Journal: microPublication Biology

Article Title: Characterization of temperature-sensitive alleles of the septation initiation network protein Mob1 in Schizosaccharomyces pombe

doi: 10.17912/micropub.biology.001595

Figure Lengend Snippet: (A) The mutations encoded by each mob1 allele are listed. (B) The indicated strains were grown in liquid YE media at 25°C until they reached mid-log phase and then adjusted to the same cell concentrations measured by optical density (Moreno et al., 1991). Next, 10-fold serial dilutions were made and 2.5 µL of each was spotted on YE agar plates and incubated at the indicated temperatures for 2-3 days prior to imaging. The spot assays were done twice and a representative is shown. (C) The indicated strains were grown in liquid YE media at 25˚C. Samples were collected before and again after growing the cells for an additional 3.5 hours at 36˚C. The cells were then fixed and stained with DAPI and methylene blue. Representative images are shown. The experiment was performed in duplicate. Scale bar, 5 µm. (D) The number of nuclei per cell (top), and the percentage of septated cells (middle) and lysed cells (bottom) were quantified at 36°C from the same experiments as in C. N≥200 cells of each genotype. (E) Ribbon diagram of a structural model of S. pombe Mob1 using AlphaFold3 (Abramson et al., 2024). The positions of the N- and C-termini and the positions of the mutated residues in the mob1 alleles are indicated. (F) Schematic of sid2 gene product (drawn to scale). Numbers indicate amino acid position (top). Ribbon diagram of a structural model of S. pombe Mob1 bound to the Sid2 regulatory region and kinase domain using AF3. Mob1 is in magenta, the Sid2 regulatory region is in green, and the Sid2 kinase domain is in cyan (bottom).

Article Snippet: AlphaFold3 structural prediction Protein structure predictions were generated with the AlphaFold3 server (Abramson et al., 2024) and visualized using the PyMOL molecular graphics system (version 3.0, Schrodinger, LLC).

Techniques: Comparison, Incubation, Imaging, Staining

Journal: Nature Structural & Molecular Biology

Article Title: A heterotrimeric protein complex assembles the metazoan V-ATPase upon dissipation of proton gradients

doi: 10.1038/s41594-025-01610-9

Figure Lengend Snippet: a , Purification protocol for the mRAVE complex. b , SEC profile (absorbance at 280 nm). Fractions containing the main or shoulder peaks were pooled. c , Aliquots of the main and shoulder peaks were subjected to SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and Coomassie blue staining. Molecular weight markers were run and their size in kDa is shown on the right. d , Immunoblotting of the two peaks with antibodies to the indicated subunits of the V 1 complex. e , Negative-stain electron microscopy (EM) of the purified mRAVE complex. Top: individual particles. Bottom: 2D averages. f , Top: AlphaFold3-predicted model of the mRAVE complex in two different views. Middle and bottom: 3D model derived from the negative-stain data in e overlaid with the AlphaFold model.

Article Snippet: Raw sequencing data and AlphaFold3 models that support the findings of this study were deposited to Mendeley data and were made available at 10.17632/85b999zcdz.1 (ref. ).

Techniques: Purification, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Molecular Weight, Western Blot, Electron Microscopy, Derivative Assay

Journal: Nature Structural & Molecular Biology

Article Title: A heterotrimeric protein complex assembles the metazoan V-ATPase upon dissipation of proton gradients

doi: 10.1038/s41594-025-01610-9

Figure Lengend Snippet: ( a ) AlphaFold3 models of the indicated RAVE complexes. The boxed regions are shown in a magnified view on the right. The helical bundle of DMXL2/Rav1p is highlighted. ( b ) Predicted Aligned Error (PAE) plots for each model, with a darker green color showing increased prediction confidence. D, DMXL; W, WDR7; R, ROGDI; R1, Rav1p; R2, Rav2p; S, Skp1p.

Article Snippet: Raw sequencing data and AlphaFold3 models that support the findings of this study were deposited to Mendeley data and were made available at 10.17632/85b999zcdz.1 (ref. ).

