Identification, cloning and characterisation of a novel copper-metallothionein in Tetrahymena pigmentosa. sequencing of cDNA and expression

The protist Tetrahymena pigmentosa accumulates large amounts of metal ions, particularly cadmium and copper. This capability is linked to the inductio n of metallothioneins (MTs), cysteine-rich metal-binding proteins found in protists, plants and animals. The present study focuses on a novel inducibl e MT isoform isolated from Tetrahymena after exposure to a non-toxic dose o f copper. The cDNA sequence was determined utilising the partial peptide se quence of purified protein. The Cu-MT cDNA encodes 96 amino acids containin g 28 cysteine residues (29%) arranged in motifs characteristic of the metal -binding regions of vertebrate and invertebrate MTs. Both the amino acid an d nucleotide sequences differ, not only from other animal MTs, but also fro m the previously characterised Tetrahymena Cd-MT. Both MTs contain the stru ctural pattern GTXXXCKCXXCKC, which may be proposed as a conservative seque nce of Tetrahymena MTs. Cu-dependent regulation of MT expression was also i nvestigated by measuring MT-mRNA and MT levels. MT synthesis occurs very qu ickly and MT contents increase with Cu accumulation. The induction of Cu-MT mRNA is very rapid, with no observable lag period, and is characterised by transient fluctuation, similar to that described for Cd-MT mRNA. The data reported here indicate that, also in the unicellular organism Tetrahymena, two very different MT isoforms, which perform different biological function s, are expressed according to the inducing metal, Cu or Cd.