Expression of the type 2 metallothionein-like gene MT2 from Arabidopsis thaliana in Zn(2+)-metallothionein-deficient Synechococcus PCC 7942: putative role for MT2 in Zn2+ metabolism.
Zn2+ proteins pervade metabolism and are essential for gene expression. However, no proteins have been ascribed the central roles of Zn2+ donation to, or removal from, metalloproteins, or Zn2+ storage in vegetative plant tissue. In animals, such functions have been proposed for metallothioneins. Plants contain multiple metallothionein-like genes but their predicted products, which differ significantly from animal metallothioneins, remain to be isolated from vegetative tissue and their roles are uncertain. The type 2 metallothionein-like gene from Arabidopsis, MT2, was expressed under the control of Zn2+-responsive elements derived from the cyanobacterial metallothionein divergon, smt. Zn2+-dependent expression of MT2 transcripts in Synechococcus PCC 7942 was confirmed by northern analysis. The Arabidopsis MT2 gene partly complemented Zn2+ hypersensitivity in mutants of Synechococcus PCC 7942 which are functionally deficient in an endogenous Zn2+-metallothionein gene, smtA. MT2 was also expressed as a recombinant fusion protein in Escherichia coli, purified and shown to bind Zn2+ in vitro. The mean pH of half displacement of Zn2+ from MT2 was estimated to be 5.05. This suggests that MT2 has a greater affinity for Zn2+ than phytochelatins. The results presented here reveal that MT2 is capable of binding Zn2+ in vitro, conferring tolerance to elevated [Zn2+] in vivo within cyanobacteria and is likely to compete with other polypeptides for cellular Zn2+ in planta.
Robinson
N J
NJ
Department of Biochemistry and Genetics, Medical School, University of Newcastle, Newcastle-upon-Tyne, UK.
Wilson
J R
JR
Turner
J S
JS
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000235:genetics*; D018076:DNA, Complementary; D005982:Glutathione Transferase / Q000235:genetics; D006859:Hydrogen; D008668:Metallothionein / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000378:metabolism*
D029681:Arabidopsis Proteins; D018076:DNA, Complementary; C100789:MT2A protein, Arabidopsis; D011993:Recombinant Fusion Proteins; C021267:zinc thionein; D006859:Hydrogen; D008668:Metallothionein; D005982:Glutathione Transferase; D015032:Zinc