The roles of sugar phosphates and thiol-reducing systems in the control of reticulocyte protein synthesis.

Abstract

The study of gel-filtered rabbit reticulocyte lysates and lysates which have been passed through 2′5′ ADP-Sepharose columns has shown that the presence of a sugar phosphate and a reducing system is necessary to maintain maximum rates of protein synthesis and to prevent an early inhibition. In this paper it is shown that the inhibition is due to a reduced rate of initiation of translation and a decrease in the level of methionyl-tRNAf· 40S-ribosomal-subunit complexes. Sugar phosphates and reducing agents are shown to act co-operatively to prevent these changes: the absence of either sugar phosphates or reducing agents leads to a decrease in polysomes and in methionyl-tRNA · 40S-subunit complexes. The requirements for reducing power can be satisfied by either dithiothreitol or by an NADPH-generating system together with a functional thioredoxin/thioredoxin reductase system; evidence is presented to show that these are required for the reduction of disulphide bonds. Incubation of lysates in the absence of a suitable reducing system leads to disulphide bond formation in a limited number of proteins present in the lysate, but no disulphide bonds could be detected in initiation factor elF-2, the protein which catalyses the formation of methionyl-tRNA · 40S-subunit complexes. On the other hand, when these lysates were incubated under conditions in which protein synthesis is inhibited, elF-2 was phosphorylated in the smallest of its three polypeptide chains (eIF-2α). The phosphorylation of eIF-2α is shown to be controlled by the presence or absence of a suitable reducing system but not by sugar phosphates; it appears to be caused by activation of a protein kinase rather than through regulation of the rate of dephosphorylation of this protein. It is concluded that sugar phosphates do not control the phosphorylation of eIF-2 but play some role as an activating cofactor affecting the rate of initiation of protein synthesis. The presence of a suitable reducing system is required to prevent or reverse disulphide bond formation in some critical protein(s) in the lysate; the oxidation of sulphydryl groups in this protein leads to activation of an eIF-2 kinase and hence to inhibition of initiation.