Characterization of the specialized role of the ribosome-associated J-protein, Jjj1, in ribosome biogenesis.

摘要

Molecular chaperones are a diverse group of proteins that are well known for interacting with newly synthesized polypeptides to aid in their proper folding. In Saccharomyces cerevisiae, the ribosome-associated J-protein, Zuo1, functions in concert with the Hsp70 Ssb in the protection of nascent chains as they exit the ribosome. However, specialized chaperones exist that function in a wide variety of additional roles, including modification of protein:protein interactions.;Like Zuo1, here I show that the J-protein Jjj1 is also ribosome-associated and can functionally replace Zuo1. However, in contrast to Zuol, Jjj1 associates primarily with free 60S ribosomal subunits and functions with the Hsp70 Ssa. Furthermore, deletion of JJJ1 (but not of ZUO1) results in accumulation of aberrant ribosome formations called half-mers, which are accompanied by a decrease in total 60S subunits. Deletion of either JJJ1 or the known 60S-biogensis factor, REI1, results in cytosolic accumulation of the normally nuclear pre-60S maturation factor Arx1. Together, these data implicate Jjj1 in 60S ribosomal subunit maturation, specifically in the removal or recycling of Arx1 to the nucleus.;As both Jjj1 and Rei1 are involved in the removal or recycling of Arx1, I sought to determine if Jjj1 and Rei1 interact. Purified Jjj1 and Rei1 bound to one another in the absence of additional factors in an in vitro assay. Furthermore, the domains responsible for interaction of Jjj1 with Rei1 include two zinc fingers as well as a region of highly charged amino acids located at the C-terminus of Jjj1. Disruption of this interaction by deletion of the charged region results in accumulation of half-mer ribosomes and concomitant accumulation of Arx1 in the cytosol, however the mutant Jjj1 protein remains ribosome-bound. Together this suggests that the interaction between Jjj1 and Rei1 is important for 60S subunit biogenesis. In addition, I present further evidence that Jjj1, Rei1, and Ssa cooperate in the physical removal of Arx1 from the pre-60S subunits, facilitating its cytosolic maturation.