title: Numb directs the subcellular localization of EAAT3 through binding the YxNxxF motif.
abstract: Excitatory amino acid transporter type 3 (EAAT3, also known as SLC1A1) is a high-affinity, Na(+)-dependent glutamate carrier that localizes primarily within the cell and at the apical plasma membrane. Although previous studies have reported proteins and sequence regions involved in EAAT3 trafficking, the detailed molecular mechanism by which EAAT3 is distributed to the correct location still remains elusive. Here, we identify that the YVNGGF sequence in the C-terminus of EAAT3 is responsible for its intracellular localization and apical sorting in rat hepatoma cells CRL1601 and Madin-Darby canine kidney (MDCK) cells, respectively. We further demonstrate that Numb, a clathrin adaptor protein, directly binds the YVNGGF motif and regulates the localization of EAAT3. Mutation of Y503, N505 and F508 within the YVNGGF motif to alanine residues or silencing Numb by use of small interfering RNA (siRNA) results in the aberrant localization of EAAT3. Moreover, both Numb and the YVNGGF motif mediate EAAT3 endocytosis in CRL1601 cells. In summary, our study suggests that Numb is a pivotal adaptor protein that mediates the subcellular localization of EAAT3 through binding the YxNxxF (where x stands for any amino acid) motif.
authors.affiliation: Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou 310058, China.
authors.forename: Jin-Feng
authors.initials: JF
authors.lastname: Su
authors.affiliation: College of Life Sciences, Wuhan University, Wuhan 430072, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Wei
authors.affiliation: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Pei-Shan
authors.initials: PS
authors.lastname: Li
authors.affiliation: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Hong-Hua
authors.initials: HH
authors.lastname: Miao
authors.affiliation: College of Life Sciences, Wuhan University, Wuhan 430072, China.
authors.forename: Yong-Chao
authors.initials: YC
authors.lastname: Ma
authors.affiliation: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Yu-Xiu
authors.initials: YX
authors.lastname: Qu
authors.affiliation: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Qin
authors.affiliation: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Bo-Liang
authors.initials: BL
authors.lastname: Li
authors.affiliation: College of Life Sciences, Wuhan University, Wuhan 430072, China.
authors.forename: Bao-Liang
authors.initials: BL
authors.lastname: Song
authors.affiliation: Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou 310058, China zpxu@zju.edu.cn jieluo@whu.edu.cn.
authors.forename: Zheng-Ping
authors.initials: ZP
authors.lastname: Xu
authors.affiliation: College of Life Sciences, Wuhan University, Wuhan 430072, China zpxu@zju.edu.cn jieluo@whu.edu.cn.
authors.forename: Jie
authors.identifier: 0000-0003-1899-3111
authors.initials: J
authors.lastname: Luo
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D004285:Dogs; D004705:Endocytosis; D050580:Excitatory Amino Acid Transporter 3 / Q000737:chemistry* / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008297:Male; D008807:Mice, Inbred BALB C; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D021381:Protein Transport; D051381:Rats; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D050580:Excitatory Amino Acid Transporter 3; D047908:Intracellular Signaling Peptides and Proteins; C559994:numb protein, rat
keywords: EAAT3; Endocytosis; NPC1L1; Numb; PTB; YxNxxF