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Nathalie Bal

Functional study of CadA, the cadmium ATPase from Listeria monocytogenes

Published on 13 December 2002


Thesis presented December 13, 2002

Abstract:
P1-type ATPases are membrane proteins responsible for the active transport of heavy metal ions against their electrochemical gradient, across the membrane. This is achieved by using the energy of hydrolysis of ATP.
The goal of this thesis was to elucidate the functional properties of CadALM, a P1-type ATPase involved in Listeria monocytogenes resistance to cadmium.
Our research began with the expression of CadALM in Sf9 cells using a Baculovirus mediated system. The study of CadALM allowed us to determine its main enzymatic characteristics, revealing a mechanism similar to those of the P2-type ATPases, the most well-known ATPases of the P-type family. This work also clarified the ionic specificity of CadALM, confirming that this ATPase can be classified with the sub-group of P1-type ATPases that translocate cadmium, zinc or lead. By studying a modified CadALM truncated of its cytoplasmic N-terminal metal-binding domain - the main structural characteristic of the P1-type ATPases - we concluded that this domain is not essential for CadALM function. Nevertheless, a regulatory role of the activation of CadALM by metal ions has been assigned to this domain. Lastly, the study of the CPC motif, another distinguishing feature of heavy metal transport ATPases which is located in the sixth transmembrane helix, revealed that this motif can be part of the membranous transport site(s). Beside this biochemical characterisation of CadALM, a new phenotype of the yeast Saccharomyces cerevisiae was discovered when expressing CadALM. This phenotype was used as a screening system to select non-functional mutants. It can therefore be used to determine the essential amino acids for CadALM functioning, helping to understand the structure-function relationships of the ATPase.

Keywords:
CadA, P1-type APTase, Listeria monocytogenes, active transport, heavy metals fixation site, Baculovirus/Sf9, Saccharomyces cerevisiae

Download this thesis (Intranet link).