A guanine nucleotide exchange factor (GEF) limits Rab GTPase-driven membrane fusion

Abstract

The identity of organelles in the endomembrane system of any eukaryotic cell critically depends on the correctly localized Rab GTPase, which binds effectors and thus promotes membrane remodeling or fusion. However, it is still unresolved which factors are required and therefore define the localization of the correct fusion machinery. Using SNARE-decorated proteoliposomes that cannot fuse on their own, we now demonstrate that full fusion activity can be achieved by just four soluble factors: a soluble SNARE (Vam7), a guanine nucleotide exchange factor (GEF, Mon1-Ccz1), a Rab-GDP dissociation inhibitor (GDI) complex (prenylated Ypt7-GDI), and a Rab effector complex (HOPS). Our findings reveal that the GEF Mon1-Ccz1 is necessary and sufficient for stabilizing prenylated Ypt7 on membranes. HOPS binding to Ypt7-GTP then drives SNARE-mediated fusion, which is fully GTP-dependent. We conclude that an entire fusion cascade can be controlled by a GEF.

Keywords: GDI; Mon1-Ccz1, Ypt7, Rab7, GDI; Rab; endosome; guanine nucleotide exchange factor (GEF); lysosome; membrane fusion.

Figure 1.

Establishment of in vitro prenylation and functionality of Ypt7 and Vps21. A, prenylation of Ypt7 and Vps21 in vitro. 3 μm Ypt7 or Vps21, respectively, 3 μm REP (Mrs6), 1 μm geranylgeranyltransferase (GGTase, Bet2-Bet4) were incubated with or without geranylgeranyl pyrophosphate (GGPP) at 30 °C for 30 min. Rab–GDI complexes were obtained as described previously (10). Samples were analyzed by SDS-PAGE and Coomassie staining. B, membrane association of Rabs after prenylation. Vps21 (top) and Ypt7 (bottom) were incubated with the prenylation machinery in either the presence or absence of GGPP as described in A. Where indicated, liposomes were added. To allow for membrane insertion after prenylation, the samples were incubated for another 30 min at 30 °C and then centrifuged for 30 min at 20,000 × g. The supernatant and pellet were analyzed by SDS-PAGE and Coomassie staining. C, analysis of GEF activity. 50 pmol of MANT–GDP–loaded Rabs were incubated with the respective GEF as described under “Materials and methods.” Loss of fluorescence was monitored in a plate reader after addition of GTP to a final concentration of 0.1 mm for 30 min at 30 °C. GEF activity of the corresponding GEF toward prenylated (REP) and unprenylated (REP (−GGPP)) Rab–REP complexes was measured in the presence of liposomes. GEF assays with soluble Rabs were performed in the absence of liposomes using the corresponding GEF. C-terminally His-tagged Ypt7 and Vps21 (His) were incubated with their respective GEF in the presence of liposomes carrying DOGS-NTA. A summary of k_cat/K_m values was obtained from three different experiments. D, model of Mon1–Ccz1–dependent Rab recruitment to membranes. For details see text. E and F, analysis of RPL fusion. 160 nm prenylated or non-prenylated Ypt7–REP complex was added in the presence of 100 nm Mon1–Ccz1 and either GDP or GTP prior to starting fusion reactions to dye-loaded proteoliposomes. Proteoliposomes carried either the vacuolar SNAREs Vam3 and Vti1 or the R-SNARE Nyv1. The SNARE:lipid ratio was 1:10,000 (see “Materials and methods”). Fusion reactions were carried out in a plate reader for 30 min at 27 °C in the presence of a fusion mixture containing HOPS, Sec17, Sec18, Vam7, and ATP. After addition of the fusion mixture to each reaction, the FRET signal as a result of content mixing was recorded (n = 3).

Figure 2.

Reconstitution of GEF-driven Ypt7–REP activation and membrane fusion. A, GEF-mediated activation of prenylated Ypt7 in fusion. Top panels, 100 nm Mon1–Ccz1 complex was added to proteoliposomes and increasing amounts of prenylated Ypt7–REP and preincubated for 15 min at 27 °C before addition of the fusion mix. Bottom panels, 500 nm GDI was added 10 min after starting the nucleotide exchange. B, EDTA-driven activation of prenylated Ypt7 in fusion. 2 mm EDTA and, after 10 min incubation, 3 mm MgCl₂ were used to drive nucleotide exchange. Fusion was measured in the absence (top panels) or presence (bottom panels) of 500 nm GDI as in A. Curves shown for content mixing assays are representatives of at least three independent experiments, using differing amounts of Ypt7, which led to the same overall result.

Figure 3.

Reconstitution of GEF-driven Ypt7–GDI activation and membrane fusion. A and B, the Ypt7–GDI complex was titrated into the fusion assay as in Fig. 2, and nucleotide exchange was either driven by Mon1–Ccz1 (top panels) or EDTA/MgCl²⁺ (bottom panels). GTP or GDP was added as indicated. C, membrane association of pYpt7 in complex with REP (top panel) or GDI (bottom panel). To allow for membrane insertion, samples were incubated in either the presence or absence of liposomes, GDP (D) or GTP (T), and, where indicated, with Mon1–Ccz1 for 30 min at 30 °C and then centrifuged for 30 min at 20,000 × g. The supernatant and pellet were analyzed by SDS-PAGE and Coomassie staining (representative example, n = 3).

Figure 4.

Reconstituted membrane fusion depends on membrane association and activation of Ypt7. A and B, fusion was performed in the presence of 80 nm pYpt7–GDI and either GDP or GTP and the full fusion mixture or in the absence of one of the components critical for fusion. C and D, controls of the heterotypic fusion reaction. Fusion was measured as in A with 80 nm Ypt7–GDI, leaving out either Sec17, Sec18, or ATP from the fusion mixture. E, fusion reactions after 30 min of fusion time as in A were fractionated by centrifugation for 30 min at 20,000 × g. Proteins in the pellet and supernatant were analyzed by SDS-PAGE and Western blotting, detecting the TAP tag on Vps41 and Ccz1, respectively, and Ypt7 (n = 2).

Figure 5.

Mon1–Ccz1 drives activation of Ypt7 and, therefore, fusion. A, the amount of Mon1–Ccz1 was titrated down in the preincubation time of the fusion assay. As before, fusion was started after allowing for nucleotide exchange of 80 nm pYpt7–GDI for 15 min. B, quantification of two independent fusion reactions.