In eukaryotic cells, binding of initiator methlonyl-tRNA to the ribosome during mRNA translation is regulated by a G-protein, eIF2, and its associated guanine nucleotide exchange factor (GEF), e!F2B. In contrast to other GEFs, which are often single subunit proteins, eIF2B consists of five dissimilar subunits. In order to study the regulation of eIF2B and the function of its individual subunits, we have used the baculovirus expression vector system to co-express the five subunits in Sf9 cells. Using this system, we have reconstituted the five subunit complex in vivo and purified it to greater than 98% homogeneity. In addition, by expressing the single subunits in Sf9 cells, eIF2Bc was identified as the catalytic subunit of the complex. Of the five subunits, it alone displayed GEF activity. However, the activity of eIF2Bf was approximately 30-fold lower than that of the complex containing all five subunits. The eIF2B complex was also reconstituted in vitro by mixing crude cell lysates containing different subunits. The ability to reconstitute the five subunit complex provides a rapid system for structure/function analyses of the catalytic and regulatory properties of the individual subunits of eIF2B. Using this approach, deletion mutants of the eIF2Br subunit were expressed and combined with the other four wildtype subunits of eIF2B. Two distinct regions of the f-subunit were identified which are required for catalysis (guanine nucleotide exchange on eIF2) and for complex formation. Supported by MH Grant DK13499.
|Published - Dec 1 1998
All Science Journal Classification (ASJC) codes
- Molecular Biology