Functional characterization of the recycling mechanism for the 60S nuclear export adapter Nmd3p in yeast
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Ribosomes are essential macromolecular machines that translate, through a messenger RNA intermediate, the information encoded in the DNA sequence of all cells into proteins. Because of their fundamental role in cell survival, an enormous amount of cellular resources must be dedicated to ribosome synthesis. Eukaryotic cells must contend with transport of materials between two compartments, the nucleus and cytoplasm. During ribosome biogenesis, these cells assemble ribosomal subunits at a specific subnuclear structure called the nucleolus and only release them into the nucleoplasm upon completion of initial assembly. The subunits must then traverse the nucleoplasm before reaching the nuclear pore complex (NPC) where they are exported to the cytoplasm to act in translation. Processing of the small (40S) subunit through this pathway occurs relatively quickly. However, processing of the large subunit (60S) involves a greater number of maturation steps. One of the last of these steps being export of the large subunit through the NPC, mediated through the export adapter protein Nmd3. The large ribosomal subunit protein Rpl10 is required for 60S export and subunit joining in yeast. It is believed that the role of Rpl10p in export is to provide the 60S binding site for Nmd3p in the nucleus. Through examination of rpl10 mutant effects on the 60S export pathway, I’ve instead found that the role of Rpl10p is indirect. This work shows that disruption of either Rpl10p or the Rpl10p 60S loading factor, Sqt1p, leads to a block in export due to entrapment of Nmd3p on 60S subunits in the cytoplasm. For rpl10 mutants these effects are suppressed by specific alleles of NMD3 that restore recycling to the nucleus. To gain a better understanding of the export function of Nmd3p, this work also examines the NES and 60S binding domains of Nmd3p and, in light of the Rpl10p results shown here, establishes an assay to identify other 60S components required for this binding. From these findings, I propose the model that Rpl10p is required for the release of Nmd3p from subunits in the cytoplasm to support further rounds of 60S export and to provide a final “quality control” step in 60S maturation prior to 40S joining.