Biochemical analysis of the eIF2beta gamma complex reveals a structural function for eIF2alpha 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 alpha-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 (eIF2betagamma) completely devoid of the alpha-subunit to further study the role of eIF2alpha in eIF2 function. These studies utilized a yeast strain genetically altered to bypass a deletion of the normally essential eIF2alpha structural gene (SUI2). Removal of the alpha-subunit did not appear to significantly alter binding of guanine nucleotide or Met-tRNA(i)(Met) ligands by eIF2 in vitro. Qualitative assays to detect 43 S initiation complex formation and eIF5-dependent GTP hydrolysis revealed no differences between eIF2betagamma and the wild-type eIF2 heterotrimer. However, steady-state kinetic analysis of eIF2B-catalyzed nucleotide exchange revealed that the absence of the alpha-subunit increased K(m) for eIF2betagamma.GDP by an order of magnitude, with a smaller increase in V(max). These data indicate that eIF2alpha 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.
Nika
J
J
Department of Molecular and Cell Biology, The University of Texas at Dallas, Richardson, Texas 75083, USA.
Rippel
S
S
Hannig
E M
EM
D002384:Catalysis; D020717:Eukaryotic Initiation Factor-2B / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D040061:Eukaryotic Initiation Factor-5; D017353:Gene Deletion; D005800:Genes, Fungal; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D010448:Peptide Initiation Factors / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics
D020717:Eukaryotic Initiation Factor-2B; D040061:Eukaryotic Initiation Factor-5; D010448:Peptide Initiation Factors; D020033:Protein Isoforms; D012358:RNA, Transfer, Met; D006160:Guanosine Triphosphate