Pivotal role of the P1 N-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 P0, an insoluble protein anchoring P1 and P2 to the ribosome. Here, rat recombinant P0 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 P0. Furthermore, the use of truncated mutants of P1 and P2 indicated that residues 1-63 in P1 connected P0 to residues 1-65 in P2. Additional experiments resulted in the conclusion that P1 and P2 bound one another, either connected with P0 or free, as found in the cytoplasm. Accordingly, a model of association for the P-proteins in the stalk is proposed. Recombinant P0 in complex with phosphorylated P2 and either P1 or its (1-63) domain efficiently restored the proteosynthetic activity of 60 S subunits deprived of native P-proteins. Therefore, refolded P0 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.