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