Chapter 12 : Structure of the Yeast Ribosomal Stalk

Models of acidic protein distribution in the yeast ribosomal stalk. (A) Heterogeneous model. Ribosomes carry one dimer of a P1 and another of a P2 protein type. Four associations of the four acidic proteins are possible: P1α-P2α, P1α-P2β, P1β-P2α, P1β-P2β. (B) Homogeneous model. All ribosomes carry one monomer of the four acidic proteins simultaneously.

Cross-linking of P1α-cys62. S. cerevisiae D7, with a disrupted P1α gene, was transformed with plasmid pFL37-P1α-cys62 carrying a P1α protein in which a unique cysteine was introduced in position 62. Ribosomes (20 μg) from the transformed strain were treated in 10 μl of buffer with o-phenanthroline-Cu2+ ( Kobashi, 1968 ; Oleinikov et al., 1993 ) to oxidize the cysteine SH groups, and the ribosomes were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (lane 2). Similarly, a preparation of purified recombinant P1α-cys62 protein (5 μg) was treated in the same way and resolved in the same gel (lane 3). As a control, ribosomes from untransformed S. cerevisiae D7 were treated in the same way (lane 1). Proteins were blotted to nitrocellulose membranes and detected with P1α-specific antibodies.

Affinity chromatography of S. cerevisiae ribosomes in Ni2+ (Ni-nitrilotriacetic acid) columns. (A) A P2α-HisT protein was constructed by fusing a six-His tail to the C-terminal end of protein P2α in plasmid pFL392α-His6. The tagged protein was expressed in S. cerevisiae D4 lacking P2α, and a ribosome preparation from the transformed strain was loaded into a 1-ml Ni2+ affinity column. The column was washed with 10 ml of 10 mM Tris-HCl (pH 7.4), 10 mM MgCl2, and 100 mM KCl buffer containing increasing concentrations of imidazol. Aliquots of the eluted fractions were resolved in sodium dodecyl sulfatepolyacrylamide gels, and the presence of ribosomes in the fractions was checked by estimating the stalk protein P0 by Western blotting with a specific monoclonal antibody. (B) Ribosomes from a control strain carrying a wild-type P2α protein were obtained and processed as for panel A. Lanes: 1, excluded volume; 2 to 6, fractions eluted with buffer containing 0, 10, 40, 70, and 100 mM imidazol.

Isoelectrofocusing of ribosomes from the affinity chromatography columns. An aliquot of the loaded sample (L) and of the 100 mM imidazol-eluted fraction (R) were resolved in polyacrylamide gels in a 2.5 to 5.0 pH gradient as previously described ( Rodriguez-Gabriel et al., 1998 ). The gels were silver stained. The positions of the different acidic proteins are marked. The upper band corresponds in each case to the phosphorylated form of the protein.

Isoelectrofocusing of ribosomes from S. cerevisiae disrupted strains lacking acidic proteins. Ribosomes (0.5 mg) from S. cerevisiae D4, D5, D6, and D7 lacking proteins P2ö, P2β, P1β, and P1α, respectively, were resolved as for Fig. 4. wt, wild type.

Isoelectrofocusing of ribosomes reconstituted with recombinant acidic proteins. Ribosomes from S. cerevisiae D4567 lacking the four acidic proteins were incubated at 30°C in 10 mM Tris-HCl (pH 7.4), 10 mM MgCl2, and 100 mM KCl with a fourfold molar excess of recombinant protein P1α (lane 3), recombinant protein P2β (lane 4), and recombinant protein P1α plus P2β (lane 5). The reconstituted ribosomes were centrifuged through two layers of 20 and 40% sucrose in 10 mM Tris-HCl (pH 7.4), 100 mM MgCl2, and 500 mM KCl buffer. Ribosomes incubated in the absence of proteins (lane 2) and a mixture of both free recombinant proteins (lane 1) were included as controls. Isoelectrofocusing was carried out as for Fig. 4.

Activity of S. cerevisiae D4567 ribosomes reconstituted with recombinant proteins. Ribosomes reconstituted as indicated in the legend to Fig. 6 were tested in a poly(U)-dependent polymerization assay ( Sanchez-Madrid et al., 1979 ). Bars: 1, D4567 ribosomes; 2, D4567 ribosomes plus SP fraction; 3, D4567 ribosomes plus P1α; 4, D4567 ribosomes plus P2β; 5, D4567 ribosomes plus P1α plus P2β. The SP fraction corresponds to an NH4Cl-ethanol wash of wild-type ribosomes containing the four acidic proteins ( Sanchez-Madrid et al., 1979 ). Control ribosomes (sample 2) polymerized 10.1 pmol of Phe per pmol of ribosomes.

Analysis of ribosomes from S. cerevisiae D45 overexpressing protein P1β. (A) Ribosomes from the P2α-P2β-deficient S. cerevisiae D45 strain transformed with the multicopy plasmid pP1b were analyzed before (lane 2) and after (lane 3) centrifugation through a sucrose gradient as for Fig. 6. Ribosomes from S. cerevisiae W303 (lane 1) were included as a control. (B) Ribosomes from S. cerevisiae D67 (lane 2), lacking proteins P1α and P1β and containing an accumulation protein P2β, in the cytoplasm ( Remacha et al., 1992 ) were analyzed as for panel A. Lane 1, control ribosomes as in panel A.

Probable distribution of the four acidic proteins in the yeast ribosomal stalk.