The Essential ATP-binding Cassette Protein RLI1 Functions in Translation by Promoting Preinitiation Complex Assembly

Abstract

RLI1 is an essential yeast protein closely related in sequence to two soluble members of the ATP-binding cassette family of proteins that interact with ribosomes and function in translation elongation (YEF3) or translational control (GCN20). We show that affinity-tagged RLI1 co-purifies with eukaryotic translation initiation factor 3 (eIF3), eIF5, and eIF2, but not with other translation initiation factors or with translation elongation or termination factors. RLI1 is associated with 40 S ribosomal subunits in vivo, but it can interact with eIF3 and -5 independently of ribosomes. Depletion of RLI1 in vivo leads to cessation of growth, a lower polysome content, and decreased average polysome size. There was also a marked reduction in 40 S-bound eIF2 and eIF1, consistent with an important role for RLI1 in assembly of 43 S preinitiation complexes in vivo. Mutations of conserved residues in RLI1 expected to function in ATP hydrolysis were lethal. A mutation in the second ATPbinding cassette domain of RLI1 had a dominant negative phenotype, decreasing the rate of translation initiation in vivo, and the mutant protein inhibited translation of a luciferase mRNA reporter in wild-type cell extracts. These findings are consistent with a direct role for the ATP-binding cassettes of RLI1 in translation initiation. RLI1-depleted cells exhibit a deficit in free 60 S ribosomal subunits, and RLI1-green fluorescent protein was found in both the nucleus and cytoplasm of living cells. Thus, RLI1 may have dual functions in translation initiation and ribosome biogenesis. Most of the proteins belonging to the ATP-binding cassette (ABC)1 superfamily of proteins are membrane transporters that use ATP hydrolysis to transport solute molecules against a concentration gradient (1). Typically, they contain two ABCs and a transmembrane domain. The ABC contains a nucleotide binding domain with Walker A and B motifs and an ␣-helical domain bearing the "LSGGQ" signature motif that, along with several other conserved features, distinguishes ABC proteins from other ATPases. ABCs bind ATP as a dimer, with the LSGGQ motif of one cassette capping the ATP molecule bound to the Walker motifs of the second cassette in the dimer. This produces an "ATP sandwich" with two ATP molecules bound to two hybrid active sites formed by the dimerized cassettes. ATP hydrolysis destabilizes the dimer, and it was proposed that a cycle of dimerization driven by ATP binding and hydrolysis can perform mechanical work. For ABC transporters, this would entail opening and closing a solute channel in the membrane through conformational changes in the transmembrane domains (2-6).The yeast proteins GCN20 and YEF3 are soluble ABC proteins whose functions are connected with the binding of tRNAs to ribosomes. YEF3 (eEF3) is an essential translation elongation factor that stimulates release of deacylated tRNA from the ribosomal E-site concurrent with the recruitment of aminoacylated tRNA to the ribosomal A-site (7). GCN20 is a positive regulator of GCN2, a protein kinase that reg...

The biological regulation of ABCE1

“…Because the interferon-dependent 2-5A/RNase L system is not found outside of mammals, whereas ABCE1 protein is universally found in eukarya and archaea, it must have other functions (32). This evidence, no doubt, opens a door for the further study of function of the protein.…”

Rustless translation

“…Studies in flies then showed that ABCE1 (known as Pixie in Drosophila) binds the 40S ribosome in an ATPdependent manner and depletion leads to an increase in in empty 80S ribosomes (Andersen and Leevers , 2007 ). ABCE1 interacts with the translation initiation factor eIF3 through the Hcr1 component in yeast (Dong et al , 2004 ), and with eIF2 and eIF5 in vertebrates (Chen et al , 2006 ). Hcr1 or eIF3 have additional functions, including also the coordination between termination and initiation, ribosomal subunit dissociation and rRNA maturation (Valasek et al , 2001 ), which made the functional interpretation of these interactions difficult.…”

“…For instance, it will be interesting to see whether the FeS domain has more direct functions in splitting as well and/or interaction with the small subunit after splitting. Because ABCE1 also interacts with initiation factors and especially the HCR1 component in yeast (Dong et al , 2004 ), the functional role of ABCE1 on recycled small subunits is another interesting road of investigation.…”

“…First insights into its core function emerged, when the Baillie, Dean, Hinnebusch, Leevers and Marygold labs found ABCE1 (Rli1p) to be involved in the assembly of the translation pre-initiation machinery, to interact with the translation initiation machinery and polysomes in yeast and to be important for translation and in worms, flies and vertebrates (Dong et al , 2004 ;Zhao et al , 2004 ;Coelho et al , 2005 ;Chen et al , 2006 ). At the same time, the Lill and Hurt labs found a functional link between the iron-sulfur clusters of ABCE1 and ribosome biogenesis (Kispal et al , 2005 ;Yarunin et al , 2005 ) while we could crystallize and determine the crystal structure of the twin ABC cassette of archaeal ABCE1 bound to ADP (Karcher et al , 2005 ) ( Figure 2).…”

“…The phylogenetic distribution suggested that ABCE1 is involved in a rather fundamental process in life that is conserved in archaea and eukaryotes, but mechanistically distinct in bacteria. In support of a core role in eukaryotic and archaeal cell physiology, ABCE1 turned out to be essential in all organisms tested (Dong et al , 2004 ;Zhao et al , 2004 ;Coelho et al , 2005 ;Karcher et al , 2005 ;Kispal et al , 2005 ;Yarunin et al , 2005 ).…”