Figure 3. Rab5 proteins can activate the Mon1-Ccz1 GEF on membranes.

(A) Scheme of Rab5-dependent Rab7 activation. Rab5-proteins in complex with the REP were chemically activated to bring protein to membranes. Addition of MANT-GDP-loaded Ypt7:GDI was followed by Mon1-Ccz1 addition. For details see text. (B) Recruiter GEF-assay of Mon1-Ccz1. 250 nM MANT-GDP-loaded Ypt7 activation was followed by change in fluorescence over time. Liposomes composed of a vacuolar mimicking lipid mix were incubated with 1.5 µM recruiter GTPase as indicated or buffer as a control (no recruiter). For activation of the recruiter, 200 µM GTP was added in the presence of 1.5 mM EDTA for 15 min at room temperature. Afterwards the EDTA was quenched by addition of 3 mM MgCl₂. After pre-activation of the recruiter, 250 nM prenylated Ypt7:GDI was added. Increasing amounts of Mon1-Ccz1 (as indicated) were added to start the reaction. Trace of no recruiter sample was plotted into all graphs for reference (grey line) (C) Protein complexes used for recruiter assay. Prenylated Ypt7 in complex with GDI and heterodimeric Mon1-Ccz1 complex were purified as described in the method section, and analyzed by SDS-PAGE and Coomassie staining. (D) Enzymatic parameters as derived from the recruiter assays in (B). Values were calculated from at least two independent measurements. Error bars represent standard deviation. For details see methods. (E) Membrane association of Rab-GTPases in the recruiter-assay. Samples from the recruiter-assay were recovered after 1000 s, and soluble and membrane fraction were separated by centrifugation for 20 min at 20,000 g. Fractions were analyzed by SDS-PAGE and Coomassie staining.

Figure 3—figure supplement 1. Prenylation of yeast Rab-GTPases for the Recruiter GEF-assay.

(A) Rab-GTPases were preloaded with GDP (Sigma Aldrich, Germany) and incubated with equimolar amounts of Rab Escort Protein (REP) in the presence of Geranylgeranyltransferase and Geranylgeranylpyrophosphat for 1 hr at 30°C. Successful prenylation was judged by downshift of the Rab-GTPase by SDS-PAGE analysis and Coomassie staining. (B) Prenylation of Ypt7 and complex formation with GDI. Ypt7 was prenylated in the presence of GDI and substoichiometric amounts REP as described above. Prenylated Ypt7:GDI was further purified by size exclusion chromatography (SEC). Fractions of Ypt7:GDI were pooled.

Figure 3—figure supplement 2. Membrane bound but not soluble Ypt10 activates Mon1-Ccz1.

Recruiter GEF-assay of Mon1-Ccz. 250 nM MANT-GDP-loaded Ypt7 activation was followed by change in fluorescence over time. Liposomes composed of a vacuolar mimicking lipid mix were incubated with (A) 1.5 µM prenylated Ypt10, (B) increasing amounts of soluble Ypt10 as indicated or (C) buffer as a control (no recruiter). For activation of Ypt10, 200 µM GTP was added in the presence of 1.5 mM EDTA for 15 min at room temperature. Afterwards the EDTA was quenched by addition of 3 mM MgCl₂. After pre-activation of the recruiter or mock treatment (no recruiter), 250 nM prenylated Ypt7:GDI was added. 100 nM Mon1-Ccz1 was added to start the reaction.