A role for theSaccharomyces cerevisiaeABCF protein New1 during translation termination

Abstract

Translation on the ribosome is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related ABCF ATPase, New1, is encoded by a non-essential gene with a cold sensitivity and ribosome assembly defect knock-out phenotype. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide ribosome assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed a combination of yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3.The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 associated ATPases belonging to the ATP-binding cassette (ABC) type F (ABCF) protein family.Eukaryotic representatives of this protein family include initiation factor ABCF1/ABC50 (7,8), elongation factor eEF3 (9,10) as well as Gcn20 -a key component of general amino acid control pathway (11).Elongation factor eEF3 is the most well-studied eukaryotic ribosome-associated ABCF ATPase.While early analyses of the evolutionary distribution of eEF3 concluded that it is a fungi-specific translational factor (12) eEF3-like homologues have more recently been found in non-fungal species, such as oomycete Phytophthora infestans (13), choanoflagellates and various algae (14). eEF3 is essential both for viability of the yeast Saccharomyces cerevisiae (10) and for peptide elongation in a reconstituted yeast translational system (15,16). While biochemical experiments suggest a secondary function for eEF3 in ribosome recycling (17), ribosome profiling analysis of eEF3-depleted S. cerevisiae suggests that translation elongation is the primary function of eEF3 in the cell (18). A cryo-electron microscopy (cryo-EM) reconstruction of eEF3 on the ribosome localizes the factor within the vicinity of the ribosomal E-site (9), providing a structural explanation for the biochemical observations of E-site tRNA displacement from the ribosome in the presence of eEF3 and ATP (19).The S. cerevisiae genome encodes 29 ABC ATPases, of which five belong to the ABCF subfamily (20). Of these, eEF3, Hef3/eEF3B and New1 together form a group with a distinctive subdomain architecture relative to the other ABCFs (14). Hef3/eEF3B is the closest homologue of eEF3 -84% identical on the amino acid level (21) -and is, most likely, a second copy of eEF3 originating from whole-genome duplication in the Saccharomyces lineage (22) (Figure 1A). The second closest homologue is New1 (20) -encoded by a non-essential gene with a cold sensitivity knock-out phenotype (23) (Figure 1A)...

Identification of phosphosites that alter protein thermal stability

“…2c). NEW1 is a translation factor that binds to the ribosome at a position analogous to eEF3 and fine-tunes the efficiency of translation termination 11 . The identified phosphosite fits the CK2 consensus motif, is located within the acidic C-terminal sequence of NEW1, and is highly conserved.…”