title: The N-terminal degenerated metal-binding domain is involved in the heavy metal transport activity of TaHMA2.
abstract: We identified key residues of TaHMA2, and the N- and C-terminal regions of the protein have different roles in its transport function when heterologously expressed in yeast. TaHMA2, a P1B-type ATPase from wheat (Triticum aestivum L.), plays an important role in heavy metal homeostasis in plants. A previous study showed that overexpressing TaHMA2 in rice (Oryza sativa L.), Arabidopsis thaliana, or tobacco (Nicotiana tabacum L.) resulted in various responses to heavy metals. Here, we report the heterologous expression of TaHMA2 in the yeast Saccharomyces cerevisiae. TaHMA2 expression increased the yeast's sensitivity to Cd, but not to Zn, Pb or Co, and increased Cd accumulation was concurrently observed. The eGFP-TaHMA2 fusion protein was localized to the plasma membrane and showed a discontinuous pattern. Mutagenesis of the cysteine and glutamate residues in the N-terminal metal-binding domain (N-MBD) impaired the function of TaHMA2. Deletion of most of the C terminus (TaHMA2ΔC, 712-1003) partially abolished the protein's function, whereas deletion of the N terminus (TaHMA2ΔN, 2-699) completely abolished Cd sensitivity. These data suggest that cysteine and glutamate residues are important for the metal-binding/translocation function of TaHMA2. Additional studies are needed to further understand the selectivity of TaHMA2 in planta.
authors.affiliation: College of Life Science, University of the Chinese Academy of Sciences, Beijing, China.
authors.forename: Shuqin
authors.initials: S
authors.lastname: Xiang
authors.forename: Shanshan
authors.initials: S
authors.lastname: Feng
authors.forename: Yuxiu
authors.initials: Y
authors.lastname: Zhang
authors.forename: Jinjuan
authors.initials: J
authors.lastname: Tan
authors.forename: Shuang
authors.initials: S
authors.lastname: Liang
authors.forename: Tuanyao
authors.initials: T
authors.lastname: Chai
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001692:Biological Transport; D002104:Cadmium / Q000633:toxicity; D002462:Cell Membrane / Q000378:metabolism; D019216:Metals, Heavy / Q000378:metabolism*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism; D016415:Sequence Alignment; D013054:Spectrophotometry, Atomic; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000378:metabolism; D014908:Triticum / Q000378:metabolism*
chemical_list: D019216:Metals, Heavy; D010940:Plant Proteins; D002104:Cadmium