Biochemical analysis of the eIF2βγ complex reveals a structural function for eIF2α in catalyzed nucleotide exchange

Eukaryotic translation initiation factor eIF2 is a heterotrimer that binds and delivers Met-tRNA(i)(Met) to the 40 S ribosomal subunit in a GTP-dependent manner. Initiation requires hydrolysis of eIF2-bound GTP, which releases an eIF2 GDP complex that is recycled to the GTP form by the nucleotide exchange factor eIF2B. The ru-subunit of eIF2 plays a critical role in regulating nucleotide exchange via phosphorylation at serine 51, which converts eIF2 into a competitive inhibitor of the eIF2B-catalyzed exchange reaction. We purified a form of eIF2 (eIF2 beta gamma) completely devoid of the alpha -subunit to further study the role of eIF2 alpha in eIF2 function. These studies utilized a yeast strain genetically altered to bypass a deletion of the normally essential eIF2 alpha structural gene (SU12), Removal of the alpha -subunit did not appear to significantly alter binding of guanine nucleotide or Met-tRN(i)(Met) ligands by eIF2 in vifro. Qualitative assays to detect 43 S initiation complex formation and eIF5-dependent GTP hydrolysis revealed no differences between eIF2 beta gamma and the wild-type eIF2 heterotrimer. However, steady-state kinetic analysis of eIF2B-catalyzed nucleotide exchange revealed that the absence of the a-subunit increased K-m for eIF2 beta gamma (.)GDP by an order of magnitude, with a smaller increase in V-max. These data indicate that eIF2 alpha is required for structural interactions between eIF2 and eIF2B that promote wild-type rates of nucleotide exchange, We suggest that this function contributes to the ability of the alpha -subunit to control the rate of nucleotide exchange through reversible phosphorylation.