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Isabelle Morin

Inquiry into copper delivery to the secretory pathway in yeast. Role of the metallo-chaperone Atx1 and the copper ATPase Ccc2

Published on 29 November 2005


Thesis presented November 29, 2005

Abstract:
The main goal of my PhD is to investigate in yeast how copper is transferred from the cytosolic metallochaperone Atx1 to Ccc2, the ATPase embedded in the Golgi membranes that is responsible for copper delivery to the secretory pathway. An interaction was previously shown between Atx1 and Ccc2 N-terminus which encloses two Atx1-like domains, denoted M1 and M2. To study copper transfer, complementation assays were performed in an atx1-
Δ yeast strain: several structural homologues (such as M1 and M2 produced as soluble proteins) could replace Atx1 in delivering copper to Ccc2. To increase the sensitivity of these phenotypic tests, a yeast expression vector was engineered to produce hardly detectable amounts of Ccc2. Using this new system of expression, we co-expressed, in an atx1-Δ ccc2-Δ yeast strain, Atx1 (or an homologue) and a modified Ccc2 bearing a mutated or truncated N-terminus. Such complementation assays were analyzed to assess copper transfer to either M1 or M2 tethered to Ccc2. Atx1 and some homologues transfer copper either to M1 or to M2 tethered to Ccc2. Complementation was less efficient with homologues than with Atx1, enhancing the special properties of Atx1. Once on the N-terminus, copper is thought to be delivered to another copper binding site of the protein, potentially localized​ on the large cytosolic loop of Ccc2.

Keywords:
Cu(I), Golgi, Saccharomyces cerevisiae, ATPase Ccc2, Metallochaperone Atx1, Complementation

Download this thesis (Intranet link).