The Uso1 globular head interacts with SNAREs to maintain viability even in the absence of the coiled-coil domain
- Department of Cellular and Molecular Biology, CSIC Centro de Investigaciones Biológicas, Spain
- Instituto de Tecnologías Biomédicas, Hospital Universitario Nuestra Señora de Candelaria, Spain
- Department of Infectious Diseases, Faculty of Medicine, Flowers Building, Imperial College, United Kingdom
- Department of Structural and Chemical Biology, CSIC Centro de Investigaciones Biológicas, Spain
- Division of Cell Biology, MRC Laboratory of Molecular Biology, Francis Crick Avenue, United Kingdom
Figures
Figure 1 with 1 supplement
Characterization of mutations bypassing the essential role of RAB1.
(A). Genetic map in the region surrounding uso1 with genetic markers used as landmarks for mapping. (B). Molecular identification of the nucleotide changes in suArab1ts strains. (C) and (D): growth …
Figure 1—figure supplement 1
E6K/G540S do not rescue lethality resulting from arf1∆, sly1∆, or sed5∆.
Top, uso1 is an essential gene. Singly-nucleated conidiospores derived from a heterokaryotic strain in which one class of nuclei carries a deficient pyrG uracil biosynthetic gene whereas the second …
Figure 2 with 1 supplement
Localization of the amino acid substitutions within the Uso1 AlphaFold2 structure.
(A). AlphaFold2 cartoon representations of A. nidulans Uso1 dimer. Note that the depiction of the coiled-coil domain (CCD) is highly speculative. It is included with the sole purpose of visually …
Figure 2—figure supplement 1
AlphaFold2 predictions of Uso1.
Ribbon representation of AlphaFold 2-predicted structures of the full-length Uso1 dimer (A) and the Uso1 globular-head-domain(GHD), represented as a monomer (B), color-coded by pLDDT values. Graphs …
Figure 3 with 1 supplement
Determining molecular masses and oligomerization status of the different Uso1 constructs by velocity sedimentation analysis.
The different panels display the sedimentation profiles of the protein being analyzed, with % of the main species, scheme of the corresponding constructs (color matching those in Figure 2), and …
Figure 3—figure supplement 1
Globular-head-domain (GHD) is a monomer across a range of concentrations.
(A) and (B). Sedimentation velocity experiments with wild-type and E6K/G540S mutant GHD, respectively, show that they behave as monomers at concentrations up to 5 μM. (C). Sedimentation velocity …
Figure 4 with 1 supplement
Subcellular localization of Uso1.
(A). Uso1-GFP localizing to punctate cytoplasmic structures, 3D shaded by software. (B). Sections of a deconvolved Z-stack and its corresponding maximal intensity projection (MIP). Uso1-GFP in …
Figure 4—figure supplement 1
Methodology for tracking the half-life of Uso1-GFP on punctate structures.
3D movies were acquired by streaming pictures to the computer RAM at 2 fps. Appropriate reduction of excitation light intensity permitted the acquisition of 400 frames without apparent …
Figure 5
Uso1 puncta do not colocalize with ER exit sites (ERESs).
(A). Low extent co-localization of Sec13 ERES and Uso1 structures. Z-stacks for the two channels were acquired simultaneously, deconvolved, and represented as maximal intensity projections (MIPs). …
Figure 6
Uso1 localizes to Rer1-containing Golgi cisternae.
(A). Tip cells showing Uso1 colocalization with the indicated subcellular markers. Images are maximal intensity projections (MIPs) of deconvolved Z-stacks. (B). Magnified images of the color-coded …
Figure 7
Uso1 localization to punctate structures is dependent on RAB1.
(A). Complete de-localization of Uso1-GFP to the cytosol by rab1ts and relocalization by E6K/G540S. (B). Uso1-GFP and rab1ts show a synthetic negative interaction that is rescued by the E6K/G540S …
Figure 8 with 1 supplement
Genetic evidence shows that the C-terminal region (CTR) region of Uso1 contributes to its recruitment to membranes.
(A). Top, scheme of the predicted interactions. Bottom, engineering a gene-replaced allele lacking the CTR domain by homologous recombination. (B). The Bug1 C-terminal residues fit into the groove …
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Figure 8—source data 1
Raw images for western blots in panel D and uncropped pictures with used exposures and regions indicated.
- https://cdn.elifesciences.org/articles/85079/elife-85079-fig8-data1-v2.zip
Figure 8—figure supplement 1
AlphaFold2 modeling of Grh1-Bug1.
(A). Cartoon, with α-helices shown as cylinders, of the nearly N-terminal PDZ domains of Grh1. (B). AlphaFold 2 prediction of a 1:1 Grh1-Bug1 complex, trimmed of disordered regions. (C). complete …
Figure 9
The globular-head-domain (GHD) of Uso1 is sufficient to support cell viability.
(A). Gene-replaced uso1GHD allele carrying the double E6K/G540S substitution is sufficient to rescue viability at 30 °C, but not at higher temperatures. (B). The Uso1 GHD is a monomer in cell …
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Figure 9—source data 1
For panels B, C, D; raw images for western blots and uncropped pictures with used exposures and regions indicated.
- https://cdn.elifesciences.org/articles/85079/elife-85079-fig9-data1-v2.zip
Figure 10
Screening the preferential association of proteins acting in the ER/Golgi interface with E6K/G540S Uso1.
