Journal: Nature Communications

Article Title: Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes

doi: 10.1038/s41467-021-21695-2

Figure Lengend Snippet: a , b GUV-based activity with membrane-tethered Rab5a. The reaction progress curves and initial rates are shown. Micrographs: AF647-PX signals at the end of reactions. Scale bars: 5 μm. Bar graphs: initial rates of the reaction curves (AF647-PX fluorescence change/min in arbitrary units, AU). a Complex II is greatly activated by membrane-tethered Rab5a in a GTP-dependent manner. b Complex I is only modestly activated by membrane-attached Rab5a–GTP. c Complex II is recruited to Rab5a-decorated LUVs in a GTP-dependent manner as measured by a flotation assay. Here, complex II is mixed with LUVs and the mixture is added on top of a sucrose gradient and centrifuged. Membrane-bound proteins float up to the top of the gradient as seen in gel lanes (Top). Gel quantification can be found in Supplementary Fig. . d Mapping the Rab5a–GTP binding site by proximity-triggered crosslinking of complex II. SDS-PAGE gel of crosslinking reactions, products indicated by green arrows. e Mapping the Rab5a binding site on complex II by the HDX-MS. HDX changes are displayed on a model of human complex II created with PyMOL. Rab5a binding protects (coloured in cyan and blue) the VPS34 C2 helical hairpin insertion (C2HH) and the VPS15 SGD and WD40 domains. The VPS34 C2 (E202) that is crosslinked by unnatural amino acid Rab5a-84BrCO6K is coloured green and shown in an expanded panel. Source data are provided as a Source Data file.

Article Snippet: The purified PX domain was labelled using AF647 C2 Maleimide kit (Life Technologies, A20347), and the labelled protein was purified using a heparin column.

Techniques: Activity Assay, Membrane, Fluorescence, Binding Assay, SDS Page

Journal: Nature Communications

Article Title: Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes

doi: 10.1038/s41467-021-21695-2

Figure Lengend Snippet: a , b GTP-locked Rab5a (Q79L) and all four components of complex II, containing either C-terminally EGFP-tagged WT ( a ) or C2HH mutant REIE > AAAA VPS34 ( b ), were coexpressed in HEK293T cells. Confocal images show cellular localisation of mCherry–Rab5a-Q79L (magenta) and VPS34-EGFP (cyan). To the right of each panel, traces for the fluorescence along the transect indicated by the line in the micrograph are shown. The C2HH REIE mutant markedly increased colocalization of complex II with Rab5a ( b , e ) compared to VPS34 WT ( a , e ). c , d GTP-locked Rab5a (Q79L) and all four components of complex I, containing either C-terminally EGFP-tagged WT ( c ) or mutant REIE > AAAA VPS34 ( d ), were coexpressed in HEK293T cells. The mutant had no impact on the colocalization of complex I with Rab5a ( c – e ), quantitated as described in methods. Error bars: standard deviation. ***: p < 0.0001; n.s.: p > 0.05. Scale bars: 10 μm. f GUV assay of activity of complex II shows that the VPS34 C2HH REIE > AAAA mutation greatly potentiates the activation of complex II by Rab5a–GTP, without affecting the basal activity of complex II. Micrographs: AF647-PX signals at the end of reactions. Scale bars: 5 μm. g The initial rates in the GUV assay in f (AF647-PX fluorescence change/min in arbitrary units, AU) are depicted. ***: p < 0.001; n.s.: p > 0.05. Source data are provided as a Source Data file.

Article Snippet: The purified PX domain was labelled using AF647 C2 Maleimide kit (Life Technologies, A20347), and the labelled protein was purified using a heparin column.

Techniques: Mutagenesis, Fluorescence, Standard Deviation, Activity Assay, Activation Assay

Journal: Nature Communications

Article Title: Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes

doi: 10.1038/s41467-021-21695-2

Figure Lengend Snippet: a Immunoblot of streptavidin precipitates from cells in which MitoID had been carried out for Rab1a WT, QL, and SN mutants (left) or Rab5 QL (right). Rab1a interacts specifically with components of complex I (VPS34, VPS15, Beclin 1, ATG14L) but not with the complex II-specific UVRAG. Rab5a shows only a weak interaction with the complex I-specific ATG14L. b Complex I was potently activated by membrane-attached Rab1a in a GTP-dependent manner. c No activation of complex II by either membrane-attached Rab1a–GTP or Rab1a–GDP was detected. b , c Micrographs: AF647-PX signals at the end of reactions. Scale bars: 5 μm. Bar graphs: initial rates of the reaction curves. d Lipid flotation assays showing complex I recruitment to Rab1a-decorated membranes in a GTP-dependent manner. Gel quantification is in Supplementary Fig. . e Mapping Rab1a binding site on complex I by HDX-MS. Rab1a binding increases protection (coloured in cyan and blue) of the VPS34 C2 insertion (C2HH) and decreases protection (coloured yellow and red) of Beclin 1 CC2. Source data are provided as a Source Data file.

Article Snippet: The purified PX domain was labelled using AF647 C2 Maleimide kit (Life Technologies, A20347), and the labelled protein was purified using a heparin column.

Techniques: Western Blot, Membrane, Activation Assay, Binding Assay

Journal: Nature Communications

Article Title: Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes

doi: 10.1038/s41467-021-21695-2

Figure Lengend Snippet: a , b GTP-locked Rab1a (Q70L) and all four components of complex I, containing either C-terminally EGFP-tagged WT ( a ) or mutant REIE > AAAA VPS34 ( b ), were coexpressed in HEK293T cells. Confocal images show the localisation of mCherry-Rab1a–Q70L (magenta) and VPS34-GFP (cyan). The REIE mutant in VPS34 reduces colocalization of complex I with Rab1a ( e , quantitated as described in ‘Methods’ section). c , d GTP-locked Rab1a (Q70L) and all four components of complex II, containing either C-terminally EGFP-tagged WT ( c ) or mutant REIE > AAAA VPS34 ( d ), were coexpressed in HEK293T cells. Complex II shows no significant colocalization with Rab1a for either WT or mutant VPS34 ( e , quantitated as described in the methods. Error bars: standard deviation. ***: p < 0.0001; n.s.: p > 0.05). Scale bars: 5 μm. f GUV assay of activity of complex I shows that the VPS34 C2HH REIE > AAAA mutation eliminates activation of complex I by Rab1-GTP, without affecting the basal activity. Micrographs: AF647-PX signals at the end of reactions. Scale bars: 5 μm. g The initial rates in the GUV assay (AF647-PX fluorescence change/min in arbitrary units, AU) in f are depicted. ***: p < 0.001; n.s.: p > 0.05. Source data are provided as a Source Data file.

Article Snippet: The purified PX domain was labelled using AF647 C2 Maleimide kit (Life Technologies, A20347), and the labelled protein was purified using a heparin column.

Techniques: Mutagenesis, Standard Deviation, Activity Assay, Activation Assay, Fluorescence