Protein P0 together with proteins P1 and P2 form the stalk in eukaryotic ribosomes. P0 has a carboxyl-terminal domain about 100 amino acids long that has high sequence similar to the ribosomal proteins P1 and P2. By sequential deletion of this region, a series of Saccharomyces cerevisiae truncated P0 genes have been constructed that encode proteins lacking 21, 87, and 132 amino acids from the carboxyl terminus, respectively. These constructions have been used to transform yeast P0 conditional null mutants to test their capacity to restore cell growth. Removal of only the last 21 amino acids causes a small effect on cell growth in wild-type strains; however, this deletion is lethal in strains having P protein-deficient ribosomes. A P0 lacking 87 amino acids allows cell growth at a low rate, and ribosomes bind P proteins with much less affinity. Lastly, removal of 132 amino acids totally inactivates P0; this deleted protein is unable to bind to the particles, causing a deficiency in active 60 S subunits and making the cell nonviable. These results indicate that at least one out of the five protein P-like carboxyl termini present in the ribosome has to be firmly bound to the particle for protein synthesis and cell viability, and this structure can be provided by protein P0. The part of P0 from around positions 230-290 is important for the interaction of proteins P1/P2 with the ribosome, but it is not essential for protein synthesis. Finally, the region including from residues 185 to 230 is required for the interaction of P0 with the rRNA.

The eukaryoic ribosomal stalk is thought to consist of the phosphoproteins P1 and P2, which form a complex with protein PO. This complex interacts at the GTPase domain in the large subunit rRNA, overlapping the binding site of the protein L11-like eukaryotic counterpart (Saccharomyces cerevisiae protein L15 and mammalian protein L12). An unusual pool of the dephosphorylated forms of proteins P1 and P2 is detected in eukaryotic cytoplasm, and an exchange between the proteins in the pool and on the ribosome takes place during translation. Quadruply disrupted yeast strains, carrying four inactive acidic protein genes and, therefore, containing ribosomes totally depleted of acidic proteins, are viable but grow with a doubling time threefold higher than wild-type cells. The in vitro translation systems derived from these stains are active but the two-dimensional gel electrophoresis pattern of proteins expressed in vivo and in vitro is partially different. These results indicate that the P1 and P2 proteins are not essential for ribosome activity but are able to affect the translation of some specific mRNAs. Protein PO is analogous to bacterial ribosomal protein L10 but carries an additional carboxyl domain showing a high sequence homology to the acidic proteins P1 and P2, including the terminal peptide DDDMGFGLFD. Successive deletions of the PO carboxyl domain show that removal of the last 21 amino acids from the PO carboxyl domain only slightly affects the ribosome activity in a wild-type genetic background; however, the same deletion is lethal in a quadruple disruptant deprived of acidic P1/P2 proteins. Additional deletions affect the interaction of PO with the P1 and P2 proteins and with the rRNA. The experimental data available support the implication of the eukaryotic stalk components in some regulatory process that modulates the ribosomal activity.

In Saccharomyces cerevisiae, the Mrt4 protein is a component of the ribosome assembly machinery that shares notable sequence homology to the P0 ribosomal stalk protein. Here, we show that these proteins can not bind simultaneously to ribosomes and moreover, a chimera containing the first 137 amino acids of Mrt4 and the last 190 amino acids from P0 can partially complement the absence of the ribosomal protein in a conditional P0 null mutant. This chimera is associated with ribosomes isolated from this strain when grown under restrictive conditions, although its binding is weaker than that of P0. These ribosomes contain less P1 and P2 proteins, the other ribosomal stalk components. Similarly, the interaction of the L12 protein, a stalk base component, is affected by the presence of the chimera. These results indicate that Mrt4 and P0 bind to the same site in the 25S rRNA. Indeed, molecular dynamics simulations using modelled Mrt4 and P0 complexes provide further evidence that both proteins bind similarly to rRNA, although their interaction with L12 displays notable differences. Together, these data support the participation of the Mrt4 protein in the assembly of the P0 protein into the ribosome and probably, that also of the L12 protein.

Pleurotus ostreatus is a hetertothallic homobasidiomycete whose mating is controlled by a bifactorial tetrapolar genetic system. Although this mechanism is well accepted, there is a lack of knowledge about its molecular basis, as the incompatibility loci have not been cloned and sequenced. As a first step towards the elucidation of the molecular structure of the A-type incompatibility locus, molecular markers have been isolated which correspond to genomic sequences present in different strains of P. ostreatus but not in other higher basidiomycetae. These markers reveal single-copy genetic regions in which some degree of genetic variability can be detected.