Abstract
RACK1 serves as a scaffold protein for a wide range of kinases and membrane-bound receptors. It is a WD-repeat family protein and is predicted to have a β-propeller architecture with seven blades like a Gβ protein. Mass spectrometry studies have identified its association with the small subunit of eukaryotic ribosomes and, most recently, it has been shown to regulate initiation by recruiting protein kinase C to the 40S subunit. Here we present the results of a cryo-EM study of the 80S ribosome that positively locate RACK1 on the head region of the 40S subunit, in the immediate vicinity of the mRNA exit channel. One face of RACK1 exposes the WD-repeats as a platform for interactions with kinases and receptors. Using this platform, RACK1 can recruit other proteins to the ribosome.
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Acknowledgements
We thank J. Enghild and I. Thøgersen, as well as C. Oxvig and S. Glerup, for mass spectrometry data, T. Heick Jensen for providing the S. cerevisiae RACK1-deleted strain, and B. Jensen for the T. lanuginosus strain. We are grateful to L. Kristensen for help with N-terminal sequencing. We thank M. Watters for preparing the illustrations, W. Li and B. Rath for their advice on the use of the fitting program and D. Lalor for her help with three-dimensional image processing. This work was supported by Howard Hughes Medical Institute and US National Institutes of Health grants R37 GM29169, R01 GM55440, P41 RR01219 and NSF BIR 9219043 (to J.F.), and by an Ole Rømer research grant and the Danish Research Council. the Human Frontier Science Program and the EMBO Young Investigator Program (to P.N.).
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Supplementary information
Supplementary Fig. 1
Purification of T. lanuginosus 80S ribosomes on hydrophobic column. (PDF 118 kb)
Supplementary Fig. 2
Fourier Shell Correlation (FSC) curves for three ribosome samples. (PDF 56 kb)
Supplementary Fig. 3
Sequence alignment for the S. cerevisiae RACK1 protein and G-β protein. (PDF 116 kb)
Supplementary Fig. 4
Stereo view of the homolog model fitted into the cryo-EM density of the S. cerevisiae 11.7 Å 80S map in the 40S subunit head region. (PDF 138 kb)
Supplementary Fig. 5
Interaction of RACK1 with ribosomal components. (PDF 223 kb)
Supplementary Table 1
Mass spectrometry identification of RACK1 in T. lanuginosus 80S ribosome. (PDF 23 kb)
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Sengupta, J., Nilsson, J., Gursky, R. et al. Identification of the versatile scaffold protein RACK1 on the eukaryotic ribosome by cryo-EM. Nat Struct Mol Biol 11, 957–962 (2004). https://doi.org/10.1038/nsmb822
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DOI: https://doi.org/10.1038/nsmb822