Human Copper Transporter 1 Lacking O-Linked Glycosylation Is Proteolytically Cleaved in a Rab9-positive Endosomal Compartment

Abstract

The human copper transporter hCTR1 is a homotrimer composed of a plasma membrane protein of 190 amino acids that contains three transmembrane segments. The extracellular 65-amino acid amino terminus of hCTR1 contains both N-linked (at Asn 15 ) and O-linked (at Thr 27 ) sites of glycosylation. If O-glycosylation at Thr 27 is prevented, hCTR1 is efficiently cleaved, removing ϳ30 amino acids from the amino terminus. We have now investigated (i) the site of this cleavage, determining which peptide bonds are cleaved, (ii) the mechanism by which glycosylation prevents cleavage, and (iii) where in the cell the proteolytic cleavage takes place. Cleavage occurs in the sequence Ala-Ser-His-Ser-His (residues 29 -33), which does not contain previously recognized protease cleavage sites. Using a series of hCTR1 mutants, we show that cleavage occurs preferentially between residues Ala 29 -Ser 30 -His 31 . We also show that the O-linked polysaccharide at Thr 27 blocks proteolysis due to its proximity to the cleavage site. Moving the cleavage site away from the Thr 27 polysaccharide by insertion of as few as 5 amino acids allows cleavage to occur in the presence of glycosylation. Imaging studies using immunofluorescence in fixed cells and a functional green fluorescent protein-tagged hCTR1 transporter in live cells showed that the cleaved peptide accumulates in punctate structures in the cytoplasm. These puncta overlap compartments were stained by Rab9, indicating that hCTR1 cleavage occurs in a late endosomal compartment prior to delivery of the transporter to the plasma membrane.Copper is acquired by eukaryotic cells through transporters in the plasma membrane known as CTR proteins (1). Copper is an essential enzymatic cofactor in numerous proteins, many of which perform electron transfer reactions in which the metal cycles (2, 3) between the redox states (Cu ϩ and Cu 2ϩ ) (4). This readily occurring redox reaction can make copper ions toxic to cells through the generation of reactive oxygen species. The free copper concentration in cells is extremely low (less than 1 fmol), and there is essentially no free copper in serum. Hence, copper transporters receive copper from copper-binding substrates in the serum, translocate it across the membrane, and transfer it to intracellular chaperones for delivery to target proteins (5).Human copper transporter 1 (hCTR1) 2 and orthologous proteins throughout eukaryotes have three transmembrane segments (6, 7) and form homotrimeric, membrane complexes (8, 9) that carry out the high affinity transport of monovalent copper (see Fig. 1, inset). The human hCTR1 gene was discovered by its ability to complement Saccharomyces cerevisiae yCtr mutants, demonstrating that high affinity copper transport is a conserved function among the CTR1 proteins (10). The CTR1 proteins range in size from 200 to 400 amino acids (1, 11), but share methionine-and histidine-rich motifs in the extracellular amino terminus, as well as conserved sequences in transmembrane segments (1, 12).Little is known about the detai...

Ctr1 Is an Apical Copper Transporter in Mammalian Intestinal Epithelial Cells in Vivo That Is Controlled at the Level of Protein Stability

“…5). Immunoblotting with an antibody raised against the Ctr1 amino terminus (30) showed cross-reactivity with the 35-37-kDa glycosylated form of Ctr1 and not the ϳ17-kDa amino-terminally truncated form (Fig. 5D), supporting the assignment of these polypeptide species.…”

“…5C). Although exposure of HEK293T cells to copper resulted in clear reductions in the steady-state levels of the full-length glycosylated form of Ctr1, steady-state levels of a previously characterized Ctr1 species that was truncated 29 -33 residues from the amino terminus (25,30) were not significantly altered in response to any metal tested (Fig. 5).…”

Rate and Regulation of Copper Transport by Human Copper Transporter 1 (hCTR1)

“…Confocal Imaging-Cells were grown on glass coverslips and treated in 12-well culture plates prior to fixing and staining with antibodies, exactly as described previously (29). Images of HEK293 cells were collected using a Zeiss LSM 700 confocal microscopy system equipped with a 100ϫ objective, controlled with Zen TM software.…”

“…The H139R and C-terminal mutants were derived from the FLAGtagged hCTR1 clone pEM94 (Ref. 29, available on request). H139R was made as described previously (29) with a site-directed mutagenesis kit (Stratagene, Carlsbad CA).…”

“…H139R Mutant and Copper Uptake-Although previous studies of hCTR1 in mammalian cells have examined the role of residues in the N terminus and on the extracellular side of the pore (22,23,28,29), little has been reported on the role of residues on the intracellular side of the pore. We next characterized an hCTR1 mutant in HEK293 cells having a histidine to arginine substitution at position 139 (H139R) in the second transmembrane segment, which we previously showed to have an elevated rate of copper transport when expressed in insect cells (10).…”