title: The Adaptor Complex AP-4 Regulates Vacuolar Protein Sorting at the trans-Golgi Network by Interacting with VACUOLAR SORTING RECEPTOR1.
abstract: Adaptor protein (AP) complexes play critical roles in protein sorting among different post-Golgi pathways by recognizing specific cargo protein motifs. Among the five AP complexes (AP-1-AP-5) in plants, AP-4 is one of the most poorly understood; the AP-4 components, AP-4 cargo motifs, and AP-4 functional mechanism are not known. Here, we identify the AP-4 components and show that the AP-4 complex regulates receptor-mediated vacuolar protein sorting by recognizing VACUOLAR SORTING RECEPTOR1 (VSR1), which was originally identified as a sorting receptor for seed storage proteins to target protein storage vacuoles in Arabidopsis (Arabidopsis thaliana). From the vacuolar sorting mutant library GREEN FLUORESCENT SEED (GFS), we isolated three gfs mutants that accumulate abnormally high levels of VSR1 in seeds and designated them as gfs4, gfs5, and gfs6. Their responsible genes encode three (AP4B, AP4M, and AP4S) of the four subunits of the AP-4 complex, respectively, and an Arabidopsis mutant (ap4e) lacking the fourth subunit, AP4E, also had the same phenotype. Mass spectrometry demonstrated that these four proteins form a complex in vivo. The four mutants showed defects in the vacuolar sorting of the major storage protein 12S globulins, indicating a role for the AP-4 complex in vacuolar protein transport. AP4M bound to the tyrosine-based motif of VSR1. AP4M localized at the trans-Golgi network (TGN) subdomain that is distinct from the AP-1-localized TGN subdomain. This study provides a novel function for the AP-4 complex in VSR1-mediated vacuolar protein sorting at the specialized domain of the TGN.
authors.affiliation: Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan (K.F., M.S., Y.S., T.K., Y.K., H.T., I.H.-N., T.S.); andPlant Global Educational Project, Nara Institute of Science and Technology, Ikoma 630-0192, Japan (Y.F.).
authors.forename: Kentaro
authors.initials: K
authors.lastname: Fuji
authors.affiliation: Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan (K.F., M.S., Y.S., T.K., Y.K., H.T., I.H.-N., T.S.); andPlant Global Educational Project, Nara Institute of Science and Technology, Ikoma 630-0192, Japan (Y.F.).
authors.forename: Makoto
authors.initials: M
authors.lastname: Shirakawa
authors.affiliation: Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan (K.F., M.S., Y.S., T.K., Y.K., H.T., I.H.-N., T.S.); andPlant Global Educational Project, Nara Institute of Science and Technology, Ikoma 630-0192, Japan (Y.F.).
authors.forename: Yuki
authors.initials: Y
authors.lastname: Shimono
authors.affiliation: Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan (K.F., M.S., Y.S., T.K., Y.K., H.T., I.H.-N., T.S.); andPlant Global Educational Project, Nara Institute of Science and Technology, Ikoma 630-0192, Japan (Y.F.).
authors.forename: Tadashi
authors.initials: T
authors.lastname: Kunieda
authors.affiliation: Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan (K.F., M.S., Y.S., T.K., Y.K., H.T., I.H.-N., T.S.); andPlant Global Educational Project, Nara Institute of Science and Technology, Ikoma 630-0192, Japan (Y.F.).
authors.forename: Yoichiro
authors.initials: Y
authors.lastname: Fukao
authors.affiliation: Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan (K.F., M.S., Y.S., T.K., Y.K., H.T., I.H.-N., T.S.); andPlant Global Educational Project, Nara Institute of Science and Technology, Ikoma 630-0192, Japan (Y.F.).
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Koumoto
authors.affiliation: Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan (K.F., M.S., Y.S., T.K., Y.K., H.T., I.H.-N., T.S.); andPlant Global Educational Project, Nara Institute of Science and Technology, Ikoma 630-0192, Japan (Y.F.).
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Takahashi
authors.affiliation: Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan (K.F., M.S., Y.S., T.K., Y.K., H.T., I.H.-N., T.S.); andPlant Global Educational Project, Nara Institute of Science and Technology, Ikoma 630-0192, Japan (Y.F.) ihnishi@gr.bot.kyoto-u.ac.jp tshimada@gr.bot.kyoto-u.ac.jp.
authors.forename: Ikuko
authors.identifier: http://orcid.org/0000-0001-8814-1593
authors.initials: I
authors.lastname: Hara-Nishimura
authors.affiliation: Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan (K.F., M.S., Y.S., T.K., Y.K., H.T., I.H.-N., T.S.); andPlant Global Educational Project, Nara Institute of Science and Technology, Ikoma 630-0192, Japan (Y.F.) ihnishi@gr.bot.kyoto-u.ac.jp tshimada@gr.bot.kyoto-u.ac.jp.
authors.forename: Tomoo
authors.initials: T
authors.lastname: Shimada
mesh_terms: D033964:Adaptor Protein Complex 4 / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D030821:Plants, Genetically Modified; D021381:Protein Transport / Q000502:physiology*; D012639:Seeds / Q000235:genetics / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism; D021601:trans-Golgi Network / Q000378:metabolism*
chemical_list: D033964:Adaptor Protein Complex 4; D029681:Arabidopsis Proteins; C106564:ELP protein, Arabidopsis; D049452:Green Fluorescent Proteins