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A study of the interaction of a 67kDa glycoprotein (p67) with eukaryotic initiation factor 2 (eIF-2) and other cellular proteins
Abstract
The eukaryotic initiation factor 2 (eIF-2) is a hetero-trimer, $\alpha\beta\gamma.$ The eIF-2 kinases (HRI and PKR) phosphorylate specifically the $\alpha$ subunit of eIF-2. This inactivates eIF-2 activity and inhibits protein synthesis. A 67 kDa glycoprotein (p$\sp{67})$ protects eIF-2 $\alpha$ subunit from inhibitory phosphorylation and thus promotes protein synthesis in the presence of active eIF-2 kinase(s). We have now studied the interactions between the eIF-2 subunits and p67 using the yeast two-hybrid system. We expressed the cDNAs for each subunits of eIF-2 and p$\sp{67}$ in yeast as a fusion with the DNA binding or the transcription activation domain of the yeast GAL4 transcription activator. We report that (i) eIF-2 $\gamma$ interacted with all three polypeptides eIF-2 $\alpha,\ \beta$ and p$\sp{67},$ (ii) No interaction was observed between eIF-2 $\alpha,$ eIF-2 $\beta$ and p$\sp{67},$ (iii) Only p$\sp{67}$ formed an oligomer. eIF-2 $\alpha,$ eIF-2 $\beta$ and eIF-2 $\gamma$ did not form oligomers. Molecular weight determination using Sephacryl S-300 gel filtration chromatography revealed that p$\sp{67}$ exits as a dimer. The two hybrid system was also used to screen for binding proteins of the eIF-2 associated 67 kDa glycoprotein, p$\sp{67}$. The plasmid pGBT9-p$\sp{67}$, having a full length rat p$\sp{67}$ fused to the DNA binding domain of GAL4 transcription factor, was prepared. The two hybrid mouse liver cDNA library containing cDNAs fused to the GAL4 trans-activation domain was used. Yeast cells (HF7c) were transformed with the plasmids and screened for clones, whose translational products interact with p$\sp{67}.$ Positive clones were obtained and their DNA sequences were partially determined. Two clones (clone 1 and 4) showed strong sequence identity with rat p$\sp{67}.$ Sequence analysis revealed that the clone 1 shares 98% sequence identity with the amino acid sequence 39-110 of rat p$\sp{67}$ and the clone 4 shares 97% sequence identity with the amino acid sequence 87-151 of rat p$\sp{67}.$ One clone (clone 3) showed 93% sequence identity to the heat shock cognate protein HSc 70 (319-406 aa). The significance of p$\sp{67}$: HSc 70 interaction was further studied using in vitro experiments. The results show: (i) p$\sp{67}$ bound to HSc 70 and was co-immunoprecipitated with the HSc 70 monoclonal antibodies. (ii) A p$\sp{67}$ deglycosylase activated in heme-deficient reticulocyte lysate, rapidly deglycosylated exogenously added p$\sp{67}$. Addition of HSc 70 significantly protected p$\sp{67}$ from this deglycosylation reaction. We suggest that HSc 70 binds to p$\sp{67}$ inside the cells and protects p$\sp{67}$ against deglycosylation and subsequent degradation.
Subject Area
Molecular biology|Biochemistry
Recommended Citation
Chattopadhyay, Ansuman, "A study of the interaction of a 67kDa glycoprotein (p67) with eukaryotic initiation factor 2 (eIF-2) and other cellular proteins" (1996). ETD collection for University of Nebraska-Lincoln. AAI9720836.
https://digitalcommons.unl.edu/dissertations/AAI9720836