Loading Drg1

The ribosome is the cellular organelle responsible for the correct translation of mRNA into the amino acid sequence of proteins and is essential for all living cells. The 80S ribosome of eukaryotes is composed of one 40S and one 60S subunit which contain different ribosomal RNA species and ribosomal proteins. The biogenesis of the ribosomal subunits is one of the most energy consuming processes in each cell. The process is best understood in yeast where it involves about 200 transacting factors that interact more or less transiently with the pre-ribosomal particles and perform specific functions like for example processing of pre-rRNA, assembly of ribosomal proteins or recruitment of further maturation factors. Many of these transacting factors assemble with the pre-ribosomal particles already in the nucleolus and are released again during a later maturation step within the nucleus. However, some of them accompany the pre-ribosomal particles into the cytoplasm, where they have to be released and recycled into the nucleus to ensure an ongoing formation of ribosomal subunits. This is achieved by a number of different cytoplasmic maturation factors. We showed previously that the AAA-ATPase Drg1 binds to cytoplasmic pre-60S particles and is essential for the release of such shuttling proteins in the cytoplasm. Our unpublished results show that inactivation of Drg1 results in accumulation of all up to now known shuttling proteins and export factors on cytoplasmic pre-60S particles, suggesting that Drg1 catalyzes the very first step in cytoplasmic pre-60S maturation. In addition, we found that the N-domain of Drg1 interacts with nucleoporins localized at the cytoplasmic surface of the nuclear pore complex in vivo and in vitro. Thus, Drg1 might be loaded onto pre-60S particles as soon as they appear at the cytoplasmic face of the NPC during export. Mutant variants of Drg1 that are defective in nucleoporin interaction show decreased levels of Drg1 on pre-60S particles, which indicates that this interaction is required for efficient loading of the AAA-protein onto the pre-ribosome. In this project, we will investigate in detail how Drg1 interacts with the nuclear pore proteins and what alterations are induced in the protein to facilitate binding to nascent pre-60S particles and to activate the function of Drg1 in the release of shuttling proteins. For this purpose we will examine how the binding of nucleoporins to the N-domain of Drg1 is communicated to the ATPase domains. This aim will be achieved by a combination of biochemical, structural and genetic investigations. The importance of communication between N-terminal domain and ATPase domains in AAA ATPases is underscored by the observation that allelic forms of the Drg1 relative p97 that are defective in this communication cause the hereditary IBMPFD disease in humans. The correlation of the structural and enzymatic properties of Drg1 mutant proteins with those observed for p97 variants (IBMPFD) extends the significance of our results to understanding molecular mechanisms underlying human diseases.

Ribosomes perform the translation of mRNA into the amino acid sequence of proteins. They consist of a large and a small subunit and are composed of ribosomal RNAs and ribosomal proteins. The formation of the ribosomal subunits is one of the major activities in each cell and follows a complicated maturation pathway that involves more than 200 non-ribosomal proteins. Although most of the rRNAs for both subunits are initially present on a common pre-rRNA, the precursors for the large and small ribosomal subunit undergo maturation in separate pathways and are exported independently into the cytoplasm where the final maturation steps take place. These steps include the release and recycling of shuttling proteins and export factors as well as the incorporation of the last ribosomal proteins. A key player of these final maturation steps is the protein Drg1, an AAA-ATPase composed of an N-terminal domain and two ATPase domains, which binds to pre-60S particles shortly after their export into the cytoplasm. This project addressed the function of Drg1 and how the protein is recruited to the pre-60S particle. We could show that Drg1 is recruited to the pre60S particle by direct interaction with the shuttling protein Rlp24. Rlp24 joins the pre-60S particle in the nucleolus and is released and recycled in the cytoplasm. Binding of Drg1 to the C-terminal domain of Rlp24 is required and sufficient to recruit the AAA-ATPase to the pre-ribosome. The interaction with Rlp24 results in a strong stimulation of the ATPase activity in both AAA-domains of Drg1. ATP hydrolysis in the D2 domain turned out to be essential to extract Rlp24 from the pre-60S particle, while ATP hydrolysis in the D1 domain is required to liberate Rlp24 from Drg1 for recycling. The release of Rlp24 from pre-60S particles by Drg1 could be investigated in detail by establishing an in vitro release experiment, using purified pre-60S particles, ATP and a short fragment of the nucleoporin Nup116. The release of Rlp24 by Drg1 initiates cytoplasmic pre-60S maturation and is a prerequisite for all downstream maturation steps. Inactivation of Drg1 therefore results in a failure to release other shuttling proteins and export factors and prevents loading of late joining factors and ribosomal proteins. This fact makes drg1 mutants to valuable tools to identify hitherto unrecognized shuttling proteins. The results of our project therefore not only allowed us to gain mechanistic insights into the substrate recognition and activation process of AAA-ATPases but also highlight to importance of cytoplasmic maturation steps for the formation of functional ribosomes.