Elevated intracellular copper induces CTR1 monomerization and prevents copper uptake

Copper is an essential element involved in various biochemical processes, such as mitochondrial energy production and antioxidant defense, but improper regulation can lead to cellular toxicity and disease. Copper Transporter 1 (CTR1) plays a key role in copper uptake and maintaining cellular copper homeostasis. Although CTR1 endocytosis was previously thought to reduce copper uptake when levels are high, it was unclear how rapid regulation is achieved. Using single-molecule localization microscopy and single-molecule neighbor density assays, we discover that elevated intracellular copper induces monomerization of the wild-type trimeric CTR1 prior to endocytosis, a response blocked in the endocytosis-deficient CTR1 (M150L) mutant. This monomerization correlates with a rapid halt in copper uptake. We propose that changes in CTR1 oligomerization state may regulate endocytosis and copper homeostasis. Elevated intracellular Cu converts CTR1 trimers to monomers at Rab5⁺ sites prior to endocytosis, rapidly halting Cu uptake; an endocytosis-deficient mutant fails to mount this response.