Techniques:

Journal: Nature Structural & Molecular Biology

Article Title: A heterotrimeric protein complex assembles the metazoan V-ATPase upon dissipation of proton gradients

doi: 10.1038/s41594-025-01610-9

Figure Lengend Snippet: a , The main and shoulder fractions from Fig. were individually treated with two bifunctional crosslinkers (BS3 and E-EDC), and crosslinked tryptic peptides were identified by mass spectrometry. Each line is an observed crosslink between the indicated regions of the components. Intra- and intermolecular crosslinks are shown in gray and green, respectively. b , AlphaFold3-predicted mRAVE model, with the highest confidence BS3 and E-EDC crosslinks between the components indicated by red and purple broken lines, respectively. The square indicated by broken lines is magnified on the right with ROGDI residues at the interface labeled. c , HEK-293T cells were transfected with cDNAs coding for WDR7-FLAG, ROGDI-HA and untagged DMXL2 or WDR7, as indicated. Cell lysates were subjected to IP with Flag antibodies. The samples were analyzed by SDS–PAGE and immunoblotting with the indicated antibodies. d , The same as in c , with expression of the indicated proteins. ROGDI-HA was immunoprecipitated with HA antibodies, and coprecipitated proteins were analyzed by SDS–PAGE and immunoblotting with the indicated antibodies. e , The same as in d , using ROGDI mutants in which residues at the interface to DMXL2 ( b ) were altered. E/W/L mut is a combination of the three single mutants. f , The same as in c , with expression of the indicated proteins. Flag-immunoprecipitates were analyzed by SDS–PAGE and immunoblotting with the indicated antibodies.

Article Snippet: Raw sequencing data and AlphaFold3 models that support the findings of this study were deposited to Mendeley data and were made available at 10.17632/85b999zcdz.1 (ref. ).

Techniques: Mass Spectrometry, Labeling, Transfection, SDS Page, Western Blot, Expressing, Immunoprecipitation

Journal: Nature Structural & Molecular Biology

Article Title: A heterotrimeric protein complex assembles the metazoan V-ATPase upon dissipation of proton gradients

doi: 10.1038/s41594-025-01610-9

Figure Lengend Snippet: ( a ) AlphaFold predictions of ROGDI and Rav2p are shown individually and overlayed. ( b ) The terminal helical bundle domain of DMXL2 and Rav1p are shown individually and overlayed. ( c ) AlphaFold3 models of the indicated RAVE complexes, highlighting the β propellers and ROGDI or Rav2p. The square indicated by broken lines shows the interactions. ( d ) Magnification of the squares in ( c ).

Article Snippet: Raw sequencing data and AlphaFold3 models that support the findings of this study were deposited to Mendeley data and were made available at 10.17632/85b999zcdz.1 (ref. ).

Techniques:

Journal: Nature Structural & Molecular Biology

Article Title: A heterotrimeric protein complex assembles the metazoan V-ATPase upon dissipation of proton gradients

