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Aurélie Gardarin

Cadmium, P-type ATPase, yeast. From transport to toxicity

Published on 19 December 2007


Thesis presented December 19, 2007

Abstract:
Two projects has been developed during my PhD. One consisting in the functional study of CadA, the Cd2+-ATPase from Listeria monocytogenes, the other one was focused on the toxicity of cadmium and the associated response of the yeast Saccharomyces cerevisiae. This two studies used a a phenotype of sensitivity to cadmium induced by CadA expression in yeast. This phenotype was used as a screening tool to identify essential amino acids of Cd transport by CadA and to study cadmium toxicity and the corresponding yeast cellular response. CadA actively transports Cd using ATP hydrolysis as energy source. Directed mutagenesis of the membranous polar, sulphur and charged amino-acids revealed that Cd transport pathway implied four transmembrane segments (Tm) and more precisely the cysteine C354, C356 and proline P355 of the CPC motif located in Tm6, aspartate D692 in Tm8, glutamate E164 in Tm4 and methionine M149 in Tm5. From our studies, 2 Cd ions would be translocated for each hydrolysis ATP. Expression of CadA in the yeast Saccharomyces cerevisiae induces an hypersensitivity to Cd. A wild type cell can grow up to 100µM cadmium whereas CadA expressing yeast cannot grow with 1µM cadmium in the culture medium. This cadmium sensitivity was due to the localization​ of CadA in the endoplasmic reticulum membrane. Transport of cadmium in this compartment produces an accumulation of misfolded proteins that induces the Unfolded Protein Response (UPR). As UPR also occurs in a wild type yeast exposed to low Cd concentration, one can point out endoplasmic reticulum as a extremely sensitive cellular compartment. UPR also appears as an early response to Cd as it happens far before any visible signs of toxicity.

Keywords:
CadA, ATPase-P, Cadmium, membrane transport sites​, Saccharomyces cerevisiae, Unfolded Protein Response

Download this thesis (Intranet link).