Characterization of wheat germ protein synthesis initiation factor eIF-4C and comparison of eIF-4C from wheat germ and rabbit reticulocytes.

R T Timmer,S R Lax,D L Hughes,W C Merrick,J M Ravel,K S Browning

doi:10.1016/s0021-9258(19)74544-x

Abstract

Eukaryotic protein synthesis initiation factor (eIF)-4C was purified from wheat germ and the molecular weight was calculated to be approximately 19,000 by SDS-polyacrylamide gel electrophoresis. A similar molecular weight was determined by gel filtration chromatography indicating that wheat germ eIF-4C is functional as a single polypeptide chain. An efficient in vitro translation system dependent upon the addition of eIF-4C was developed. This system was used to determine the concentrations of eIF-4C required for the half-maximal rate of translation of satellite tobacco necrosis virus RNA, alfalfa mosaic virus RNA 4, and barley alpha-amylase mRNA. No significant differences in the concentrations of eIF-4C required for the translation of these mRNAs were observed, although differences were noted for eIF-4A and eIF-4F. This finding suggests that eIF-4C is not involved in the binding of mRNA to 40 S ribosomal subunits. In heterologous assays, rabbit reticulocyte eIF-4C was as active as wheat germ eIF-4C in the wheat germ eIF-4C-dependent system. In addition, wheat germ eIF-4C substituted for rabbit reticulocyte eIF-4C in in vitro assay systems from rabbit reticulocytes. These results indicate that eIF-4C from wheat and rabbit contain conserved functional domains.

Highlights

From the Departmentof Chemistry and Biochemistry, Universityof Texas, Austin, Texas78712 and the $Departmentof Biochemistry, Case Western Reserve University Schoolof Medicine, Cleveland, Ohio44106
A similar molecular weight was determined by gel filtrationchromatography indicating that wheat germ eIF-4C is functional as a singlepolypeptide chain
Rabbit reticulocyte eIF-4Cwas asactiveas wheat germ eIF-4C in the wheat germ eIF-4C-dependribosomes, and methionyl-puromycin synthesis

Summary

SeDhacrvl s-200

’A unit is defined as that amountwhich supports the incorporation of 1nmol of leucine into polypeptide under the conditions described under “ExperimentalProcedures.”. The pooled and concentrated phosphocellulose fraction (-1 ml containing about mg of protein) was applied to a 35-ml Sephacryl S-200 column Buffer B-100; fractions of 0.35 ml were collected, and aliquotsof the fractions were analyzed by SDS-PAGE High amounts of eIF-4C were pooled (generally 10 fractions)and stored a t -70 “C in small aliquots. Preparation of a Wheat Germ 40-70% Ammonium Sulfate Fraction Deficient in eZF-4C-A 5-ml aliquot of the 40-70% ammonium sulfate fraction was brought to 500 mM KC1 by the addition of 1.15 ml of Buffer B containing 2.5 M KCI. Wheat germ eIF-4C eluted from a Sephacryl S200 column a t a volume expected for a 19-kDa protein, indi-. Very little cross-reactivitybetween theantiformed as described previously (1,17)

RESULTS

Findings

DISCUSSION