Molecular Characterization of hCTR1, the Human Copper Uptake Protein

Abstract

We have expressed hCTR1, the human copper transporter, in Sf9 cells using a baculovirus-mediated expression system, and we observed greatly enhanced copper uptake. Western blots showed that the protein is delivered to the plasma membrane, where it mediates saturable copper uptake with a K m of ϳ3.5 M. We also expressed functional transporters where the N-linked glycosylation sites were substituted, and we provided evidence for the extracellular location of the amino terminus. Accessibility of amino-terminal FLAG epitope to antibody prior to permeabilization and of carboxyl-terminal FLAG only after permeabilization confirmed the extracellular location of the amino terminus and established the intracellular location of the carboxyl terminus. Tryptic digestion of hCTR1 occurred within the cytoplasmic loop and generated a 10-Da carboxyl-terminal peptide; cleavage was prevented by the presence of copper. hCTR1 mutants where Cys-161 and Cys-189, the two native cysteines, were replaced with serines also mediated copper uptake, indicating that neither cysteine residue was essential for transport. However, the mutants provided evidence that these residues may stabilize hCTR1 oligomerization. Western blots of hCTR1 in Sf9 cells showed expression levels 100-fold higher than in mammalian (HepG2) cells. The high level of functional expression and the low level of endogenous copper uptake will enable future structure-function analysis of this important protein.

Reciprocal functions of Cryptococcus neoformans copper homeostasis machinery during pulmonary infection and meningoencephalitis

“…5A,B), suggesting that these Cu þ transporters undergo post-translational modification. In mammals, the Ctr1 Cu þ transporter is glycosylated at two positions within the N terminus [20][21][22][23] . We used a protein deglycosylation enzyme mix that removes both N-and O-glycosylation to determine whether C. neoformans Cu transporters are glycosylated.…”

“…Interestingly, a strong candidate for an N-glycosylation site was detected in Ctr4 near the putative amino-terminal extracellular domain Cu-binding motifs. The glycosylation of Ctr4 resembles that of mammalian Ctr1, in which glycosylation influences Cu uptake 20 . Although the N19Q mutant restored Cu uptake in ctr1Dctr4D cells, complemented cells exhibited reduced growth compared with ctr1Dctr4D cells expressing wild-type CTR4, implying that N-glycosylation is involved in facilitating Ctr4 function in an undefined manner.…”

Oligomerization of the Na,K-ATPase in Cell Membranes

“…Baculovirusmediated co-expressions in High Five cells were carried out with the tagged ␣-subunits and the unrelated membrane protein, hCTR1. hCTR1 is the human copper transporter that has been functionally expressed recently in insect cells using the baculovirus system (27). The hCTR1 protein does not specifically associate with the Na,K-ATPase, but it is delivered to the PM, like the Na,K-ATPase.…”

“…The complex pattern of bands seen with hCTR1 is due to glycosylation and the presence of some degradation products (18 kDa and below) of this protein (27). Total membrane preparations of cells co-expressing hCTR1 with ␣-His/␤ or ␣-FLAG/␤ were subjected to the same 2% DDM solubilization protocol as used in Fig.…”

Posttranslational regulation of copper transporters

“…hCTR1 was identified as a highaffinity CTR by functional complementation of a yeast strain deficient in high-affinity copper uptake [29][30][31][32]. The characterization of CTR1 as a high-affinity CTR marked the start of extensive studies on the structure, function, and cellular localization of the CTR protein family [2,6,7,[33][34][35][36][37][38][39][40][41][42]. Overexpression of hCTR1 in several cell lines results in a substantial, specific, and saturable induction of cellular copper import with a K m of approximately 1-5 lM [7,35,39].…”