Mutational Analysis of the Ribosomal Protein Rpl10 from Yeast

Anne Hofer,Cyril Bussiere,Arlen W Johnson

doi:10.1074/jbc.m705057200

Anne Hofer, Cyril Bussiere + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m705057200

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Abstract

Yeast Rpl10 belongs to the L10e family of ribosomal proteins. In the large (60 S) subunit, Rpl10 is positioned in a cleft between the central protuberance and the GTPase-activating center. It is loaded into the 60 S subunit at a late step in maturation. We have shown previously that Rpl10 is required for the release of the Crm1-dependent nuclear export adapter Nmd3, an event that also requires the cytoplasmic GTPase Lsg1. Here we have carried out an extensive mutational analysis of Rpl10 to identify mutations that would allow us to map activities to distinct domains of the protein to begin to understand the molecular interaction between Rpl10 and Nmd3. We found that mutations in a central loop (amino acids 102-112) had a significant impact on the release of Nmd3. This loop is unstructured in the crystal and solution structures of prokaryotic Rpl10 orthologs. Thus, the loop is not necessary for stable interaction of Rpl10 with the ribosome, suggesting that it plays a dynamic role in ribosome function or regulating the association of other factors. We also found that mutant Rpl10 proteins were engineered to be unable to bind to the ribosome accumulated in the nucleus. This was unexpected and may suggest a nuclear role for Rpl10.

Highlights

Contacts with other ribosomal proteins, it makes extensive contacts with 23 S rRNA along helices H38 and H89 and minor contacts with 5 S rRNA
Release of Nmd3 depends on the cytoplasmic GTPase Lsg1, and we have suggested that the GTPase activity of Lsg1 drives the accommodation of Rpl10 into the subunit, facilitating the release of Nmd3 [12, 16]
In an attempt to disrupt Rpl10 binding to the ribosome without significantly altering the structure of Rpl10, we mutated two regions of basic amino acids that were likely important for electrostatic interaction with rRNA

Summary

EXPERIMENTAL PROCEDURES

Yeast Strains and Media—All of the strains were grown at 30 °C in rich medium (yeast extract peptone) or synthetic dropout medium [20] containing either 2% glucose or 1% galactose as the carbon source. PAJ1199 (RPL10[R153E R154E]-myc LEU2-CEN) was made by fusion PCR using the oligonucleotide pairs AJO264 and AJO744, and AJO743 and AJO722 (Table 2). PAJ1319 (NMD3[I112T, I362T]-myc URA3 2␮) was made by moving the NMD3-containing HindIII-EagI fragment from pAJ1315 [16] into the same sites of pRS426. Western Blotting—Extracts were prepared from 5-ml cultures of AJY2104 containing mutant Rpl plasmids grown in LeuϪ galactose medium. Immunoprecipitations—For immunoprecipitations (IPs) of mutant myc-tagged Rpl proteins, 25-ml cultures were grown to A600 ϳ 0.5 in LeuϪ galactose medium. The cell pellets were washed with 2 ml of polysome buffer (10 mM TrisHCl, pH 7.6, 100 mM KCl, 10 mM MgCl2, 6 mM ␤-mercaptoethanol, 150 ␮g/ml cycloheximide, 1 mM phenylmethylsulfonyl fluoride, and 1 ␮M each leupeptin and pepstatin A), resuspended in one volume of the same buffer and disrupted by vortexing with glass beads.

Oligonucleotides used for PCR and sequencing

RESULTS

Wild type ϩϩϩϩ no

DISCUSSION