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

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 Asn¹⁵) and O-linked (at Thr²⁷) sites of glycosylation. If O-glycosylation at Thr²⁷ is prevented, hCTR1 is efficiently cleaved, removing ∼30 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 Ala²⁹–Ser³⁰–His³¹. We also show that the O-linked polysaccharide at Thr²⁷ blocks proteolysis due to its proximity to the cleavage site. Moving the cleavage site away from the Thr²⁷ 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⁺ and Cu²⁺) (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).Open in a separate windowFIGURE 1.Extracellular amino terminus of hCTR1. Location of N- and O-linked glycosylation at Asn¹⁵ and Thr²⁷, and the end points of 3 truncation mutants in gray: H22, A29, and G34. In the absence of O-glycosylation at Thr²⁷, hCTR1 is efficiently cleaved between A29 and G34 (black triangles). Location of the FLAG epitope tag is shown. Inset shows the complete 190-amino acid hCTR1 protein, with extracellular NH₂ terminus, three membrane spanning domains, intracellular loop, and COOH-terminal tail. The 5 amino acids in which cleavage occurs are shown in black. Three hCTR1 polypeptides form a symmetrical trimer in the copper transporter (8, 9).Little is known about the details of the copper transport mechanism in CTR1 proteins. Mutational studies of hCTR1 have identified a number of residues important for copper transport (12–14), such as methionine residues within the extracellular amino terminus, and two transmembrane segments that were important for ⁶⁴Cu uptake in cultured cells (12). A study of hCTR1 mutants expressed in insect cells identified residues in or near the transmembrane domains that affect K_m and or V_max of ⁶⁴Cu uptake (14). These results and recent structural studies suggest that copper transits a pore lined by transmembrane segments two and three in the homotrimeric complex (8, 9). Another mechanism based on endocytosis and degradation of hCTR1 has also been proposed (15).Vertebrate CTR1 proteins are widely expressed, and may play other roles in addition to copper transport. Mice homozygous for mCtr1 knock-out alleles die during midgestation, which was thought to reflect an early requirement for copper transport during development. However, a recent study showed that xCTR1 was part of a fibroblast growth factor signaling complex in Xenopus embryos active in Ras/extracellular signal-regulated kinase (ERK) signaling. The signaling role, which affects embryonic development in Xenopus and ES cell differentiation in mammalian cells, appears to be independent of the copper transport activity of CTR1 (16).In previous structure/function studies of hCTR1 we found that the extracellular amino terminus of ∼65 amino acids is modified by N- and O-linked glycosylation at Asn¹⁵ and Thr²⁷, respectively (6, 17) (see Fig. 1). N-Linked glycans at Asn¹⁵ increase the predicted mass of the hCTR1 polypeptide by about 9 kDa. Removing N-linked polysaccharides by a N15Q mutation does not significantly affect the expression or function of the transporter (6, 17). O-Linked polysaccharides at Thr²⁷ that terminate in sialic acid residues increase the mass of the polypeptide by 1–2 kDa, (17). In the absence of O-linked glycosylation, the polypeptide undergoes very efficient cleavage near Thr²⁷, leaving a 17-kDa hCTR1 protein lacking about 30 amino acids from the extracellular amino terminus (Fig. 1). The truncated (17 kDa) hCTR1 protein was efficiently delivered to the plasma membrane, but exhibited only 50–60% of the copper transport activity of wild-type hCTR1 (17).In recent years, an impressive variety of proteases have been characterized in the secretory pathway and plasma membrane (18–22). Many of these proteases perform some kind of regulatory cleavage, from maturation of pre-proteins, (including proteases), to membrane proteases involved in shedding of ectodomains. Presumably, cleavage of hCTR1 lacking O-linked glycosylation must occur after the addition of the O-linked sugars would have occurred in the golgi (23). Cleavage of the unglycosylated hCTR1 protein could thus occur while the transporter is en route to the plasma membrane (23, 24), after delivery to the surface, or, as in the case of some receptors, during recycling between the plasma membrane and interior compartments (25–28).In this report, we show that inhibition of cleavage by O-linked glycosylation at Thr²⁷ requires close proximity of the polysaccharide to the site of cleavage. Moving the cleavage site away from Thr²⁷ polysaccharides allowed cleavage. In mutants lacking O-glycosylation, hCTR1 is cleaved within amino acids 29–33 (ASHSH), preferentially between Ala²⁹–Ser³⁰–His³¹. Live cell imaging of GFP-tagged mutant hCTR1 and staining of fixed cells overexpressing FLAG-tagged hCTR1 shows that the cleaved amino-terminal peptides accumulate in punctate structures that partially overlap Rab9, a late endosome marker, suggesting that cleavage occurs after transit through the golgi, but prior to delivery to the plasma membrane.