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J. Biol. Chem., Vol. 279, Issue 10, 8946-8956, March 5, 2004

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The j-Subunit of Human Translation Initiation Factor eIF3 Is Required for the Stable Binding of eIF3 and Its Subcomplexes to 40 S Ribosomal Subunits in Vitro^*

Christopher S. Fraser, Jennifer Y. Lee, Greg L. Mayeur, Martin Bushell, Jennifer A. Doudna¶, and John W. B. Hershey||

From the Department of Biological Chemistry, School of Medicine, University of California, Davis, California 95616

Eukaryotic initiation factor 3 (eIF3) is a 12-subunit protein complex that plays a central role in binding of initiator methionyl-tRNA and mRNA to the 40 S ribosomal subunit to form the 40 S initiation complex. The molecular mechanisms by which eIF3 exerts these functions are poorly understood. To learn more about the structure and function of eIF3 we have expressed and purified individual human eIF3 subunits or complexes of eIF3 subunits using baculovirus-infected Sf9 cells. The results indicate that the subunits of human eIF3 that have homologs in Saccharomyces cerevisiae form subcomplexes that reflect the subunit interactions seen in the yeast eIF3 core complex. In addition, we have used an in vitro 40 S ribosomal subunit binding assay to investigate subunit requirements for efficient association of the eIF3 subcomplexes to the 40 S ribosomal subunit. eIF3j alone binds to the 40 S ribosomal subunit, and its presence is required for stable 40 S binding of an eIF3bgi subcomplex. Furthermore, purified eIF3 lacking eIF3j binds 40 S ribosomal subunits weakly, but binds tightly when eIF3j is added. Cleavage of a 16-residue C-terminal peptide from eIF3j by caspase-3 significantly reduces the affinity of eIF3j for the 40 S ribosomal subunit, and the cleaved form provides substantially less stabilization of purified eIF3–40S complexes. These results indicate that eIF3j, and especially its C terminus, play an important role in the recruitment of eIF3 to the 40 S ribosomal subunit.

Received for publication, November 21, 2003 , and in revised form, December 18, 2003.

^* This work was supported by Grant GM-22135 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Molecular and Cell Biology, and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720.

Supported by Grant 063233/B/00/Z from the Wellcome Trust while in the laboratory of Dr. P. Sarnow. Present address: Dept. of Microbiology and Immunology, Stanford University School of Medicine, 299 Campus Dr., Stanford, CA 94305.

^¶ Present address: Dept. of Molecular and Cell Biology, and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720.

^|| To whom correspondence should be addressed: Dept. of Biological Chemistry, School of Medicine, University of California, Davis, CA 95616. Tel.: 530-752-3235; Fax: 530-752-3516; E-mail: jwhershey{at}ucdavis.edu.

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