The recruitment of the 40S ribosomal subunit to the mRNA start codon is thought to be the rate-limiting step in eukaryotic translation. This process requires the assembly of a ribonucleoprotein complex, which joins mRNAs with some 30 different polypeptides referred to as eukaryotic initiation factors (eIFs) (Hershey and Merrick, 2000
). 40S ribosomes associate with the eIF2·GTP/Met-tRNA ternary complex, the multisubunit eIF3 complex, and several other eIFs to form the 43S pre-initiation complex. This complex then binds to a second protein assembly organized around eIF4G, resulting in the 43S initiation complex. eIF4G interacts with both the cap-binding protein eIF4E and the poly-A binding protein, thus presumably circularizing the mRNA. eIF4G also recruits the eIF4A helicase assisting the 43S complex in scanning along the mRNA. Once the start codon is identified, the 43S complex is converted into the 48S initiation complex, which forms a stable interaction with the initiator AUG. At this point, eIF2-bound GTP is hydrolyzed, leading to dissociation of eIFs, thus allowing the 60S ribosomal subunit to join for productive protein synthesis.
eIF3 is the most complex translation initiation factor and plays several important roles that were revealed by in vitro reconstitution experiments (Dong and Zhang, 2006
; Hinnebusch, 2006
). First, eIF3 binds to the 40S ribosome and facilitates loading of the eIF2·GTP/Met-tRNA ternary complex to form the 43S pre-initiation complex. Subsequently, eIF3 assists in recruiting mRNAs to the 43S complex, presumably involving the RNA recognition motifs found in some of its subunits. Lastly, eIF3 binding to the 40S ribosome prevents the joining of the 60S subunit until the start codon is identified, eIF2-bound GTP is hydrolyzed by eIF5, and all eIFs are released. While these discreet reaction steps were deciphered extensively in vitro, it remained unclear how they are coordinated in vivo in order to ensure efficient translation.
Whereas human eIF3 consists of 13 subunits, consecutively named eIF3a – m (Damoc et al., 2007
; Unbehaun et al., 2004
; Zhou et al., 2008
), budding yeast contains only five stochiometric subunits, which are orthologs of human eIF3a, b, c, g, and eIF3i, and the substoichiometric eIF3j. These subunits may constitute a core complex, as all are essential for viability (Asano et al., 1997
; Phan et al., 1998
). In the fission yeast, Schizosaccharomyces pombe, eIF3 contains the same five core subunits, in addition to the non-core subunits eIF3d, e, f, g, h, i, and m (Akiyoshi et al., 2001
; Bandyopadhyay et al., 2002
; Burks et al., 2001
; Crane et al., 2000
; Dunand-Sauthier et al., 2002
; Ray et al., 2008
; Zhou et al., 2005
). Two distinct eIF3 complexes were identified in fission yeast that contain an overlapping set of core subunits but are distinguished by the presence of the related eIF3e and eIF3m proteins (Zhou et al., 2005
). The eIF3m containing complex appears to mediate the translation of the bulk of cellular mRNAs, whereas the eIF3e containing complex associates with a far more restricted set of mRNAs. Distinct eIF3 complexes may therefore contribute to mRNA specificity of translation.
eIF3 also has functions that are apparently independent of its role in translation initiation. For example, some eIF3 subunits interact with the 26S proteasome (Dunand-Sauthier et al., 2002
; Hoareau Alves et al., 2002
; Paz-Aviram et al., 2008
; Yen et al., 2003b
). The significance of this interaction was revealed in S. pombe, where deletion of the non-essential eIF3d/Moe1p and eIF3e/Yin6p confers a series of cellular phenotypes that indicate a defect in proteasomal protein degradation (Yen et al., 2003b
). This defect was pinpointed to a role of these eIF3 subunits in the nuclear accumulation and assembly of the 26S proteasome. However, the molecular mechanisms underlying eIF3-directed proteasome localization and assembly remained unknown.
To clarify this issue and to further elucidate the functions of eIF3, we performed a high sensitivity mass spectrometry analysis of eIF3 complexes purified from S. pombe. This eIF3 interactome suggests an extensive repertoire of eIF3 roles in protein synthesis and degradation thus establishing a molecular link between these processes.