title: Transport of the cholera toxin B-subunit from recycling endosomes to the Golgi requires clathrin and AP-1.
abstract: The retrograde pathway is defined by the transport of proteins and lipids from the plasma membrane through endosomes to the Golgi complex, and is essential for a variety of cellular activities. Recycling endosomes are important sorting stations for some retrograde cargo. SMAP2, a GTPase-activating protein (GAP) for Arf1 with a putative clathrin-binding domain, has previously been shown to participate in the retrograde transport of the cholera toxin B-subunit (CTxB) from recycling endosomes. Here, we found that clathrin, a vesicle coat protein, and clathrin adaptor protein complex 1 (AP-1) were present at recycling endosomes and were needed for the retrograde transport of CTxB from recycling endosomes to the Golgi, but not from the plasma membrane to recycling endosomes. SMAP2 immunoprecipitated clathrin and AP-1 through a putative clathrin-binding domain and a CALM-binding domain, and SMAP2 mutants that did not interact with clathrin or AP-1 could not localize to recycling endosomes. Moreover, knockdown of Arf1 suppressed the retrograde transport of CTxB from recycling endosomes to the Golgi. These findings suggest that retrograde transport is mediated by clathrin-coated vesicles from recycling endosomes and that the role of the coat proteins is in the recruitment of Arf GAP to transport vesicles.
authors.affiliation: Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Tatsuyuki
authors.initials: T
authors.lastname: Matsudaira
authors.affiliation: Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Niki
authors.affiliation: Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan Pathological Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan tom_taguchi@mol.f.u-tokyo.ac.jp harai@mol.f.u-tokyo.ac.jp.
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Taguchi
authors.affiliation: Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan Pathological Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan tom_taguchi@mol.f.u-tokyo.ac.jp harai@mol.f.u-tokyo.ac.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Arai
mesh_terms: D020823:ADP-Ribosylation Factor 1 / Q000235:genetics; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D002772:Cholera Toxin / Q000378:metabolism*; D002966:Clathrin / Q000235:genetics* / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000235:genetics* / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006056:Golgi Apparatus / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000235:genetics* / Q000378:metabolism
chemical_list: D002966:Clathrin; D020690:GTPase-Activating Proteins; D008565:Membrane Proteins; C553893:SMAP2 protein, human; D018808:Transcription Factor AP-1; D002772:Cholera Toxin; D020823:ADP-Ribosylation Factor 1
keywords: Cholera toxin; Clathrin; Endosomes; Retrograde transport; SMAP2