Evidence of the Presence of an Additional Copper Uptake Transporter in the Plasma Membrane of Mammalian Cells
Authors:Ramin Farhad, Rebecca Vargas
Mentor:Maria Linder, Professor of Biochemistry , California State University, Fullerton
Copper is an essential trace element for all living organisms as a co-factor for many enzymes/proteins supportive of life. Blood plasma proteins such as ceruloplasmin (Cp), α2-macroglobulin, and albumin are the direct sources of Cu for mammalian cells, as demonstrated in previous studies from our laboratory. Copper transporter 1 (CTR1) is the only Cu uptake transporter known to date on the plasma membrane of cells that take up Cu from these plasma proteins. However, the CTR1 inhibitor (Ag+) did not restrain uptake of Cu by these cultured cells. To determine whether Cu is taken up by an additional mechanism, rates of uptake from the different Cu carrying plasma proteins and Cu-histidine were compared in cells that did and did not express CTR1. siRNA knocked down expression of CTR1 in human mammary epithelial and hepatic cell models, but this had no effect on uptake of Cu(II) from the plasma proteins. Mouse embryonic fibroblasts (MEF) that did not express CTR1 took up Cu (II) bound to albumin or Cp almost as well as those expressing CTR1. Uptake rates from mouse albumin reached a 2-4 fold higher Vmax (with a lower Km) than from human albumin. Maximum uptake rates for Cu(I) versus Cu(II)-histidine were significantly higher, suggesting mediation by a reductase. All of this evidence indicates that there is at least one additional Cu transporter that I am working to identify. The unidentified transporter will be captured by a cross-linking method involving biotinylation. Cells incubated with the Cp delivering Cu are cross-linked, creating a covalent bond between the protein on the cell surface (unknown transporter) and the Cp. Upon further treatment of the cell membrane this results in biotinylation of the transporter, which can then be precipitated with Streptavidin-HRP, further separated and sequenced.