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.

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.Biogenesis of the ribosomal subunits in Saccharomyces cerevisiae starts with transcription of the 35S pre-rRNA, which is a common precursor for the individual rRNAs for both the small and large ribosomal subunits. This pre-rRNA assembles with ribosomal and nonribosomal proteins to form a 90S preribosomal particle (for recent reviews on ribosome biogenesis, see references 5, 6, and 32). During maturation, preribosomal particles undergo substantial changes in protein composition, which are accompanied by a series of pre-rRNA processing events (see reference 5). Separation of the biogenesis pathways for the 40S and 60S subunits occurs when the 32S precursor rRNA is cleaved into the 20S and 27SA2 pre-rRNAs, the precursors for the small-and large-subunit rRNAs, respectively.

Background: Diazaborine is the only known inhibitor of eukaryotic ribosome biogenesis, but its target is unknown. Results: Diazaborine binds the AAA-ATPase Drg1 and inhibits its ATP hydrolysis, thereby blocking release of Rlp24 from pre-60S particles. Conclusion: Diazaborine blocks ribosome biogenesis by inhibiting the physiological activity of Drg1. Significance: Our results highlight the potential of the ribosome biogenesis pathway as target for novel inhibitors.

LysM motifs are carbohydrate-binding modules found in prokaryotes and eukaryotes. They bind to N-acetylglucosamine-containing carbohydrates, such as chitin, chitio-oligosaccharides and peptidoglycan. In this review, we summarize the features of the protein architecture of LysM-containing proteins in fungi and discuss their so far known biochemical properties, transcriptional profiles and biological functions. Further, based on data from evolutionary analyses and consensus pattern profiling of fungal LysM motifs, we show that they can be classified into a fungal-specific group and a fungal/bacterial group. This facilitates the classification and selection of further LysM proteins for detailed analyses and will contribute to widening our understanding of the functional spectrum of this protein family in fungi. Fungal LysM motifs are predominantly found in subgroup C chitinases and in LysM effector proteins, which are secreted proteins with LysM motifs but no catalytic domains. In enzymes, LysM motifs mediate the attachment to insoluble carbon sources. In plants, receptors containing LysM motifs are responsible for the perception of chitin-oligosaccharides and are involved in beneficial symbiotic interactions between plants and bacteria or fungi, as well as plant defence responses. In plant pathogenic fungi, LysM effector proteins have already been shown to have important functions in the dampening of host defence responses as well as protective functions of fungal hyphae against chitinases. However, the large number and diversity of proteins with LysM motifs that are being unravelled in fungal genome sequencing projects suggest that the functional repertoire of LysM effector proteins in fungi is only partially discovered so far.

SummaryChitin is an important structural constituent of fungal cell walls composed of N‐acetylglucosamine (GlcNAc) monosaccharides, but catabolism of GlcNAc has not been studied in filamentous fungi so far. In the yeast C andida albicans, the genes encoding the three enzymes responsible for stepwise conversion of GlcNAc to fructose‐6‐phosphate are clustered. In this work, we analysed GlcNAc catabolism in ascomycete filamentous fungi and found that the respective genes are also clustered in these fungi. In contrast to C . albicans, the cluster often contains a gene for an Ndt80‐like transcription factor, which we named RON1 (regulator of N‐acetylglucosamine catabolism 1). Further, a gene for a glycoside hydrolase 3 protein related to bacterial N‐acetylglucosaminidases can be found in the GlcNAc gene cluster in filamentous fungi. Functional analysis in T richoderma reesei showed that the transcription factor RON1 is a key activator of the GlcNAc gene cluster and essential for GlcNAc catabolism. Furthermore, we present an evolutionary analysis of Ndt80‐like proteins in Ascomycota. All GlcNAc cluster genes, as well as the GlcNAc transporter gene ngt1, and an additional transcriptional regulator gene, csp2, encoding the homolog of N eurospora crassa  CSP2/GRHL, were functionally characterised by gene expression analysis and phenotypic characterisation of knockout strains in T . reesei.