title: Dynamic internalization and recycling of a metal ion transporter: Cu homeostasis and CTR1, the human Cu⁺ uptake system.
abstract: Cu ion (Cu) entry into human cells is mediated by CTR1 (also known as SLC31A1), the high-affinity Cu transporter. When extracellular Cu is raised, the cell is protected against excess accumulation by rapid internalization of the transporter. When Cu is lowered, the transporter returns to the membrane. We show in HEK293 cells overexpressing CTR1 that expression of either the C-terminal domain of AP180 (also known as SNAP91), a clathrin-coat assembly protein that sequesters clathrin, or a dominant-negative mutant of dynamin, decreases Cu-induced endocytosis of CTR1, as does a dynamin inhibitor and clathrin knockdown using siRNA. Utilizing imaging, siRNA techniques and a new high-throughput assay for endocytosis employing CLIP-tag methodology, we show that internalized CTR1 accumulates in early sorting endosomes and recycling compartments (containing Rab5 and EEA1), but not in late endosomes or lysosomal pathways. Using live cell fluorescence, we find that upon extracellular Cu removal CTR1 recycles to the cell surface through the slower-recycling Rab11-mediated pathway. These processes enable cells to dynamically alter transporter levels at the plasma membrane and acutely modulate entry as a safeguard against excess cellular Cu.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Illinois College of Medicine, Chicago, IL 60607, USA.
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Clifford
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Illinois College of Medicine, Chicago, IL 60607, USA.
authors.forename: Edward B
authors.initials: EB
authors.lastname: Maryon
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Illinois College of Medicine, Chicago, IL 60607, USA kaplanj@uic.edu.
authors.forename: Jack H
authors.identifier: http://orcid.org/0000-0002-7048-6574
authors.initials: JH
authors.lastname: Kaplan
mesh_terms: D027682:Cation Transport Proteins / Q000378:metabolism*; D002966:Clathrin / Q000235:genetics / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D000079923:Copper Transporter 1; D034281:Dynamins / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D057809:HEK293 Cells; D006706:Homeostasis; D006801:Humans; D017136:Ion Transport; D034041:Monomeric Clathrin Assembly Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D021381:Protein Transport; D034741:RNA, Small Interfering; D033921:Vesicular Transport Proteins / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
chemical_list: D027682:Cation Transport Proteins; D002966:Clathrin; D003300:Copper; D000079923:Copper Transporter 1; D034281:Dynamins; D034041:Monomeric Clathrin Assembly Proteins; D034741:RNA, Small Interfering; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C490079:SLC31A1 protein, human; C439023:clathrin assembly protein AP180; C093846:early endosome antigen 1
keywords: CTR1; Cu transporter recycling; Metal ion homeostasis; Regulatory endocytosis