PROTEIN SYNTHESIS INITIATION IN EUKARYOTES: PURIFICATION OF EIF-2 AND EIF-2-ANCILLARY PROTEIN FACTOR FROM YEAST SACCHAROMYCES CEREVISIAE AND COMPARISON OF THEIR ACTIVITIES WITH SIMILAR ACTIVITIES FROM RABBIT RETICULOCYTES

Two peptide chain initiation factor activities, eIF-2(,y) and Co-eIF-2A(,y)(\u2720) were purified from the yeast Saccharomyces Cerevisiae and their properties were studied. (1) eIF-2(,y) was composed of two subunits (M(,r), 54K and 36K). Co-eIF-2A(,y)(\u2720) was a single polypeptide (M(,r) 20K). (2) The molecular weight of eIF-2(,y) and Co-eIF-2A(,y)(\u2720) determined using a density gradient centrifugation method were approximately 140K and 20K respectively. (3) Co-eIF-2A(,y)(\u2720) stimulated (2-3 fold) GTP dependent ternary complex (TC) formation by eIF-2(,y) and eIF-2(,r). (4) Co-eIF-2A(,y)(\u2720) stimulated eIF-2(,y) promoted Met-tRNA(,f) binding to 40S ribosomes. (5) Co-eIF-2A(,y)(\u2720 )activity was heat labile and NEM sensitive. (6) Antibodies prepared against homogeneous Co-eIF-2A(,y)(\u2720) inhibited protein synthesis in yeast cell-free system and completely blocked Co-eIF-2A(,y)(\u2720) stimulation of Met-tRNA(,f)(.)40S initiation complex formation. (7) Unlike eIF-2(,r), purified eIF-2(,y) did not contain bound GDP. (8) Purified eIF-2(,y) preparation contained GTPase activity and dephosphorylated GTP to GDP. (9) An anti-eIF-2(,r) preparation which predominately precipitated the (gamma)-subunit (54K) of eIF-2(,r) also precipitated the larger subunit (54K) of eIF-2(,y). (10) Unlike eIF-2(,r), TC formation by eIF-2(,y) was not inhibited by Mg(\u272+). (11) Co-eIF-2A(,y)(\u2720) and Co-eIF-2(,r) significantly enhanced Met-tRNA(,f) binding to eIF-2(,y) and again Mg(\u272+) did not have any effect on this stimulated Met-tRNA(,f) binding to eIF-2(,y). (12) eIF-2(,y) bound (\u273)H GDP and this binding was significantly enhanced in the presence of Mg(\u272+). Co-eIF-2A(,y) had no effect on GDP binding to eIF-2(,y) nor on GDP exchange reactions. (13) (\u273)H GDP in preformed (and purified) eIF-2(,y)(.) (\u273)H GDP complex was rapidly and spontaneously lost upon standard TC formation condition with or without Mg(\u272+). (14) Reticulocyte HRI, which phosphorylated the (alpha)-subunit (38K) of eIF-2(,r), also phosphorylated the smaller subunit (36K) of eIF-2(,y). However such phosphorylation had no significant effect on TC formation, GDP binding and GDP exchange reactions