In this study, we demonstrated that yeast lacking the ribosome-associated protein Stm1p exhibited increased sensitivity to specific protein synthesis inhibitors, e.g. anisomycin and cycloheximide, especially when propagated in minimal medium. The effect of STM1 deletion on the sensitivity of the cells to anisomycin provides good in vivo evidence of a role for Stm1p in translational elongation. We also found that ribosomes lacking Stm1p had elevated levels of the yeast-specific elongation factor eEF3 associated with them as compared with wild-type ribosomes. In addition, we found that when ribosomes had elevated levels of associated Stm1p, they exhibited decreased levels of eEF3 association. Similarly, overexpression of eEF3 in cells lacking Stm1p resulted in an enhanced growth defect, which notably correlated strongly with elevated levels of eEF3 associated with ribosomes. Taken together, these genetic and biochemical data suggest that Stm1p is important for eEF3 function, presumably by affecting the proper association of eEF3 with 80S ribosomes.
One of the best ways of determining how Stm1p affects translation in vivo
is to investigate Stm1p-dependent changes in ribosome distribution following sucrose gradient ultracentrifugation. We observed that the absence of Stm1p caused a notable increase in polysomes, which was exacerbated by eEF3 overexpression. Similarly, depletion or inactivation of the yeast translation factor eIF5A was shown to cause a pronounced increase in polysomes and inhibit translation elongation (37
). Given that our methionine incorporation experiments indicated decreased protein synthesis in stm1
Δ yeast that was exacerbated by eEF3 overexpression, these data strongly support the contention that the absence of Stm1p affects translation elongation in a cooperative fashion with eEF3. In addition, overexpression of Stm1p caused both an increase in heavy polysomes and a decrease in protein synthesis. Such is reminiscent of human ribosomal S5 protein expression in yeast, which demonstrated an increase in heavy polysomes and a decrease in eEF3-ribosome interactions, thereby affecting translation elongation (39
). Thus, there may be an ideal range of Stm1p and eEF3 concentrations that permit optimal translation elongation.
Previously, we found that Stm1p exists in a 1:1 complex with 80S ribosomes and interacts with both the 40S and 60S ribosomal subunits (9
). The interface between ribosomal subunits is well-known to be very important for ribosomal function, as it is the interaction site for aminoacyl-tRNA and the canonical elongation factors eEF1A and eEF2. As mutations or deletions of genes directly involved in translation elongation usually lead to a lethal phenotype or substantial alterations in protein synthesis (40–45
), we postulate that Stm1p most likely does not have an overlapping interaction site with eEF1A and eEF2. Cryo-electron microscopy of the eEF3-80S ribosome complex has indicated that eEF3 interacts with both the large and small ribosomal subunits but at a different site than those recognized by eEF1A and eEF2 (27
). The fact that eEF3 has a completely different binding site from the canonical elongation factors is consistent with the observation that in the absence of Stm1p more eEF3 associates with ribosomes. It is of interest to note that sequence alignments between S. cerevisiae
eEF3 (Yef3p) and Stm1p show some degree of sequence similarity, especially in the C-terminal regions of each protein (Pickering,B., unpublished data). However, this sequence similarity is not conserved in homologs of these proteins found in other yeasts and lower fungi. In fact, the most significant sequence homology between these different eEF3 and Stm1p proteins maps to the C-terminal end of eEF3, which is rich in arginine and lysine residues, and basic patches within the C-terminal region of Stm1p. Thus, it is tempting to speculate that eEF3 and Stm1p share overlapping ribosome-binding sites recognized by these basic domains.
Translocation of tRNAs in yeast ribosomes during translation elongation requires two canonical elongation factors, eEF1A and eEF2 (14
). eEF1A facilitates cognate aminoacyl-tRNA binding to the A-site in 80S ribosomes. Following the peptidyl transferase reaction, eEF2 facilitates the translocation of deacylated tRNA from the P- to the E-site and aminoacyl-tRNA from the A- to the P-site. Afterwards, eEF3, which is thought to interact with 80S ribosomes near the E-site, facilitating dissociation of deacyl-tRNA from the ribosome (23
). These findings are consistent with the allosteric model of translation, whereby release of tRNA from the E-site in turn facilitates occupancy of the A-site with cognate aminoacyl-tRNA, thereby permitting efficient translation elongation. Andersen et al.
) have proposed a detailed model for the specific role of eEF3 in the translation elongation cycle (). This model involves the following steps: (i) weak binding of eEF3 to post-translocation ribosomes with the deacyl-tRNA ‘locked’ in the ribosome E-site by an ‘in’-position L1 stalk and the 40S head; (ii) a conformational change in eEF3, which is caused by ATP binding, which results in higher-affinity ribosome binding and the repositioning of the L1 stalk to an ‘out’ position; and (iii) eEF3 conformation-promoted ATP hydrolysis, resulting in eEF3 dissociation from the ribosome, E-site opening and unlocking of the 40S head. This last step allows the eEF1A–GTP–aminoacyl-tRNA complex to bind to the ribosomal A-site and deacyl-tRNA to be released from the E-site, allowing a subsequent round of elongation to ensue. Our data are consistent with a model in which ribosome-bound Stm1p tempers binding of eEF3 to 80S ribosomes. This could occur through Stm1p directly competing with eEF3-ribosome binding, promoting eEF3 ATPase activity and/or facilitating an eEF3 or ribosome conformational change that permits efficient dissociation of eEF3 from ribosomes following ATP hydrolysis. In the absence of Stm1p, eEF3 may not efficiently dissociate from ribosomes, potentially leading to an unproductive cycle of ATP binding, eEF3 conformational change and ATP hydrolysis. The failure of eEF3 to dissociate from the ribosome could prevent the release of deacyl-tRNA from the E-site and binding of eEF1A–GTP–aminoacyl-tRNA to the A-site, thereby retarding these final steps in the cycle of translation elongation. In addition, these effects are likely be exacerbated when concentrations of deacyl-tRNA are elevated and/or aminoacyl-tRNAs are depressed, exactly the circumstances that occur during nutrient deprivation (14
). Similarly, given that the net effect of anisomycin on protein synthesis is to block aminoacyl-tRNA access to the ribosome peptidyltransferase center (32
), this provides an explanation for the observed increased sensitivity of yeast lacking Stm1p or overexpressing eEF3 to this particular antibiotic. We believe that Stm1p, when bound to the ribosome, facilitates the release of the ATP-hydrolyzed conformation of eEF3, thereby permitting efficient translation elongation. However, high concentrations of Stm1p could also inhibit eEF3-80S ribosome binding and thereby reduce translation elongation, which we observed. The exact mechanism by which Stm1p binds to the ribosome and affects the function of eEF3 awaits further structural data and is currently under investigation.
Figure 5. Role of Stm1p in translation elongation. Shown is a model of the yeast elongation cycle. Starting at the top left, Stm1p at high concentrations can inhibit the association of eEF3 (green ellipse) to post-translocation ribosomes. After ATP binding, which (more ...)