Cd2+- or Hg2+-binding proteins can replace the Cu+-chaperone Atx1 in delivering Cu+ to the secretory pathway in yeast

AbstractCopper delivery to Ccc2 – the Golgi Cu+-ATPase – was investigated in vivo, replacing the Cu+-chaperone Atx1 by various structural homologues in an atx1-Δ yeast strain. Various proteins, displaying the same ferredoxin-like fold and (M/L)(T/S)CXXC metal-binding motif as Atx1 and known as Cu+-, Cd2+- or Hg2+-binding proteins were able to replace Atx1. Therefore, regardless of their original function, these proteins could all bind copper and transfer it to Ccc2, suggesting that Ccc2 is opportunistic and can interact with many different proteins to gain Cu+. The possible role of electrostatic potential surfaces in the docking of Ccc2 with these Atx1-homologues is discussed

The dual personality of ionic copper in biology

International audienceBiological copper is mainly involved in electron transport to catalyse essential oxido-reduction processes. It is an essential trace element which is extremely toxic because exchangeable intracellular copper is Cu(I) which generates reactive oxygen species. To handle this paradox the evolution has led to a fine homeostasis in which copper ions are never free. Intracellular Cu(I) instead is bound to numerous proteins forming specific cascades towards its targets

Dissecting the role of the N-terminal metal-binding domains in activating the yeast copper ATPase in vivo

International audienceIn 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 atx1Δccc2Δ 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

Interplay between glutathione, Atx1 and copper. 1. Copper(I) glutathionate induced dimerization of Atx1.

International audienceCopper is both an essential element as a catalytic cofactor and a toxic element because of its redox properties. Once in the cell, Cu(I) binds to glutathione (GSH) and various thiol-rich proteins that sequester and/or exchange copper with other intracellular components. Among them, the Cu(I) chaperone Atx1 is known to deliver Cu(I) to Ccc2, the Golgi Cu-ATPase, in yeast. However, the mechanism for Cu(I) incorporation into Atx1 has not yet been unraveled. We investigated here a possible role of GSH in Cu(I) binding to Atx1. Yeast Atx1 was expressed in Escherichia coli and purified to study its ability to bind Cu(I). We found that with an excess of GSH [at least two GSH/Cu(I)], Atx1 formed a Cu(I)-bridged dimer of high affinity for Cu(I), containing two Cu(I) and two GSH, whereas no dimer was observed in the absence of GSH. The stability constants (log beta) of the Cu(I) complexes measured at pH 6 were 15-16 and 49-50 for CuAtx1 and Cu (2) (I) (GS(-))(2)(Atx1)(2), respectively. Hence, these results suggest that in vivo the high GSH concentration favors Atx1 dimerization and that Cu (2) (I) (GS(-))(2)(Atx1)(2) is the major conformation of Atx1 in the cytosol

An apparatus for stopped-flow X-ray scattering

A stopped-flow apparatus and control system, designed for the study of rapid reaction kinetics in solution by X-ray scattering, is described. Inspired from a commercial stopped-flow unit used with UV and visible light, the X-ray device has a dead-time of 80 ms. Results are presented for the polymerization of the coat protein of Brome mosaic virus following a pH jump, using a small angle X-ray scattering instrument at Hasylab (Hamburg).Cet article décrit un appareil de « stopped-flow » et son système de contrôle destiné à l'étude, par diffusion des rayons X, de cinétiques rapides de réactions en solution. Inspiré d'un appareil commercial utilisant les UV et la lumière visible, l'appareil à rayons X a un temps mort de 80 ms. Des résultats sont présentés pour la polymérisation de la protéine de conque du virus de la mosaïque du Brome par saut de pH, en utilisant l'installation de diffusion de rayons X à petits angles de Hasylab (Hambourg)

An apparatus for stopped-flow X-ray scattering

A stopped-flow apparatus and control system, designed for the study of rapid reaction kinetics in solution by X-ray scattering, is described. Inspired from a commercial stopped-flow unit used with UV and visible light, the X-ray device has a dead-time of 80 ms. Results are presented for the polymerization of the coat protein of Brome mosaic virus following a pH jump, using a small angle X-ray scattering instrument at Hasylab (Hamburg)