CONICET | Buscador de Institutos y Recursos Humanos

Cu+ transporting PIB-ATPases are heavy metal membrane transporting proteins that have an important role in cooper homeostasis and detoxification. The most known member of this class is the Cu+-ATPase from Archaeglobus fulgidus (AfCopA). In a previous work we have characterized the temperature and chemical effect on folding and activity of this thermophilic protein (Cattoni et al., 2008 and Roman et al., 2010). The comparison of these properties with that corresponding to mesophilic homologs of AfCopA would provide new insight to understand the adaptation of membrane proteins to different conditions of temperature. Recently the first atomic structure of a PIB-ATPases, CopA from Legionella pneumophila (LpCopA), was determined by X-ray crystallography at 3.2 A resolution (Gourdon et al., 2011). The structure is similar to that Ca2+-ATPase but reveals two N-terminal transmembrane helices which are specific for this class and provides a framework to analyze the molecular pathophysiology of Menkes and Wilsons diseases. Thus it constitutes a good model for the proposed study. Here we report the production of LpCopA in Saccharomyces cerevisiae. Using a galactose induced system and GFP as an indicator we optimized the conditions to express LpCopA measuring fluorescence in whole cells. Maximal fluorescence was obtained growing the transformed cells in media containing 0.67% YNB, 0.08% Complete supplement mixture-URA, 0.1% glucose, 0.6% yeast extract and 0.1% peptone and induced with 2% galactose for 24hs at 28oC. Separation of proteins from isolated membranes by SDS-PAGE showed a main band with a molecular mass corresponding to the fusion protein LpCopA-GFP-his8 by in gel fluorescence. Purification of LpCopA-GFPhis8 will be performed by affinity chromatography. This will allow to compare the regulation of catalytic activity and stability of this protein with that corresponding to the thermophilic counterpart.