The Novel ATP-Binding Cassette Protein ARB1 Is a Shuttling Factor That Stimulates 40S and 60S Ribosome Biogenesis

Abstract

ARB1 is an essential yeast protein closely related to members of a subclass of the ATP-binding cassette (ABC) superfamily of proteins that are known to interact with ribosomes and function in protein synthesis or ribosome biogenesis. We show that depletion of ARB1 from Saccharomyces cerevisiae cells leads to a deficit in 18S rRNA and 40S subunits that can be attributed to slower cleavage at the A0, A1, and A2 processing sites in 35S pre-rRNA, delayed processing of 20S rRNA to mature 18S rRNA, and a possible defect in nuclear export of pre-40S subunits. Depletion of ARB1 also delays rRNA processing events in the 60S biogenesis pathway. We further demonstrate that ARB1 shuttles from nucleus to cytoplasm, cosediments with 40S, 60S, and 80S/90S ribosomal species, and is physically associated in vivo with TIF6, LSG1, and other proteins implicated previously in different aspects of 60S or 40S biogenesis. Mutations of conserved ARB1 residues expected to function in ATP hydrolysis were lethal. We propose that ARB1 functions as a mechanochemical ATPase to stimulate multiple steps in the 40S and 60S ribosomal biogenesis pathways.Ribosome synthesis takes place primarily in the nucleus of eukaryotic cells and involves the processing of a large precursor rRNA containing the sequences of 18S rRNA, found in mature 40S subunits, and the 5.8S and 25S rRNAs found in 60S subunits. A large ensemble of trans-acting factors, the U3 snoRNP complex, and ribosomal proteins (mostly of the 40S subunit), interact with the pre-rRNA to form the 90S preribosome, or small subunit processome. Three cleavage reactions that excise the 20S precursor of 18S rRNA from the 35S rRNA precursor (at sites A0, A1, and A2) (see Fig. 2G) take place in the Ͼ2-MDa 90S complex (reviewed in references 15, 25, and 70). The pre-40S particle released after cleavage at sites A0-A1-A2 is transported to the cytoplasm with a relatively small number of factors, some of which also reside in the 90S complex, while others appear to join with the pre-40S particle after its separation from the pre-60S particle (62). Cleavage of 20S pre-rRNA to mature 18S rRNA occurs following export of the pre-40S particle to the cytoplasm. The pre-60S particle contains 27SA pre-rRNA (see Fig. 2G) and a large array of trans-acting factors not found in the 90S particle. It undergoes a series of RNA cleavage reactions that excise 25S and 5.8S from the 27SA pre-rRNA before being exported from the nucleus. Only a small number of factors present in the 90S particle also occur in pre-60S particles, and it is thought that such factors dissociate from the pre-60S particle early in its maturation. Hence, there are few factors found stably associated with both pre-40S and pre-60S particles (52, 70).

Domain and Nucleotide Dependence of the Interaction between Saccharomyces cerevisiae Translation Elongation Factors 3 and 1A

“…However, the soluble members of this family, including Gcn20p, RL11 (41), eEF3, and the recently reported ARB1 (42) in yeast are also implicated in functions related to protein synthesis, ribosome biogenesis, and translation elongation. The crystal structure of several members of the class I ATPases clearly establish the phenomena of homodimerization of two ABC proteins to sandwich two ATP molecules utilizing the Walker A and B motifs of the one monomer (43,44) and Walker C or the conserved LSGGQ motif, characteristic of only the ABC members of the ATPases superfamily, from the other monomer.…”

Powering through ribosome assembly

“…In a highly unusual manner, Arb1 and Rli1 are required for LSU and SSU rRNA processing and maturation; they are both shuttling proteins that mainly reside in the cytoplasm and are both required for export of nascent subunits (Dong et al 2005;Kispal et al 2005;Yarunin et al 2005). Furthermore, while Arb1 binds to pre-60S ribosomes, its depletion reduces the amount of 40S subunits relative to 60S.…”

“…While Arb1 is an essential protein, its depletion only leads to moderate defects in SSU and LSU rRNA processing (Dong et al 2005), suggesting either that it acts after rRNA processing is complete, or that its essential role is in a different cellular process (or both). Consistent with both of these possibilities, Arb1 interacts with the very-late cytoplasmic 60S assembly factors Tif6 (see Fig.…”

“…1, shown in cyan; Dong et al 2004Dong et al , 2005Kispal et al 2005;Yarunin et al 2005). Mutation of conserved residues predicted to be involved in ATP binding and hydrolysis is lethal for both Rli1 and Arb1, suggesting that ATPase activity is central to the essential roles of both proteins (Dong et al 2004(Dong et al , 2005.…”

“…1, shown in cyan; Dong et al 2004Dong et al , 2005Kispal et al 2005;Yarunin et al 2005). Mutation of conserved residues predicted to be involved in ATP binding and hydrolysis is lethal for both Rli1 and Arb1, suggesting that ATPase activity is central to the essential roles of both proteins (Dong et al 2004(Dong et al , 2005. Rli1 was initially identified as a factor involved in translation initiation and copurifies with the multi-subunit initiation factor eIF3 via direct interaction with the Hcr1 subunit (Dong et al 2004;Kispal et al 2005;Yarunin et al 2005;Andersen and Leevers 2007).…”