Molecular analysis of initiation factor complexes contain eIF3
EMSL Project ID
30391
Abstract
The large multi-subunit eukaryotic initiation factor eIF3 is an essential component of translation initiation in all cells. In mammalian cells it is responsible for recruiting the eIF2-GTP-tRNA(i)met ternary complex, mRNA and other factors to the 40S ribosomal subunit. Although we know eIF3 is composed of 13 different subunits (a-m), much is unknown about each subunit, their function, how they interact with other factors and how post-translational modifications may regulate their function through various signaling pathways. We have been investigating the formation of eIF3 complexes and how these are regulated by signals involved in the increase in protein synthesis during the mitogenic stimulation of human T lymphocytes. Translation rates rapidly increase in T lymphocytes when activated with ionomycin and phorbol myristate acetate (I+PMA). Our prior studies show that eIF3 subunits form a number of complexes that contain different combinations of eIF3 subunits and other factors and change with experimental conditions. A number of biochemical methods (2D PAGE and immunoblotting) and proteomic analysis were used to identify the protein composition of these complexes. Different activation conditions of human T lymphocytes show distinct corresponding changes in the 2D PAGE location of eIF3 subunit isoforms. In particular, eIF3a p170, the largest subunit has a considerable number of protein isoforms that are likely phosphorylated. However, the heterogeneity in the multiple isoforms, which have a broad pH range, makes this subunit particularly difficult to analyze by conventional proteomic methods. The fact that we are using primary, native human lymphocytes that are untransformed and have intact signaling systems (unlike most cell lines grown in culture) also makes this project difficult. The physical (pI and mass) changes of eIF3a can be observed by 2D PAGE and immunoblotting, but we are limited by material and our ability to analyze the type and location of the post-translation modifications for each subunit. The capabilities and resources of EMSL are well suited for this project. We propose to characterize the post-translation modifications of eIF3a and eIF3c in activating lymphocytes using free-flow electrophoresis and high-resolution nano-LC connected to a 12T FTICR mass spectrometer with electron transfer dissociation capability, using a top-down, intact protein LC-MS approach. At the same time a bottoms up LC MS/MS approach using a linear ion trap quadrupole mass spectrometer equipped with electron transfer dissociation will be used to obtain the type, site and sequence identification of post-translational modification of two eIF3 subunits, eIF3a and eIF3c. The information gained will be valuable for identifying the different isoforms for eIF3a and eIF3c and also identify the biologically relevant phosphorylation and cleavage sites of each molecule. Finding the sites will also help us to identify the potential kinase or protease that is involved with regulation of these factors. Identifying the relevant kinases or proteases will then allow us to connect post-translational modifications of eIF3a and eIF3c to signaling pathways and translation control in the activating lymphocytes. These results will be the basis for future molecular studies that will be needed to verify the connection between these modifications and translation in these cells.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2009-01-19
End Date
2010-01-24
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members