Dual protection by Bcp1 and Rkm1 ensures incorporation of uL14 into pre-60S ribosomal subunits.

Eukaryotic ribosomal proteins contain extended regions essential for translation coordination. Dedicated chaperones stabilize the associated ribosomal proteins. We identified Bcp1 as the chaperone of uL14 in Saccharomyces cerevisiae. Rkm1, the lysine methyltransferase of uL14, forms a ternary complex with Bcp1 and uL14 to protect uL14. Rkm1 is transported with uL14 by importins to the nucleus, and Bcp1 disassembles Rkm1 and importin from uL14 simultaneously in a RanGTP-independent manner. Molecular docking, guided by crosslinking mass spectrometry and validated by a low-resolution cryo-EM map, reveals the correlation between Bcp1, Rkm1, and uL14, demonstrating the protection model. In addition, the ternary complex also serves as a surveillance point, whereas incorrect uL14 is retained on Rkm1 and prevented from loading to the pre-60S ribosomal subunits. This study reveals the molecular mechanism of how uL14 is protected and quality checked by serial steps to ensure its safe delivery from the cytoplasm until its incorporation into the 60S ribosomal subunit.

Puf6 and Loc1 Are the Dedicated Chaperones of Ribosomal Protein Rpl43 in Saccharomyces cerevisiae.

Ribosomal proteins are highly expressed, and the quality of ribosomal proteins must be rigorously controlled to build up a functional ribosome. Rpl43, ribosomal protein large subunit 43, is located nearby the E-site of ribosomes. In our previous study, we found that Puf6, Loc1, and Rpl43 form a trimeric complex in Saccharomyces cerevisiae. Rpl43 protein levels are under-accumulated in the absence of PUF6 or LOC1. However, why the loss of Puf6 or Loc1 decreased the protein levels of Rpl43 remained unclear. In the present study, we further dissected the connections among these three proteins and found that the processing defects of pre-ribosomal RNA in puf6Δ and loc1Δ are similar to those of the mutant with depletion of Rpl43. The stability of newly synthesized Rpl43 protein decreased slightly in puf6Δ and significantly in loc1Δ. We also found that Puf6 and Loc1 could interact with nascent Rpl43 co-translationally via the N-terminus of Rpl43. While the association and dissociation of Rpl43 with karyopherins did not depend on Puf6 and Loc1, Puf6 and Loc1 interacted with nascent Rpl43 in collaboration. While the N-terminus of Puf6 contained nuclear localization signals for transport, the PUF (Pumilio) domain was essential to interaction with Loc1, Rpl43, and 60S subunits. The C-terminus of Loc1 is more important for interaction with Puf6 and Rpl43. In this study, we found that Puf6 and Loc1 are the dedicated chaperones of ribosomal protein Rpl43 and also analyzed the potential interaction domains among the three proteins. Correct formation of the Puf6, Loc1, and Rpl43 ternary complex is required to properly proceed to the next step in 60S biogenesis.