doi: 10.1038/s41594-025-01610-9

Figure Lengend Snippet: a , AlphaFold3-predicted interactions of DMXL2 with subunits of the V 1 complex, using a model containing single copies of subunits A, B2, D, E1 and G2. The numbers following the letters refer to the dominant isoforms in HEK-293T cells. The square indicated by broken lines contains the interactions between the helical bundle of DMXL2 with subunits A and D of the V 1 complex. b , Top left: magnification of the square in a (subunits A and D in yellow and purple, respectively). The indicated amino acids of DMXL2 were mutated. Bottom left: the same as in the top panel, but in a rotated view. Top right: observed crosslinks with BS3 or E-EDC shown as red and green broken lines, respectively. Bottom right: conservation of the C-terminal tail of subunit D calculated by ConSurf (scale at the bottom). c , Flag-tagged WT DMXL2 or the indicated mutants carrying substitutions at the interface with subunit A of the V 1 complex were coexpressed with WDR7-Flag and untagged or HA-tagged ROGDI. Cell lysates were subjected to IP with HA antibodies. The samples were analyzed by SDS–PAGE and immunoblotting with the indicated antibodies. d , The same as in c , but with DMXL2 mutants carrying substitutions at the interface with subunit D of the V 1 complex. e , The same as in c , but with cells expressing only the WT or mutant helical bundle of DMXL2. f , DMXL1 -KO cells expressing endogenous DMXL2-dTAG-3xHA were engineered to stably overexpress WT or mutant HA-DMXL2 defective in the interaction with the V 1 complex (for expression levels, see Extended Data Fig. ). The cells were treated with or without the degrader dTAG13. After treatment with BafA1, the drug was removed, and the cells were recovered in fresh medium. Cells were then stained with LysoTracker and analyzed by flow cytometry. Shown are the means and standard deviation of the median fluorescence intensity from n = 3 biological replicates. Data were analyzed using ordinary one-way analysis of variance followed by Dunnett’s multiple-comparisons test ( P < 0.0001). MS, mass spectrometry.

Article Snippet: Raw sequencing data and AlphaFold3 models that support the findings of this study were deposited to Mendeley data and were made available at 10.17632/85b999zcdz.1 (ref. ).

Techniques: SDS Page, Western Blot, Expressing, Mutagenesis, Stable Transfection, Staining, Flow Cytometry, Standard Deviation, Fluorescence, Mass Spectrometry

Journal: Nature Structural & Molecular Biology

Article Title: A heterotrimeric protein complex assembles the metazoan V-ATPase upon dissipation of proton gradients

doi: 10.1038/s41594-025-01610-9

Figure Lengend Snippet: ( a ) AlphaFold3-predicted interactions of Rav1p with subunits of the yeast V 1 complex, using a model containing single copies of subunits A, B, D, E, and G. The square indicated by broken lines shows the interactions between the helical bundle of Rav1p with subunits A and D of the V 1 complex. ( b ) Upper panel: magnification of the square in ( a ) (subunits A and D in yellow and purple, respectively). Lower panel: as in the upper panel, but in a rotated view. ( c ) Predicted Aligned Error (PAE) plot, with a darker green color showing increased prediction confidence. ( d ) DMXL1 knockout cells expressing endogenous DMXL2-dTAG-3xHA were engineered to stably overexpress wild-type or mutant HA-DMXL2 defective in the interaction with the V 1 complex. Cell lysates were analyzed by SDS-PAGE followed by immunoblotting with HA antibodies. Blotting for actin served as a loading control.

Article Snippet: Raw sequencing data and AlphaFold3 models that support the findings of this study were deposited to Mendeley data and were made available at 10.17632/85b999zcdz.1 (ref. ).

Techniques: Knock-Out, Expressing, Stable Transfection, Mutagenesis, SDS Page, Western Blot, Control

Journal: Nature Structural & Molecular Biology

Article Title: A heterotrimeric protein complex assembles the metazoan V-ATPase upon dissipation of proton gradients

doi: 10.1038/s41594-025-01610-9

Figure Lengend Snippet: (a) Cryo-EM structure of the V-ATPase holoenzyme (PDB code: 9BRA) . Subunit A of V 1 is colored yellow, and V O is shown in black. The arrow points to the domain in subunit A that interacts with DMXL2. ( b ) Overlay of the cryo-EM structure with the AlphaFold3-predicted structure of DMXL2-V 1 complex (see Fig. ). DMXL2 is shown in blue, subunit D of V 1 in purple, and subunit B of V 1 in red. (c) As in ( a ), but in a rotated view. (d) As in ( b ), but in a rotated view.

Article Snippet: Raw sequencing data and AlphaFold3 models that support the findings of this study were deposited to Mendeley data and were made available at 10.17632/85b999zcdz.1 (ref. ).

Techniques: Cryo-EM Sample Prep