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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    ABSTRACT: SNARE proteins catalyze membrane fusion events in the secretory and endolysosomal systems, and all SNARE-mediated fusion processes require cofactors of the Sec1/Munc18 (SM) family. Vps33 is a SM proteins and subunit of the Vps-C complexes CORVET and HOPS, which are central regulators of endocytic traffic. Here, we present biochemical studies of interactions between Saccharomyces cerevisiae vacuolar SNAREs and the HOPS holocomplex or Vps33 alone. HOPS binds the N-terminal H(abc) domain of the Qa-family SNARE Vam3, but Vps33 is not required for this interaction. Instead, Vps33 binds the SNARE domains of Vam3, Vam7, and Nyv1. Vps33 directly binds vacuolar quaternary SNARE complexes, and the affinity of Vps33 for SNARE complexes is greater than for individual SNAREs. Through targeted mutational analyses, we identify missense mutations of Vps33 that produce a novel set of defects including cargo missorting and the loss of Vps33-HOPS association. Together, these data suggest a working model for membrane docking in which HOPS associates with N-terminal domains of Vam3 and Vam7 through Vps33-independent interactions, followed by binding of Vps33, the HOPS SM protein, to SNARE domains and finally to the quaternary SNARE complex. Our results also strengthen the hypothesis that SNARE complex binding is a central conserved attribute of SM proteins.
    Molecular biology of the cell 10/2012; 23(23). DOI:10.1091/mbc.E12-05-0343 · 5.98 Impact Factor
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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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    ABSTRACT: Traffic through late endolysosomal compartments is regulated by sequential signaling of small G proteins of the Rab5 and Rab7 families. The Saccharomyces cerevisiae Vps-C protein complexes CORVET (class C core vacuole/endosome tethering complex) and HOPS (homotypic fusion and protein transport) interact with endolysosomal Rabs to coordinate their signaling activities. To better understand these large and intricate complexes, we performed interaction surveys to assemble domain-level interaction topologies for the eight Vps-C subunits. We identified numerous intersubunit interactions and up to six Rab-binding sites. Functional modules coordinate the major Rab interactions within CORVET and HOPS. The CORVET-specific subunits, Vps3 and Vps8, form a subcomplex and physically and genetically interact with the Rab5 orthologue Vps21. The HOPS-specific subunits, Vps39 and Vps41, also form a subcomplex. Both subunits bind the Rab7 orthologue Ypt7, but with distinct nucleotide specificities. The in vivo functions of four RING-like domains within Vps-C subunits were analyzed and shown to have distinct functions in endolysosomal transport. Finally, we show that the CORVET- and HOPS-specific subunits Vps3 and Vps39 bind the Vps-C core through a common region within the Vps11 C-terminal domain (CTD). Biochemical and genetic experiments demonstrate the importance of these regions, revealing the Vps11 CTD as a key integrator of Vps-C complex assembly, Rab signaling, and endosomal and lysosomal traffic.
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