Roles of 67 kDa polypeptide in the regulation of protein synthesis in animal cells and possible mechanism of its activity

Addition of partially purified exogenous eIF-2 or the cell supernatant factor (RF), enriched in GEF activity reverses protein synthesis inhibition in heme deficient reticulocyte lysate. This partially purified eIF-2 contains regular three subunits ($\alpha$, $\beta$ and $\gamma$) together with a 67 kDa polypeptide. This 67 kDa polypeptide protects eIF-2 $\alpha$-subunit from eIF-2 kinase catalyzed phosphorylation. This p$\sp{67}$ is a glycoprotein, contains multiple GlcNAc residues. In heme deficient reticulocyte lysate p$\sp{67}$ is deglycosylated and becomes inactive to protect eIF-2 $\alpha$-subunit from phosphorylation. HRI is active in both heme-supplemented and heme-deficient reticulocyte lysate. It is the p$\sp{67}$ which becomes inactive due to deglycosylation in heme-deficient reticulocyte lysate. In heme supplemented reticulocyte lysate p$\sp{67}$ is stable and active to protect $\alpha$-subunit. p$\sp{67}$ is degradable and inducible protein. In quiescent tumor hepatoma cells (KRC-7) p$\sp{67}$ is completely disappeared and becomes prominent in an hour after addition of TPA (phorbol ester). The levels of other polypeptides and interestingly the level of dSI remains essentially same. The level of p$\sp{67}$ correlates directly with the protein synthesis of the cells. At the confluent stage, the protein synthesis rate is maximum, the phosphorylation of eIF-2 $\alpha$-subunit is very low and the p$\sp{67}$ level is very high. At the quiescent stage the protein synthesis rate is very low, phosphorylation of eIF-2 $\alpha$-subunit is maximum and p$\sp{67}$ is almost nonexistent. So the availability of p$\sp{67}$ directly controls the rate of protein synthesis in animal cells. p$\sp{67}$ has to react with eIF-2 for its activity. p$\sp{67}$ reacts with eIF-2 through $\gamma$-subunit. This interaction is protein-protein in nature. The interaction of p$\sp{67}$ with eIF-2 is essential for its activity to protect eIF-2 $\alpha$-subunit from eIF-2 kinase catalyzed phosphorylation. p$\sp{67}$ activity is sensitive to Mg$\sp{+2}$ concentration. At physiological Mg$\sp{+2}$ concentration (1mM-2mM) p$\sp{67}$ is active and is co-immunoprecipitated with eIF-2. At high Mg$\sp{+2}$ concentration (3mM-5mM) the interaction of p$\sp{67}$ with eIF-2 is inhibited and is no longer active to protect eIF-2 $\alpha$-subunit. p$\sp{67}$ activity is very specific. It protects only eIF-2 $\alpha$-subunit from HRI or dSI kinase catalyzed phosphorylation. It is unable to protect eIF-2 $\beta$-subunit from casein kinase or histones from dSI kinase catalyzed phosphorylation. The glycosyl residues on p$\sp{67}$ may be involved in p$\sp{67}$ activity because preincubation of p$\sp{67}$ with WGA inhibits p$\sp{67}$ activity to protect eIF-2 $\alpha$-subunit from phosphorylation. The possible mechanism by which p$\sp{67}$ protects eIF-2 $\alpha$-subunit from phosphorylation is by masking the $\alpha$-subunit with the help of glycosyl residues on p$\sp{67}$