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. 2021 Mar 10;12:1564. doi: 10.1038/s41467-021-21695-2

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — 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.