Ubiquitin binding by the CUE domain promotes endosomal localization of the Rab5 GEF Vps9

Abstract

Vps9 and Muk1 are guanine nucleotide exchange factors (GEFs) in Saccharomyces cerevisiae that regulate membrane trafficking in the endolysosomal pathway by activating Rab5 GTPases. We show that Vps9 is the primary Rab5 GEF required for biogenesis of late endosomal multivesicular bodies (MVBs). However, only Vps9 (but not Muk1) is required for the formation of aberrant class E compartments that arise upon dysfunction of endosomal sorting complexes required for transport (ESCRTs). ESCRT dysfunction causes ubiquitinated transmembrane proteins to accumulate at endosomes, and we demonstrate that endosomal recruitment of Vps9 is promoted by its ubiquitin-binding CUE domain. Muk1 lacks ubiquitin-binding motifs, but its fusion to the Vps9 CUE domain allows Muk1 to rescue endosome morphology, cargo trafficking, and cellular stress-tolerance phenotypes that result from loss of Vps9 function. These results indicate that ubiquitin binding by the CUE domain promotes Vps9 function in endolysosomal membrane trafficking via promotion of localization.

FIGURE 1:

Vps9 is the yeast Rab5 GEF that drives the biogenesis of late endosomal compartments. (A–C, E–G) Electron micrographs; A′, B′, E′, and F′ show higher magnification of MVBs or class E compartments. Bars, 100 nm (A′, B′, E′, F′), 500 nm (A, B, C, E, F, G). (D, H) Quantification of MVBs or class E compartments in 50 cell profiles.

FIGURE 2:

CUE-domain binding to ubiquitin drives Vps9 accumulation at class E compartments. Confocal fluorescence micrographs of FM 4-64–stained cells expressing GFP-tagged versions of Vps9 from a high-copy (2 μ) plasmid in wild-type (A) and vps4∆ (B–H) cells. Arrowheads indicate FM 4-64–labeled class E compartments at which GFP either does (closed arrowheads) or does not (open arrowheads) colocalize. Bar, 2 μm. (I) Model of Vps9 domains. (J) Fluorescence intensity ratio (F_R) of GFP localization at class E compartments relative to cytosol. Error bars are 95% confidence interval from at least 10 cells. (K) Western blot of total yeast cell extracts showing expression of mutant GFP-Vps9 proteins. IB, immunoblot.

FIGURE 3:

Ubiquitin binding by Vps9 is not required for MVB or class E compartment biogenesis. EM quantification of the number of MVBs (A) or class E compartments (B) in 50 cell profiles of vps9∆ or vps9∆ muk1∆ cells transformed with low-copy (CEN) plasmids expressing the indicated vps9 alleles.

FIGURE 4:

The CUE and GEF domains have distinct roles in Vps9 function. (A) Limiting dilutions of yeast after 45 h of growth on minimal synthetic agar medium at 30 vs. 37°C. (B) Analysis of a chimeric CPY-Inv reporter enzyme in extracellular and total cellular fractions. Bars represent means of four independent experiments, except for column 3, which represents three independent experiments. (C, D) LUCID analysis of FLuc-Cps1 sorting in the MVB pathway. Signal from luciferase-tagged Cps1 is inversely proportional to the efficiency of sorting into ILVs. Alleles of VPS9 expressed from a CEN plasmid were analyzed for function in cells lacking only Vps9 (vps9∆; C) and cells lacking both Vps9 and Muk1 (vps9∆ muk1∆; D). Cells lacking Vps4 (vps4∆) served as a positive control for defective MVB cargo sorting. Bars represent the means of four biological replicates each. Cps1, carboxypeptidase S.

FIGURE 5:

Fusion of the CUE domain to Muk1 rescues Vps9 function at late endosomes. (A) Fluorescence micrographs of FM 4-64–stained cells expressing GFP-Muk1 chimeras from a CEN plasmid. Arrowheads indicate FM 4-64–labeled class E compartments at which GFP either does (closed arrowheads) or does not (open arrowheads) colocalize. (B) Quantification of CPY-Inv in extracellular and total cellular fractions from vps9∆ cells expressing the indicated alleles from a CEN plasmid. Bars represent means of three independent biological replicates each. Unpaired one-way ANOVA: p < 0.0001 overall; ***p < 0.001. (C) LUCID quantification of luciferase-tagged Cps1 in vps9∆ cells expressing the indicated alleles from a CEN plasmid. Bars represent means of four independent biological replicates each. Unpaired one-way ANOVA: p < 0.0001 overall; **p < 0.01; ***p < 0.001. (D) EM quantification of MVBs (black) and class E compartments (red) in 50 cell profiles. (E, F) Growth curves of yeast lacking both Rab5 GEFs (E) or only Vps9 (F) carrying plasmid-borne VPS9 or alleles of MUK1. Cells were shaken at 30°C in synthetic medium supplemented with 3 mM zinc sulfate. Plotted points represent the mean value of three replicate cultures each.

FIGURE 6:

Model of ubiquitin-mediated recruitment of Vps9 to endosomes. (A) During normal endosomal transport, the CUE domain of Vps9 promotes recruitment of Vps9 to endosomes and endocytic vesicles through interaction with ubiquitinated transmembrane cargo proteins, thereby promoting Vps21 nucleotide exchange and endosomal localization. Dimerization of the Vps9 CUE domain might increase Vps9 recruitment to endosomes. Sorting and deubiquitination of cargo by ESCRTs reduces ubiquitin at the endosomal surface, promoting the dissociation of Vps9. (B) Disruption of ESCRT function leads to an accumulation of ubiquitinated cargo at the endosomal surface, thereby aberrantly maintaining Vps9 at endosomes and prolonging Vps21 activity. Vps9-mediated Vps21 hyperactivation leads to the increased endosomal membrane fusion that creates class E compartments.