Guanine nucleotide exchange factors (GEFs) have a critical but not exclusive role in organelle localization of Rab GTPases

Abstract

Membrane fusion at eukaryotic organelles is initiated by Rab GTPases and tethering factors. Rabs in their GDP-bound form are kept soluble in the cytoplasm by the GDP dissociation inhibitor (GDI) chaperone. Guanine nucleotide exchange factors (GEFs) are found at organelles and are critical for Rab function. Here, we surveyed the overall role of GEFs in Rab localization. We show that GEFs, but none of the proposed GDI displacement factors, are essential for the correct membrane localization of yeast Rabs. In the absence of the GEF, Rabs lost their primary localization to the target organelle. Several Rabs, such as vacuolar Ypt7, were found at the endoplasmic reticulum and thus were still membrane-bound. Surprisingly, a Ypt7 mutant that undergoes facilitated nucleotide exchange localized to vacuoles independently of its GEF Mon1-Ccz1 and rescued vacuole morphology. In contrast, wild-type Ypt7 required its GEF for localization and to counteract the extraction by GDI. Our data agree with the emerging model that GEFs are critical for Rab localization but raise the possibility that additional factors can contribute to this process.

Keywords: GAP; GDF; Guanine Nucleotide Exchange Factor (GEF); Membrane Fusion; Rab; Rab Proteins; Subcellular Organelles.

FIGURE 1.

Loss of the Mon1-Ccz1 complex alters Rab Ypt7 distribution. A, localization of Ypt7 upon relocalization of the Mon1-Ccz1 complex to the mitochondria. DIC, differential interference contrast. B, subcellular fractionation of cells expressing GFP-tagged Ypt7 in wild-type (wt) and ccz1Δ cells. P13 is the pellet obtained after centrifugation at 13,000 × g for 15 min at 4 °C, and P100 and S100 are the pellet and supernatant obtained after centrifugation at 100,000 × g for 30 min at 4 °C, respectively. Protein distribution was analyzed by Western blotting and antibody decoration. Note that Vac8 was used as a vacuole marker. C, localization of GFP-tagged Ypt7 in the wild-type, mon1Δ, ccz1Δ, and vps39Δ strains. D, co-localization of Ypt7 and C-terminally mRFPmars-tagged Sec63 in the wild-type and GEF deletion strains. Scale bars = 5 μm.

FIGURE 2.

GEF activity determines the correct localization of Rabs. A, GFP-tagged Ypt6 distribution in the wild-type (wt) and GEF deletion strains. Sec63 was used as an ER marker. B, analysis of Vps21 co-localization with Sec63 in the wild-type and indicated deletion strains. C, GFP-tagged Ypt6 (left panel) and Vps21 (right panel) remained membrane-bound in the absence of their corresponding GEFs. Subcellular fractionation was performed as described for Fig. 1B. Arc1 was used as a cytosolic marker. D, subcellular targeting of Ypt1, Ypt31, and Ypt32 in the wild-type, bet3, and trs130 temperature-sensitive (ts) mutants grown at 26 and 37 °C for 1.5 h. E, the localization of GFP-Sec4 was examined in the sec2 temperature-sensitive mutant grown at 25 and 37 °C for 1 h, followed by fluorescence microscopy. Scale bars = 5 μm.

FIGURE 3.

Mutation K127E in Ypt7 bypasses the need for GEF activity. A, representation of Ypt7-Gpp(NH)p (Protein Data Bank entry 1ky2; generated with YASARA), with the p-loop and switch regions indicated in yellow and green, respectively. The surface of Lys-127 is shown in gray. B, analysis of vacuole morphology via the lipophilic dye FM4-64 in the wild-type (wt) and GEF deletion strains expressing Ypt7 or the Ypt7 K127E mutant from a centromeric (CEN) plasmid. C, localization of the Ypt7 K127E mutant in wild-type cells and in cells lacking the Ypt7 GEF complex. D, microscopy of the GFP-tagged Ypt7 Q68L mutant in the wild-type and ccz1Δ strains. Scale bars = 5 μm. DIC, differential interference contrast.

FIGURE 4.

Contribution of Yip proteins and methylation to Rab localization. A, the yip1-1 mutant carrying GFP-Ypt7 was grown at 25 and 37 °C for 1 h and analyzed by fluorescence microscopy. B, subcellular distribution of GFP-tagged Ypt7 in the indicated single and multiple deletion strains. C, microscopy of GFP-tagged Ypt7, Ypt6, and Vps21 was performed in the wild-type (wt) and ste14Δ strains. D, localization of Vps21 and Ypt7 mutants containing the C-terminal Cys-Cys (CC) prenylation motif. Scale bars = 5 μm. DIC, differential interference contrast.

FIGURE 5.

Mon1-Ccz1 GEF activity for Ypt7 counteracts GDI. A, membrane targeting of GFP-tagged Ypt7 was monitored in wild-type cells (wt) and in cells expressing Gdi1 or Mrs6 under the control of the GAL1 promoter. B, subcellular fractionation of cells expressing GFP-Ypt7 and Gdi1 under the control of the GAL1 promoter. The assay was performed as described for Fig. 1B. Vac8 was used as a vacuole marker. C, localization of GFP-Ypt7 in diploids cells overexpressing Gdi1 together with the Ypt7 GAPs (Gyp7 and Msb3) or Mon1 and Ccz1 was analyzed by fluorescence microscopy. ER labeling is indicated by white arrowheads in cells overexpressing the Ypt7 GAPs. Scale bars = 5 μm. DIC, differential interference contrast.