PIB-ATPases are membrane proteins that transport heavy metal ions across biological membranes upon ATP-hydrolysis. These enzymes contribute to metal homeostasis in archaeal, prokaryotic and eukaryotic cells. Typically, most PIB-ATPases have eight transmembrane segments, one or more metal binding domains in the cytoplasmic N-terminal region and a series of amino acids conserved in all the members of this family. By sequence homology analysis, the metal specificity for most ATPases has been predicted. Here, we report studies on PIB-ATPases from different organisms. The first part of this work focuses in a group of ATPases from Arabidopsis thaliana plants. Transcription levels of HMA3, 4 and 8 were analyzed in different plant organs and in seedlings upon metal exposure. Tissue specificity was studied for HMA8 by generation of transgenic plants carrying a reporter gene downstream its promoter region. Attempts to determine metal specificity of proteins expressed in yeast cells were performed. Finally, in order to study the effects of removing the genes products from the plants, HMA4 and 8 mutant plants were identified. The second part describes a novel Pb-transport ATPase from a thermophilic archaeabacterium, Aeropyrum pernix. This enzyme is predicted to have only six transmembrane segments, no regulatory metal binding domains and unusual metal specificity. PbTP was cloned, expressed in Escherichia coli and partially purified. The enzyme retained its thermophilicity characteristics when isolated from its native lipid environment. The metal dependent ATPase activity was determined in the presence of different metals at 75ºC. The enzyme was highly activated by Pb2+ (Vmax: 23.6 µmol Pi/mg/h) and to a lesser extent by Zn2+, Hg2+ and Cd2+. Lead interacts with PbTP with high apparent affinity (K1/2: 4.6 µM). The enzymatic ATP hydrolysis was independent of cysteine or glutathione, suggesting direct interaction of the metal ions with the transmembrane transport sites.
Worcester Polytechnic Institute
Chemistry & Biochemistry
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Orofino, Maria J., "Heavy Metal ATPases from Archaeabacteria to Plants" (2006). Masters Theses (All Theses, All Years). 662.
PIB-ATPases, plants, archaebacteria