Dynamic internalization and recycling of a metal ion transporter: Cu homeostasis and hCTR1, the human Cu uptake system

Abstract

Cu entry into human cells is mediated by hCTR1, the high affinity Cu transporter. When extracellular Cu is raised, the cell is protected against excess accumulation by rapid internalization of the transporter. When Cu is lowered, the transporter returns to the membrane. We show in HEK293 cells overexpressing hCTR1, that expression of either, the C-terminal domain of AP180, a clathrin-coat assembly protein that sequesters clathrin, or a dominant negative mutant of dynamin, decreases Cu-induced endocytosis of hCTR1, as does a dynamin inhibitor and clathrin knockdown via siRNA. Utilizing imaging, siRNA techniques, and a new high through-put assay for endocytosis employing CLIP-tag methodology, we show that internalized hCTR1 accumulates in early sorting endosomes and recycling compartments (containing Rab5 and EEA1), but not in late endosomes or lysosomal pathways. Using live cell fluorescence, we find upon extracellular Cu removal, hCTR1 recycles to the cell surface through the slower recycling Rab11-mediated pathway. These processes enable cells to dynamically alter transporter levels at the plasma membrane and acutely modulate entry as a safeguard against excess cellular Cu.