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 pla ys a major role in translation, It contains PO, an insoluble protein anchor ing P1 and P2 to the ribosome, Here, rat recombinant PO was overproduced in inclusion bodies and solubilized in complex with the other P-proteins. Thi s method of solubilization appeared suitable to show protein complexes and revealed that P1, but not P2, interacted with PO. Furthermore, the use of t runcated 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 conclusio n that P1 and P2 bound one another, either connected with PO or free, as fo und in the cytoplasm. Accordingly, a model of association for the P-protein s in the stalk is proposed. Recombinant PO in complex with phosphorylated P 2 and either PI or its (1-63) domain efficiently restored the proteosynthet ic activity of 60 S subunits deprived of native P-proteins. Therefore, refo lded PO was functional and residues 1-63 only in P1 were essential, Further more, our results emphasize that the refolding principle used here is worth considering for solubilizing other insoluble proteins.