P1 and P2 protein heterodimer binding to the P0 protein of Saccharomyces cerevisiae is relatively non-specific and a source of ribosomal heterogeneity

Abstract

The ribosomal stalk is formed by four acidic phosphoproteins in Saccharomyces cerevisiae, P1α, P1β, P2α and P2β, which form two heterodimers, P1α/P2β and P1β/P2α, that preferentially bind to sites A and B of the P0 protein, respectively. Using mutant strains carrying only one of the four possible P1/P2 combinations, we found a specific phenotype associated to each P1/P2 pair, indicating that not all acidic P proteins play the same role. The absence of one P1/P2 heterodimer reduced the rate of cell growth by varying degrees, depending on the proteins missing. Synthesis of the 60S ribosomal subunit also decreased, particularly in strains carrying the unusual P1α-P2α or P1β-P2β heterodimers, although the distinct P1/P2 dimers are bound with similar affinity to the mutant ribosome. While in wild-type strains the B site bound P1β/P2α in a highly specific manner and the A site bound the four P proteins similarly, both the A and B binding sites efficiently bound practically any P1/P2 pair in mutant strains expressing truncated P0 proteins. The reported results support that while most ribosomes contain a P1α/P2β-P0-P1β/P2α structure in normal conditions, the stalk assembly mechanism can generate alternative compositions, which have been previously detected in the cell.

Figure 1.

Response of S. cerevisiae stalk mutants to temperature and osmotic stress. Serial dilutions of cells from the four double mutants (D46, D47, D56 and D57), and of the parental W303 strain, were grown for 4 days on YEPD agar plates in the presence or absence of 0.3 M NaCl at 20, 30 or 37°C.

Figure 2.

Total cell extracts from the strains indicated grown to mid-logarithmic phase (OD₆₀₀ = ∼0.5), were resolved in 10–50% sucrose gradients under the conditions described in ‘Materials and Methods’ section, and the A₂₆₀ of the fractions was measured. The position of the 40S subunits, 60S subunits, 80S monosomes and polysomes containing increasing numbers of ribosomes is indicated.

Figure 3.

Total amount of ribosomal subunits in cell extracts resolved by sucrose gradients in dissociating conditions. The A₂₆₀ profile of the mutant extracts (continuous lines) is superimposed upon that of the parental W303 strain (discontinuous line). The position of the 40S and 60S subunits is indicated.

Figure 4.

2D electrophoresis of total extracts from mutant strains D46, D47, D56 and D57. Some of the differential spots that appear in one sample but not in others are indicated.

Figure 5.

Analysis of stalk proteins in purified ribosomes from mutant strains. Ribosomes purified from the indicated mutant strains and the parental W303 (W) strain were resolved by 15% SDS–PAGE and the indicated proteins were detected in immunoblots probed with specific monoclonal antibodies.

Figure 6.

Ribosomal stalk composition of mutant yeast strains carrying truncated P0 proteins. Ribosomes were purified from the wild-type W303 strain (A), from the D46 (B) and D57 (D) mutants containing canonical heterodimers, and from the D47 (C) and D56 (E) mutants containing non-canonical heterodimers, expressing either wild-type P0 (P0wt) or truncated P0 proteins ΔA, ΔB and ΔAB (see Supplementary Figure S1). The ribosomes were resolved by isoelectrofocusing in a pH range of 2.0–5.0 and the proteins were detected by silver staining. As a control, purified ribosomes from the respective non-transformed strain were included in the corresponding gels. In gel A, ribosomes from two clones from two different transformations of W303dGP0 with protein P0-B (ΔB₁ and ΔB₂) were included. The upper and lower bands correspond to the phosphorylated and non-phosphorylated forms of each protein.