Dom34-Hbs1 mediated dissociation of inactive 80S ribosomes promotes restart of translation after stress

Abstract

Following translation termination, ribosomal subunits dissociate to become available for subsequent rounds of protein synthesis. In many translation-inhibiting stress conditions, e.g. glucose starvation in yeast, free ribosomal subunits reassociate to form a large pool of non-translating 80S ribosomes stabilized by the 'clamping' Stm1 factor. The subunits of these inactive ribosomes need to be mobilized for translation restart upon stress relief. The Dom34-Hbs1 complex, together with the Rli1 NTPase (also known as ABCE1), have been shown to split ribosomes stuck on mRNAs in the context of RNA quality control mechanisms. Here, using in vitro and in vivo methods, we report a new role for the Dom34-Hbs1 complex and Rli1 in dissociating inactive ribosomes, thereby facilitating translation restart in yeast recovering from glucose starvation stress. Interestingly, we found that this new role is not restricted to stress conditions, indicating that in growing yeast there is a dynamic pool of inactive ribosomes that needs to be split by Dom34-Hbs1 and Rli1 to participate in protein synthesis. We propose that this provides a new level of translation regulation.

Figure 1

Dom34 stimulates restart of translation in yeast recovering from glucose starvation stress. A, B Dom34 stimulates the rapid reappearance of polysomes in cells recovering from glucose starvation stress. Polysome profiles of wild-type (A) or dom34Δ (B) yeast grown in glucose-rich medium (left graph), after 10 min of glucose starvation (second graph), and 5 and 30 min after glucose addition (third and fourth graph) at 16°C. C Quantification of 80S over polysome ratios, corresponding to the first, second and fourth graphs in (A) and (B). Means ± standard deviations of two independent experiments are shown. D, E Dom34 stimulates protein production in cells recovering from glucose starvation stress. Wild-type (D) and dom34Δ (E) yeast depleted of glucose or grown in glucose-rich medium for 10 min at 16°C was resuspended in glucose-rich medium containing ³⁵S-methionine, followed by incubation at 16°C. ³⁵S-methionine incorporation was measured at the indicated time points. Means ± standard deviations of 3 independent experiments are shown.

Figure 2

Dom34-Hbs1 and Rli1 participate in the dissociation of inactive, Stm1-containing 80S ribosomes.

1. Dom34-Hbs1 and Rli1 dissociate ribosomes from glucose-starved yeast in vitro. ³²P-labeled 80S ribosomes purified from glucose-starved yeast were incubated with the indicated proteins in the presence of ATP and GTP or GDPNP. After 15 min of incubation dissociation was monitored by sucrose density gradient centrifugation and scintillation counting of collected fractions.

2. Observed rate constants were determined by monitoring the fraction of dissociated ribosomes in (A) over time on a native gel system (see Supplementary Fig S2). Rate constants were normalized for endpoints. Means ± standard deviations of three independent experiments are shown.

Figure 3

Weakening ribosome subunit interaction reduces the need for Dom34 during restart of translation after glucose starvation stress. A, B Polysome profiles of stm1Δ (A) and dom34Δstm1Δ (B) yeast grown in glucose-rich medium (left graph), exposed to glucose starvation (second graph), and 5 and 30 min after glucose readdition (third and fourth graphs) at 16°C. C Quantification of 80S over polysome ratios, corresponding to the first, second and fourth graphs in (A) and (B) as well as in Fig 1A and B. Means ± standard deviations of two independent experiments are shown.

Figure 4

Restart of translation after glucose depletion stress requires Hbs1 GTPase activity but not Dom34-Hbs1 interaction or the Hbs1 N-terminus. A, B Polysome profiles of hbs1Δ (A) or dom34 Δ (B) yeast transformed with plasmid expressing the indicated mutants, grown in glucose-rich medium (left graph), exposed to glucose starvation (middle graph) and after glucose readdition (right graph) at 16°C.

Figure 5

The Dom34-Hbs1 complex stimulates translation in non-stress-related conditions. A, B Inactive 80S ribosomes accumulate in dom34Δ yeast. Polysome profiles were obtained from wild-type and dom34Δ yeast in low-salt (100 mM KCl) (A) and high-salt (400 mM KCl) (B) conditions. Yeast strains were grown at 30°C. C Quantification of 80S over polysome ratios corresponding to (A) and (B). Means ± standard deviations of three independent experiments are shown. The indicated significance level was calculated using a Mann–Whitney U-test. D Dom34-Hbs1 stimulates translation by ribosomes that were not exposed to starvation stress. A firefly luciferase mRNA was translated for 1 h in cell extract obtained from a dom34Δhbs1Δ strain, after which luciferase activity was measured. Addition of increasing amounts of recombinant Dom34-Hbs1 complex, but not of Hbs1 or Dom34 alone, stimulated luciferase production. Means and SDs of 3 independent experiments are shown. * indicates that at these time points the result obtained in presence of both Dom34 and Hbs1 significantly differ (P < 0.001) from the result obtained adding Dom34 or Hbs1 alone (Student's t-test).