Itt1p, a novel protein inhibiting translation termination in Saccharomyces cerevisiae

Abstract

Background: Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRFl and eRF3. eRFl recognizes nonsense codons UAA, UAG and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRFl - dependent manner. Recent studies has shown that proteins interacting with these release factors can modulate the efficiency of nonsense codon readthrough.

Results: We have isolated a nonessential yeast gene, which causes suppression of nonsense mutations, being in a multicopy state. This gene encodes a protein designated Itt1p, possessing a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes. Overexpression of Itt1p decreases the efficiency of translation termination, resulting in the readthrough of all three types of nonsense codons. Itt1p interacts in vitro with both eRFl and eRF3. Overexpression of eRFl, but not of eRF3, abolishes the nonsense suppressor effect of overexpressed Itt1p.

Conclusions: The data obtained demonstrate that Itt1p can modulate the efficiency of translation termination in yeast. This protein possesses a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes, and this is a first observation of such protein being involved in translation.

Figure 1

A comparison of ammo acid sequences of the Itt1 protein of S. cerevisiae and its best homologues from Schizosaccharomyces pombe (GenBank CAB65614.1), man (androgen receptor activator ARA54, NP_004281.1) and C. elegans (AAB52683.1). Sequences were aligned by introducing gaps (...). Conservative amino acids are highlighted in black (cysteines and histidines) and gray and shown as "consensus". The putative nuclear import signal is underlined. The similarities between the second and third double zinc finger elements are shown by lines. In the third element, the residues presumably involved in zinc binding are numbered. In the earlier assignment [17], these are 1,2, &,$ , 3, 4, 5, 6, with $ corresponding to histidine of some other TRIAD sequences.

Figure 2

The levels of nonsense suppression related to Itt1p overexpression. Suppression efficiency was determined in the strain 1A-H8-B2 (itt1::LEU2) and its derivative, 1A-H8-B2-R (itt1::LEU2 SUP35-C). The strains carried either one of the plasmids YCplac22-ITT1 (centromeric ITT1), YEplac112-ITT1 (multicopy ITT1) or pITT1-SUP45 (multicopy plasmid with both ITT1 and SUP45), in combinations with pUKC815, pUKC817, pUKC818 or pUKC819. The levels of readthrough of UAA (a), UAG (b) or UGA (c) nonsense codons were determined as described in Materials and Methods. Values are the mean of three independent assays ± SD. The expression status of ITT1 and other relevant genes is shown below the graph: Single, ITT1 expressed from centromeric plasmid; Multi, expression of ITT1 or SUP45 from multicopy plasmids; C, chromosomal SUP35-C deletion allele; WT, chromosomal SUP35 or SUP45 wild-type alleles.

Figure 3

Itt1p interacts with both eRF3 and eRF1. All proteins were isolated from E. coli, except eRF3 (S.c.), isolated from S. cerevisiae. The indicated proteins were incubated with GST-Itt1p or GST proteins immobilized on glutathione-Sepharose 4B. Following washing, bound proteins were eluted and analyzed by Western blotting with polyclonal antibodies against eRF3 and eRF1.

Figure 4

Overexpression of eRF1 and N-terminal truncation of eRF3 inhibits nonsense suppressor phenotype of transformants with multicopy ITT1 plasmid. The strain 5V-H19 harboring pairs of plasmids YEplac181-ITTl/YEplacl95, YEplac181/YEplac195, YEplacl81- ITTl/YEplacl95-SUP45 or YEplacl81-ITTl/YEplacl95-SUP35 and the strain 1-5V-H19 (SUP35-C) with YEplac181- ITT1/YEplac19 5 or YEplac181/YEplac195 were grown on medium selective for plasmids, patched on SC-Ade and SC+Ade plates and incubated for three days. The growth of three independently obtained transformants of each strain is shown.