Regulatory sites in the Mon1-Ccz1 complex control Rab5 to Rab7 transition and endosome maturation

Abstract

Maturation from early to late endosomes depends on the exchange of their marker proteins Rab5 to Rab7. This requires Rab7 activation by its specific guanine nucleotide exchange factor (GEF) Mon1-Ccz1. Efficient GEF activity of this complex on membranes depends on Rab5, thus driving Rab-GTPase exchange on endosomes. However, molecular details on the role of Rab5 in Mon1-Ccz1 activation are unclear. Here, we identify key features in Mon1 involved in GEF regulation. We show that the intrinsically disordered N-terminal domain of Mon1 autoinhibits Rab5-dependent GEF activity on membranes. Consequently, Mon1 truncations result in higher GEF activity in vitro and alterations in early endosomal structures in Drosophila nephrocytes. A shift from Rab5 to more Rab7-positive structures in yeast suggests faster endosomal maturation. Using modeling, we further identify a conserved Rab5-binding site in Mon1. Mutations impairing Rab5 interaction result in poor GEF activity on membranes and growth defects in vivo. Our analysis provides a framework to understand the mechanism of Ras-related in brain (Rab) conversion and organelle maturation along the endomembrane system.

Keywords: Mon1; Rab–cascade; endosomal maturation; endosome; lysosome.

Fig. 1.

The disordered N-terminal region of Drosophila Mon1 regulates Mon1–Ccz1 GEF activity. (A) Overview of the Rab5-Rab7 cascade on endosomes. Active Rab5 (green) recruits the Mon1–Ccz1 GEF complex (blue) to PI3P-positive (red) endosomal membranes and promotes Rab7 (gray) recruitment and activation. For details, see text. (B) The N-terminal region of D.m. Mon1 is disordered. The disorder probability of each residue of D.m. Mon1 was determined using the IUPred2A web interface (24, 25). Longin domains (LDs) 1 to 3 are depicted in gray shades. Values >0.5 are considered disordered. (C) Truncation of Mon1 does not affect the complex stability of the trimeric Mon1^Δ1-50–Ccz1–Bulli complex (∆50) compared to wild-type complex (wt). GEF complexes were purified as described in the Materials and Methods section and analyzed by SDS-PAGE and Coomassie staining. (D) On liposome GEF assay of Mon1^wt and Mon1^Δ1-50 containing trimer. Liposomes were preloaded with 150 nM prenylated Rab5 in the presence of 200 µM GTP and 1.5 mM Ethylenediaminetetraacetic acid (EDTA). The nucleotide was stabilized using 3 mM MgCl₂. 250 nM Mant-GDP-loaded Rab7:GDI was added, and nucleotide exchange was triggered by adding 6.25 nM wild-type (blue) or mutant (green) GEF complex. The decrease in fluorescence was measured over time and normalized to fluorescence prior to GEF addition. (E) Comparison of fold-change in GEF activity of various Mon1 truncations. GEF assays were performed as in D, and k_obs of each curve was determined as described in the Materials and Methods section. k_obs values of mutants were normalized to the corresponding wild-type value in the respective experiment. Bar graphs represent average fold-change to the respective wild-type value, and dots represent individual changes from at least three experiments. (P value: *P < 0.05 and **P < 0.01, using a two-sample Student’s t test assuming equal variances). For kinetic constants of all GEF complexes, see SI Appendix, Table S3. (F) GEF activity of wild type and Mon1^Δ1-100 containing trimer in solution. 2 µM nonprenylated Rab was incubated with increasing amounts of GEF. After baseline stabilization, nucleotide release was triggered by adding 0.1 mM GTP final. For details of data fitting and statistics, see the Materials and Methods section. The k_cat/K_M (M⁻¹s⁻¹) value for Mon1^Δ1-100 containing trimer was normalized to the value of Mon1^wt. Bar graphs represent average fold-change, and dots represent individual changes from three experiments. (P value: n.s. using a two-sample Student’s t test assuming equal variances). (G) Multiple sequence alignment of a hydrophobic patch in Mon1. The N-terminal regions of Mon1 from Drosophila melanogaster (D.m.), Saccharomyces cerevisiae (S.c.), Homo sapiens (H.s.), Mus musculus (M.m.), Caenorhabditis elegans (C.e.), and Schizosaccharomyces pombe (S.p.) were aligned using th Clustal omega web interface (26, 27). The hydrophobic patch is marked by a yellow box. Conservation was determined using Jalview (28). (H) Comparison of fold-change in GEF activity of Mon1^{I47, 48A}. GEF assays were performed as in D, and k_obs of each curve was determined as described in the Materials and Methods section. k_obs values of mutants were normalized to the corresponding wild-type value in the respective experiment. Bar graphs represent average fold-change to the respective wild-type value, and dots represent individual changes from three experiments (P value: *P < 0.05, using a two-sample Student’s t test assuming equal variances). For kinetic constants of all GEF complexes, see SI Appendix, Table S3.

