title: Trafficking of the Menkes copper transporter ATP7A is regulated by clathrin-, AP-2-, AP-1-, and Rab22-dependent steps.
abstract: The transporter ATP7A mediates systemic copper absorption and provides cuproenzymes in the trans-Golgi network (TGN) with copper. To regulate metal homeostasis, ATP7A constitutively cycles between the TGN and plasma membrane (PM). ATP7A trafficking to the PM is elevated in response to increased copper load and is reversed when copper concentrations are lowered. Molecular mechanisms underlying this trafficking are poorly understood. We assess the role of clathrin, adaptor complexes, lipid rafts, and Rab22a in an attempt to decipher the regulatory proteins involved in ATP7A cycling. While RNA interference (RNAi)-mediated depletion of caveolin 1/2 or flotillin had no effect on ATP7A localization, clathrin heavy chain depletion or expression of AP180 dominant-negative mutant not only disrupted clathrin-regulated pathways, but also blocked PM-to-TGN internalization of ATP7A. Depletion of the μ subunits of either adaptor protein-2 (AP-2) or AP-1 using RNAi further provides evidence that both clathrin adaptors are important for trafficking of ATP7A from the PM to the TGN. Expression of the GTP-locked Rab22aQ64L mutant caused fragmentation of TGN membrane domains enriched for ATP7A. These appear to be a subdomain of the mammalian TGN, showing only partial overlap with the TGN marker golgin-97. Of importance, ATP7A remained in the Rab22aQ64L-generated structures after copper treatment and washout, suggesting that forward trafficking out of this compartment was blocked. This study provides evidence that multiple membrane-associated factors, including clathrin, AP-2, AP-1, and Rab22, are regulators of ATP7A trafficking.
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, United Kingdom.
authors.forename: Zoe G
authors.initials: ZG
authors.lastname: Holloway
authors.forename: Antonio
authors.initials: A
authors.lastname: Velayos-Baeza
authors.forename: Gareth J
authors.initials: GJ
authors.lastname: Howell
authors.forename: Clotilde
authors.initials: C
authors.lastname: Levecque
authors.forename: Sreenivasan
authors.initials: S
authors.lastname: Ponnambalam
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Sztul
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Monaco
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000235:genetics* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D027682:Cation Transport Proteins / Q000235:genetics* / Q000378:metabolism; D002413:Cations, Divalent; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000235:genetics* / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000378:metabolism / Q000648:ultrastructure; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism / Q000648:ultrastructure; D003300:Copper / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D004705:Endocytosis; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D021381:Protein Transport; D015398:Signal Transduction; D018808:Transcription Factor AP-1 / Q000235:genetics* / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D033962:Adaptor Protein Complex 2; D027682:Cation Transport Proteins; D002413:Cations, Divalent; D002966:Clathrin; C484191:RAB22A protein, human; D018808:Transcription Factor AP-1; D003300:Copper; D000251:Adenosine Triphosphatases; D020691:rab GTP-Binding Proteins; C081184:ATP7A protein, human; D000073840:Copper-Transporting ATPases