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Vol. 15, Issue 3, 1197-1210, March 2004
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* Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242;
Cambridge Institute for Medical Research and Department of Clinical Biochemistry, University of Cambridge, Addenbrookes Hospital, Cambridge CB2 2XY, United Kingdom
Submitted June 4, 2003;
Revised October 27, 2003;
Accepted October 27, 2003
Monitoring Editor: Juan Bonifacino
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.
Abbreviations used: Av-ALP, avidin-alkaline phosphatase conjugate; EEA1, early endosome antigen-1; GST, glutathione S-transferase; HOPS, homotypic protein sorting and vacuolar fusion complex; MBP, maltose binding protein; PNS, postnuclear supernatant; S/M, Sec1/Munc like protein; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor(s); TfR, transferrin receptor; Vps, vacuole protein sorting.
Corresponding author. E-mail address: piperr{at}physiology.uiowa.edu.
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