title: Clathrin-dependent mechanisms modulate the subcellular distribution of class C Vps/HOPS tether subunits in polarized and nonpolarized cells.
abstract: Coats define the composition of carriers budding from organelles. In addition, coats interact with membrane tethers required for vesicular fusion. The yeast AP-3 (Adaptor Protein Complex 3) coat and the class C Vps/HOPS (HOmotypic fusion and Protein Sorting) tether follow this model as their interaction occurs at the carrier fusion step. Here we show that mammalian Vps class C/HOPS subunits and clathrin interact and that acute perturbation of clathrin function disrupts the endosomal distribution of Vps class C/HOPS tethers in HEK293T and polarized neuronal cells. Vps class C/HOPS subunits and clathrin exist in complex with either AP-3 or hepatocyte growth factor receptor substrate (Hrs). Moreover, Vps class C/HOPS proteins cofractionate with clathrin-coated vesicles, which are devoid of Hrs. Expression of FK506 binding protein (FKBP)-clathrin light chain chimeras, to inhibit clathrin membrane association dynamics, increased Vps class C/HOPS subunit content in rab5 endosomal compartments. Additionally, Vps class C/HOPS subunits were concentrated at tips of neuronal processes, and their delivery was impaired by expression of FKBP-clathrin chimeras and AP20187 incubation. These data support a model in which Vps class C/HOPS subunits incorporate into clathrin-coated endosomal domains and carriers in mammalian cells. We propose that vesicular (AP-3) and nonvesicular (Hrs) clathrin mechanisms segregate class C Vps/HOPS tethers to organelles and domains of mammalian cells bearing complex architectures.
authors.affiliation: Department of Cell Biology, Emory University, Atlanta, GA 30322, USA.
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Zlatic
authors.forename: Karine
authors.initials: K
authors.lastname: Tornieri
authors.forename: Steven W
authors.initials: SW
authors.lastname: L'Hernault
authors.forename: Victor
authors.initials: V
authors.lastname: Faundez
mesh_terms: D033963:Adaptor Protein Complex 3 / Q000378:metabolism; D033981:Adaptor Protein Complex delta Subunits / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D016764:Cell Polarity; D002966:Clathrin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000187:drug effects / Q000378:metabolism; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D046912:Multiprotein Complexes / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000494:pharmacology; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism
chemical_list: C463061:AP20187; C491904:AP3D1 protein, human; D033963:Adaptor Protein Complex 3; D033981:Adaptor Protein Complex delta Subunits; D002966:Clathrin; D046912:Multiprotein Complexes; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D016559:Tacrolimus