Minisymposium 13: Heavy metals

Abs # 26002: Functional role of HMA2, a Zn transporting P-type ATPase

Presenter:	Argüello, José M., arguello@wpi.edu
Authors	Eren, Elif  (A)   Orofino, María J. (A)   Argüello, José M. (A)
Affiliations:	(A): Dept. of Chemistry and Biochemistry, Worcester Polytechnic Institute
Web Site:	http://users.wpi.edu/~arguello/

P_IB-type ATPases transport heavy metal ions (Cu⁺, Cu²⁺, Zn²⁺, Cd²⁺, Co²⁺, etc.) across biological membranes. In Arabidopsis thaliana, there are eight genes (HMA1-8) encoding for members of this subfamily. Previous studies using plant gene knock-out and yeast functional complementation have shown that HMA6 (PAA1) and HMA7 (RAN1) are involved in Cu homeostasis. HMA4 and HMA3, two putative Zn²⁺-ATPases confer resistance to Cd²⁺ in yeast. Higher plants appear to be the only eukaryotes where Zn²⁺-ATPases are present. Analysis of conserved amino acids in the transmembrane region (i.e., likely involved in metal binding) suggests that HMA2 is also a Zn²⁺-ATPase. To understand its functional role, we have cloned HMA2, expressed it in yeast and, using membrane preparations, determined its enzymatic properties. HMA2 is a Zn²⁺-ATPase also activated by Cd²⁺ and to a lesser extent by other divalent heavy metals (Pd²⁺, Ni²⁺, Cu²⁺). HMA2 interacts with Zn²⁺ and Cd²⁺ with high affinity (Zn²⁺ K_1/2=0.11 µM and Cd²⁺ K_1/2 = 0.031 µM). However, its activity is dependent on millimolar concentrations of cysteine in the assay media. As all P-type ATPases, in the presence of the outwardly transported metal and ATP, HMA2 forms an acid stable phosphorylated intermediate. Similarly, the enzyme is inhibited by vanadate. In agreement with phosphorylation measurements, Zn²⁺ transport determinations indicate that HMA2 drives the outward transport of metals from the cell cytoplasm. To evaluate the role of HMA2 in plant, we measured its transcript levels in various tissues and its regulation in response to metal exposure. This showed that the enzyme is likely present in all plant organs and its transcript levels do not change after exposure to various metals. Further evidence of HMA2 role in Zn²⁺ homeostasis was provided by hma2-2 and hma2-3 plants carrying two mutant alleles of the gene (T-DNA inserts). No morphological or developmental alteration was observed in these plants. However, removal of HMA2 transcript results in Zn²⁺ accumulation in plant tissues. hma2 plants also accumulate Cd²⁺ when exposed to this metal. These results suggest that HMA2 is a housekeeping enzyme located in the plasma membrane and responsible for Zn²⁺ efflux from the cells. Moreover, because of its broad metal specificity, HMA2 might also play a key role preventing high levels of non-physiological heavy metals.