Saccharomyces cerevisiae strains with either three inactivated genes (triple disruptants) or four inactivated genes (quadruple disruptants) encoding the four acidic ribosomal phosphoproteins, YP1␣, YP1␤, YP2␣, and YP2␤, present in this species have been obtained. Ribosomes from the triple disruptants and, obviously, those from the quadruple strain do not have bound P proteins. All disrupted strains are viable; however, they show a cold-sensitive phenotype, growing very poorly at 23؇C. Cell extracts from the quadruple-disruptant strain are about 30% as active as the control in protein synthesis assays and are stimulated by the addition of free acidic P proteins. Strains lacking acidic proteins do not have a higher suppressor activity than the parental strains, and cell extracts derived from the quadruple disruptant do not show a higher degree of misreading, indicating that the absence of acidic proteins does not affect the accuracy of the ribosomes. However, the patterns of protein expressed in the cells as well as in the cell-free protein system are affected by the absence of P proteins from the particles; a wild-type pattern is restored upon addition of exogenous P proteins to the cell extract. In addition, strains carrying P-protein-deficient ribosomes are unable to sporulate but recover this capacity upon transformation with one of the missing genes. These results indicate that acidic proteins are not an absolute requirement for protein synthesis but regulate the activity of the 60S subunit, affecting the translation of certain mRNAs differently.The presence of a set of strongly acidic proteins is a universal feature of ribosomes from all organisms. These proteins have been involved in interactions with different supernatant factors during protein synthesis in prokaryotic as well as eukaryotic systems (see reference 16 for a review).In eukaryotic organisms, the acidic ribosomal proteins, called generically P proteins, are capable of being phosphorylated, and this modification drastically affects the interaction of the polypeptides with the ribosome in vitro (11,14). In fact, this process might be part of a control mechanism that, by regulating the amount of bound protein, can affect the activity of the particles in relation to the metabolic requirements of the cell (24).In Saccharomyces cerevisiae, four acidic ribosomal proteins have been reported and characterized; according to structural and functional considerations, they can be classified in two groups, YP1␣/␤ and YP2␣/␤, corresponding to the two types of acidic polypeptides present in mammalian ribosomes (35). Using gene disruption techniques, we previously found that the individual elimination of each polypeptide, and even the double disruption of any two of the four proteins, is not a lethal event for the cell, although the generation time is affected to different extents (20, 21). The effect is especially important when the genes encoding the two members of the same group are disrupted, since in this case the remaining acidic proteins present in the c...

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.

The Saccharomyces cerevisiae ribosomal stalk is made of five components, the 32-kDa P0 and four 12-kDa acidic proteins, P1␣, P1␤, P2␣, and P2␤. The P0 carboxyl-terminal domain is involved in the interaction with the acidic proteins and resembles their structure. Protein chimeras were constructed in which the last 112 amino acids of P0 were replaced by the sequence of each acidic protein, yielding four fusion proteins, P0-1␣, P0-1␤, P0-2␣, and P0-2␤. The chimeras were expressed in P0 conditional null mutant strains in which wild-type P0 is not present. In S. cerevisiae D4567, which is totally deprived of acidic proteins, the four fusion proteins can replace the wild-type P0 with little effect on cell growth. In other genetic backgrounds, the chimeras either reduce or increase cell growth because of their effect on the ribosomal stalk composition. An analysis of the stalk proteins showed that each P0 chimera is able to strongly interact with only one acidic protein. The following associations were found: P0-1␣⅐P2␤, P0-1␤⅐P2␣, P0-2␣⅐P1␤, and P0-2␤⅐P1␣. These results indicate that the four acidic proteins do not form dimers in the yeast ribosomal stalk but interact with each other forming two specific associations, P1␣⅐P2␤ and P1␤⅐P2␣, which have different structural and functional roles.