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Contributions à l'étude de la détoxication de la levure par les transporteurs ABC: 1 - étude biochimique de Yor1p; 2 - rôle des thiols dans la toxicité du sélénium.

Abstract : The ABC-proteins form a large family of proteins present in all the living organisms. These proteins use the energy of ATP hydrolysis to transport a wide range of substances across biological membranes. In humans, some of these proteins have significant implications in pathology. For example, dysfunction of CFTR causes cystic fibrosis, and the overproduction of MRP1 is often associated with multidrug resistance phenomena. The yeast Saccharomyces cerevisiae has a cluster of 6 proteins (Y! or1p, Ycf1p, Bpt1p, Ybt1p, Vmr1p, Nft1p) homologous to CFTR and MRP1. This family can thus serve as a model for the study of human proteins. The first part of this work was a biochemical study of the yeast protein Yor1p. We added a histidine tag to the YOR1 gene and placed this construction under the control of a promoter allowing an overproduction in yeast. We showed that the histidine tagged Yor1p was produced in a functional form and, subsequently, we developed a method allowing the solubilization and the purification of Yor1p in a single step by metal affinity chromatography. In a second part of this work, we produced in an isolated form in Escherichia coli the two NBD domains of Yor1p that are involved in ATP binding and hydrolysis and purified them to homogeneity. We studied ATP binding to these two domains, and were able to conclude that these domains are well structured. Thus, they can now be used for structural studies. Finally, we considered the role of Ycf1p in selenite detoxification. We observed that the selenite toxicity for yeast was considerably increased in the presence of thiols. The formation of reactive oxygen species is probably at the origin of this hypertoxicity.
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https://pastel.archives-ouvertes.fr/pastel-00001994
Contributor : Ecole Polytechnique <>
Submitted on : Wednesday, July 28, 2010 - 4:00:14 PM
Last modification on : Wednesday, July 29, 2020 - 4:10:05 PM
Long-term archiving on: : Friday, October 29, 2010 - 10:10:41 AM

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Ioana Grigoras. Contributions à l'étude de la détoxication de la levure par les transporteurs ABC: 1 - étude biochimique de Yor1p; 2 - rôle des thiols dans la toxicité du sélénium.. Biochimie, Biologie Moléculaire. Ecole Polytechnique X, 2005. Français. ⟨pastel-00001994⟩

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