Purified yeast translational initiation factor eIF-3 is an RNA-binding protein complex that contains the PRT1 protein.

T Naranda,S E Macmillan,J W Hershey

doi:10.1016/s0021-9258(18)31633-8

T Naranda, S E Macmillan + Show 1 more

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https://doi.org/10.1016/s0021-9258(18)31633-8

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Abstract

Eukaryotic initiation factor-3 (eIF-3) plays a pivotal role in the initiation phase of protein synthesis where it promotes dissociation of 80 S ribosomes into subunits, stabilizes methionyl-tRNAi binding to 40 S ribosomal subunits, and is required for mRNA binding. Mammalian eIF-3 is comprised of eight subunits, but no mammalian cDNA encoding these proteins has been cloned and sequenced, nor has the corresponding factor been characterized in yeast. Since many initiation factors are strongly conserved between mammalian and yeast systems, we employed a mammalian assay for initiation, the synthesis of methionyl-puromycin, to detect eIF-3 activity in yeast subcellular fractions. Yeast eIF-3 was purified from the high salt wash of ribosomes by Superose 6 molecular sieve and MonoS ion exchange chromatography. Yeast eIF-3 contains eight subunits with masses of 16, 21, 29, 33, 39, 62, 90, and 135 kilodaltons all of which coelute with an apparent mass of 550 kilodaltons from the Superose 6 column. Immunoblotting shows that the 90-kDa subunit corresponds to the product of the PRT1 gene whose mutant form, prt1-1, exhibits destabilization of methionyl-tRNAi binding to 40 S ribosomal subunits. eIF-3, and specifically the 62-kDa subunit, bind to RNA. These biochemical approaches to defining yeast eIF-3 complement genetic methods so far used in characterizing the initiation factors and provide another route to defining the yeast translational machinery.

Highlights

Eukaryotic initiation factor-3 plays a pivotal tion factorsso far identified in mammaliancells (3).A detailed role in the initiation phase of protein synthesis where it biochemical characterization of the initiation pathway in yeast promotes dissociation of 80 S ribosomes into subunits, is lacking, and no functional role for these putative initiation stabilizes methionyl-tRNA,bindingto 40 S ribosomal factors has been demonstrated.a number of subunits, and is required for mRNA binding
Thehigh extent of sequence relatedness and the ability of many mam- Purification of the Yeast Eukaryotic initiation factor-3 (eIF-3) ComplexS. cereuisiae strain W303 malian cDNAs to function in vivoin place of the corresponding (MATa, leu2-3, his3-11, ade2-1, trpl-1, canl-100) was grown to an yeast genes indicate that the initiationprocess is highly conserved between mammalian and yeast cells
Optimized amounts of HeLa initiation factors were added to the assay as follows: 0.9 pg of eIF-2, 0.6 pg of eIF-5, 0.27 pg of eIF-lA, 0.96 pgof eIF-5A,and either3.12 pgof eIF-3 or the yeast eIF-3 preparation being about 3-fold bypurified HeLa eIF-3 (Fig. 1B).To test for stimulation by putative yeast eIF-3, we first fractionated the yeast high salt wash fraction on a Superose-12column which separates proteins according to size

Summary

MATERIALSAND METHODS

Extent of sequence relatedness and the ability of many mam- Purification of the Yeast eIF-3 ComplexS. cereuisiae strain W303 malian cDNAs to function in vivoin place of the corresponding (MATa, leu, his, ade, trpl-1, canl-100) was grown to an yeast genes indicate that the initiationprocess is highly conserved between mammalian and yeast cells. The abbreviationsused are: eIF, eukaryotic initiation factor; FPLC, fast protein liquid chromatography; AEBSF4, -(2-aminoethyl)-benzenesulfonylfluoride; PAGE, polyacrylamidegel electrophoresis; B-RNA,b1otinylated RNA Met-PM, methionyl-puromycin;CAPS, 3-(cyclohexylamino)propanesulfonic acid. 2 mM Mg(OAc),, 2 mM dithiothreitol) containing protease inhibitors temperature for 5 min with buffer containing 20 mM HEPES, pH 7.5,. The ribosomal high salt wash BiotinylatedRNA Binding-For making biotinylated RNA (B-RNA), enriched in initiation factors was dialyzed against buffer A containing 100 p~ allylamine-UTPwas added to the standard SP6 transcription protease inhibitors, quickfrozen in liquid nitrogen, and stored at mixture described aboveA. The allylamine groups of the RNA transcripts were treated with 6 gel filtration column of a fast protein liquid chromatography system the reagent Enzotin (Enzo-Diagnostics) accordintgo the manufacturer's (FPLC, Pharmacia LKB Biotechnology Inc.) in batches of 6 mg of pro- protocol. RNAcomplexeswere separated from free eIF-3 by rapid tified by the methionyl-puromycinsynthesis assay (17)and were pooled centrifugation of the beads, followed by SDS-PAGE and Western blot together. Column (FPLC, Pharmacia LKB Biotechnology Inc.) by elution with a linear 100450m~ KC1gradient in buffer C

RESULTS

Factor tested in

DISCUSSION