The proteasomal ATPase ring, comprising Rpt1-Rpt6, associates with the heptameric α ring of the proteasome core particle (CP) in the mature proteasome, with the Rpt C-terminal tails inserting into pockets of the α ring1–4. Rpt ring assembly is mediated by four chaperones, each binding a distinct Rpt subunit5–10. We report that the base subassembly of the proteasome, which includes the Rpt ring, forms a high affinity complex with the CP. This complex is subject to active dissociation by the chaperones Hsm3, Nas6, and Rpn14. Chaperone-mediated dissociation was abrogated by a nonhydrolyzable ATP analog, indicating that chaperone action is coupled to nucleotide hydrolysis by the Rpt ring. Unexpectedly, synthetic Rpt tail peptides bound α pockets with poor specificity, except for Rpt6, which uniquely bound the α2/α3 pocket. Although the Rpt6 tail is not visualized within an α pocket in mature proteasomes2–4, it inserts into the α2/α3 pocket in the base-CP complex and is important for complex formation. Thus, the Rpt-CP interface is reconfigured when the lid complex joins the nascent proteasome to form the mature holoenzyme.

In eukaryotic translation initiation, the eIF2 . GTP/MettRNA iMet ternary complex (TC) binds the eIF3/eIF1/eIF5 complex to form the multifactor complex (MFC), whereas eIF2 . GDP binds the pentameric factor eIF2B for guanine nucleotide exchange. eIF5 and the eIF2Be catalytic subunit possess a conserved eIF2-binding site. Nearly half of cellular eIF2 forms a complex with eIF5 lacking Met-tRNA i Met , and here we investigate its physiological significance. eIF5 overexpression increases the abundance of both eIF2/eIF5 and TC/eIF5 complexes, thereby impeding eIF2B reaction and MFC formation, respectively. eIF2Be mutations, but not other eIF2B mutations, enhance the ability of overexpressed eIF5 to compete for eIF2, indicating that interaction of eIF2Be with eIF2 normally disrupts eIF2/eIF5 interaction. Overexpression of the catalytic eIF2Be segment similarly exacerbates eIF5 mutant phenotypes, supporting the ability of eIF2Be to compete with MFC. Moreover, we show that eIF5 overexpression does not generate aberrant MFC lacking tRNA i Met , suggesting that tRNA iMet is a vital component promoting MFC assembly. We propose that the eIF2/eIF5 complex represents a cytoplasmic reservoir for eIF2 that antagonizes eIF2B-promoted guanine nucleotide exchange, enabling coordinated regulation of translation initiation.

Eukaryotic translation initiation factor (eIF) 5 is crucial for the assembly of the eukaryotic preinitiation complex. This activity is mediated by the ability of its C-terminal HEAT domain to interact with eIF1, eIF2, and eIF3 in the multifactor complex and with eIF4G in the 48S complex. However, the binding sites for these factors on eIF5-C-terminal domain (CTD) have not been known. Here we present a homology model for eIF5-CTD based on the HEAT domain of eIF2B. We show that the binding site for eIF2␤ is located in a surface area containing aromatic and acidic residues (aromatic͞ acidic boxes), that the binding sites for eIF1 and eIF3c are located in a conserved surface region of basic residues, and that eIF4G binds eIF5-CTD at an interface overlapping with the acidic area. Mutations in these distinct eIF5 surface areas impair GCN4 translational control by disrupting preinitiation complex interactions. These results indicate that the eIF5 HEAT domain is a critical nucleation core for preinitiation complex assembly and function.general amino acid control ͉ ribosome preinitiation complex ͉ translation initiation ͉ translational control I n eukaryotic translation initiation, the 40S ribosomal subunit binds Met-tRNA i Met , 5Ј-capped mRNA, and the 60S subunit in a coordinated manner, setting up the 80S initiation complex with the anticodon of Met-tRNA i Met base-paired at the ribosomal P site to the first start codon of the mRNA (for review, see ref. 1). At least 11 eukaryotic initiation factors (eIFs) mediate this process. MettRNA iMet binds the 40S subunit in a ternary complex (TC) with eIF2 and GTP to form the 43S preinitiation complex. Subsequent joining of the 43S particle to the mRNA͞eIF4F assembly produces the 48S preinitiation complex, which then scans for the first AUG codon. Correct AUG pairing with the Met-tRNA i Met anticodon triggers eIF5-dependent GTP hydrolysis for eIF2, leading to dissociation of the eIFs and formation of the 40S initiation complex. The GDPbound eIF2 that is released after GTP hydrolysis is recycled to eIF2-GTP by the pentameric guanylate exchange factor eIF2B.The C-terminal domain (CTD) of eIF5 is an important nucleation core of the preinitiation complex assembly and mediates formation of the multifactor complex (MFC) with eIF1, eIF2 TC, and eIF3 (2, 3). It contains unique aromatic͞acidic boxes (AA boxes) 1 and 2. These are also found in the CTDs of eIF2B (the catalytic subunit of eIF2B) and mammalian eIF4G (4). The AA boxes in eIF5 and eIF2B are required for binding to the lysine-rich segment [lysine box (K box)] present in the N-terminal domain of the common substrate, the ␤ subunit of eIF2 (4). The ability of eIF5-CTD to bind eIF3c is strongly enhanced by its interaction with the eIF2␤ K box, then leading to rapid and tight MFC assembly (5).The integrity of the translation initiation machinery is critical for proper cellular response to different stress stimuli (6). In yeast, amino acid starvation activates Gcn2p kinase to phosphorylate eIF2, rendering eIF2 a competitive inhibit...

