Supplying copper to the cuproenzyme peptidylglycine alpha-amidating monooxygenase.
We explored the role of known copper transporters and chaperones in delivering copper to peptidylglycine-alpha-hydroxylating monooxygenase (PHM), a copper-dependent enzyme that functions in the secretory pathway lumen. We examined the roles of yeast Ccc2, a P-type ATPase related to human ATP7A (Menkes disease protein) and ATP7B (Wilson disease protein), as well as yeast Atx1, a cytosolic copper chaperone. We expressed soluble PHMcc (catalytic core) in yeast using the yeast pre-pro-alpha-mating factor leader region to target the enzyme to the secretory pathway. Although the yeast genome encodes no PHM-like enzyme, PHMcc expressed in yeast is at least as active as PHMcc produced by mammalian cells. PHMcc partially co-migrated with a Golgi marker during subcellular fractionation and partially co-localized with Ccc2 based on immunofluorescence. To determine whether production of active PHM was dependent on copper trafficking pathways involving the CCC2 or ATX1 genes, we expressed PHMcc in wild-type, ccc2, and atx1 mutant yeast. Although ccc2 and atx1 mutant yeast produce normal levels of PHMcc protein, it lacks catalytic activity. Addition of exogenous copper yields fully active PHMcc. Similarly, production of active PHM in mouse fibroblasts is impaired in the presence of a mutant ATP7A gene. Although delivery of copper to lumenal cuproproteins like PAM involves ATP7A, lumenal chaperones may not be required.
El Meskini
Rajaâ
R
Department of Neuroscience, University of Connecticut Health Center, Farmington, Connecticut 06030-3401, USA.
Culotta
Valeria Cizewski
VC
Mains
Richard E
RE
Eipper
Betty A
BA
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D003300:Copper / Q000494:pharmacology*; D000079922:Copper Transport Proteins; D000073840:Copper-Transporting ATPases; D005347:Fibroblasts / Q000166:cytology; D015971:Gene Expression Regulation, Enzymologic; D006056:Golgi Apparatus / Q000378:metabolism; D051379:Mice; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology
C093341:ATX1 protein, S cerevisiae; C486201:Atp7a protein, mouse; C092974:CCC2 protein, S cerevisiae; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D000079922:Copper Transport Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D003300:Copper; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D000251:Adenosine Triphosphatases; C486202:Atp7a protein, rat; D000073840:Copper-Transporting ATPases