Deletion analysis of the SUP35 gene of the yeast Saccharomyces cerevisiae reveals two non‐overlapping functional regions in the encoded protein

Abstract

SUP35 is an omnipotent suppressor gene of Saccharomyces cerevisiae coding for a protein consisting of a C-terminal part similar to the elongation factor EF-1 alpha and a unique N-terminal sequence of 253 amino acids. Twelve truncated versions of the SUP35 gene were generated by the deletion of fragments internal to the coding sequence. Functional studies of these deletion mutants showed that: (i) only the EF-1 alpha-like C-terminal part of the Sup35 protein is essential for the cell viability; (ii) overexpression of either the N-terminal part of the Sup35 protein or the full-length Sup35 protein decreases translational fidelity, resulting in omnipotent suppression and reduced growth of [psi+] strains; (iii) expression of the C-terminal part of the Sup35 protein generates an antisuppressor phenotype; and (iv) both the N- or C-terminal segments of the Sup35 protein can bind to 80S ribosomes. Thus, the data obtained define two domains within the Sup35 protein which are responsible for different functions.

[PSI+] prion generation in yeast: characterization of the ‘strain’ difference

“…A series of pEMBL-yex4-based plasmids (Cesareny and Murray, 1987) containing either the complete SUP35 gene or its 3k-deletion alleles (Figure 1) has been described previously (Ter-Avanesyan et al, 1993). The pUKC815 plasmid carrying the PGK±lacZ gene fusion, and pUKC817, which is a pUKC815 derivative carrying an in-frame TAA termination codon at the junction of the PGK and lacZ genes, were described by Stans®eld et al (1995).…”

“…Sup35p is a yeast member of the eRF3 family of translation termination factors (Zhouravleva et al, 1995;Stans®eld et al, 1995) and is composed of the amino-terminal region and carboxy-terminal (C) domain of 253 and 432 amino acids, respectively (Kushnirov et al, 1988;Ter-Avanesyan et al, 1993, 1994. The evolutionarily conserved C domain of Sup35p is responsible for its function in translation termination and is essential for cell viability, while the N-terminal region is neither conserved nor essential.…”

The ribosome-associated complex antagonizes prion formation in yeast

“…4B, top panels), consistent with previous reports. 21,59 However, in cells lacking RAC function, expression of either 62Q or Sup35NM substantially inhibits growth, and toxicity is exacerbated by [PSI C ] (Fig. 4B, bottom panels).…”

Dominant Gain-of-Function Mutations in Hsp104p Reveal Crucial Roles for the Middle Region

“…The change between these states is regulated by interactions between the N-terminal domain and Hsp104p (Patino et al, 1996;Schirmer and Lindquist, 1997;Cashikar et al, 2002). When Sup35p prion conversion is too efficient it can be toxic (Ter-Avanesyan et al, 1993;Derkatch et al, 1996;Li and Lindquist, 2000), because the essential translation-termination activity of the C-terminal domain is inhibited when the protein is in the [PSI ϩ ] state. Thus, it is possible that the gain-of-function mutant HSP104 phenotypes were due to excess conversion of Sup35p to the prion state.…”

“…Sup35p, a translation-termination factor, is the protein determinant of the yeast prion [PSI ϩ ] (reviewed in Serio and Lindquist, 2000). The C-terminal region of Sup35p contains the essential translation activity (Ter-Avanesyan et al, 1993), and the N-terminal region confers upon Sup35p the capacity to assume distinct prion and nonprion conformations. The change between these states is regulated by interactions between the N-terminal domain and Hsp104p (Patino et al, 1996;Schirmer and Lindquist, 1997;Cashikar et al, 2002).…”