Assembly of Saccharomyces cerevisiae Ribosomal Stalk:  Binding of P1 Proteins Is Required for the Interaction of P2 Proteins

Abstract

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.

Asymmetric Interactions between the Acidic P1 and P2 Proteins in the Saccharomyces cerevisiae Ribosomal Stalk

“…Protein P0 seems to be able to directly interact only with the P1 proteins as shown previously by ribosome reconstitution tests (58) and double hybrid data (36), and it is possible, therefore, that a direct interaction between P0 and the P2 proteins may not exist in the ribosome. There are at least two ways in which P1␣⅐P2␤ and P1␤⅐P2␣ can be assembled in the stalk (Fig.…”

The puzzling lateral flexible stalk of the ribosome

“…There is also a consensus for an anchorage of the P1/P2 dimer by a direct interaction of the P1 N-terminal domain, but not the P2 N-terminal domain with P0 (Gonzalo et al, 2001;Shimizu et al, 2002;Zurdo et al, 2000).…”

Mass Spectrometry of Ribosomes from Saccharomyces cerevisiae

“…P0 has the same 11-amino acid C-terminal sequence as P1 and P2, and the phosphorylation site, Ser-302 in P0, is within this conserved C terminus. Examination of the stalk assembly both in vivo and in vitro has revealed that P2␤ is unable to bind to the ribosome in the absence of P1␣; in contrast P1␣ is capable of binding to the particle in the absence of P2␤ (13). These results have been interpreted to suggest that assembly of the stalk complex is a coordinated process in which P1 proteins provide anchorage to the ribosome for the P2 proteins, whereas the P2 proteins confer functionality to the complex (13).…”

“…Examination of the stalk assembly both in vivo and in vitro has revealed that P2␤ is unable to bind to the ribosome in the absence of P1␣; in contrast P1␣ is capable of binding to the particle in the absence of P2␤ (13). These results have been interpreted to suggest that assembly of the stalk complex is a coordinated process in which P1 proteins provide anchorage to the ribosome for the P2 proteins, whereas the P2 proteins confer functionality to the complex (13). Recent evidence has also shown that the heterodimer P1␣-P2␤ is a stable entity in solution; this observation led to the proposal that the P proteins form dimers in the cytoplasm prior to assembly on the ribosome (14).…”