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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-tRNAi 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 α-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 (eIF2βγ) completely devoid of the α-subunit to further study the role of eIF2α in eIF2 function. These studies utilized a yeast strain genetically altered to bypass a deletion of the normally essential eIF2α structural gene (SUI2). Removal of the α-subunit did not appear to significantly alter binding of guanine nucleotide or Met-tRNAi Metligands by eIF2 in vitro. Qualitative assays to detect 43 S initiation complex formation and eIF5-dependent GTP hydrolysis revealed no differences between eIF2βγ and the wild-type eIF2 heterotrimer. However, steady-state kinetic analysis of eIF2B-catalyzed nucleotide exchange revealed that the absence of the α-subunit increased K m for eIF2βγ·GDP by an order of magnitude, with a smaller increase inV max. These data indicate that eIF2α 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 α-subunit to control the rate of nucleotide exchange through reversible phosphorylation.