The essential ATP-binding cassette protein RLI1 functions in translation by promoting preinitiation complex assembly.
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 ATP-binding 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.
Laboratory of Gene Regulation and Development, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
Jinsheng
J
Dong
Ruby
R
Lai
Klaus
K
Nielsen
Christie A
CA
Fekete
Hongfang
H
Qiu
Alan G
AG
Hinnebusch
D018528:ATP-Binding Cassette Transporters / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000378:metabolism; D039621:Eukaryotic Initiation Factor-3 / Q000378:metabolism; D039642:Eukaryotic Initiation Factors / Q000378:metabolism*; D005656:Fungal Proteins / Q000502:physiology*; D046911:Macromolecular Substances; D011485:Protein Binding; D014176:Protein Biosynthesis; D012270:Ribosomes; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism / Q000502:physiology*
D018528:ATP-Binding Cassette Transporters; D018797:Cell Cycle Proteins; D039621:Eukaryotic Initiation Factor-3; D039642:Eukaryotic Initiation Factors; D005656:Fungal Proteins; D046911:Macromolecular Substances; C492990:RLI1 protein, S cerevisiae; C111889:RPG1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins