The ribosomal P0 protein, the orthologue of bacterial ribosomal L10 protein, is the central component of the eukaryotic ribosomal stalk. This structure is formed by the association of P0 with two heterodimers of the P1 and P2 acidic phosphoproteins, the counterparts of prokaryotic protein L7/L12. Although the stalk seems to essentially fulfill the same role in the translational machinery of all organisms, which is related to the interaction and activity of several soluble factors (1
), the eukaryotic P0/(P1–P2)2
complex has some peculiar properties that are not attributed to the prokaryotic structure. These differences have led to the proposal that this complex might modulate ribosomal activity, which does not yet appear to be the case in prokaryotes (2
). Among these peculiarities, the eukaryotic domain seems to undergo a process of assembly/disassembly during translation, which can be detected as an exchange between the ribosome bound P1/P2 proteins and the free proteins present in the cytoplasm (3–6
). It is not clear when this exchange occurs but it seems most likely to take place during ribosome dissociation, after the termination step. It is possible that as a result of this process, the Saccharomyces cerevisiae
ribosome population does not have a homogeneous P1/P2 content. Thus, particles containing different amounts of these acidic proteins, or even totally lacking them, can be detected in the cell (7
). Since the proposed modulatory role of the ribosome stalk is based on the relative proportion of ribosomes with different P1/P2 content, it is fundamental to understand how this ribosome domain is assembled and how the assembly/disassembly process is controlled.
The stalk must be assembled as part of the ribosome and ribosome assembly in eukaryotes is an extremely complicated process that remains far from being understood (9
). A large number of proteins and small rRNA molecules, generically known as trans
-acting or assembly factors, participate in the assembly process. Many of these components have been identified in S. cerevisiae
, although their specific function is only known in a limited number of cases. In yeast, ribosome biogenesis starts in the nucleolus with the transcription of the pre-rRNAs, which bind to a considerable number of trans
-acting factors and ribosomal proteins in order to form the 90S particle (10
). After three consecutive processing steps, the 90S pre-ribosomal particle, which contains the 35S pre-rRNA, yields a pre-40S subunit (12
) and a pre-60S subunit (13
). These two particles follow independent pathways in the nucleus that include the formation of a still poorly defined number of intermediates, especially in the case of the pre-60S. Late pre-ribosomal particles are then exported into the cytoplasm where they undergo the final maturation steps.
The information available regarding stalk assembly is still scarce. The P0 protein has been detected in different pre-ribosomal complexes (14–17
). However, additional information will be required before a conclusion can be reached in regard to stage in which the protein is assembled. Similarly, there is no direct evidence that the P1/P2 stalk proteins are present in pre-ribosomal particles and it is generally assumed that they are assembled in the cytoplasm.
Significant sequence homology has been found between a number of ribosomal proteins and several assembly factors. Thus, the L24 ribosomal protein (Rpl24) is homologous to Rlp24, as is L7 (Rpl7) to Rlp7 and Rps9 to Imp3 (9
). In the case of the Rpl24/Rlp24 pair, it has been proposed that the ribosomal protein might replace the assembly factor in the late pre-ribosome particles as one of the last maturation steps that take place in the cytoplasm (13
Interestingly, the P0 protein and Mrt4 share notable amino acid homology. Mrt4 was initially thought to be involved in mRNA turnover (21
) but it was later found in pre-ribosomes, and it is now considered a component of the ribosome assembly machinery (22
). The Mrt4 protein has been detected in particles purified using tagged proteins involved in the early and mid-stages of 60S ribosomal subunit assembly (9
), suggesting that it fulfills a specific role at the beginning of the 60S ribosomal subunit biogenesis pathway. However, Mrt4 has recently been associated with factors involved in the late stages of 60S assembly (23
), making its true role far from clear.
Here, we have investigated the functional relationship between P0 and Mrt4, finding that both proteins can interact with the same region in the rRNA but that they are mutually exclusive in the ribosome. These data support the hypothesis that Mrt4 binds first to pre-60S ribosomal particles, occupying the P0 site in the earlier assembly steps, and that it is then later displaced by the ribosomal protein.