Interaction of endogenous Pab1p with recombinant eRF3. (A) Full-length eRF3 interacts with endogenous Pab1p. The lysate of strain 33G-D373 (lane 1; 1/30 of the extract used per assay was loaded) was incubated with His-tagged eRF3 proteins (full length [aa 1 to 685; lane 2], 2ATG [aa 124 to 685; lane 3], or 3ATG [aa 232 to 685; lane 4]), with eRF1 (lane 5), or with no recombinant protein (lane 6) as a control. Recombinant eRF3 and associated factors were purified by metal affinity chromatography. Bound proteins were eluted and assayed for Pab1p (upper panel) and then eRF3 (lower panel) by immunoblotting. (B) Association between eRF3 and Pab1p is not affected by RNase treatment. Recombinant eRF3 was incubated with yeast lysate in the presence or absence of RNAses. Bound proteins were eluted and assayed for eRF3 (upper panel) and then for Pab1p (lower panel) by immunoblotting. Numbers at left of panels are molecular masses in kilodaltons.

The N+M part of eRF3 interacts with the C part of yeast Pab1 protein. (A) Two-hybrid interactions. Strain L40 carrying different PAB1 deletions in pBTM116 plasmid (69) was transformed with pGADGH/SUP35 (FL), pGADGH/SUP35-Bcl (BclI), pGADGH/SUP35-N123 (N123), pGADGH/SUP35-2ATG (2ATG), pGADGH/SUP35-3ATG (3ATG) (see Materials and Methods), and pACT2/SUP35N (N113) (4). The interactions were tested by the extent of resistance to 3-AT and by an X-Gal filter-lifting assay. Symbols for β-galactosidase assay: ++++, very strong; +++, strong; ++, moderate; +, weak; −, no activity. For the 3-AT assay the highest concentration of 3-AT that still allowed yeast cell growth is noted. Extent of β-galactosidase activity is shown in parentheses. At least five independent transformants were tested in each case. (B) Minimal domain of Pab1p participating in the interaction with yeast eRF3. Strain HF7C was cotransformed with pGADGH/SUP35 (FL), pGADGH/SUP35-Bcl (BclI), pGADGH/SUP35-N123 (N123), pGADGH/SUP35-2ATG (2ATG), pACT2/SUP35N (N113), pGBT9/PAB1 C (C), and pGBT9/PAB1 CΔ24 (ΔC24). The extent of 3-AT resistance is shown; −His corresponds to the growth on His⁻ medium. (C) Minimal domain of eRF3 participating in the interaction with the C-terminal domain of Pab1p. Strain HF7C bearing pGADGH/PAB1 P+C plasmid was transformed with pAS-SUP35N (N113), pAS-SUP35N (N113) Δ22-69 (N-Δ22-69), or pAS/SUP35N-PNM2 (N-PNM2). The interactions were tested in the same way as for panel B. (D) Interactions of yeast Pab1p with human and Xenopus eRF3 in the two-hybrid system. Strain HF7C bearing pGADGH/PAB1 P+C plasmid was transformed with plasmids pGBT9/hGSPT1 (human FL), pAST-eRF3 h-N (human N), and pGBT9/xeRF3 (Xenopus FL), and the resulting cotransformants were assayed for interactions in the same way as for panel B.

Antisuppressor effect of overexpressed PAB1 in the strain bearing the sup35-21 mutation. (A) The 2-33G-D373 strain was transformed with plasmid pSTR7 bearing the wild-type SUP35 gene (eRF3), the vector pFL44 alone (vector), pFL44/PAB1 bearing the PAB1 gene (Pab1p), and pFL44/PAB1ΔC (Pab1pΔC) encoding the C-terminally deleted Pab1p (containing aa 1 to 472). Growth was assessed under permissive (25°C) and nonpermissive (37°C) conditions on the rich medium (YPD) and on the YPD medium containing 0.5 mg of paromomycin (Par)/ml after 5 days of incubation at 25°C. Drops of yeast suspension of the same density were used. Five independent transformants with each plasmid were tested, and representative results are shown. (B) Antisuppressor effect of overexpressed Pab1p. Strain 2-33G-D373 was cotransformed with plasmids bearing the indicated stop codon in frame with the lacZ coding sequence in combination with pYX242 (vector) or pYX242/PAB1 (Pab1p) plasmid. Percent misreading was quantified as a ratio of β-galactosidase activity in cells harboring lacZ with the stop codon to that in cells harboring lacZ (wt). Results are the means of at least three separate experiments. (C) Basal and overexpressed level of Pab1p. Lysates prepared from the 2-33G-D373 strain transformed with pYX242 (vector) or pYX242/PAB1 (Pab1p) were analyzed by immunoblotting using anti-Pab1p or anti-α-tubulin antibodies. Pab1 protein encoded by pYX242/PAB1 is 8 aa longer than endogenous Pab1p. Numbers at left are molecular masses in kilodaltons. (D) Overexpression of Pab1p does not promote decay of nonsense-containing mRNAs. Total RNA was isolated from 2-33G-D373[pYX242/PAB1] cells harboring the plasmid with lacZ (wt) (WT) or bearing the indicated stop codon. Twenty-five micrograms of RNA was separated on a 1% agarose gel and subjected to quantitative Northern blot analysis. Following hybridization with the lacZ probe, the blot was reprobed for actin to standardize for loading differences. The lacZ/actin ratio was calculated for each sample.