Eukaryotic initiation factor 4G (eIF4G) promotes mRNA recruitment to the ribosome by binding to the mRNA cap-and poly(A) tail-binding proteins eIF4E and Pap1p. eIF4G also binds eIF4A at a distinct HEAT domain composed of five stacks of antiparallel ␣-helices. The role of eIF4G in the later steps of initiation, such as scanning and AUG recognition, has not been defined. Here we show that the entire HEAT domain and flanking residues of Saccharomyces cerevisiae eIF4G2 are required for the optimal interaction with the AUG recognition factors eIF5 and eIF1. eIF1 binds simultaneously to eIF4G and eIF3c in vitro, as shown previously for the C-terminal domain of eIF5. In vivo, cooverexpression of eIF1 or eIF5 reverses the genetic suppression of an eIF4G HEAT domain Ts ؊ mutation by eIF4A overexpression. In addition, excess eIF1 inhibits growth of a second eIF4G mutant defective in eIF4E binding, which was also reversed by cooverexpression of eIF4A. Interestingly, excess eIF1 carrying the sui1-1 mutation, known to relax the accuracy of start site selection, did not inhibit the growth of the eIF4G mutant, and sui1-1 reduced the interaction between eIF4G and eIF1 in vitro. Moreover, a HEAT domain mutation altering eIF4G moderately enhances translation from a non-AUG codon. These results strongly suggest that the binding of the eIF4G HEAT domain to eIF1 and eIF5 is important for maintaining the integrity of the scanning ribosomal preinitiation complex.

In the human genome, translation initiation from non-AUG codons plays an important role in various gene regulation programs. However, mechanisms regulating the non-AUG initiation rate remain poorly understood. Here, we show that the non-AUG initiation rate is nearly consistent under a fixed nucleotide context in various human and insect cells. Yet, it ranges from <1% to nearly 100% compared to AUG translation, depending on surrounding sequences, including Kozak, and possibly additional nucleotide contexts. Mechanistically, this range of non-AUG initiation is controlled in part, by the eIF5-mimic protein (5MP). 5MP represses non-AUG translation by competing with eIF5 for the Met-tRNAi-binding factor eIF2. Consistently, eIF5 increases, whereas 5MP decreases translation of NAT1/EIF4G2/DAP5, whose sole start codon is GUG. By modulating eIF5 and 5MP1 expression in combination with ribosome profiling we identified a handful of previously unknown non-AUG initiation sites, some of which serve as the exclusive start codons. If the initiation rate for these codons is low, then an AUG-initiated downstream ORF prevents the generation of shorter, AUG-initiated isoforms. We propose that the homeostasis of the non-AUG translatome is maintained through balanced expression of eIF5 and 5MP.

The translation factor eIF5 is an important partner of eIF2, directly modulating its function in several critical steps. First, eIF5 binds eIF2/GTP/Met-tRNAiMet ternary complex (TC), promoting its recruitment to 40S ribosomal subunits. Secondly, its GTPase activating function promotes eIF2 dissociation for ribosomal subunit joining. Finally, eIF5 GDP dissociation inhibition (GDI) activity can antagonize eIF2 reactivation by competing with the eIF2 guanine exchange factor (GEF), eIF2B. The C-terminal domain (CTD) of eIF5, a W2-type HEAT domain, mediates its interaction with eIF2. Here, we characterize a related human protein containing MA3- and W2-type HEAT domains, previously termed BZW2 and renamed here as eIF5-mimic protein 1 (5MP1). Human 5MP1 interacts with eIF2 and eIF3 and inhibits general and gene-specific translation in mammalian systems. We further test whether 5MP1 is a mimic or competitor of the GEF catalytic subunit eIF2Bε or eIF5, using yeast as a model. Our results suggest that 5MP1 interacts with yeast eIF2 and promotes TC formation, but inhibits TC binding to the ribosome. Moreover, 5MP1 is not a GEF but a weak GDI for yeast eIF2. We propose that 5MP1 is a partial mimic and competitor of eIF5, interfering with the key steps by which eIF5 regulates eIF2 function.

Eukaryotic initiation factor (eIF) 1 is a small protein (12 kDa) governing fidelity in translation initiation. It is recruited to the 40 S subunit in a multifactor complex with Met-tRNA i Met , eIF2, eIF3, and eIF5 and binds near the P-site. eIF1 release in response to start codon recognition is an important signal to produce an 80 S initiation complex. Although the ribosome-binding face of Dev. 21, 1217-1230). Interestingly, eIF1-KH includes the altered hydrophobic residues. Thus, eIF5 is an excellent candidate for the direct partner of eIF1-KH that mediates the critical link. The direct interaction at eIF1-KH also places eIF5 near the decoding site of the 40 S subunit.

Summary During eukaryotic translation initiation, eIF3 binds the solvent-accessible side of the 40S ribosome and recruits the gate-keeper protein eIF1 and eIF5 to the decoding center. This is largely mediated by the N-terminal domain (NTD) of eIF3c, which can be divided into three parts: 3c0, 3c1 and 3c2. The N-terminal part, 3c0, binds eIF5 strongly, but only weakly to the ribosome-binding surface of eIF1, whereas 3c1 and 3c2 form a stoichiometric complex with eIF1. 3c1 contacts eIF1 through Arg-53 and Leu-96, while 3c2 faces 40S protein uS15/S13, to anchor eIF1 to the scanning pre-initiation complex (PIC). We propose that the 3c0:eIF1 interaction diminishes eIF1 binding to the 40S, whereas 3c0:eIF5 interaction stabilizes the scanning PIC by precluding this inhibitory interaction. Upon start codon recognition, interactions involving eIF5, and ultimately 3c0:eIF1 association facilitate eIF1 release. Our results reveal intricate molecular interactions within the PIC, programmed for rapid scanning-arrest at the start codon.