hsMis18α77-187 elutes at 15.5 ml as a dimer (31.3 kDa), hsMis18β56-183 elutes at 17.3 ml as a monomer (15 kDa). Purified hsMis18α77-187 and hsMis18β56-183 when mixed together elute at 15.4 ml as a heterodimer (27.5 kDa).

D. Cartoon representation of the structure of the homology-modelled human Mis18α77-187 - Mis18β56-183 heterodimer using the crystal structure of spMis181-120 reported here as a template. Residues mutated in this study are highlighted with circles. Modelling was carried out using Phyre2 web server (www.sbg.bio.ic.ac.uk/phyre2/).

E. SDS-PAGE analysis of the GST-pull down assay where wt and dimer disrupting mutants of hsMis18α77-187 and hsMis18β56-183 were co-expressed as His- and GST-tagged proteins, respectively in E. coli. While wt GST-hsMis18β56-183 showed interaction with wt His-hsMis18α77-187, hsMis18 proteins harbouring dimer-disrupting mutations, GST-hsMis18β56-183 V77E/Y172D and His-hsMis18α77-187 V82E/Y176D did not show noticeable interaction. The corresponding Ni-NTA pull downs showing the expression of His-tagged proteins are shown in Fig EV2D.
Centromere Localization and Function of Mis18 Requires 'Yippee-like' Domain- Mediated Oligomerization (2016)
Figure 3

Figure 3. spMis18fl forms a tetramer in vitro.

A & B. SEC-MALS analysis of His-GFP-spMis18fl and His-spMis18ΔC, respectively. Refractive index (RI, left Y-axis) and molecular mass (kDa, right Y-axis) profiles show that both His-GFP-spMis18fl (predicted MW of a monomer is 51.97 kDa) and His-spMis18ΔC (Predicted MW of a monomer 21.8 kDa) predominantly exist as tetramers (187.6 kDa and 78.6 kDa, respectively) with a small population of dimer (128.5 kDa and 45.8 kDa, respectively).

C. SEC profile of a sample containing an equimolar mixture of purified His-GFP-spMis18C-term-α and His-spMis18MeDiY (upper panel) and SDS-PAGE analysis of SEC-fractions (bottom panel). His-GFP-spMis18C-term-α and His-spMis18MeDiY eluted separately at 12.3 and 15.3 ml, respectively, demonstrating the inability of these domains to interact with each other.

D. SEC-MALS analysis of His-GFP-spMis18C-term-α shows that it exists as a homotrimer with a measured molecular weight of 119.8 kDa. (Theoretically calculated MW of a monomer is 38.3 kDa).