Article

HOPS Interacts with Apl5 at the Vacuole Membrane and Is Required for Consumption of AP-3 Transport Vesicles

Department of Biochemistry, University of Washington, Seattle, WA 98195-3750, USA.
Molecular biology of the cell (Impact Factor: 5.98). 10/2009; 20(21):4563-74. DOI: 10.1091/mbc.E09-04-0272
Source: PubMed

ABSTRACT Adaptor protein complexes (APs) are evolutionarily conserved heterotetramers that couple cargo selection to the formation of highly curved membranes during vesicle budding. In Saccharomyces cerevisiae, AP-3 mediates vesicle traffic from the late Golgi to the vacuolar lysosome. The HOPS subunit Vps41 is one of the few proteins reported to have a specific role in AP-3 traffic, yet its function remains undefined. We now show that although the AP-3 delta subunit, Apl5, binds Vps41 directly, this interaction occurs preferentially within the context of the HOPS docking complex. Fluorescence microscopy indicates that Vps41 and other HOPS subunits do not detectably colocalize with AP-3 at the late Golgi or on post-Golgi (Sec7-negative) vesicles. Vps41 and HOPS do, however, transiently colocalize with AP-3 vesicles when these vesicles dock at the vacuole membrane. In cells with mutations in HOPS subunits or the vacuole SNARE Vam3, AP-3 shifts from the cytosol to a membrane fraction. Fluorescence microscopy suggests that this fraction consists of post-Golgi AP-3 vesicles that have failed to dock or fuse at the vacuole membrane. We propose that AP-3 remains associated with budded vesicles, interacts with Vps41 and HOPS upon vesicle docking at the vacuole, and finally dissociates during docking or fusion.

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    • "tethering (CORVET) complex along with two HOPS-specific subunits , VPS39 and 41 (Angers and Merz, 2009; Cabrera et al., 2009; Plemel et al., 2011; Swetha et al., 2011). Although the analysis in S2 cells did not reveal a role for other HOPS subunits in sorting to the regulated pathway, we also knocked down in PC12 cells two components of the core complex (VPS11 and 18) and HOPS-specific subunit VPS39. "
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    Developmental Cell 11/2013; DOI:10.1016/j.devcel.2013.10.007 · 10.37 Impact Factor
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    • "Analysis was performed by SDS–PAGE and Western blotting. Antibodies were prepared as previously described (Angers and Merz, 2009). All pull downs were repeated a minimum of three times; representative results are shown. "
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    • "Vacuole morphology was evaluated by pulse-chase labeling with the endocytic tracer FM4-64 (Invitrogen) as described (Brett et al., 2008). Subcellular fractionation by differential centrifugation was performed as described (Angers and Merz, 2009) Fluorescence microscopic analysis of GFP-CPS localization in FM4-64 labeled cells employed the plasmid pGO47 (Odorizzi et al., 1998). "
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