A (re)initiation‐dependent cell‐free protein‐synthesis system from mouse erythroleukemia cells

Abstract

Cultured mouse erythroleukemia cells (MEL cells) can be induced in vivo to erythroid differentiation which is marked by the onset of globin mRNA and haemoglobin synthesis. When these cells are briefly exposed to hypertonic growth medium prior to lysis, the resulting post-mitochondrial supernatants show a high in vitro protein-synthesis activity. Amino acid incorporation is linear up to 60 min; more than 80% of this is due to (re)initiation, as shown by the inhibition with edeine. Extracts from induced cells reach only a third of overall incorporation as compared to extracts from uninduced cells. This reduction of the protein-synthesizing capacity is also observed in vivo. Polyacrylamide gel electrophoresis shows that extracts from uninduced cells faithfully translate their endogenous mRNA, whereas in extracts from induced cells, non-globin protein synthesis is reduced and globin is preferentially synthesized. Haemin (40 microM) as well as purified eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocytes enhance amino acid incorporation in both kinds of extracts, which suggests that both uninduced and induced MEL cells contain a haemin-controlled eIF-2 alpha kinase. This system should be useful for studying the mechanisms controlling protein synthesis in a nucleated differentiating cell.