Pab1p has an antisuppressor effect on [PSI⁺]-mediated suppression. (A) [PSI⁺] causes sensitivity to paromomycin at 18°C. The sensitivity of the BSC783/4a [PSI⁺] strain and the resistance of its [psi⁻] derivative to different concentrations of paromomycin (milligrams per milliliter) at 18°C were assayed. Drops of yeast suspensions with the same cell concentration were placed on YPD medium with paromomycin and incubated at 18°C for 8 days. (B) Distribution of eRF3 and Pab1p between the soluble and aggregated forms. Lysates from BSC783/4a [PSI⁺] and [psi⁻] strains were fractionated through a sucrose cushion. Cytosol (C), sucrose fraction (S), and pellet (P) were analyzed by immunoblotting. (C) Overexpression of Pab1p restores the paromomycin resistance of the [PSI⁺] strain at 18°C. Strain BSC783/4a [PSI⁺] was transformed with plasmid pSTR7 bearing the wild-type SUP35 gene (eRF3), the vector pFL44 alone (vector), pFL44/PAB1 bearing the PAB1 gene (Pab1p), and pFL44/PAB1ΔC (Pab1pΔC) encoding Pab1p with the C-terminal region deleted (containing aa 1 to 472). The sensitivity to paromomycin was tested in the same way as for panel A. (D) Pab1p overexpression does not lead to eRF3 solubilization from high-molecular-weight aggregates. The eRF3 and Pab1p distribution of transformants described for panel C was performed as described for panel B. (E) Antisuppressor effect of overexpressed PAB1. BSC783/4a [PSI⁺] was cotransformed with plasmids bearing the wild-type copy of the lacZ gene or with lacZ (UAA), lacZ (UAG), or lacZ (UGA) in combination with pYX242 or pYX242/PAB1 plasmid. Percent misreading was quantified as a ratio of β-galactosidase activity in cells harboring lacZ (UAA) to that in cells harboring lacZ (wt). Results are the means of at least three separate experiments. Numbers at right of panels B and D are molecular masses in kilodaltons.

Excess Pab1p acts against phenotypic suppression. Strain 33G-D373 was transformed with pYX242 and pYX242/PAB1 plasmids. Drops of yeast suspensions with the same cell concentration were plated on selective medium with paromomycin and incubated at 25°C. The His⁺ phenotype indicated phenotypic suppression, and the Leu⁺ phenotype confirmed the plasmid transformation. Ten independent transformants with each plasmid were tested. Representative results are shown.

Network of interactions between molecules interacting with eRF3 or involved in RNA stability or degradation and translation (initiation and termination). Most of the interactions shown here were demonstrated between two molecules examined separately. Further analysis must be carried out in vivo to determine whether these interactions occur singly or together. Isolation and analysis of complexes have to be carried out under precise and well-defined conditions for translation and RNA stability or degradation.

Secondary structure prediction for the PABP-interacting region in various eRF3 homologues. Alignment of the N-terminal parts of Xenopus (xeRF3, aa 51 to 87), human (hGSPT1, aa 48 to 85), mouse (mGSPT2, aa 35 to 76), and yeast (yeRF3, aa 109 to 149) eRF3 proteins was performed with the MULTALIN (17) program (http://npsa-pbil.ibcp.fr). The PABP-interacting consensus sequence (LNVNAKPFVP) (57) is shown in boldface. Sec, secondary structure predicted by SOPM algorithm (37); c, random coil; e, extended strand; t, beta turn. The α-helical region (h) is underlined.