The P1/P2 Protein Heterodimers Assemble to the Ribosomal Stalk at the Moment When the Ribosome Is Committed to Translation but Not to the Native 60S Ribosomal Subunit in Saccharomyces cerevisiae

Abstract

The four structural acidic ribosomal proteins that dissociate from P1A/P2B and P1B/P2A heterodimers of Saccharomyces cerevisiae were searched in the 60S ribosomal subunit, the 80S monosome, and the polysomal fractions after ribosome profile centrifugation in sucrose gradients in TMN buffer, and after dissociation of monosomes and polysomes to small and large ribosomal subunits in LMS buffer. Analysis by isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting of these fractions or the purified acidic protein samples showed eight bands that correspond to the acidic ribosomal proteins in the 60S dissociated subunits of the 80S monosome and polysomes. After samples had been radiolabeled with (32)P, four bands were shown to correspond to the phosphorylated form of the acidic ribosomal proteins located in the 80S monosome and the polysomes. Surprisingly, native 60S subunits have no acidic ribosomal proteins. Altogether, these findings indicate that P1/P2 heterodimers bind to P0 when both ribosomal subunits are joined and committed to translation, and they detached from the stalk, just after the small and large ribosomal subunits were separated from the mRNA. Evidence that the phosphorylated and unphosphorylated P1 and P2 acidic ribosomal proteins are part of the functional stalk is also presented.

Functions of Ribosomal Proteins in Assembly of Eukaryotic Ribosomes In Vivo

“…This reaction enables the irreversible binding of P0 (128). In contrast, the P1 and P2 r-proteins, which together with P0 form the stalk structure (reviewed in Reference 138), cycle on and off of mature 60S subunits (139). Assembly of the P stalk is necessary for binding and activation of the GTPase Efl1 (35).…”

Neuronal ribosomes dynamically exchange ribosomal proteins in a context-dependent manner

“…This indicates that either they rapidly and persistently associate with mature ribosomes or that they are replaced (exchanged) by nascent (but un-labeled) proteins synthesized during the chase. Supporting the idea of RP exchange, we noted the presence of RPLP1 and 2 among the proteins with the lowest fold change after the chase; these proteins are the only two known RPs to transiently associate and dissociate from mature ribosomes (Tsurugi and Ogata, 1985; Zinker, 2014). Taken together, these data reveal that the binding kinetics of RPs to neuronal ribosomes are not homogeneous, and that a subset of RPs can rapidly and dynamically incorporate into neuronal ribosomes.…”

An “alert state” ribosome population acts as a master regulator of cytokine-mediated processes

“…This pentameric stalk (also known as the P-stalk) is the central component of the GTPase-associated center of the ribosome 30 . Interestingly, the P1/P2 heterodimer has long been known not to be essential for translation 31 , and, unlike most other RPs, dynamically associates with the ribosome 32 . To understand whether the cytokine-mediated changes we have detected are P1 specific, or rather a result in an increase in the P-stalk, we tested whether P2 is also increased in translating ribosomes following cytokine treatment.…”