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: 4.47). 10/2009; 20(21):4563-74. DOI: 10.1091/mbc.E09-04-0272
Source: PubMed


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.

    • "Beside the Rab GTPase Ypt7 and SNARE proteins additional interactors of the HOPS complex could be identified. One prominent example is the Apl5 motif of the AP-3 delta subunit, which directly binds Vps41 [60] [61]. The interaction depends on the conserved hydrophobic residues L100 and L101 within Vps41 [38]. "
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    ABSTRACT: The HOPS multisubunit tethering factor (MTC) is a macromolecular protein complex composed of six different subunits. It is one of the key components in the perception and subsequent fusion of multivesicular bodies and vacuoles. Electron microscopy studies indicate structural flexibility of the purified HOPS complex. Inducing higher rigidity into HOPS by biochemically modifying the complex declines the potential to mediate SNARE-driven membrane fusion. Thus, we propose that integral flexibility seems to be not only a feature, but of essential need for the function of HOPS. This review focuses on the general features of membrane tethering and fusion. For this purpose, we compare the structure and mode of action of different tethering factors to highlight their common central features and mechanisms. Copyright © 2015. Published by Elsevier B.V.
    No preview · Article · Jun 2015 · FEBS letters
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    • "Thus the results we present here do not rule out— and indeed point to—the involvement of interactions between HOPS and other proteins and/or lipids that contribute to Ypt7p-and HOPS-mediated membrane tethering. One possibility is the AP-3 cargo adapter subunit Apl5p, which binds HOPS via its Vps41p subunit (Angers and Merz, 2009). We next measured the size of our liposomes. "
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    ABSTRACT: Many Rab GTPase effectors are membrane tethering factors, that is, they physically link two apposed membranes prior to intracellular membrane fusion. Here, we investigate the distinct binding factors needed on apposed membranes for Rab effector-dependent tethering. We show that the homotypic fusion and protein sorting/class C vacuole protein sorting (HOPS/class C Vps) complex can tether low-curvature membranes, i.e. liposomes with a diameter of ∼100 nm, only when the yeast vacuolar Rab GTPase Ypt7p is present in both tethered membranes. When HOPS is phosphorylated by the vacuolar casein kinase I, Yck3p, tethering only takes place when GTP-bound Ypt7p is present in both tethered membranes. When HOPS is not phosphorylated, however, its tethering activity shows little specificity for the nucleotide-binding state of Ypt7p. These results suggest a model for HOPS-mediated tethering in which HOPS tethers membranes by binding to Ypt7p in each of the two tethered membranes. Moreover, because vacuole-associated HOPS is presumably phosphorylated by Yck3p, our results suggest that nucleotide exchange of Ypt7p on multivesicular bodies (MVB's)/late endosomes must take place before HOPS can mediate tethering at vacuoles. © 2015 by The American Society for Cell Biology.
    Preview · Article · May 2015 · Molecular biology of the cell
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    • "Indeed, Vps41 contains a membrane-binding amphipathic lipid packaging sensor (ALPS) motif, which likely supports its interaction with endosomes.23,43 In addition, Vps41 harbors a conserved interaction site for the AP-3 coat and may bind to incoming AP-3 vesicles.23,24 HOPS likely uses the available Ypt7-pool to efficiently bind to the vacuole. "
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    ABSTRACT: Endosomal and vacuole fusion depends on the two homologous tethering complexes CORVET and HOPS. HOPS binds the activated Rab GTPase Ypt7 via two distinct subunits, Vps39 and Vps41. To understand the participation and possible polarity of Vps41 and Vps39 during tethering, we used an in vivo approach. For this, we established the ligand-induced relocalization to the plasma membrane, using the Mon1-Ccz1 GEF complex that activates Ypt7 on endosomes. We then employed slight overexpression to compare the mobility of the HOPS-specific Vps41 and Vps39 subunits during this process. Our data indicate an asymmetry in the Rab-specific interaction of the two HOPS subunits: Vps39 is more tightly bound to the vacuole, and relocalizes the entire vacuole to the plasma membrane, whereas Vps41 behaved like the more mobile subunit. This is due to their specific Rab binding, as the mobility of both subunits was similar in ypt7∆ cells. In contrast, both HOPS subunits were far less mobile if tagged endogenously, suggesting that the entire HOPS complex is tightly bound to the vacuole in vivo. Similar results were obtained for the endosomal association of CORVET, when we followed its Rab-specific subunit Vps8. Our data provide in vivo evidence for distinct Rab specificity within HOPS, which may explain its function during tethering, and indicate that these tethering complexes are less mobile within the cell than previously anticipated.
    Full-text · Article · Apr 2014
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