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. 2021 Aug 12;220(9):e202105120. doi: 10.1083/jcb.202105120

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© 2021 Borchers et al.

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Figure 4. — Approaches to determine GEF activity in vitro. Methods to determine GEF activity for Mon1-Ccz1. In all approaches, Rab7 is preloaded with fluorescent MANT-GDP. Fluorescence decreases upon GEF-mediated nucleotide exchange. (A) GEF assays. (Ai) In-solution Rab GEF assay. Mon1-Ccz1 (blue, Bulli/Rmc1/C18orf8 subunit, indicated by unlabeled hexagon) and Rab7 (gray) are freely diffusible in the test tube, which results in random collision and Rab activation. (Aii) GEF-mediated activation of artificially recruited Rab7 on liposomes. Rab7 with a C-terminal 6xHis-tag is permanently immobilized on membranes containing the cationic lipid DOGS-NTA. Mon1-Ccz1 unspecifically binds to this membrane surface and activates Rab7. Diffusion is limited to the membrane surface, thus increasing chances of interactions. (Aiii) Reconstitution of Rab5-mediated Rab7 activation by Mon1-Ccz1 on liposomes. Chemically activated, prenylated Rab5 (green), delivered to the membrane by the Rab Escort Protein (REP), allows Mon1-Ccz1 recruitment and Rab7 activation from the GDI complex (see text for further details). (B) Summary of Ai–Aiii. pren., prenylation.