Pivotal role of the P1N-terminal domain in the assembly of the mammalian ribosomal stalk and in the proteosynthetic activity

In the 60 S ribosomal subunit, the lateral stalk made of the P-proteins plays a major role in translation, It contains PO, an insoluble protein anchoring P1 and P2 to the ribosome, Here, rat recombinant PO was overproduced in inclusion bodies and solubilized in complex with the other P-proteins. This method of solubilization appeared suitable to show protein complexes and revealed that P1, but not P2, interacted with PO. Furthermore, the use of truncated mutants of PI and P2 indicated that residues 1-68 in P1 connected PO to residues 1-65 in P2. Additional experiments resulted in the conclusion that P1 and P2 bound one another, either connected with PO or free, as found in the cytoplasm. Accordingly, a model of association for the P-proteins in the stalk is proposed. Recombinant PO in complex with phosphorylated P2 and either PI or its (1-63) domain efficiently restored the proteosynthetic activity of 60 S subunits deprived of native P-proteins. Therefore, refolded PO was functional and residues 1-63 only in P1 were essential, Furthermore, our results emphasize that the refolding principle used here is worth considering for solubilizing other insoluble proteins.