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Molecular and Cellular Biology, March 2009, p. 1626-1634, Vol. 29, No. 6
0270-7306/09/$08.00+0     doi:10.1128/MCB.01718-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Direct Link between RACK1 Function and Localization at the Ribosome In Vivo

Scott M. Coyle,² Wendy V. Gilbert,^2, and Jennifer A. Doudna^1,2,3,4*

Howard Hughes Medical Institute,¹ Department of Molecular and Cell Biology,² Department of Chemistry, University of California, Berkeley, Berkeley, California 94720,³ Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720⁴

Received 7 November 2008/ Returned for modification 15 December 2008/ Accepted 20 December 2008

The receptor for activated C-kinase (RACK1), a conserved protein implicated in numerous signaling pathways, is a stoichiometric component of eukaryotic ribosomes located on the head of the 40S ribosomal subunit. To test the hypothesis that ribosome association is central to the function of RACK1 in vivo, we determined the 2.1-Å crystal structure of RACK1 from Saccharomyces cerevisiae (Asc1p) and used it to design eight mutant versions of RACK1 to assess roles in ribosome binding and in vivo function. Conserved charged amino acids on one side of the β-propeller structure were found to confer most of the 40S subunit binding affinity, whereas an adjacent conserved and structured loop had little effect on RACK1-ribosome association. Yeast mutations that confer moderate to strong defects in ribosome binding mimic some phenotypes of a RACK1 deletion strain, including increased sensitivity to drugs affecting cell wall biosynthesis and translation elongation. Furthermore, disruption of RACK1's position at the 40S ribosomal subunit results in the failure of the mRNA binding protein Scp160 to associate with actively translating ribosomes. These results provide the first direct evidence that RACK1 functions from the ribosome, implying a physical link between the eukaryotic ribosome and cell signaling pathways in vivo.

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* Corresponding author. Mailing address: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720. Phone: (510) 643-0225. Fax: (510) 643-0080. E-mail: doudna{at}berkeley.edu

Published ahead of print on 29 December 2008.

Present address: Dept. of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.

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Molecular and Cellular Biology, March 2009, p. 1626-1634, Vol. 29, No. 6
0270-7306/09/$08.00+0     doi:10.1128/MCB.01718-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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