Figure 7. The VINE complex exhibits characteristics of a membrane sorting complex.

(A) Time-lapse imaging of cells over-expressing GFP-Vin1 and Vrl1 show tubules emanating from Did2-labeled endosomes. Images were uniformly enlarged using a bicubic expansion function to show detail. Solid arrowheads mark a tubule, open arrowhead marks a scission event. (B) Normalized intensity line scan analysis performed on images from A along the yellow dotted line. (C) Punctate localization of GFP-tagged Mrl1, but not other endosomal recycling cargo, is decreased in cells expressing VRL1. (D) Quantification of GFP-tagged puncta in WT and vrl1 strains in C. Two tailed Welch’s t tests; n=3, cells/strain/replicate ≥902; not significant, n.s.=p > 0.05, *=p < 0.05. (E) Mutation of the D373 residue required for VPS9 GEF activity does not prevent Vrl1 from redistributing Mrl1. (F) Quantification of Mrl1-mScI puncta per cell in E. One-way ANOVA with Tukey’s multiple comparison test; n=3, cells/strain/replicate ≥1788; not significant, n.s.=p > 0.05, *=p < 0.05. (G) Schematic of Vps10 cytosolic tail mutant and Mrl1 cytosolic tail chimera tested for VINE-mediated sorting in H, I. (H) The Mrl1 cytosolic tail is sufficient to confer VINE-mediated redistribution. (I) Percent of cells showing punctate localization of indicated GFP-tagged constructs in H. Blind scoring of GFP signal was conducted manually. One-way ANOVA with Tukey’s multiple comparison test; n=3, cells/strain/replicate ≥237; not significant, n.s.=p > 0.05, ***=p < 0.001, ****=p < 0.0001. (J) Mrl1-mScI puncta are reduced in a snx4∆ strain. (K) Quantification of Mrl1-mScI puncta per cell in J. One-way ANOVA with Tukey’s multiple comparison test; n=3, cells/strain/replicate ≥1036; not significant, n.s.=p > 0.05, *=p < 0.05, **=p < 0.01, ***=p < 0.001, ****=p < 0.0001. (L) Model for VINE activity and redistribution of Mrl1. VINE promotes its own recruitment to endosomes through a positive feedback loop involving Vrl1 GEF activity and local PI3P production. VINE-coated tubules then recycle cargo, such as Mrl1, from endosomes. VINE may target Mrl1 to the Golgi for subsequent delivery to the vacuolar membrane by the AP-3 complex. Mrl1 is then returned to the endosome by Snx4-containing complexes. See text for details. Scale bars, 2 µm. Error bars report SEM. OE, over-expressed. TM, transmembrane. WT, wild type.

Figure 7—figure supplement 1. Vrl1 redistributes Mrl1 puncta from endosomes.

(A) Mrl1-mScI puncta colocalize with Did2-Envy and sfGFP-Vps21-labeled endosomes, but not with sfGFP-Ypt7 puncta that label vacuolar sites. Insets are scaled to match other images in the same channel (see Materials and methods for details). (B) Quantification of colocalization as the percentage of Mrl1 puncta overlapping GFP puncta in A. One-way ANOVA with Tukey’s multiple comparison test; n=3, cells/strain/replicate ≥2286; **=p < 0.01, ****=p < 0.0001. (C) Localization of Vin1-mScI in BY4741 (vrl1) and an isogenic strain with the vrl1 mutation corrected at the endogenous VRL1 locus using CRISPR-Cas9 gene editing technology. Vin1-mScI localizes to puncta in a CRISPR-corrected VRL1 strain. (D) Mrl1-mScI is redistributed from puncta in the CRISPR-corrected VRL1 strain. (E) Quantification of large, bright Mrl1-mScI puncta per cell in D. Two tailed equal variance t test; n=3, cells/strain/replicate ≥1183; **=p < 0.01. Scale bars, 2 µm. Error bars report SEM.