Protein synthesis in yeast. Isolation of variant forms of elongation factor 1 from the yeast Saccharomyces cerevisiae

Abstract

Two species of the elongation factor 1 (EF-1) differing in molecular weight, subunit composition, and isoelectric point have been isolated from cell-free extracts of the yeast Saccharomyces cerevisiae. The ratio of these two forms of EF-1 activity (EF-1 alpha and EF-1H) seem to vary in different strains and upon the growth phase from which the cells have been isolated. The log phase cells of a protease negative yeast strain EJ101 show a distribution of EF-1 alpha and EF-1H in the ratio of 3:1. Another laboratory yeast strain, D-587-4B, shows a distribution pattern of 4:1. The two forms of EF-1 are completely separable by ion exchange, gel permeation, and hydrophobic and affinity chromatography. Yeast EF-1 alpha is a single polypeptide of molecular weight 50,000 and has an isoelectric point of 8.9. The newly identified form of the yeast EF-1 (EF-1H) has a molecular weight of 200,000. The isoelectric point of this protein is around 5.5. Electrophoresis of the partially purified EF-1H in polyacrylamide gel containing sodium dodecyl sulfate indicates the presence of three nonidentical polypeptides having molecular weights of 50,000, 47,000, and 33,000. The three polypeptides are present in the ratio of 2:1:1. EF-1H is readily converted to EF-1 alpha and EF-1 beta gamma on anion exchange columns. The 50,000 dalton component of EF-1H immunologically cross-reacts with the antibody to EF-1 alpha. The other two polypeptides do not. On the basis of molecular weight, EF-1H is 2-3-fold more active than EF-1 alpha in poly(U)-dependent polyphenylalanine synthesis. EF-1H exchanges nucleotide (GDP----GTP) at a faster rate than EF-1 alpha. Both EF-1 alpha and EF-1H exhibit similar binding constants for GDP and GTP although the affinity of EF-1 alpha for guanine nucleotides is several-fold higher than that of EF-1H. The 33,000-dalton component of EF-1H appears to be functionally analogous to EF-1 beta (Ts) isolated from other eukaryotic sources. The function of EF-1 gamma is unknown.