Nascent 60S ribosomal subunits enter the free pool bound by Nmd3p.
Nmd3p from yeast is required for the export of the large (60S) ribosomal subunit from the nucleus (Ho et al., 2000). Here, we show that Nmd3p forms a stable complex with free 60S subunits. Using an epitope-tagged Nmd3p, we show that free 60S subunits can be coimmunoprecipitated with Nmd3p. The interaction was specific for 60S subunits; 40S subunits were not coimmunoprecipitated. Using this coprecipitation technique and pulse-chase labeling of ribosomal subunit proteins we showed that Nmd3p bound nascent subunits, consistent with its role in export. However, under conditions in which ribosome biogenesis was inhibited (e.g., inhibition of transcription with thiolutin, inhibition of transcription of ribosomal protein and RNA genes in a sly1-1 mutant at nonpermissive temperature, and inhibition of translation in a conditional prt1 mutant), Nmd3p remained associated with 60S subunits. In addition, Nmd3delta120, a truncated protein that lacked a nuclear localization signal, retained 60S binding. These results suggest that Nmd3p recruits nascent 60S subunits into the pool of free 60S subunits and exchanges on 60S subunits as they recycle during translation.
Ho
J H
JH
Section of Molecular Genetics and Microbiology and The Institute for Cellular and Molecular Biology, The University of Texas at Austin, 78712-1095, USA.
Kallstrom
G
G
Johnson
A W
AW
D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000502:physiology*; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D051938:Munc18 Proteins; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D014176:Protein Biosynthesis; D012331:RNA, Fungal / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D012270:Ribosomes / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D014158:Transcription, Genetic
D002352:Carrier Proteins; D005656:Fungal Proteins; D051938:Munc18 Proteins; C117894:NMD3 protein, S cerevisiae; D009838:Oligodeoxyribonucleotides; D012331:RNA, Fungal; D012335:RNA, Ribosomal; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C072520:SLY1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D008715:Methionine