Fig. 2.

Loss of the disordered Mon1 N-terminal region affects localization of endosomal Rab GTPases and Rab5-dependent GEF activity. (A) Localization of endogenous Rab5 in Drosophila nephrocytes from 3rd instar larvae expressing wild type or Mon1^∆100 under the control of the handC-GAL4 driver. Antibodies against Rab5 were used. Optical sections show the distribution of Rab5 in detail. Size bar: 10 µm. (B) Analysis of the mean size of Rab5 dots per cell from 15 cells from three animals for wild-type Mon1 and 13 cells from four animals for Mon1^∆100. (P value ****< 0.0001 using an unpaired, two-sample Student’s t test). (C–F) Truncation of the Mon1 N-terminal affects Ypt7 and Vps21 localization. (C) Localization of mNEON-Ypt7 under its endogenous promotor in yeast cells in the presence of endogenously expressed wild type or Mon1^Δ100 using fluorescence microscopy. Vacuoles were stained with FM4-64. Size bar, 2 µm, arrows show representative Ypt7 accumulations. (D) Quantification of Ypt7-positive dots in C. Cells (n ≥ 50) were counted from three independent experiments; (P value ***< 0.001 using a two-sample Student’s t test assuming equal variances). For image processing details, see the Materials and Methods section. (E) Localization of mCherry-tagged Vps21 in cells expressing mNeon-Ypt7 and endogenously expressed wild type or Mon1^Δ100 using fluorescence microscopy. Size bar 2 µm, arrows show representative Vps21 accumulations. (F) Quantification of Vps21-positive dots in E. Cells (n ≥ 50) were counted from three independent experiments. (P value ***< 0.001 using a two-sample Student’s t test assuming equal variances). For image processing details, see the Materials and Methods section. (G) Effect on cell growth by Mon1 truncations. Strains endogenously expressing wild type or Mon1^Δ100 were grown to the same OD₆₀₀ in Yeast extract peptone dextrose (YPD) media and spotted in serial dilutions onto agar plates containing YPD or YPD supplemented with 4 mM ZnCl₂. Plates were incubated for several days at 30 °C. (H) A hydrophobic patch mutation in the N-terminal part of Mon1 affects GEF activity. Liposomes were loaded with 150 nM prenylated Ypt10 in the presence of 200 µM GTP and 1.5 mM EDTA. The nucleotide was stabilized using 3 mM MgCl₂. 250 nM Mant-GDP-loaded Ypt7:GDI was added, and the reaction was triggered by adding 12.5 nM wild-type (blue) or mutant complex expressing Mon1^L95A,L96A (green). The decrease in fluorescence detected over time is normalized to initial fluorescence. (I) Comparison of fold-change in GEF activity of wild-type and Mon1^L95A,L96A mutant GEF complexes on liposomes and in solution. For details of in solution GEF assay, see the Materials and Methods section. For the GEF assay on liposomes, k_obs of each curve was determined as described in the Materials and Methods section. k_obs values were normalized to the wild-type value. For in solution assays, k_cat/K_m values were determined and normalized to the wild-type value. Bar graphs represent average fold-change, and dots represent individual changes from three experiments. (P value: *P < 0.05 using a two-sample Student’s t test assuming equal variances).

Fig. 3.

