The constant supply of ribosomal proteins is crucial for a growing cell to maintain a maximal rate of ribosome synthesis. Hence, it is conceivable that mechanisms exist which ensure that ribosomal proteins are not only synthesized in high amounts, but also remain soluble and are efficiently targeted to the ribosome. In this study, we discovered an anti-aggregation function of the non-ribosomal protein Yar1, which it exhibits exclusively on the small ribosomal subunit protein Rps3. Hence we suggest that Yar1 functions as a specific chaperone for Rps3.
Because of their extensive interactions with ribosomal RNA, ribosomal proteins usually contain a high proportion of positive charges, which are known to cause aggregation in the presence of polyanions such as RNA (10
). Yar1 is composed of two ankyrin repeats. Ankyrin repeats are helix-turn-helix motifs of 33 amino acid residues that exhibit an L-shaped topology and exclusively function in mediating protein-protein interactions (27
). Since Yar1 is a small protein (22 kDa) that does not contain any further domains, it is likely that the main function of Yar1 is to bind Rps3, thereby preventing its aggregation. Protection from aggregation with RNA could be achieved by shielding the positive charges of the basic Rps3 protein (pI 10.2). It is tempting to speculate that Yar1 could act as an RNA mimic for Rps3 considering the high content of negative charges (pI 4.2) found in Yar1.
Newly translated Rps3 travels from the cytoplasm through the nuclear pores and into the nucleus where it assembles with pre-ribosomal particles. Therefore, it needs to be protected from aggregation along this entire path. Consistently, we found that Yar1 is localized both in the cytoplasm and the nucleus. Yar1 shows a predominantly cytoplasmic steady-state localization, however accumulation is observed in the nucleus after inhibition of the export receptor Xpo1. This is in contrast to previous data from the Lycan laboratory, where nuclear accumulation of Yar1-GFP was not observed upon leptomycin B treatment of an LMB-sensitive xpo1
). An explanation for this discrepancy may be that Seiser et al.
used a wild-type strain containing Yar1-GFP on a plasmid, probably resulting in competition between the chromosomal wild-type copy and plasmid encoded GFP-tagged Yar1. According to our data, Yar1 is a shuttling protein that binds Rps3 in the cytoplasm and is presumably imported into the nucleus in complex with Rps3, possibly via the N-terminal NLS of Rps3. The low amount of Yar1 detected in the nucleus under steady-state conditions indicates that it is quickly exported into the cytoplasm after dissociation from Rps3, where it can encounter a new Rps3 molecule ().
Apparently, the requirement for Yar1 is indirectly proportional to the cellular concentration of Rps3: When Rps3 levels are high, the function of Yar1 is not required for optimal growth. This is probably because sufficient soluble Rps3 is present in the cell to reach pre-ribosomal particles even in the absence of a chaperone. When wild-type levels of Rps3 are expressed, the absence of Yar1 reduces the amount of soluble Rps3, resulting in 40S export defects and a reduced production of mature 40S subunits, eventually leading to reduced growth rates. When Rps3 is not fully functional (as in the case of the rps3-1 mutant), the absence of Yar1 is lethal. This may be due to an insufficient amount of Rps3 reaching pre-ribosomal particles in order to ensure synthesis of the critical number of 40S subunits necessary for growth.
Recent reports have highlighted the importance of the general chaperone network in assisting ribosome biogenesis (reviewed in Ref. 12
). Ribosomal proteins and ribosome biogenesis factors have been found in aggregates in strains deleted for the Hsp70 chaperone SSB (11
). Furthermore, Zuo1, which is involved in stimulation of SSB, and its homologue Jjj1 have been shown to bind to pre-ribosomal particles and participate in ribosome biogenesis (29
). However, the exact role of the general chaperone network in ribosome biogenesis remains unclear, and as to date, no direct substrates have been described. Additionally, importins have been shown to protect the human ribosomal proteins S7, S3a, L4, L6, and L18a from aggregation with RNA and were suggested to function as general chaperones for ribosomal proteins (10
The existence of a specific anti-aggregation factor for Rps3 suggests that for some proteins, the general chaperone network of the cell is insufficient for production of the required amounts of soluble protein. The need for additional, more specific factors makes particular sense for ribosomal proteins, which are not only highly expressed but beyond that also prone to aggregation. For these reasons, it is likely that Rps3 is not the only ribosomal protein with a specific chaperone. Chaperone-like functions have also been suggested for Rrb1, a non-ribosomal binding partner of the ribosomal protein Rpl3, Sqt1, an assembly factor for the ribosomal protein Rpl10, as well as Rpf2 and Rrs1, which are involved in the recruitment of 5S rRNA and the ribosomal proteins Rpl5 and Rpl11 into pre-60S subunits (33
). It remains open to future investigations to address whether similar aggregation-preventing mechanisms also exist for other ribosomal proteins.