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. 2020 May 11;9:e56090. doi: 10.7554/eLife.56090

Figure 5. Rab5 can trigger Rab7-dependent fusion.

(A) Scheme of the Rab5-dependent fusion assay. Rab5-proteins in complex with the REP protein were chemically activated. Addition of MANT-GDP-loaded Ypt7:GDI was followed by Mon1-Ccz1 addition. After allowing for nucleotide exchange of Ypt7, fusion machinery was added and fusion reaction was finally triggered by addition of Vam7. (B) Fusion depends on the recruiter GTPase. SNARE-bearing proteoliposomes were primed either with chemically activated pYpt10 or mock-treated for 10 min at 27°C. 250 nM Ypt7:GDI and 12.5 nM Mon1-Ccz1 was added, and the reaction was incubated for another 15 min, allowing for nucleotide exchange of Ypt7. Fusion reaction was triggered by addition of 50 nM HOPS, 0.6 µM Sec17, 50 nM Sec18 and 20 nM Vam7. Fusion rate was followed by a content mixing assay, where FRET of enclosed fluorophores is followed (see Methods). (C) Fusion rates of (B) as measured after 30 min. Error bars represent standard deviation. (D) Membrane association of Mon1-Ccz1 and HOPS in fusion assay. Samples of fusion experiments as in (B) were recovered after 30 min of measurement. Membrane-bound and soluble fraction were separated by centrifugation for 20 min at 20,000 g, and analyzed by SDS-PAGE and Western-Blot. Proteins were detected by using an antibody against the TAP-tag on Vps41 and Ccz1 in the HOPS- and GEF-complex, respectively. (E) Densiometric analysis of Western-Blot signals of Vps41 and Ccz1 as shown in (D). The ratio of Vps41 over Ccz1-signal is shown for fusion reactions in the presence or absence of a recruiter-GTPase. Signals have been normalized to the Ccz1 signal in the pellet fraction. Error bars represent standard deviation.

Figure 5—source data 1. Quantification Figure 5C and Figure 5—figure supplement 1B.
Figure 5—source data 2. Quantification Figure 5E.

Figure 5.

Figure 5—figure supplement 1. Fusion of reconstituted proteoliposomes in dependence of Mon1-Ccz1.

Figure 5—figure supplement 1.

(A) SNARE-bearing proteoliposomes were incubated with 250 nM pYpt7:GDI in presence of indicated amounts of Mon1-Ccz1 and GTP. After allowing for nucleotide exchange for 15 min, fusion was triggered by addition of 50 nM HOPS, 0.6 µM Sec17, 50 nM Sec18 and 20 nM Vam7. Fusion was followed by a content mixing assay, where FRET of enclosed fluorophores is followed (see Materials and methods). (B) Fusion rates of (A) as determined after 30 min.