Defining the Order in Which Nmd3p and Rpl10p Load onto Nascent 60S Ribosomal Subunits

Abstract

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.In eukaryotic cells, ribosomes are assembled in the nucleolus and are transported across the nuclear membrane through nuclear pore complexes to the cytoplasm, where they are activated for translation. Many rRNA processing events must coincide with the assembly of proteins onto nascent ribosomal subunits during their biogenesis (25,34,35). The nascent 60S particle is preassembled in the nucleolus from locally transcribed and processed rRNAs and ribosomal proteins imported from the cytoplasm. The assembly events are highly organized and require over 150 trans-acting factors, many of which reside solely in the nucleolus. Much work has been done in elucidating the various intermediate forms of the pre-60S particle during its maturation in the nucleolus and nucleoplasm (references 7, 8, and 34 and references therein). Most of the assembly factors are removed from the preassembled complex before it leaves the nucleolus, and the complement of trans-acting factors is further reduced in the nucleoplasm prior to export (2, 29).Nuclear export of the large subunit requires the adapter protein Nmd3p, which provides the nuclear export signal (NES) for the subunit (9, 15). 60S export also depends on the export receptor Crm1p, which recognizes the leucine-rich NES of Nmd3p (9,15). This export pathway is conserved in humans, where it has also been suggested that Nmd3p binds to 60S subunits in the nucleolus and may be required for their release into the nucleoplasm (17,32,33).NMD3 functionally interacts with the ribosomal protein RPL10. The interaction of NMD3 and RPL10 was first observed in a ge...

Plant L10 Ribosomal Proteins Have Different Roles during Development and Translation under Ultraviolet-B Stress

“…RPL10 is a ubiquitous protein that participates in joining the 40S and 60S ribosomal subunits into a functional 80S ribosome; it organizes the union site to aminoacyl-tRNA; also, its incorporation into the 60S subunit is a prerequisite for the union of subunits and the initiation of translation (Eisinger et al, 1997;Loftus et al, 1997;West et al, 2005;Hofer et al, 2007). In addition, increasing evidence indicates that RPL10 protein from various organisms has multiple extraribosomal functions, besides being a constituent of the ribosome and participating in protein synthesis (Chan et al, 1996;Wool, 1996;Nika et al, 1997;Mills et al, 1999;Chávez-Ríos et al, 2003;Wen et al, 2005).…”

“…In yeast, a mutation in Rpl10 demonstrated that it is essential for viability, as RPL10 organizes the union site to aminoacyl-tRNA; also, its incorporation into the 60S subunit is a prerequisite for the union of subunits and the initiation of translation (West et al, 2005;Hofer et al, 2007). Besides its role in translation, L10 has additional cellular roles.…”

Mutations in the ribosomal protein gene RPL10 suggest a novel modulating disease mechanism for autism

“…The latter is functionally linked to Nmd3 via the Xpo1/exportin export receptor pathway. 16,17 Studies in yeast show that in the cytoplasm, Rpl10 is necessary for joining the 60S and 40S subunits in a late step of translation initiation, 18 a functional reflection of its location at the interface between the two ribosomal subunits adjoining the A and P sites as well as the transpeptidyl transferase center. The high level of sequence conservation of RPL10 in all organisms and the location close to regulatory and catalytic sites of the ribosome indicate the importance of the structural and functional integrity of this protein in ribosome assembly and function, respectively.…”

Bcp1 Is the Nuclear Chaperone of Rpl23 in Saccharomyces cerevisiae

“…In addition to their classic role of protecting from proteolysis, chaperones are also involved in adjusting the loading or transport processes. Yar1 directly interacts with Rps3 (S3) and acts as an anti-aggregation factor (35,37), and Sqt1 is a chaperone for Rpl10 (uL16) that facilitates loading of Rpl10 into 60S subunits (34,42). Rrb1 has direct interaction with Rpl3 (uL3), and overexpression of Rrb1 increased the level and nuclear accumulation of Rpl3 (36).…”

“…It has been shown that karyopherins or chaperones interact with ribosomal proteins to maintain their solubility and also facilitate their loading into nascent ribosomal subunits (33)(34)(35)(36)(37)(38)(39)(40)(41)(42).…”