The Menkes disease ATPase (ATP7A) is internalised via a Rac1-regulated, clathrin- and caveolae-independent pathway

Abstract

The Menkes disease gene encodes a P-type transmembrane ATPase (MNK) that translocates cytosolic copper ions across intracellular membranes of compartments along the secretory pathway. MNK moves from the trans-Golgi network (TGN) to the cell surface in response to exogenously added copper ions and recycles back to the TGN upon copper removal. The protein contains a C-terminal di-leucine motif necessary for internalization from the cell surface. In this study we show that MNK is internalized by a novel pathway that is independent of clathrin-mediated endocytosis. Expression of dominant-negative mutants of the dynamin-I and dynamin-II proteins that block clathrindependent endocytosis of the transferrin receptor, do not inhibit internalization of endogenous MNK, or a CD8-MCF1 reporter molecule. Similarly, inhibitors of caveolae-mediated uptake do not affect MNK internalization whilst preventing uptake of PODIPY-ganglioside GM1, a caveolae marker. In contrast, expression of a constitutively active mutant of the Rac1 GTPase inhibits plasma membrane internalization of both the MNK and transferrin receptor transmembrane proteins. Potential downstream effectors of Rac1-regulated MNK internalization are also analyzed. These findings define a novel route required for MNK internalization and delivery to endosomes.