Tetrahymena Metallothioneins Fall into Two Discrete Subfamilies

Abstract

BackgroundMetallothioneins are ubiquitous small, cysteine-rich, multifunctional proteins which can bind heavy metals.Methodology/Principal FindingsWe report the results of phylogenetic and gene expression analyses that include two new Tetrahymena thermophila metallothionein genes (MTT3 and MTT5). Sequence alignments of all known Tetrahymena metallothioneins have allowed us to rationalize the structure of these proteins. We now formally subdivide the known metallothioneins from the ciliate genus Tetrahymena into two well defined subfamilies, 7a and 7b, based on phylogenetic analysis, on the pattern of clustering of Cys residues, and on the pattern of inducibility by the heavy metals Cd and Cu. Sequence alignment also reveals a remarkably regular, conserved and hierarchical modular structure of all five subfamily 7a MTs, which include MTT3 and MTT5. The former has three modules, while the latter has only two. Induction levels of the three T. thermophila genes were determined using quantitative real time RT-PCR. Various stressors (including heavy metals) brought about dramatically different fold-inductions for each gene; MTT5 showed the highest fold-induction. Conserved DNA motifs with potential regulatory significance were identified, in an unbiased way, upstream of the start codons of subfamily 7a MTs. EST evidence for alternative splicing in the 3′ UTR of the MTT5 mRNA with potential regulatory activity is reported.Conclusion/SignificanceThe small number and remarkably regular structure of Tetrahymena MTs, coupled with the experimental tractability of this model organism for studies of in vivo function, make it an attractive system for the experimental dissection of the roles, structure/function relationships, regulation of gene expression, and adaptive evolution of these proteins, as well as for the development of biotechnological applications for the environmental monitoring of toxic substances.

Cloning, characterization, and gene expression analysis of a novel cadmium metallothionein gene in Tetrahymena pigmentosa

“…The Tpig-MT1 gene is unusually long (521 bp), uses TGA as the stop codon, and is intronless. The deduced amino acid sequence of the protein reveals a high degree of internal similarity, as seen for other Tetrahymena MTs (Boldrin et al, 2003;Diaz et al, 2007).…”

“…Thus, it contains the well conserved CCC and CC clusters found in the five previously reported members of the Tetrahymena MT subfamily 7a (Diaz et al, 2007). The common structural pattern GTXXXCKCXXCKC found in TpMT-1, MTT1, and MT-2 genes of the three Tetrahymena species was proposed to be a conserved sequence of Tetrahymena MTs (Boldrin et al, 2003).…”

“…MT or MT-like proteins were reported to be induced by Pb in rats, human, and fish (Ikebuchi et al, 1986;Church et al, 1993;Cheung et al, 2005). The MTT5 gene in T. thermophila showed stronger induction to Pb than Cd (Diaz et al, 2007). Tpig-MT1 mRNA was expressed at its highest level after a short exposure to either Hg or Pb, not Cd (Fig.…”

“…However, since the average GC content of the Tetrahymena genome is 22% and intergenic sequences tend to be even more ATrich (Eisen et al, 2006), such AT-rich motifs are easily found throughout the genome. So far, none of those motifs has been experimentally demonstrated to have a functional role in any gene in any species of this genus, and thus remain hypothetical (Diaz et al, 2007).…”

“…According to their phylogenetic relationships, the pattern of clustering of Cys residues, and which metal inducer they preferentially respond to, they were grouped into Cd-MTs (subfamily 7a) or Cu-MTs (subfamily 7b), showing little sequence identity with one another. The Tetrahymena isoforms so far characterized show unusually longer peptide chains than almost all MTs reported in other organisms and showed only limited similarities with a unique internal homology (Boldrin et al, 2003;Diaz et al, 2007). Previous studies indicated that the two different Tetrahymena-MT polypeptides carry out different biological functions in the process of metal homeostasis and detoxification (Boldrin et al, 2002).…”