title: The Menkes disease ATPase (ATP7A) is internalized via a Rac1-regulated, clathrin- and caveolae-independent pathway.
abstract: The Menkes disease gene encodes a P-type transmembrane ATPase (ATP7A) that translocates cytosolic copper ions across intracellular membranes of compartments along the secretory pathway. ATP7A 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 ATP7A is internalized by a novel pathway that is independent of clathrin-mediated endocytosis. Expression of dominant-negative mutants of the dynamin-I, dynamin-II and Eps15 proteins that block clathrin-dependent endocytosis of the transferrin receptor do not inhibit internalization of endogenous ATP7A, or an ATP7A reporter molecule (CD8-MCF1). Similarly, inhibitors of caveolae-mediated uptake do not affect ATP7A internalization whilst preventing uptake of PODIPY-ganglioside GM(1), a caveolae marker. In contrast, expression of a constitutively active mutant of the Rac1 GTPase inhibits plasma membrane internalization of both the ATP7A and transferrin receptor transmembrane proteins. These findings define a novel route required for ATP7A internalization and delivery to endosomes.
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Headington, Oxford 0X3 7BN, UK.
authors.forename: Christian
authors.initials: C
authors.lastname: Cobbold
authors.forename: Julie
authors.initials: J
authors.lastname: Coventry
authors.forename: Sreenivasan
authors.initials: S
authors.lastname: Ponnambalam
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Monaco
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism* / Q000502:physiology; D020816:Amino Acid Motifs; D000818:Animals; D001896:Boron Compounds / Q000494:pharmacology; D019556:COS Cells; D027682:Cation Transport Proteins / Q000378:metabolism* / Q000502:physiology; D021941:Caveolae / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism*; D003300:Copper / Q000378:metabolism; D000073840:Copper-Transporting ATPases; D003600:Cytosol / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007477:Ions; D007930:Leucine / Q000737:chemistry; D008164:Luminescent Proteins / Q000378:metabolism; D007706:Menkes Kinky Hair Syndrome / Q000235:genetics*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009154:Mutation; D017434:Protein Structure, Tertiary; D011990:Receptors, Transferrin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism* / Q000502:physiology; D013696:Temperature; D013997:Time Factors; D014162:Transfection; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D021601:trans-Golgi Network / Q000378:metabolism
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D027682:Cation Transport Proteins; D002966:Clathrin; D007477:Ions; D008164:Luminescent Proteins; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D003300:Copper; D000251:Adenosine Triphosphatases; D020830:rac1 GTP-Binding Protein; C081184:ATP7A protein, human; D000073840:Copper-Transporting ATPases; D007930:Leucine