Complex Formation by All Five Homologues of Mammalian Translation Initiation Factor 3 Subunits from Yeast Saccharomyces cerevisiae*
- From the Laboratory of Eukaryotic Gene Regulation, NICHD, National Institutes of Health, Bethesda, Maryland 20892
Abstract
The PRT1, TIF34, GCD10, and SUI1 proteins ofSaccharomyces cerevisiae were found previously to copurify with eukaryotic translation initiation factor 3 (eIF3) activity. Although TIF32, NIP1, and TIF35 are homologous to subunits of human eIF3, they were not known to be components of the yeast factor. We detected interactions between PRT1, TIF34, and TIF35 by the yeast two-hybrid assay and in vitro binding assays. Discrete segments (70–150 amino acids) of PRT1 and TIF35 were found to be responsible for their binding to TIF34. Temperature-sensitive mutations mapping in WD-repeat domains of TIF34 were isolated that decreased binding between TIF34 and TIF35 in vitro. The lethal effect of these mutations was suppressed by increasing TIF35 gene dosage, suggesting that the TIF34-TIF35 interaction is important for TIF34 function in translation. Pairwise in vitrointeractions were also detected between PRT1 and TIF32, TIF32 and NIP1, and NIP1 and SUI1. Furthermore, PRT1, NIP1, TIF34, TIF35, and a polypeptide with the size of TIF32 were specifically coimmunoprecipitated from the ribosomal salt wash fraction. We propose that all five yeast proteins homologous to human eIF3 subunits are components of a stable heteromeric complex in vivo and may comprise the conserved core of yeast eIF3.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Supported by the Japan Society for the Promotion of Science Research Fellowships for Japanese Biomedical and Behavioral Researchers at the National Institutes of Health.
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↵§ To whom correspondence should be addressed. Tel.: 301-496-4480; Fax: 301-496-6828; E-mail: ahinnebusch{at}nih.gov.
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↵1 The abbreviations used are: eIF, eukaryotic translation initiation factor; PCR, polymerase chain reaction; ORF, open reading frame; kb, kilobase(s); GST, glutathioneS-transferase; UTR, untranslated region; SC, synthetic complete (medium); 5-FOA, 5-fluoroorotic acid; 3-AT, 3-aminotriazole; bp, base pair(s); WCE, whole cell extract(s); RSW, ribosomal salt wash; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin.
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↵2 K. Block, H.-P. Vornlocher, K. Asano, and J. Hershey, manuscript in preparation. The amino acid sequence of human eIF3-p44 was made available by J. Hershey.
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↵3 J. Anderson, K. Asano, and A. G. Hinnebusch, unpublished observations.
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↵4 R. Cuesta, O. Calvo, J. Anderson, M. T. Garcia Barrio, A. G. Hinnebusch, and M. Tamame, manuscript in preparation.
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↵5 J. Anderson, M. Pak, L. Phan, R. Cuesta, K. Asano, M. Tamame, and A. G. Hinnebusch, submitted for publication.
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- Received January 5, 1998.
- Revision received April 13, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
