Functional divergence between the two P1-P2 stalk dimers on the ribosome in their interaction with ricin A chain

Abstract

The eukaryotic stalk, which is responsible for the recruitment of translation factors, is a pentamer containing two P1-P2 dimers with unclear modes of action. In Saccharomyces cerevisiae, P1/P2 proteins (individual P1 and P2 proteins) are organized into two distinct dimers, P1A-P2B and P1B-P2A. To investigate the functional contribution of each dimer on the ribosome, RTA (ricin A chain), which binds to the stalk to depurinate the SRL (sarcin/ricin loop), was used as a molecular probe in yeast mutants in which the binding site for one or the other dimer on P0 was deleted. Ribosome depurination and toxicity of RTA were greatly reduced in mutants containing only P1A-P2B on the ribosome, whereas those with only P1B-P2A were reduced less in depurination and were unaffected in toxicity. Ribosomes bearing P1B-P2A were depurinated by RTA at a similar level as wild-type, but ribosomes bearing P1A-P2B were depurinated at a much lower level in vitro. The latter ribosomes showed the lowest association and almost no dissociation with RTA by surface plasmon resonance. These results indicate that the P1B-P2A dimer is more critical for facilitating the access of RTA to the SRL, providing the first in vivo evidence for functional divergence between the two stalk dimers on the ribosome.

Figure 1. Analysis of the ribosomal stalk in the yeast mutants

(A) Schematic representation of the yeast stalk mutants. (B) Immunoblot analysis of the yeast stalk mutants. Ribosomes (10 pmol) and the cytosolic fraction (40 µg of total protein) isolated from the P0_ΔH1(P1B–P2A), and P0_ΔH2(P1A–P2B) mutants and the isogenic wild-type were analysed by immunoblot analysis using monoclonal antibody against the C-termini of P-proteins (IB3B) to detect the P0 protein. Monoclonal antibodies against P2A (IBE3) and P2B (IAA9) were used to detect P2A and P2B respectively. Anti-L3 and anti-Pgk1 antibodies were used as loading controls for the ribosome and cytosol fractions respectively. The immunoblot analysis was repeated three times using different ribosome preparations. (C) Growth of yeast stalk mutants on minimal medium supplemented with 2 % glucose.

Figure 2. Analysis of viability, expression and ribosome depurination by RTA in the yeast stalk mutants

(A) Viability of yeast stalk mutants expressing RTA was measured by plating serial dilutions on galactose plates. Yeast cells transformed with a plasmid carrying gene encoding pre-RTA under the GAL1 promoter were first grown SD medium supplemented with 2% glucose and then serially diluted and plated on to SD medium supplemented with 2% galactose and grown at 30°C for 3 days. Cells carrying the VC (empty vector) were used as controls. (B) Immunoblot analysis of RTA expression in yeast cells at 6 and 12 hpi. Total protein extracted from equal amount of cells (D₆₀₀ of 0.8) was analysed by immunoblot analysis using monoclonal antibodies (PB10) against RTA and IR-labelled secondary antibodies. The blot was visualized using a LI-COR IR imaging system. Monoclonal antibodies against Dpm1 were used as the loading control. (C) Depurination activity of RTA in the yeast stalk mutants determined by qRT-PCR analysis. Total RNA was purified from D₆₀₀ values of 0.3–0.8 of yeast cells using Qiagen RNeasy Mini Kit with on-column DNase digestion. Total RNA (375 ng) was used for cDNA synthesis. The relative level of depurination in cells expressing RTA was measured by qRT-PCR in comparison with cells containing the VC by the ΔΔC_T method [59]. The fold increase in depurination in yeast expressing RTA compared with yeast harbouring the VC is shown. The experiment was repeated three times, the error bars show S.D.

Figure 3. Interaction of RTA with ribosomes from the stalk mutants

(A) Interaction of RTA with ribosomes isolated from wild-type and stalk mutants was analysed with a Biacore T200 system at a 1 nM ribosome concentration. (B) Interaction of RTA with the stalk mutant ribosomes at a 1 nM concentration. (C) Interaction of RTA with the stalk mutant ribosomes at a 5 nM concentration. The interactions were measured using a Biacore T200 system with a CM3 chip. RTA was immobilized to Fc2 at 840 RU by amine coupling. Fc1 was activated and blocked as a control. Ribosomes from wild-type yeast or the stalk mutants were passed over both surfaces at 40 µl/min. Running buffer consisted of 10 mM Hepes, pH 7.6, 150 mM NaCl, 10 mM magnesium acetate, 5 µM EDTA and 0.005% surfactant P20.

Figure 4. Depurination of ribosomes from the stalk mutants

Depurination of ribosomes isolated from the stalk mutants by (A) RTA or (B) PAP. Different concentrations of RTA or PAP were used in a reaction mixture containing 40 nM yeast ribosomes and reaction buffer (10 mM Tris/HCl, pH 7.4,60 mM KCl and 10 mM MgCl₂) in a total reaction of 100 µl for 10 min at 30 °C. The extent of depurination was determined by qRT-PCR in comparison with no toxin control used by the ΔΔC_T as described previously [59]. The experiment was repeated three times, the error bars show S.D.