Three strongly acidic proteins with pIs between 3.0 and 3.5 have been detected and purified from an ammonium-ethanol extract of Saccharomyces cerevisiae ribosomes. The three proteins, called L45, L44, and L44', have a similar amino acid composition, but differences were shown by tryptic peptide analysis. Nevertheless, the three polypeptides show total cross-reaction to antisera raised against one of them. Protein L44' is very unstable in the extract when treated at the basic pH 9.2, due to an enzymatic process not yet clarified. When purified, the protein is, however, stable. In solution, the proteins are present as dimers, as verified by ultracentrifugation, column filtration, and photochemical cross-linking. The tendency to dimerization is much lower in the case of protein L44'. On the average, 3.2 copies of these proteins are detected per ribosome. The proteins are monophosphorylated when present in the ribosome. Phosphorylation seems to regulate the affinity of the polypeptides for the particles because unphosphorylated proteins bind poorly to the ribosomes deprived of the acidic proteins. Since these proteins are unphosphorylated when present in the cytoplasm [Zinker, S. (1980) Biochim. Biophys. Acta 606, 76-82; Sánchez-Madrid, F., Vidales, F. J., & Ballesta, J. P. G. (1981) Eur. J. Biochem. 114, 609-613], a regulatory mechanism of the ribosomal function based on a phosphorylation-dephosphorylation process of the acidic proteins is being studied.

The yeast ribosomal stalk is formed by a protein pentamer made of the 38 kDa P0 and four 12 kDa acidic P1/P2. The interaction of recombinant acidic proteins P1 alpha and P2 beta with ribosomes from Saccharomyces cerevisiae D4567, lacking all the 12 kDa stalk components, has been used to study the in vitro assembly of this important ribosomal structure. Stimulation of the ribosome activity was obtained by incubating simultaneously the particles with both proteins, which were nonphosphorylated initially and remained unmodified afterward. The N-terminus state, free or blocked, did not affect either the binding or reactivating activity of both proteins. Independent incubation with each protein did not affect the activity of the particles, however, protein P2 beta alone was unable to bind the ribosome whereas P1 alpha could. The binding of P1 alpha alone is a saturable process in acidic-protein-deficient ribosomes and does not take place in complete wild-type particles. Binding of P1 proteins in the absence of P2 proteins takes also place in vivo, when protein P1 beta is overexpressed in S. cerevisiae. In contrast, protein P2 beta is not detected in the ribosome in the P1-deficient D67 strain despite being accumulated in the cytoplasm. The results confirm that neither phosphorylation nor N-terminal blocking of the 12 kDa acidic proteins is required for the assembly and function of the yeast stalk. More importantly, and regardless of the involvement of other elements, they indicate that stalk assembling is a coordinated process, in which P1 proteins would provide a ribosomal anchorage to P2 proteins, and P2 components would confer functionality to the complex.

Using 125I-labeled phenol-alanine sparsomycin, an analogue of sparsomycin having higher biological activity than the unmodified antibiotic, we studied the requirements and the characteristics of its interaction with the ribosome. The drug does not bind to either isolated ribosomal subunits or reconstituted whole ribosomes. For sparsomycin binding to 70S and 80S ribosomes, the occupation of the peptidyltransferase P-site by an N-blocked aminoacyl-tRNA is a definitive requirement. The sparsomycin analogue binds to bacterial and yeast ribosomes with Ka values of around 10(6) M-1 and 0.6 x 10(6) M-1, respectively, but its affinity is probably affected by the character of the peptidyl-tRNA bound to the P-site. Chloramphenicol, lincomycin, and 16-atom ring macrolides compete with sparsomycin for binding to bacterial ribosomes, but streptogramins and 14-atom ring macrolides do not. Considering the reported low affinity of puromycin for bacterial ribosomes, this antibiotic is also a surprisingly good competitor of sparsomycin binding to these particles. In the case of yeast ribosomes, blasticidin is a relatively good competitor of sparsomycin interaction, but anisomycin, trichodermin, and narciclasin are not. As expected, puromycin is a poor competitor of the binding in this case. The results from competition studies carried out with different sparsomycin analogues reveal, in some cases, a discrepancy between the drug ribosomal affinity and its biological effects. This suggests that some intermediate step, perhaps a ribosomal conformational change, is required for the inhibition to take place.

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