Different roles of P1 and P2 Saccharomyces cerevisiae ribosomal stalk proteins revealed by cross‐linking

Abstract

The stalk is an essential domain of the large ribosomal subunit formed by a complex of a set of very acidic proteins bound to a core rRNA binding component. While in prokaryotes there is only one type acidic protein, L7/12, two protein families are found in eukaryotes, phosphoproteins P1 and P2, which presumably have different roles. To search for differences zero-length cross-linking by S-S bridge formation was applied using Saccharomyces cerevisiae mutant P1 and P2 proteins carrying single cysteine residues at various positions. The results show a more exposed location of the N-terminal domain of the P2 proteins, which in contrast to P1, can be found as dimers when the Cys is introduced in this domain. Similarly, the Cys containing C-terminal domain of mutant P2 proteins shows a notable capacity to form cross-links with other proteins, which is considerably lower in the P1 type. On the other hand, mutation at the conserved C-domain of protein P0, the eukaryotic stalk rRNA binding component, results in removal of about 14 terminal amino acids. Protein P2, but not P1, protects mutant P0 from this truncation. These results support a eukaryotic stalk structure in which P1 proteins are internally located with their C-terminals having a restricted reactivity while P2 proteins are more external and accessible to interact with other cellular components.

The Catalytic Subunit of Shiga-like Toxin 1 Interacts with Ribosomal Stalk Proteins and is Inhibited by Their Conserved C-Terminal Domain

“…These results suggest that the A 1 chain of SLT-1 binds to the C terminus of P1 and/or P2 but most likely to P2 due to its exposure in the cytosol while binding to another location on RPP0 in close proximity. [27][28][29] Our studies show that type II RIPs, the SLT-1 A 1 chain as well as the A chain of the plant toxin RTA, also recognize the conserved C terminus 17 amino acids of ribosomal stalk proteins (Fig. 5a).…”

“…19,20,[24][25][26] The P2 C terminus is left exposed. [27][28][29] RPP0 has been shown to bind to rRNA via its N-terminal region (Fig. 5b).…”

Functional divergence between the two P1–P2 stalk dimers on the ribosome in their interaction with ricin A chain

“…The function of the other ribosomal stalk P-proteins, which share the same conserved CTD, is not clear. Despite the high sequence similarity between P1/P2 proteins, their biophysical and functional properties are different [25,36]. Previous cross-linking experiments showed differences in the reactivity of P1 and P2 and suggested that the P1 protein, which displayed restricted reactivity, is internally located, whereas the P2 protein is more external and accessible to interact with the other cellular components [36].…”

“…Despite the high sequence similarity between P1/P2 proteins, their biophysical and functional properties are different [25,36]. Previous cross-linking experiments showed differences in the reactivity of P1 and P2 and suggested that the P1 protein, which displayed restricted reactivity, is internally located, whereas the P2 protein is more external and accessible to interact with the other cellular components [36]. Moreover, fluorescence correlation spectroscopy experiments suggested that the function of P1–P2 dimers is not equally distributed [36,37].…”

“…Previous cross-linking experiments showed differences in the reactivity of P1 and P2 and suggested that the P1 protein, which displayed restricted reactivity, is internally located, whereas the P2 protein is more external and accessible to interact with the other cellular components [36]. Moreover, fluorescence correlation spectroscopy experiments suggested that the function of P1–P2 dimers is not equally distributed [36,37]. A recent study on silkworm ribosomes showed that both P1 and P2 contribute to ribosomal activity dependent on eEF-1a (eukaryotic elongation factor-1a) and eEF-2, and the relative contributions to protein synthesis seemed to be different between P1 and P2 [38].…”