(A) S-tagged baits (Uso1, wt, and E6K/G540S, and the unrelated protein BapH), expressed after gene replacement, were captured with their associated polypeptides on S-protein agarose beads. Candidate …
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Figure 10—source data 1
Raw images for western blots and silver-stained gels and uncropped pictures with used exposures and regions indicated.
- https://cdn.elifesciences.org/articles/85079/elife-85079-fig10-data1-v2.zip
Figure 11 with 1 supplement
The Uso1 globular-head-domain (GHD) interacts directly with Bos1 and Bet1 SNAREs acting in the ER/Golgi interface.
(A). Purified fusion proteins in which the complete cytosolic domains of the indicated SNAREs have been fused to GST were used in pulldown experiments with His-tagged, purified wild-type and …
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Figure 11—source data 1
Raw images for western blots and Coomassie-stained gels and uncropped pictures with used exposures and regions indicated.
- https://cdn.elifesciences.org/articles/85079/elife-85079-fig11-data1-v2.zip
Figure 11—figure supplement 1
AlphaFold2 prediction of the ER/Golgi SNARE bundle.
(A) Sec5/Bos1/Bet1/Sec22 predicted SNARE bundle. (B) Quality control (pLDDT, color-coded, and predicted aligned error (PAE)) of the model.
Figure 12 with 3 supplements
AlphaFold2 models provide insight into the mechanisms of suppression by E6K and G540S.
(A). Model of full-length Uso1 bound to the ER/Golgi SNAREs Bos1 and Bet1. (B). Top, ribbon representation of the Bos1 N-terminal Habc domain and Uso1 globular-head-domain (GHD). Bottom, Inset …
Figure 12—figure supplement 1
AlphaFold2 prediction of the Bet1-GHD interaction.
(A) The putative binding surface of Bet1 and Uso1 as determined by AlphaFold2. Top images, a cartoon of Bet1-GHD interactions, colored by pLDDT score. Alignment of four independent predictions …
Figure 12—figure supplement 2
AlphaFold 2 predictions of the RAB1 binding site on the Bet1/Bos1/Uso1 globular-head-domain (GHD) complex.
(A) cartoon representations of the AlphaFold 2-predicted GHD-RAB1 complex, depicted as pipes and planks. (B): Orthogonal views of the Uso1 GHD-RAB1-Bet1-Bos1Habc structural model. The Uso1 GHD is …
Figure 12—figure supplement 3
Quality control assessment of AlphaFold2 predictions for the indicated complexes.
Figure 13 with 2 supplements
The N-terminal region of Aspergillus Uso1 containing Glu6 is required for full function.
(A) Sequence comparison of N-terminal regions up to the second helix of the first armadillo repeat including 196 Uso1 proteins of Eurotiomycetes available in https://fungidb.org/fungidb/app (B) …
Figure 13—figure supplement 1
AlphaFold prediction of N-terminal helix: model organisms.
Alpha-Fold Uso1 structures of the indicated model organisms contain an N-terminal extension which includes a short α-helix akin to that detected in Aspergillus.
Figure 13—figure supplement 2
Explanation of key genetic manipulations used for this work.
(A) Relative levels of inulinase (InuA) on different C sources, as determined by western blotting. (B) Genetically engineering of a strain conditionally expressing Uso1. The inulinase coding region …
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Figure 13—figure supplement 2—source data 1
Raw images for western blots and uncropped pictures with used exposures and regions indicated.
- https://cdn.elifesciences.org/articles/85079/elife-85079-fig13-figsupp2-data1-v2.zip
Figure 14
Over-expression of Uso1, but not of Uso1 (∆1–13) rescues the lethality of rab1∆, and rescue is stimulated by E6K.
(A) Western blot showing that all three alleles used in growth tests shown below result in similar levels of protein when driven by the inuAp on sucrose (suc). The protein detected on glucose (glu) …
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Figure 14—source data 1
Raw images for western blots and uncropped pictures with used exposures and regions indicated.
- https://cdn.elifesciences.org/articles/85079/elife-85079-fig14-data1-v2.zip
Videos
Video 1
Shaded 3D reconstruction of a hypha expressing Uso1-GFP.
Video 2
4D acquisition showing the dynamics of Uso1-GFP.
4D (x, y, z, t) in which Z-stacks were acquired at a rate of 1 frame every 2.6 s.
Video 3
Dynamics of Uso1-GFP at 2 fps 3D acquisition (200 frames) showing the dynamics of Uso1-GFP.
Time resolution, 2 fps.
Video 4
Single Uso1-GFP cisterna tracked over time Example of Uso1-GFP cisterna.
The video contains 96 photograms acquired at 2fps.
Video 5
3D reconstruction of a hypha expressing fluorescently labeled Uso1-GFP and Sec13-mCh.
There is little colocalization between Uso1-GFP and Sec13 ERES.
Video 6
4D video (1 fpm) of a hypha expressing fluorescently labeled Uso1-GFP and Sec7-mCh Uso1 does not colocalize at all with the TGN marker Sec7.
Additional files
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Supplementary file 1
‘Protein interfaces, surfaces, and assemblies’ service PISA.
- https://cdn.elifesciences.org/articles/85079/elife-85079-supp1-v2.xlsx
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Supplementary file 2
List and complete genotypes of A. nidulans strains used in this work.
- https://cdn.elifesciences.org/articles/85079/elife-85079-supp2-v2.docx
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Supplementary file 3
Primers used for PCR-based genetic manipulations.
- https://cdn.elifesciences.org/articles/85079/elife-85079-supp3-v2.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/85079/elife-85079-mdarchecklist1-v2.docx
