The eukaryotic P1 and P2 ribosomal proteins which constitute, with P0, a pentamer forming the lateral stalk of the 60 S ribosomal subunit, exhibit several differences from their prokaryotic equivalents L7 and L12; in particular, P1 does not have the same primary structure as P2 and both of them are phosphorylated, the significance of the latter remaining unclear. Rat liver P1 and P2 were overproduced in Escherichia coli cells and their interaction with elongation factor eEF-2 was studied. Both recombinant proteins were found to be required for the ribosomedependent GTPase activity of eEF-2, with P2 in the phosphorylated form. The surface plasmon resonance technique revealed that, in vitro, both proteins interact specifically with eEF-2, with a higher affinity for P1 (K d = 3.8 Â 10 28 m) than for P2 (K d = 2.2 Â 10 26 m). Phosphorylation resulted in a moderate increase (two-to four-fold) in these affinities. The interaction of both P1 and P2 (phosphorylated or not) with eEF-2 resulted in a conformational change in the factor, revealed by an increase in the accessibility of Glu554 to proteinase Glu-C. This increase was observed in both the presence and absence of GTP and GDP, which themselves produced marked opposite effects on the conformation of eEF-2. Our results suggest that the two proteins P1 and P2 both interact with eEF-2 inducing a conformational transition of the factor, but have acquired some specific properties during evolution.Keywords: acidic ribosomal proteins; ribosomal; elongation factor 2; phosphorylation.P proteins are present in the 60 S ribosomal subunits of all eukaryotic cells. In mammalian cells, there are three different proteins called P0, P1 and P2. P1 and P2 are 12-kDa acidic proteins which exhibit some similarities to and some differences from the prokaryotic ribosomal proteins L7 and L12. Among the similarities are their localization on the large ribosomal subunit forming a lateral stalk, their molecular mass and physicochemical properties, and their requirement for translational factor activity. Thus, the GTPase activity of the eukaryotic elongation factor eEF-2 which catalyses the translocation of peptidyl-tRNA from the A to the P site of the ribosome is dependent on the presence of P1 and P2 on the large ribosomal subunit [1], just as the GTPase activity of the corresponding prokaryotic factor EF-G is dependent on the presence of L7/L12 on this subunit. A direct interaction between L7/L12 and EF-G has been recently visualized directly by cryoelectron microscopy [2]. The differences between P1/P2 and L7/L12 are numerous and interesting to study in terms of evolution. First, sequence homologies between eukaryotic and prokaryotic proteins are not obvious [3]. Secondly, P1 and P2 do not have the same primary structure [3], whereas L7 and L12 do, with the only exception being an N-acetyl group. Thirdly, in contrast with L7 and L12, P1 and P2 are phosphorylated on serine residues when present on the ribosome [4], and this phosphorylation seems to be important for their function, althoug...

The acidic ribosomal proteins P1-P2 from rat liver were overproduced for the first time by expression of their cDNA in Escherichia coli. They were tested for their ability to reactivate inactive P1-P2-deficient core particles derived from 60 S ribosomal subunits treated with dimethylmaleic anhydride, in poly(U)-directed poly(Phe) synthesis. The recombinant P1-P2 were unable to reactivate these core particles although they could bind to them. When recombinant P1-P2 had been phosphorylated first with casein kinase II, they were as efficient in the reactivation process as P1-P2 extracted with ethanol/KCl from the 60 S subunits. Reconstitution experiments were carried out using all possible combinations of the two recombinant proteins phosphorylated or not. Reactivation of the core particles required the presence of both P1 and P2 with the latter in its phosphorylated form. These experiments reveal a distinct role for P1 and P2 in protein synthesis. Phosphorylated P2 produced a partial quenching of the intrinsic fluorescence of eukaryotic elongation factor 2, which was not observed with the unphosphorylated protein. This result demonstrates the existence of an interaction between phosphorylated P2 and eukaryotic elongation factor 2. P2 also quenched part of the intrinsic fluorescence of P1, due to the interaction between the two proteins.The large subunit of eukaryotic ribosomes contains 12-kDa acidic P proteins, which seem more numerous in lower eukaryotes than in higher organisms (1). In these, two types of P proteins are found, designated as P1 and P2 (2, 3). They share similar properties with prokaryotic proteins L7-L12, which form a pentamer with protein L10, (L7/L12) 4 -L10, constituting the lateral stalk of the 50 S ribosomal subunits (4). The eukaryotic P proteins, as their prokaryotic counterparts, seem to play an essential role in the interaction with elongation factors and in factor-dependent GTPase activity (5). However, some specific properties are observed with eukaryotic P proteins. First, these proteins exist on the ribosome as phosphorylated derivatives. They can be phosphorylated in vitro by either casein kinase II or by an endogenous ribosome-bound enzyme (6). Second, proteins P1-P2 present on the ribosome can exchange with a cytoplasmic pool of these unphosphorylated proteins (7). Phosphorylation of P1-P2, which appears to be necessary for ribosome activity, was originally suggested to be a requirement for the binding of these proteins to ribosomes (5), but recently this hypothesis has been challenged by new results obtained in yeast (8).We have shown previously that active rat liver 60 S ribosomal subunits could be reconstituted from inactive core particles prepared with 2,3-dimethylmaleic anhydride (DMMA) 1 (9), by adapting a method previously used with yeast ribosomes (10). Reactivation of the rat liver core particles was obtained not only with DMMA-split proteins containing several proteins including P1-P2 but also with a 50% ethanol, 0.08 M KCl extract containing P1-P2 exclusively. Dephospho...