|Parameswaran, A - UNIV KONSTANZ GERMANY|
|Leitenmaier, B - UNIV KONSTANZ GERMANY|
|Yang, M - UNIV KONSTANZ GERMANY|
|Kroneck, Pmh - UNIV KONSTANZ GERMANY|
|Welte, W - UNIV KONSTANZ GERMANY|
|Lutz, G - UNIV KONSTANZ GERMANY|
|Papoyan, A - CORNELL UNIVERSITY|
|Kupper, H - UNIV KONSTANZ GERMANY|
Submitted to: Biochemical and Biophysical Research Communications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 1, 2007
Publication Date: January 18, 2008
Citation: Parameswaran, A., Leitenmaier, B., Yang, M., Kroneck, P., Welte, W., Lutz, G., Papoyan, A., Kochian, L.V., Kupper, H. 2008. A native Zn/Cd transporting P1B ATPase from natural overexpression in a hyperaccumulator plant reveals post-translational processing. Biochemical and Biophysical Research Communications. 363:51-56. Interpretive Summary: Heavy metal contamination of soils poses serious problems worldwide, and the current technologies used to remediate soils are costly and disruptive. There is considerable interest in the use of terrestrial plants to clean up heavy metals from the soil. Several metal hyperaccumulating plant species have been identified that tolerate highly contaminated soils and accumulate these metals to high concentrations. We have been studying the mechanisms for metal hyperaccumulation in Thlaspi caerulescens, a zinc/cadmium (Zn/Cd)hyperaccumulator. We had previously shown that in Thlaspi, a specialized transporter known as HMA4 (where HMA stands for heavy metal ATPase, which is the family of transporters HMA4 belongs to)plays an important role in metal hyperaccumulation. This transporter appears to be involved in the transfer of heavy metals from the root to the shoot, which is a key aspect of metal hyperaccumulation. In the current study, we developed techniques that enabled us to purify the HMA4 protein and showed that during the process of expression of the HMA4 gene and subsequent synthesis of the HMA4 protein, modifications occur that split the protein in two pieces. This study sets the stage for investigation of the transport processes for HMA4 via study of the purified protein.
Technical Abstract: TcHMA4 is a P1B-type ATPase that is highly expressed in the Cd/Zn hyperaccumulator plant Thlaspi caerulescens and contains a C-terminal 9-histidine repeat. After isolation from roots, we purified TcHMA4 protein via metal affinity chromatography. The purified protein exhibited Cd- and Zn activated ATPase activity after reconstitution into lipid vesicles, showing that it was in its native state. Gels of crude root extract and of the purified protein revealed 2 TcHMA4-specific bands of about 50 and 60 kDa, respectively, while the TcHMA4 mRNA predicts a single protein with a size of 128 kDa. This indicates the occurrence of post-translational processing; the properties of the 2 bands were characterized by their activity and binding properties.