Stable plasma membrane levels of hCTR1 mediate cellular copper uptake.
The human copper transporter 1 (hCtr1), when heterologously overexpressed in insect cells, mediates saturable Cu uptake. In mammalian expression systems, a rapid Cu-dependent internalization of hCtr1 has been reported in cells that overexpress epitope-tagged hCtr1 when exposed to Cu in the external medium. This finding led to the suggestion that such internalization may be a step in the hCtr1 transmembrane Cu transport mechanism. We have demonstrated that preincubation in Cu-containing media of sf9 cells stably expressing hCtr1 has no effect on the initial rate of Cu transport. Furthermore, Western blot analyses of fractionated sf9 cell membranes show no evidence of a regulatory Cu-dependent internalization from the plasma membrane. In similar studies on human embryonic kidney (HEK) 293 cells, we showed that incubation with Cu does not alter the initial rate of Cu uptake mediated by endogenous levels of hCtr1 compared with untreated cells. Confirmation that hCtr1 mediates this transport is provided by specific small interfering RNA-dependent decreases in hCtr1 protein levels and in Cu transport rates. Western blot analysis and confocal microscopy of human embryonic kidney 293 cells showed that the majority of hCtr1 protein is localized at the plasma membrane and no significant internalization is detected upon Cu treatment. We concluded that internalization of hCtr1 is not a required step in the transport pathway; we suggest that oligomeric hCtr1 acts as a conventional transporter providing a permeation pathway for Cu through the membrane and that internalization of endogenous hCtr1 in response to elevated extracellular Cu levels does not play a significant regulatory role in Cu homeostasis.
Eisses
John F
JF
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
Chi
Yiqing
Y
Kaplan
Jack H
JH
D000818:Animals; D001692:Biological Transport; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D000079923:Copper Transporter 1; D004622:Embryo, Mammalian; D004625:Embryo, Nonmammalian; D006801:Humans; D007668:Kidney; D007700:Kinetics; D021381:Protein Transport; D016372:RNA, Antisense / Q000235:genetics; D034741:RNA, Small Interfering; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
D027682:Cation Transport Proteins; D000079923:Copper Transporter 1; D016372:RNA, Antisense; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C490079:SLC31A1 protein, human; D003300:Copper