Exploration of the proxiOME of large subunit ribosomal proteins reveals Acl1 and Bcl1 as cooperating dedicated chaperones of Rpl1

The eukaryotic ribosome consists of four ribosomal RNAs (rRNAs) and around 80 ribosomal proteins (r-proteins). Most r-proteins, due to their extensive interactions with rRNA, contain highly basic regions, which can predispose them to aggregation prior to their assembly. Accordingly, dedicated chaperones (DCs) and importins have been shown to play a crucial role in safeguarding newly synthesized r-proteins until their incorporation into pre-ribosomal particles. However, only relatively few DCs and interactions with importins have so far been identified for the 79 r-proteins in Saccharomyces cerevisiae . Here, we systematically explored the physical proximities of all r-proteins of the large 60S subunit by TurboID-based proximity labelling. This approach revealed importins and potential novel DCs within the proxiOME of several r-proteins. We provide evidence for a direct interaction of Kap119/Nmd5 with the r-protein Rpl30 (eL30), of Kap121/Pse1 with Rpl38 (eL38), and of Kap114 with both Rpl6 (eL6) and Rpl40 (eL40). Notably, the two previously uncharacterized proteins Acl1 (Ycr051w) and Bcl1 (Ynl035c) were found in the proxiOME of the universally conserved Rpl1 (uL1). While Acl1 appears to be a fungi-specific protein, the human orthologue of Bcl1, WDR89, was found in the proxiOME of the human Rpl1 orthologue RPL10A, suggesting that Bcl1 carries out an evolutionarily conserved function. We show that both Acl1 and Bcl1 are direct interaction partners of Rpl1 and that they have the capacity to form a trimeric complex with Rpl1 in vitro. We also report the crystal structure of a minimal Acl1-Rpl1 complex, which reveals how the ankyrin repeats of Acl1 interact with the second domain of Rpl1. Moreover, we have performed a detailed mutational analysis, based on the Acl1-Rpl1 crystal structure and the predicted Bcl1-Rpl1 structure, to verify the relevance of the interaction-mediating surfaces on Rpl1 and Acl1. In vivo, concomitant absence of Acl1 and Bcl1 leads to a synergistic growth defect and affects the subunit-joining capacity of the produced 60S subunits, presumably due to the reduced incorporation of Rpl1 into nuclear pre-60S subunits. Interestingly, both Acl1 and Bcl1 contain a functional nuclear localization signal (NLS) at their C-terminal extremity. While the exact mechanism of Acl1 import remains to be established, we show that the NLS of Bcl1 corresponds to a Kap104-interacting PY-NLS. Notably, only simultaneous absence of both NLSs abolishes the nuclear accumulation of Acl1 and Bcl1, suggesting that they can be co-imported into the nucleus as a trimeric Acl1-Rpl1-Bcl1 complex. Taken together, our data indicate that Acl1 and Bcl1 may function as cooperating DCs of Rpl1 that assure the safe nuclear transfer and efficient loading of Rpl1 onto pre-60S subunits. In addition, and besides permitting the identification of DCs and importins, our TurboID-based proximity labelling screen also proved to be highly suitable for the visualization of both transient and stable neighbourhoods of r-proteins on pre-60S and mature 60S subunits, altogether making this data set a rich resource for studying many potentially exciting physical proximities of large subunit r-proteins along their entire life cycle.