Dissecting the role of the N-terminal metal-binding domains in activating the yeast copper ATPase in vivo.
In yeast, copper delivery to the trans-Golgi network involves interactions between the metallo-chaperone Atx1 and the N-terminus of Ccc2, the P-type ATPase responsible for copper transport across trans-Golgi network membranes. Disruption of the Atx1-Ccc2 route leads to cell growth arrest in a copper-and-iron-limited medium, a phenotype allowing complementation studies. Coexpression of Atx1 and Ccc2 mutants in an atx1Delta ccc2Delta strain allowed us to study in vivo Atx1-Ccc2 and intra-Ccc2 domain-domain interactions, leading to active copper transfer into the trans-Golgi network. The Ccc2 N-terminus encloses two copper-binding domains, M1 and M2. We show that in vivo Atx1-M1 or Atx1-M2 interactions activate Ccc2. M1 or M2, expressed in place of the metallo-chaperone Atx1, were not as efficient as Atx1 in delivering copper to the Ccc2 N-terminus. However, when the Ccc2 N-terminus was truncated, these independent metal-binding domains behaved like functional metallo-chaperones in delivering copper to another copper-binding site in Ccc2 whose identity is still unknown. Therefore, we provide evidence of a dual role for the Ccc2 N-terminus, namely to receive copper from Atx1 and to convey copper to another domain of Ccc2, thereby activating the ATPase. At variance with their prokaryotic homologues, Atx1 did not activate the Ccc2-derived ATPase lacking its N-terminus.
Morin
Isabelle
I
CEA, DSV, iRTSV, Laboratoire de Chimie et Biologie des Métaux, Grenoble, France.
Gudin
Simon
S
Mintz
Elisabeth
E
Cuillel
Martine
M
D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D000079922:Copper Transport Proteins; D004789:Enzyme Activation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
C093341:ATX1 protein, S cerevisiae; C092974:CCC2 protein, S cerevisiae; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D000079922:Copper Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D003300:Copper