Identification of the Rab5-binding site in the Mon1–Ccz1 complex. (A) Identification of the putative Rab5-binding site in Mon1. Composite model of the S.c. Mon1 (blue)-Ccz1 (green)-Ypt7 (beige)-Ypt10 (pink) complex based on an Alphafold 2 prediction (31, 32) and the crystal structure of the catalytic core complex (PDB ID: 5LDD) (19, 32, 33). (B) Close-up view of the Mon1–Ypt10 binding interface. Colors are as in A. (C) Multiple sequence alignment of Mon1 β-strand in the modeled Rab5 binding region. Mon1 sequences from S.c., D.m., Caenorhabditis elegans (C.e.), and Homo sapiens (H.s.) were aligned using the Clustal omega web interface. Conservation was determined using Jalview. (D) Effect of the Mon1^W406A mutant on Rab5 binding. 75 µg purified GST-Ypt10 was loaded with GTP (T) or GDP (D) and incubated with 25 µg of either wild-type or Mon1^W406A GEF complex. Elution of bound GEF was performed with EDTA. 20% of the eluate was analyzed together with 1% input by western blotting using an anti-Mon1 antibody. 2% GST-Ypt10 was stained with Coomassie as loading control (E) Quantification of bound GEF complex to Ypt10-GTP. The band intensity of Mon1 signal in elution fraction was measured using Fiji software and compared to input signal. (P value **< 0.01 using a two-sample Student’s t test assuming equal variances). (F) Effect of the Mon1^W406 mutation on Rab5-dependent GEF activity. 250 nM Mant-GDP-loaded Ypt7:GDI was added, and Rab activation was measured by the fluorescence decrease over time. Liposomes were loaded with 150 nM prenylated Ypt10 using 200 µM GTP and 1.5 mM EDTA. The nucleotide was stabilized using 3 mM MgCl₂. The reaction was triggered by adding 25 nM GEF complex. The decrease in fluorescence was normalized to fluorescence prior to GEF addition. (G) Comparison of fold-change in GEF activity of Mon1^W406A to Mon1^wt complex on liposomes and in solution. For details of in solution GEF assay, see Materials and Methods. For GEF assay on liposomes, k_obs of each curve was determined as described in the Materials and Methods section. k_obs values of mutant were normalized to the wild-type GEF complex value. For in solution assays, k_cat/K_m values were normalized to the wild-type value. Bar graphs represent average fold-change, and dots represent individual values from three experiments. (P value ***< 0.001, using a two-sample Student’s t test assuming equal variances). (H) Growth assay. Indicated Mon1 variants in a mon1 deletion background were grown to the same OD₆₀₀ in YPD media and then spotted in serial dilutions onto agar plates containing YPD or YPD supplemented with 4 mM ZnCl₂. Plates were incubated for several days at 30 °C. (I) Localization of Ypt7 in wild-type and mutant strains. Plasmids encoding Mon1^wt, Mon1^W406A, or Mon1^W406K variants were expressed under their endogenous promoter in a mon1 deletion strain. Vacuoles were stained with FM4-64. Images were deconvolved (SOftWoRx software 5.5). Size bar, 2 µm. (J) Quantification of the ratio between FM4-64 and mNeon-Ypt7 mean intensity signal on the vacuolar rim. Mean intensity signals were determined by a line profile across the vacuole. For details of image processing and quantification, see the Materials and Methods section. Cells (n ≥ 50) were counted from three independent experiments. (P value ***< 0.001 using a one-way ANOVA).

Fig. 4.

The Rab5-binding site in Mon1 is conserved. (A) Analysis of the Drosophila Mon1–Ccz1–Bulli complex with a Mon1^W334A mutation. Stability of wild-type and Mon1^W334A trimeric GEF complexes was analyzed by SDS-PAGE and Coomassie staining. (B) GEF activity of the wild-type and Mon1^W334A trimeric GEF complex. Rab activation was measured by fluorescence decrease over time. Liposomes were loaded with 150 nM prenylated Rab5 in the presence of 200 µM GTP and 1.5 mM EDTA; bound nucleotide was stabilized with 3 mM MgCl₂. 250 nM Mant-GDP-loaded Rab7:GDI complex was added, and the reaction was triggered by adding 6.25 nM of the corresponding GEF complex. The decrease in fluorescence was normalized to fluorescence prior to GEF addition. (C) Fold-change in GEF activity of Mon1^W334A compared to wild-type complex on liposomes and in solution. For details, see Materials and Methods. Bar graphs represent average fold-change, and dots represent individual values of two experiments normalized to corresponding wild-type value. (D) Membrane association of wild-type and mutant GEF complexes. 333 µM liposomes were preloaded with 150 nM pRab5:Rab escort protein (REP) as in Fig. 4B. As control, the Rab was omitted. Liposomes were preincubated for 8 min at 30 °C; then, 12.5 nM GEF complex was added, and incubation was continued for 15 min. Liposomes were sedimented at 20,000 g for 20 min. 100% of the pellet was loaded together with 10% input on SDS gels and analyzed with anti-FLAG antibody. (E) Quantification of C. Band intensities were quantified using Fiji, and binding in the absence and presence of Rab5 was normalized to the input value. Bar graphs represent average binding, and dots represent individual values from four experiments. (F) Working model. The unstructured N-terminal part of Mon1 is folding back to the core of Mon1 resulting in autoinhibition of GEF activity. Rab5 (green) binding to a conserved site activates Mon1–Ccz1 (blue) and drives nucleotide exchange of Ypt7. For details, see text.