Defining the order in which Nmd3p and Rpl10p load onto nascent 60S ribosomal subunits.
The large ribosomal subunit protein Rpl10p is required for subunit joining and 60S export in yeast. We have recently shown that Rpl10p as well as the cytoplasmic GTPase Lsg1p are required for releasing the 60S nuclear export adapter Nmd3p from subunits in the cytoplasm. Here, we more directly address the order of Nmd3p and Rpl10p recruitment to the subunit. We show that Nmd3p can bind subunits in the absence of Rpl10p. In addition, we examined the basis of the previously reported dominant negative growth phenotype caused by overexpression of C-terminally truncated Rpl10p and found that these Rpl10p fragments are not incorporated into subunits in the nucleus but instead sequester the WD-repeat protein Sqt1p. Sqt1p is an Rpl10p binding protein that is proposed to facilitate loading of Rpl10p into the 60S subunit. Although Sqt1p normally only transiently binds 60S subunits, the levels of Sqt1p that can be coimmunoprecipitated by the 60S-associated GTPase Lsg1p are significantly increased by a dominant mutation in the Walker A motif of Lsg1p. This mutant Lsg1 protein also leads to increased levels of Sqt1p in complexes that are coimmunoprecipitated with Nmd3p. Furthermore, the dominant LSG1 mutant also traps a mutant Rpl10 protein that does not normally bind stably to the subunit. These results support the idea that Sqt1p loads Rpl10p onto the Nmd3p-bound subunit after export to the cytoplasm and that Rpl10p loading involves the GTPase Lsg1p.
West
Matthew
M
Section of Molecular Genetics and Microbiology, ESB 325, The University of Texas at Austin, Austin, TX 78712-1095, USA.
Hedges
John B
JB
Chen
Anthony
A
Johnson
Arlen W
AW
D020816:Amino Acid Motifs; D003593:Cytoplasm / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000378:metabolism; D047468:Immunoprecipitation; D009154:Mutation / Q000235:genetics; D021381:Protein Transport; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
C117894:NMD3 protein, S cerevisiae; D016601:RNA-Binding Proteins; C093333:RPL10 protein, S cerevisiae; D012269:Ribosomal Proteins; C107942:SQT1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D019204:GTP-Binding Proteins; C475026:LSG1 protein, S cerevisiae