[show abstract][hide abstract] ABSTRACT: Formation of eukaryotic ribosomes is driven by energy-consuming enzymes. The AAA-ATPase Drg1 is essential for the release of several shuttling proteins from cytoplasmic pre-60S particles and the loading of late joining proteins. However, its exact role in ribosome biogenesis has been unknown. Here we show that the shuttling protein Rlp24 recruited Drg1 to pre-60S particles and stimulated its ATPase activity. ATP hydrolysis in the second AAA domain of Drg1 was required to release shuttling proteins. In vitro, Drg1 specifically and exclusively extracted Rlp24 from purified pre-60S particles. Rlp24 release required ATP and was promoted by the interaction of Drg1 with the nucleoporin Nup116. Subsequent ATP hydrolysis in the first AAA domain dissociated Drg1 from Rlp24, liberating both proteins for consecutive cycles of activity. Our results show that release of Rlp24 by Drg1 defines a key event in large subunit formation that is a prerequisite for progression of cytoplasmic pre-60S maturation.
The Journal of Cell Biology 11/2012; 199(5):771-82. · 10.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: Allelic forms of DRG1/AFG2 confer resistance to the drug diazaborine, an inhibitor of ribosome biogenesis in Saccharomyces cerevisiae. Our results show that the AAA-ATPase Drg1 is essential for 60S maturation and associates with 60S precursor particles in the cytoplasm. Functional inactivation of Drg1 leads to an increased cytoplasmic localization of shuttling pre-60S maturation factors like Rlp24, Arx1, and Tif6. Surprisingly, Nog1, a nuclear pre-60S factor, was also relocalized to the cytoplasm under these conditions, suggesting that it is a previously unsuspected shuttling preribosomal factor that is exported with the precursor particles and very rapidly reimported. Proteins that became cytoplasmic under drg1 mutant conditions were blocked on pre-60S particles at a step that precedes the association of Rei1, a later-acting preribosomal factor. A similar cytoplasmic accumulation of Nog1 and Rlp24 in pre-60S-bound form could be seen after overexpression of a dominant-negative Drg1 variant mutated in the D2 ATPase domain. We conclude that the ATPase activity of Drg1 is required for the release of shuttling proteins from the pre-60S particles shortly after their nuclear export. This early cytoplasmic release reaction defines a novel step in eukaryotic ribosome maturation.
Molecular and Cellular Biology 11/2007; 27(19):6581-92. · 5.37 Impact Factor