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.

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    • "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.
    FEBS letters 06/2015; DOI:10.1016/j.febslet.2015.06.001 · 3.17 Impact Factor
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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.
    04/2014; 4(2):e29191. DOI:10.4161/cl.29191
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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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    ABSTRACT: The regulated release of polypeptides has a central role in physiology, behavior, and development, but the mechanisms responsible for production of the large dense core vesicles (LDCVs) capable of regulated release have remained poorly understood. Recent work has implicated cytosolic adaptor protein AP-3 in the recruitment of LDCV membrane proteins that confer regulated release. However, AP-3 in mammals has been considered to function in the endolysosomal pathway and in the biosynthetic pathway only in yeast. We now find that the mammalian homolog of yeast VPS41, a member of the homotypic fusion and vacuole protein sorting (HOPS) complex that delivers biosynthetic cargo to the endocytic pathway in yeast, promotes LDCV formation through a common mechanism with AP-3, indicating a conserved role for these proteins in the biosynthetic pathway. VPS41 also self-assembles into a lattice, suggesting that it acts as a coat protein for AP-3 in formation of the regulated secretory pathway.
    Developmental Cell 11/2013; DOI:10.1016/j.devcel.2013.10.007 · 9.71 Impact Factor
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