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 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.
Highlights
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)
RLI1 Is Associated with Specific Translation Initiation Factors in Vivo —To confirm the reported association of RLI1 with eIF5 and a subunit of eukaryotic translation initiation factor 3 (eIF3), we introduced the coding sequences for 13 tandem myc epitopes at the C terminus of chromosomal RLI1 and found that the resulting RLI1-myc strain grew indistinguishably from the parental RLI1 strain
It is possible that the association of eukaryotic translation initiation factor 2 (eIF2) with nonribosomal complexes containing RLI1, eIF3, and eIF5 is labile and does not persist in the postribosomal supernatant, which contains a lower concentration of most initiation factors compared with the whole cell extracts (WCEs)
Summary
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). 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 is an essential protein [12], and in a systematic analysis of protein complexes in yeast [13] it copurified with TAP affinity-tagged forms of the largest subunit of translation initiation factor 3 (eIF3a/TIF32) and initiation factor 5 (eIF5). These findings raised the possibility that RLI1 functions in the initiation of protein synthesis. Description pRS315 PEMBLyex YCplac111 YCplac YEplac195 YEplac181 PDH98 PDH177 PDH178 PDH181 PDH183 PDH184 PDH185 PDH201 PDH202 PDH203 P3924 P3928 PTS068 PFA6a-13Myc-His3MX6 PFA6a-GFP(S65T)-His3MX6 lc LEU2 hc URA3 sc LEU2 lc URA3 hc URA3 hc LEU2 lc LEU2 RLI1-myc in pRS315 backbone sc URA3 RLI1 in YCplac backbone sc URA3 RLI1-F in YCplac backbone sc LEU2 PGAL-UBI-R- FH-RLI1 in YCplac111 backbone sc LEU2 PGAL-UBI-M-FH-RLI1 in YCplac111 backbone sc LEU2 PGAL-UBI-M- FH-RLI1-G224D,G225D in YCplac111 backbone sc LEU2 PGAL-UBI-M-FH-RLI1-G470D,G471D in YCplac111 backbone hc URA3 PGAL-RLI1-F in pEMBLyex backbone lc LEU2 RLI1-E493Q-myc in pRS315 backbone hc URA3 PGAL-RLI1-E493Q-F in pEMBLyex backbone hc URA3 NIP1-His in YEplac195 backbone hc LEU2 NIP1-His in YEplac181 backbone lc URA3 NOP1-GFP a sc, single copy; lc, low copy; hc, high copy
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