Characterization of Mutant hCTR1 Copper Transporters Truncated by Proteolytic Cleavage in the Absence of O-Linked Glycosylation at Thr27

Abstract

The human copper transporter hCTR1 is a plasma membrane protein of 190 amino acids that contains three transmembrane segments. Three hCTR1 polypeptides form symmetrical homotrimeric complexes that contain a permeation pathway for copper transport across the plasma membrane that is formed by the transmembrane segments (1). Little is known about the role(s) of the 65 amino acid extracellular amino terminus.In previous studies (2-4) we showed that extracellular terminus of hCTR1 contains both N-linked (at Asn15) and O-linked (at Thr27) sites of glycosylation. If O-glycosylation at Thr27 is prevented by mutation or by expressing wild type hCTR1 in mutant cells that cannot initiate O-glycosylation, hCTR1 is efficiently cleaved, removing 31-32 amino acids from the amino-terminus. The cleaved amino terminal peptide accumulates in punctate structures in the cytoplasm that overlap compartments containing Rab9, indicating that hCTR1 cleavage occurs in a late golgi or late endosomal compartment. The amino-terminal truncated hCTR1 transporters are delivered to the plasma membrane, where they exhibit about 50% of the 64Cu uptake of full-length (wild type) hCTR1 transporters.We have further examined the truncated hCTR1 transporters for defects in copper transport and copper-stimulated endocytosis, and we have characterized wild type and amino terminal truncated hCTR1 transporter complexes in native gels. The truncated transporters retain some copper regulatory and transport characteristics, such as copper-stimulated endocytosis. Analysis of full-length and amino-terminal truncated complexes in native gels indicates that the truncated hCTR1 transporters may lack non-hCTR1 components present in wild type hCTR1 complexes.1. DeFeo, et.al. 2009. PNAS 106: 4237-42422. Eisses and Kaplan 2002. JBC 277: 29662-291713. Maryon, Molloy and Kaplan 2007. JBC, 282:20376-203874. Maryon, Zhang, Jellison, and Kaplan 2009. JBC, 284:28104-28114