Nuclear recycling of the pre-60S ribosomal subunit-associated factor Arx1 depends on Rei1 in Saccharomyces cerevisiae.
Arx1 and Rei1 are found on late pre-60S ribosomal particles containing the export adaptor Nmd3. Arx1 is related to methionine aminopeptidases (MetAPs), and Rei1 is a C2H2 zinc finger protein whose function in ribosome biogenesis has not been previously characterized. Arx1 and Rei1 localized predominately to the nucleus and cytoplasm, respectively, but could be coimmunoprecipitated, suggesting that they are transiently in the same 60S complex. arx1delta mutants showed a modest accumulation of 60S subunits in the nucleus, suggesting that Arx1 enhances 60S export. Deletion of REI1 led to cold sensitivity and redistribution of Arx1 to the cytoplasm, where it remained bound to free 60S subunits. However, deletion of ARX1 or the fusion of enhanced GFP (eGFP) to Rpl25 suppressed the cold sensitivity of an rei1delta mutant. The presence of eGFP on Rpl25 or its neighboring protein Rpl35 reduced the binding of Arx1 to 60S subunits, suggesting that Arx1 binds to 60S subunits in the vicinity of the exit tunnel. Mutations in Arx1 that disrupted its binding to 60S also suppressed an rei1delta mutant and restored the normal nuclear localization of Arx1. These results indicate that the cold sensitivity of rei1delta cells is due to the persistence of Arx1 on 60S subunits in the cytoplasm. Furthermore, these results suggest that Rei1 is needed for release of Arx1 from nascent 60S subunits after export to the cytoplasm but not for the subsequent nuclear import of Arx1.
Hung
Nai-Jung
NJ
Section of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology, 1 University Station, A5000, The University of Texas at Austin, Austin, Texas 78712-0162, USA.
Johnson
Arlen W
AW
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D003513:Cycloheximide / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D017353:Gene Deletion; D005800:Genes, Fungal; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D003513:Cycloheximide