Vps27-Hse1 and ESCRT-I complexes cooperate to increase efficiency of sorting ubiquitinated proteins at the endosome

Ubiquitin (Ub) attachment to cell surface proteins causes their lysosomal degradation by incorporating them into lumenal membranes of multivesicular bodies (MVBs). Two yeast endosomal protein complexes have been proposed as Ub-sorting “receptors,” the Vps27-Hse1 complex and the ESCRT-I complex. We used NMR spectroscopy and mutagenesis studies to map the Ub-binding surface for Vps27 and Vps23. Mutations in Ub that ablate only Vps27 binding or Vps23 binding blocked the ability of Ub to serve as an MVB sorting signal, supporting the idea that both the Vps27-Hse1 and ESCRT-I complexes interact with ubiquitinated cargo. Vps27 also bound Vps23 directly via two PSDP motifs present within the Vps27 COOH terminus. Loss of Vps27-Vps23 association led to less efficient sorting into the endosomal lumen. However, sorting of vacuolar proteases or the overall biogenesis of the MVB were not grossly affected. In contrast, disrupting interaction between Vps27 and Hse1 caused severe defects in carboxy peptidase Y sorting and MVB formation. These results indicate that both Ub-sorting complexes are coupled for efficient recognition of ubiquitinated cargo

Mammalian Late Vacuole Protein Sorting Orthologues Participate in Early Endosomal Fusion and Interact with the Cytoskeleton

In Saccharomyces cerevisiae, the class C vacuole protein sorting (Vps) proteins, together with Vam2p/Vps41p and Vam6p/Vps39p, form a complex that interacts with soluble N-ethylmaleimide-sensitive factor attachment protein receptor and Rab proteins to “tether” vacuolar membranes before fusion. To determine a role for the corresponding mammalian orthologues, we examined the function, localization, and protein interactions of endogenous mVps11, mVps16, mVps18, mVam2p, and mVam6. We found a significant proportion of these proteins localized to early endosome antigen-1 and transferrin receptor-positive early endosomes in Vero, normal rat kidney, and Chinese hamster ovary cells. Immunoprecipitation experiments showed that mVps18 not only interacted with Syntaxin (Syn)7, vesicle-associated membrane protein 8, and Vti1-b but also with Syn13, Syn6, and the Sec1/Munc18 protein mVps45, which catalyze early endosomal fusion events. Moreover, anti-mVps18 antibodies inhibited early endosome fusion in vitro. Mammalian mVps18 also associated with mVam2 and mVam6 as well as with the microtubule-associated Hook1 protein, an orthologue of the Drosophila Hook protein involved in endosome biogenesis. Using in vitro binding and immunofluorescence experiments, we found that mVam2 and mVam6 also associated with microtubules, whereas mVps18, mVps16, and mVps11 associated with actin filaments. These data indicate that the late Vps proteins function during multiple soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated fusion events throughout the endocytic pathway and that their activity may be coordinated with cytoskeletal function