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.
Dong
Jinsheng
J
Laboratory of Gene Regulation and Development, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
Lai
Ruby
R
Nielsen
Klaus
K
Fekete
Christie A
CA
Qiu
Hongfang
H
Hinnebusch
Alan G
AG
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