Factors Affecting Nuclear Export of the 60S Ribosomal Subunit In Vivo

Abstract

In Saccharomyces cerevisiae, the 60S ribosomal subunit assembles in the nucleolus and then is exported to the cytoplasm, where it joins the 40S subunit for translation. Export of the 60S subunit from the nucleus is known to be an energy-dependent and factor-mediated process, but very little is known about the specifics of its transport. To begin to address this problem, an assay was developed to follow the localization of the 60S ribosomal subunit in S. cerevisiae. Ribosomal protein L11b (Rpl11b), one of the ϳ45 ribosomal proteins of the 60S subunit, was tagged at its carboxyl terminus with the green fluorescent protein (GFP) to enable visualization of the 60S subunit in living cells. A panel of mutant yeast strains was screened for their accumulation of Rpl11b-GFP in the nucleus as an indicator of their involvement in ribosome synthesis and/or transport. This panel included conditional alleles of several rRNA-processing factors, nucleoporins, general transport factors, and karyopherins. As predicted, conditional alleles of rRNAprocessing factors that affect 60S ribosomal subunit assembly accumulated Rpl11b-GFP in the nucleus. In addition, several of the nucleoporin mutants as well as a few of the karyopherin and transport factor mutants also mislocalized Rpl11b-GFP. In particular, deletion of the previously uncharacterized karyopherin KAP120 caused accumulation of Rpl11b-GFP in the nucleus, whereas ribosomal protein import was not impaired. Together, these data further define the requirements for ribosomal subunit export and suggest a biological function for KAP120. INTRODUCTIONAlthough eukaryotic ribosomes function in the cytoplasm, the synthesis, processing, and assembly of the ribosomal subunits in Saccharomyces cerevisiae and higher eukaryotes occur in the nucleolus. The entire ribosome is composed of four rRNA species and ϳ75 ribosomal proteins (r-proteins) distributed between two subunits. The 18S, 5.8S, and 25S rRNAs are derived from a single 35S rRNA precursor that is synthesized by RNA polymerase I and then processed by a series of endonucleolytic and exonucleolytic cleavages (reviewed by Kressler et al., 1999;Venema and Tollervey, 1999). The 5S rRNA is synthesized separately by RNA polymerase III and associates with the 60S preribosomal subunit early in assembly. The mature 40S ribosomal subunit contains the 18S rRNA and ϳ32 r-proteins, whereas the 60S subunit is composed of the 5S, 5.8S, and 25S rRNAs and ϳ45 r-proteins. Proper assembly of each ribosomal subunit requires the coordination of several events, including the synthesis and import of r-proteins, the synthesis and processing of rRNA, and the concomitant assembly of r-proteins into the preribosomal subunits. Although a pathway for 35S rRNA maturation has been well defined through both genetic and biochemical approaches (reviewed by Kressler et al., 1999;Venema and Tollervey, 1999), less is known about the association of r-proteins with the rRNA and the export of the assembled subunits out of the nucleus.All nucleocytoplasmic transport occurs ...

Arx1 Is a Nuclear Export Receptor for the 60S Ribosomal Subunit in Yeast

“…Although the Nup84 complex is symmetrically disposed across the nuclear envelope, Nup82 and Gle2 are found on the cytoplasmic face (Suntharalingam and Wente, 2003). Previous work has shown that both the Nup84 and the Nup82 complexes are involved in 60S export (Stage-Zimmermann et al, 2000;Gleizes et al, 2001). …”

“…Subsequent cloning and sequencing of two of the synthetic lethal mutants identified them as NMD3 and NUP120. Nmd3 acts as the Crm1-dependent export adapter for the 60S subunit (Ho et al, 2000b;Gadal et al, 2001), whereas Nup120 is a nucleoporin that has previously been shown to be required for efficient 60S export (Stage-Zimmermann et al, 2000). Sequencing the genomic locus of the nmd3 mutant revealed a T-to-A transversion at nt 1514 (nmd3⌬C14), resulting in a premature stop codon that eliminates the C-terminal 14 aa.…”

“…Of the mutations known to trap Nmd3 in the nucleus, all are in factors that act late in the 60S export pathway. These include Crm1 (Ho et al, 2000b), Mex67/Mtr2 (Yao et al, 2007), proteins that regulate Ran and a subset of nucleoporins (Stage-Zimmermann et al, 2000). Thus, Arx1 itself appears to behave as an export receptor or as an effector of NPC function.…”

“…In yeast the large and small ribosomal subunits are assembled in the nucleolus and exported to the cytoplasm separately but in a Crm1-dependent manner (Moy and Silver, 1999;Ho et al, 2000b;Stage-Zimmermann et al, 2000;Gadal et al, 2001). Nmd3 is an essential nucleocytoplasmic shuttling protein that contains a leucine-rich NES and is required for Crm1-dependent export of the 60S subunit (Ho et al, 2000b;Gadal et al, 2001;Thomas and Kutay, 2003;Trotta et al, 2003).…”

Novel Interaction of the 60S Ribosomal Subunit Export Adapter Nmd3 at the Nuclear Pore Complex

“…To this end, we expressed mutant or wild-type Nmd3 proteins in cells containing GFP fusions to various nucleoporins obtained from the GFP-tagged strain collection (32). Particular emphasis was given to those nucleoporins that had previously been implicated in 60S export ( (17,33,34). For analysis of Nup120, which was absent from the GFP library, we expressed NUP120-GFP from a low copy vector in a nup120 deletion strain.…”