Nmd3p, the nuclear export adapter for the 60S ribosomal subunit: characterization of its recycling mechanism and novel interaction with the nuclear pore complex in yeast

Ribosomes are the macromolecular machines dedicated to the fundamental task of converting genetic information encoded within messenger RNAs into the vast complement of proteins that sustain cellular structure and metabolism in all organisms. Eukaryotic ribosomes are composed of two ribonucleoprotein particles, the large (60S) and small (40S) subunits. Although they function in the cytoplasm, the two subunits are initially synthesized and matured in the nucleus and, therefore, require accessory factors to mediate their nuclear export. Our lab and others have shown that export of the 60S subunit requires an adapter protein, Nmd3p, which provides the nuclear export signal (NES) for the subunit in trans. The manner in which Nmd3p is recycled to the nucleus to sustain 60S export, however, is largely unknown. We have recently shown that, in yeast, the ribosomal protein Rpl10p is required for Nmd3p’s release from exported subunits. Through mutational analysis of the cytoplasmic GTPase, Lsg1p, I have discovered that it also participates in this process, as lsg1 mutants exhibit a marked defect in 60S biogenesis and export due to the entrapment of Nmd3p on cytoplasmic subunits. Consistent with their common roles in Nmd3p recycling, lsg1 and rpl10 mutants are suppressed by dominant mutations in NMD3 that weaken its affinity for the subunit and restore nuclear recycling. I also show that lsg1 mutants accumulate a late cytoplasmic 60S biogenesis intermediate in which Rpl10p is not fully accommodated into the subunit. From these findings, I propose that Lsg1p is required for loading Rpl10p into Nmd3p-bound 60S subunits prior to translation initiation in a process that assesses the structural fidelity of the subunit and triggers the release of the export adapter for future rounds of export. This work also includes an expanded examination of the manner in which Nmd3p engages the export machinery and is translocated across the nuclear envelope through nuclear pore complexes (NPCs). This analysis was facilitated through the use of Nmd3p mutants that are deficient for 60S binding and arrest within NPCs at an intermediate stage of export complex disassembly. From this analysis, I propose that Nmd3p possesses a particularly potent NES whose interaction with the export machinery is modulated via its association with 60S subunits.