Identification of a translation initiation factor 3 (eIF3) core complex, conserved in yeast and mammals, that...

Area of Science:

• Molecular Biology
• Protein Biochemistry
• Genetics

Background:

• Human eukaryotic translation initiation factor 3 (eIF3) has nine subunits, but only five have homologs in yeast.
• Previous studies identified Prt1p and Tif34p as yeast eIF3 subunits.
• The complete composition and interactions of yeast eIF3 were not fully elucidated.

Purpose of the Study:

• To identify the subunits of the yeast eIF3 complex.
• To characterize the interaction between yeast eIF3 and other translation initiation factors.
• To investigate the role of identified subunits in ribosome function.

Main Methods:

• Purification of a polyhistidine-tagged Prt1p complex using Ni2+ affinity and gel filtration chromatography.
• Mass spectrometry to identify copurifying polypeptides.
• Biochemical assays to test the function of the purified complex in ribosome binding assays.
• Yeast two-hybrid and in vitro protein binding assays to study protein interactions.

Main Results:

• A ~600 kDa complex containing six polypeptides was purified, dependent on His-Prt1p.
• Four known yeast eIF3 homolog subunits (Tif32p, Nip1p, Tif34p, Tif35p) and eIF5 were identified.
• The purified complex rescued Met-tRNAiMet binding in defective yeast extracts, confirming eIF3 activity.
• Nip1p was found to interact with eIF5 and Sui1p, both involved in start codon recognition.

Conclusions:

• Tif32p, Nip1p, Prt1p, Tif34p, and Tif35p form a conserved core eIF3 complex.
• This core complex stably interacts with eIF5.
• eIF5 and Sui1p may be recruited to 40S ribosomes via Nip1p, contributing to accurate start codon selection.