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Roles of a 67 kDa polypeptide in the regulation of protein synthesis initiation in rabbit reticulocytes
Abstract
During heme-deficiency in reticulocyte lysates, the heme-regulated protein synthesis inhibitor, HRI phosphorylates the $\alpha$-subunit of eukaryotic initiation factor 2 (eIF-2) and thus inhibits protein synthesis. Addition of partially purified exogenous eIF-2 or the cell supernatant factor (RF) preparation enriched in GEF activity reverses protein synthesis inhibition in heme-deficient reticulocyte lysate. This partially purified eIF-2 preparation contained its regular three subunits ($\alpha$, $\beta$, and $\gamma$) together with a 67 kDa polypeptide. The cell supernatant factor, RF, contained eIF-2 $\alpha$, $\beta$- subunits, the 67 kDa polypeptide and other different polypeptides but no eIF-2 $\gamma$-subunit and that's why RF cannot form ternary complex. The addition of the isolated $\gamma$-subunit to the RF preparation restored its ternary complex activity. The 67 kDa polypeptide when associated with eIF-2 or RF, it protects eIF-2 $\alpha$-subunit from phosphorylation by eIF-2 kinases and thus eIF-2 and RF reverse protein synthesis inhibition in heme-deficient reticulocyte lysates. The 67 kDa polypeptide is a growth regulated polypeptide. When cells enter into the exponential phase from lag phase, protein synthesis increases and at the same time the level of the 67 kDa polypeptide increased compared to the lag phase. On the other hand, the phosphorylation of the eIF-2 $\alpha$-subunit decreased during exponential phase compared to the lag phase. The level of the 67 kDa polypeptide even increased when the quiescent KRC-7 cells were induced with phorbol ester (TPA), a tumor promoting agent, whereas the level of the eIF-2 $\alpha$-subunit remained constant during this TPA induction. The 67 kDa polypeptide is a glycoprotein containing O-linked GlcNAc moieties attached to the protein backbone. The mechanism by which the 67 kDa polypeptide protects eIF-2 $\alpha$-subunit from phosphorylation by eIF-2 kinases is due to its glycosidic residues which mask the phosphorylation site(s) of the eIF-2 $\alpha$-subunit.
Subject Area
Biochemistry|Molecular biology
Recommended Citation
Datta, Bansidhar, "Roles of a 67 kDa polypeptide in the regulation of protein synthesis initiation in rabbit reticulocytes" (1989). ETD collection for University of Nebraska-Lincoln. AAI8925232.
https://digitalcommons.unl.edu/dissertations/AAI8925232