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Myosin VI targeting to clathrin-coated structures and dimerization is mediated by binding to Disabled-2 and PtdIns(4,5)P2

Nature Cell Biology volume 9, pages 176–183 (2007)Cite this article

Abstract

Vesicle transport is essential for the movement of proteins, lipids and other molecules between membrane compartments within the cell. The role of the class VI myosins in vesicular transport is particularly intriguing because they are the only class that has been shown to move 'backwards' towards the minus end of actin filaments1. Myosin VI is found in distinct intracellular locations and implicated in processes such as endocytosis2,3, exocytosis, maintenance of Golgi morphology4,5 and cell movement6. We have shown that the carboxy-terminal tail is the key targeting region and have identified three binding sites: a WWY motif for Disabled-2 (Dab2) binding, a RRL motif for glucose-transporter binding protein (GIPC) and optineurin binding and a site that binds specifically and with high affinity (Kd = 0.3 μM) to PtdIns(4,5)P2-containing liposomes. This is the first demonstration that myosin VI binds lipid membranes. Lipid binding induces a large structural change in the myosin VI tail (31% increase in helicity) and when associated with lipid vesicles, it can dimerize. In vivo targeting and recruitment of myosin VI to clathrin-coated structures (CCSs) at the plasma membrane is mediated by Dab2 and PtdIns(4,5)P2 binding.

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Figure 1: Two distinct 'hot spots' for binding Dab2 and GIPC were identified on the myosin VI tail.
Figure 2: The C-terminal half of the myosin VI tail binds to liposomes and is composed of two independent domains.
Figure 3: Myosin VI binds specifically to PtdIns(4,5)P2-containing liposomes.
Figure 4: Liposome binding increases the helical structure of the C-terminal tail of myosin VI and induces dimerization.
Figure 5: Mutations in the PtdIns(4,5)P2 binding site reduce the targeting of myosin VI to clathrin-coated structures in vivo.

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Acknowledgements

We thank: B. Peter and H. McMahon for help with lipid binding assays and discussion; R. Williams, D. Veprintsev and O. Perisic for programmes and help with the FRET assay; S. Peak-Chew and F. Begum for N-terminal sequencing and mass spectrometry; and D. Owen for critical reading of the manuscript. The work was funded by a USA Royal Society Postdoctoral Fellowship (G.S.), a Croucher Foundation (Hong Kong) Student Scholarship (J.S.A.), a Wellcome Trust Senior Fellowship (F.B.) and was supported by the Medical Research Council. The Cambridge Institute for Medical Research is in receipt of a strategic award from the Wellcome Trust.

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Authors and Affiliations

  1. MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

    Giulietta Spudich, Josephine Sui-Yan Au & John Kendrick-Jones

  2. Cambridge Institute for Medical Research, University of Cambridge, Wellcome/MRC Building, Hills Road, Cambridge, CB2 2XY, UK

    Margarita V. Chibalina, Susan D. Arden & Folma Buss

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  1. Giulietta Spudich
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Each of the authors made a significant contribution to the experimental work described.

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Correspondence to John Kendrick-Jones.

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Spudich, G., Chibalina, M., Au, JY. et al. Myosin VI targeting to clathrin-coated structures and dimerization is mediated by binding to Disabled-2 and PtdIns(4,5)P2. Nat Cell Biol 9, 176–183 (2007). https://doi.org/10.1038/ncb1531

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