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

Figure 4. Mon1-Ccz1 activation by Rab5 is conserved in metazoan cells.

(A) Schematic representing the architecture of yeast and metazoan Rab7-GEF-complexes. Yeast Mon1-Ccz1 consists of a heterodimer whereas metazoan GEF-complex is a heterotrimer with Bulli (in Drosophila) or RMC1 in mammalian cells as a third subunit. A dimer of metazoan Mon1-Ccz1 may exist as well. (B) Purification and prenylation of Drosophila Rab-GTPases. Rab5 and Rab7 were prenylated and complexed with REP and GDI, respectively. (C) Purification of Drosophila dimeric and trimeric Rab7-GEF-complexes from insect cells as used for GEF-assays. (D) Recruiter GEF-assay with Drosophila dimeric and trimeric (Bulli-)Mon1-Ccz1. The recruiter GEF-assay was performed as described in Figure 3 using Drosophila protein complexes and increasing amounts of either Mon1-Ccz1 (Dimer) or Bulli-Mon1-Ccz1 (Trimer). (E) Enzymatic parameters as measured in (D). Values were calculated of two independent experiments. Error bars represent standard deviation. (F) Recruiter GEF-assay with decreasing amounts of Rab5. The recruiter-assay was performed as described above with 12.5 nM Mon1-Ccz1 and Bulli-Mon1-Ccz1, respectively, and the indicated amounts of recruiter Rab5. (G) Rate constants as derived from (F) in dependence of concentration of Rab5. Error bars represent standard deviation. For details see Methods.

Figure 4—source data 1. Quantification Figure 4E.
Figure 4—source data 2. Quantification Figure 4G.

Figure 4.

Figure 4—figure supplement 1. Purification and prenylation of Drosophila proteins.

Figure 4—figure supplement 1.

(A) Proteins as used for prenylation of Drosophila Rab5 and Rab7. For purification details see methods. (B) Prenylation of Drosophila Rab5. Drosophila Rab5 was incubated with equimolar amounts of yeast REP in the presence of yeast GGTase and GGPP for 1 hr at 30°C. As a control GGPP was omitted. Downshift of the Rab5 band indicates successful prenylation. (C) Prenylation of Drosophila Rab7 in complex with Drosophila GDI. Rab7 was prenylated as described in the method section and was further purified by (D) size exclusion chromatography. (E) Purification of Drosophila dimeric Mon1-Ccz1 complex. For details see text. (F) Size exclusion chromatography of purified d.m. Mon1-Ccz1. Mon1-Ccz1 eluted as one peak at a retention volume of 12.5 ml. (G) Purification of Drosophila trimeric Bulli-Mon1-Ccz1 complex. For details see text. (H) Size exclusion chromatography of purified d.m. Mon1-Ccz1. Bulli-Mon1-Ccz1 eluted as one peak at a retention volume of 11.5 ml. (I) Membrane association of recruiter GTPase Rab5 in assays shown in Figure 4D.